department of medical microbiology medical and health science center university of Debrecen
Dr. Krisztina Szarka – Dr. Gábor Kardos
Pharmaceutical microbiology I. Antimicrobial Procedures and Chemotherapy Lecture notes for pharmacy students
Debrecen University Press 2010 Reviewed by Prof. Dr. Lajos Gergely
ISBN 978-963-318-021-1
© Debreceni Egyetemi Kiadó Debrecen University Press, beleértve az egyetemi hálózaton belüli elektronikus terjesztés jogát is
Kiadta a Debreceni Egyetemi Kiadó Debrecen University Press Felelős kiadó: Dr. Virágos Márta Készült a DE sokszorosítóüzemében, 2010-ben 10-328 Table of contents
Sterilization and disinfection (Krisztina Szarka) ...... 1
The kinetics of the destruction of microbes ...... 1 Factors influencing the efficacy of sterilization/disinfection...... 3 Physical methods for sterilization and disinfection...... 5 High Temperature...... 5 Mechanisms of thermal injury ...... 5 Application of moist heat ...... 5 Application of dry heat ...... 6 The control of the efficacy of heat sterilization ...... 6 Cold, freezing ...... 7 Radiation ...... 7 Ultraviolet (UV) radiation ...... 7 Ionizing radiation ...... 8 Ultrasound ...... 9 Filtration ...... 9 Gas sterilization, plasma sterilization...... 9 Spectrum of efficacy of physical methods...... 10 Chemical methods for sterilization and disinfection ...... 11 Determination of the efficacy of the disinfectants ...... 11 Groups of disinfectants...... 15 Disinfectants damaging the cell membrane...... 16 Surface-active compounds ...... 16 Cation-active tensides...... 16 Anion-active tensides...... 17 Non-ionic tensides ...... 18 Amphoteric tensides ...... 18 Alcohols ...... 19 Phenol and phenolics ...... 19 Biguanides ...... 20 Protein denaturing agents...... 21 Acids and alkali compounds ...... 21 Agents modifying the functional groups of proteins and nucleic acids...... 22 Heavy metal compounds ...... 22 Oxidizing agents ...... 23 Halogen derivatives ...... 23 Ozone ...... 24 Peroxides ...... 25 Dyes ...... 25 Aniline dyes...... 26 Acridine dyes...... 26 Alkylating agents ...... 26 Aldehydes...... 27 Ethylene-oxide...... 28 β-propiolactone...... 28 Disinfectant combinations and disinfectant systems ...... 28 Spectrum of activity of disinfectants...... 29 Microbial contamination and spoilage of pharmaceuticals. Preservation of pharmaceuticals...... 30 Sensitivity of formula components to microbial degradation...... 30 Therapeutic agents (drugs) ...... 30 Surface-active agents ...... 31 Organic polymers ...... 31 Moisturizing agents ...... 31 Fats and oils ...... 31 Sweeteners, aromas and colouring agents ...... 31
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Preservatives and disinfectants ...... 32 Visible signs of microbial degradation in pharmaceutical formulae ...... 32 Factors influencing the microbial spoilage ...... 32 Type and number of colonizing microorganisms ...... 32 Nutrients ...... 33 Moist content – water activity ...... 33 Redox potential ...... 34 Storage temperature ...... 34 pH ...... 34 Packaging ...... 34 Resistance of microorganisms in pharmaceutical products ...... 35 Health risk arising from microbial contamination...... 35 Potential sources and prevention of contamination...... 36 Contamination during the manufacturing process ...... 36 Production in the hospital pharmacy – magistral formulations...... 36 Water quality ...... 36 Manufacturing environment...... 36 Packaging...... 37 Contamination during use ...... 37 Contamination of human origin ...... 37 Environmental sources...... 38 Contamination originating from devices and equipment ...... 38 Factors influencing the outcome of pharmaceutical-transmitted infections ...... 38 Type and extent of microbial contamination ...... 39 Mode of administration ...... 39 Resistance of the patients to infection ...... 39 Preservation of pharmaceutical products ...... 40 Factors affecting the efficacy of preservatives ...... 40 Control of the microbiological quality of the products ...... 41 Quality assurance during formula design and development ...... 41 Good pharmaceutical manufacturing practice ...... 42 Quality control processes ...... 42 Resistance against disinfectants...... 43 Bacterial resistance to disinfectants...... 44 Natural resistance of bacteria...... 44 Intrinsic resistance of Gram positive bacteria ...... 44 Intrinsic resistance of Gram negative bacteria ...... 45 Intrinsic resistance of mycobacteria ...... 45 Intrinsic resistance of bacterial spores ...... 45 Biofilm production as a mechanism of the intrinsic resistance ...... 46 Acquired resistance of bacteria to disinfectants...... 46 Resistance of bacteria transferred by mobile genetic elements ...... 47 Resistance of bacteria due to mutations ...... 47 Phenotypic adaptation as a part of acquired resistance ...... 47 Fungal resistance to disinfectants ...... 48 Viral resistance to disinfectants ...... 48 Protozoal resistance to disinfectants...... 49 Resistance of prions to disinfectants...... 49 Disinfectant policy...... 49
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Antimicrobial chemotherapy (Gábor Kardos) ...... 52
Classification of antimicrobial agents...... 52 Principles of antibacterial and antifungal therapy ...... 53 Resistance to antibiotics...... 55 Classification of resistance ...... 55 Development and spread of resistance...... 56 Genetic background of resistance ...... 58 Possible mechanisms of resistance ...... 59 Alteration of the chemical structure of the drug...... 59 Cleavage...... 59 Enzymatic modification ...... 60 Decrease of intracellular concentration of the drug ...... 60 Low permeability/permeability decrease...... 60 Active efflux...... 61 Changes connected with the target...... 62 Target modification...... 62 Overproduction of the target ...... 62 Production of a new target ...... 62 Development of a metabolic bypass...... 62 Protection of the target...... 63 Decreased activation...... 63 Tolerance to antibiotics ...... 63 Antibiotic susceptibility testing ...... 64 Antibiotic susceptibility testing methods ...... 65 Quantitative methods...... 66 Methods to determine MIC ...... 66 Agar dilution method...... 66 Broth dilution method...... 67 E-test®...... 68 Determination of MBC/MFC ...... 68 Determination of SBT ...... 68 Time-kill tests (curves) ...... 69 Checkerboard dilution ...... 69 Semiquantitative methods ...... 71 Breakpoint determination ...... 71 Resistance screening ...... 71 Disc-diffusion (Kirby-Bauer) method ...... 71 Semiautomated and automated susceptibility testing ...... 72 Comparison of susceptibility testing methods...... 72 Direct demonstration of resistance mechanisms...... 73 Demonstration of proteins responsible for resistance ...... 73 Demonstration of β-lactamases ...... 73 Demonstration of ESBLs...... 73 Demonstration of MBLs...... 74 Demonstration of PBP2a responsible for β-lactam resistance of MRSA ...... 74 Direct demonstration of resistance genes ...... 74 Interpretation of in vitro susceptibility...... 74 Using antibiotic susceptibility testing for diagnostic purposes ...... 75 Mathematical description of the antibiotic effect...... 75 Pharmacodynamic parameters ...... 76 Pharmacodynamic model systems ...... 78 In vitro models...... 78 Animal models...... 78 Using clinical data ...... 78 Pharmacodynamics of resistance development...... 78
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Development of antimicrobial agents...... 80 Sources of compounds with antimicrobial activity ...... 80 The drug development process ...... 81 Problems with development of new antibiotics...... 82 Antibiotic policy (antibiotic stewardship) ...... 83 Data collection...... 84 Measuring antibiotic consumption ...... 85 Regulation of antibiotic usage...... 86 Training ...... 88 Antibacterial agents ...... 89 Drugs targeting cell wall synthesis ...... 89 β-lactam antibiotics ...... 89 Penicillins ...... 90 Early penicillins ...... 90 βββ-lactamase- (penicillinase-) stable penicillins...... 90 Broad spectrum penicillins ...... 91 Aminopenicillins...... 91 Carboxypenicillins ...... 91 Ureidopenicillins...... 91 Temocillin...... 92 Mecillinam (amdinocillin) ...... 92 Penicillins protected with β-lactamase inhibitor ...... 92 Cephalosporins ...... 93 1st generation (drugs licensed before 1978) ...... 94 2nd generation (drugs licensed between 1978-1981) ...... 94 3rd generation (drugs licensed after 1981)...... 95 4th generation ...... 97 ‘5 th generation’...... 97 Cefamycins ...... 97 Carbapenems ...... 98 Monobactams ...... 99 Mechanism of resistance to β-lactam antibiotics ...... 99 Resistance due to production of β-lactamases...... 99 Important β-lactamases by functional groups...... 101 Phylogenetic classification of the clinically most important β-lactamases ...... 105 Clinically important β-lactamases by groups of bacteria ...... 105 Resistance conferred by alteration of PBPs...... 106 Resistance caused by alternative cell wall synthesis...... 107 Resistance based on decreased permeability (slower drug uptake) ...... 107 Efflux-based resistance...... 107 Glycopeptides ...... 108 Mechanisms of glycopeptide resistance ...... 109 Lipoglycopeptides ...... 110 Daptomycin ...... 111 Fosfomycin (fosfonomycin) ...... 111 Mechanisms of fosfomycin resistance ...... 111 Lipoglycodepsipeptides ...... 111 Drugs targeting the cell membrane ...... 112 Polymyxins ...... 112 Daptomycin ...... 113 Lipoglycopeptides ...... 113 Drugs targeting the ribosome...... 115 Aminoglycosides and aminocyclitoles ...... 115 Mechanisms of aminoglycoside resistance ...... 117 Tetracyclines ...... 119 Mechanisms of tetracycline resistance ...... 119 Glycylcyclines ...... 120 Chloramphenicol ...... 120
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Mechanisms of chloramphenicol resistance ...... 120 Macrolide antibiotics ...... 121 Macrolides in the narrow sense ...... 121 Azalides...... 122 Ketolides...... 123 Mechanisms of macrolide resistance ...... 123 Lincosamides ...... 125 Mechanisms of lincosamide resistance ...... 125 Streptogramins ...... 126 Mechanisms of streptogramin resistance ...... 127 Oxazolidinones ...... 128 Mechanisms of oxazolidinone resistance ...... 128 Fusidic acid ...... 128 Pleuromutilines ...... 128 Inhibitors of nucleic acid metabolism...... 129 Quinolones, fluoroquinolones ...... 129 1st generation ...... 129 Norfloxacin...... 130 2nd generation...... 130 3rd generation...... 130 4th generation ...... 131 „5 th generation”...... 131 Mechanisms of quinolone resistance ...... 131 Rifamycins ...... 133 Mechanisms of rifamycin resistance ...... 133 Drugs targeting folic acid metabolism...... 134 Sulfonamides ...... 134 Mechanisms of sulfonamide resistance ...... 134 Trimethoprim ...... 135 Mechanisms of trimethoprim resistance ...... 135 Drugs with miscellaneous mechanism of action ...... 136 Metronidazole ...... 136 Nitrofurantoin ...... 136 Methenamine ...... 136 Mupirocin ...... 137 Nitazoxanide ...... 137 Drugs inhibiting potential new targets ...... 137 The most important natural resistances according to pathogens...... 138 Antituberculotic agents ...... 139 Antituberculotic susceptibility testing...... 139 The proportion method ...... 140 The radiometric method...... 140 MGIT (Mycobacterium Growth Indicator Tube) method...... 140 First-line antituberculotics ...... 141 Isonicotinic acid-hydrazide (isoniazide, INH) ...... 141 Mechanisms of INH resistance ...... 142 Rifamycins ...... 142 Pyrazinamide ...... 143 Ethambuthol ...... 143 Streptomycin ...... 143 Second-line antituberculotics...... 143 Fluoroquinolones ...... 144 Paraamino-salicylic acid (PAS) ...... 144 Cycloserine ...... 144 Ethionamide, prothionamide ...... 144 Capreomycin, viomycin ...... 145 Amikacin, kanamycin, streptomycin ...... 145 Thioacetazone ...... 145 Principles of antituberculotic therapy...... 146
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Drugs against leprosy ...... 146 Dapsone ...... 146 Clofazimine ...... 146 Rifamycins (rifampin) ...... 147 Other drugs ...... 147 Drugs against atypical mycobacteria ...... 147 Macrolides ...... 147 Miscellaneous drugs ...... 147 Other drugs ...... 147 Antifungal agents ...... 148 Polyenes ...... 150 Amphotericin B ...... 150 Nystatin...... 151 Natamycin...... 151 Mechanisms of resistance to polyenes ...... 151 Azole antifungals ...... 152 Imidazoles ...... 152 Triazoles...... 153 Mechanisms of resistance to azole antifungals ...... 154 5-fluorocytosine (flucytosine) ...... 155 Mechanisms of flucytosine resistance ...... 155 Allylamines ...... 156 Morpholines (amorolfine) ...... 156 Griseofulvin ...... 156 Echinocandins ...... 157 Drugs active against Pneumocystis jiroveczii ( carinii ) ...... 158 Drugs used in microsporidiosis...... 158 Antiprotozoal drugs...... 159 Drugs against protozoa parasitizing body cavities...... 159 Nitroimidazoles ...... 160 Quinacrine ...... 160 Furazolidone ...... 161 Paromomycin ...... 161 Emetine ...... 161 Iodoquinol ...... 161 Diloxanide furoate ...... 161 Tetracyclines ...... 162 Nitazoxanide ...... 162 Albendazole (benzimidazoles) ...... 162 Other drugs ...... 162 Antimalarial agents...... 162 Drugs targeting folic acid metabolism...... 164 Sulfonamides and sulfones (type 1 folic acid antagonists) ...... 164 Diaminopyrimidines (type II folic acid antagonists)...... 164 Biguanides (type II folic acid antagonists) ...... 165 Drugs targeting mitochondria...... 165 Naphtoquinones ...... 165 8-aminoquinolines ...... 166 Tafenoquine ...... 166 Blood schizontocidal drugs...... 167 Quinolines and their structural analogues ...... 167 4-aminoquinolines (type 1 blood schizontocidal drugs) ...... 167 Aminoacridines (type 1 blood schizontocidal drugs)...... 168 Bis-quinolines (type 1 blood schizontocidal drugs)...... 168 Cinchona alcaloids (type 2 blood schizontocidal drugs) ...... 169 Quinoline-methanoles (type 2 blood schizontocidal drugs)...... 169 Halofantrine (type 2 blood schizontocidal drug) ...... 170 Lumefantrine (benflumetol) (type 2 blood schizontocidal drug) ...... 170
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Artemisinin-derivatives ...... 170 Rarely used antimalarials ...... 171 Tetracyclines ...... 171 Clindamycin ...... 171 Macrolides ...... 171 Antimalarial drug combinations...... 172 Treatment and chemoprophylaxis of malaria ...... 173 Drugs against Toxoplasma...... 173 Drugs against Trypanosoma spp...... 174 Diamidines (pentamidine) ...... 174 Suramin ...... 175 Organic arsenicals ...... 175 Eflornithine ...... 176 Nitrofurans (Nifurtimox) ...... 176 Nitroimidazoles ...... 176 Other drugs against Trypanosoma spp...... 177 Drugs against Leishmania spp...... 177 Organic antimony derivatives (antimonials) ...... 178 Amphotericin-B ...... 179 Paromomycin (Aminosidine)...... 179 Phosphocholines ...... 179 Azoles ...... 180 Diamidines ...... 180 Allopurinol ...... 180 Other drugs ...... 180 Drugs against free-living amoebae ...... 181 Drugs of first choice in protozoal infections...... 181 In vitro testing of the susceptibility of protozoa ...... 182 Anthelminthic drugs ...... 183 Drugs affecting the microtubular system of worms ...... 184 Benzimidazoles ...... 184 Drugs affecting the nervous system of the worms ...... 185 Drugs acting on excitatory (acetylcholinerg) synapses ...... 185 Imidazolothiazoles ...... 185 Tetrahydropyrimidines ...... 186 Bephenium ...... 186 Metrifonate...... 186 Drugs acting on inhibitory synapses ...... 186 Macrocyclic lactones (avermectins) ...... 186 Piperazine ...... 187 Drugs affecting the carbohydrate- or energy metabolism of the worms...... 187 Niclosamide ...... 187 Bithionol ...... 188 Albendazole ...... 188 Cyanine dyes ...... 188 Drugs with other or unknown mechanism of action...... 189 Diethylcarbamazine ...... 189 Praziquantel ...... 189 Oxamniquine ...... 190 Artemisinin derivatives ...... 190 Nitazoxanide ...... 190 Tribendimidine ...... 190 Spectrum of anthelminthic drugs ...... 191 Drugs of first choice in human helminthioses...... 192 Anthelminthic susceptibility testing...... 193 Drugs against ectoparasites...... 194 Pyrethroids ...... 194 Lindane ...... 195
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Ivermectin ...... 195 Organophosphates ...... 195 Other drugs ...... 195 Antiviral agents ...... 196 Resistance to antiviral agents...... 197 Determination of the antiviral susceptibility...... 197 Methods based on genotypic tests...... 197 Phenotypic testing of constructs...... 198 Phenotypic methods...... 198 Plaque reduction assays...... 198 Examination of dye uptake...... 198 Examination of nucleic acid hybridization...... 199 Direct demonstration of enzymes or enzyme activities causing resistance...... 199 Virtual phenotyping ...... 199 Drugs active against both DNA and RNA viruses...... 201 Ribavirin...... 201 α-interferon ...... 202 Drugs against DNA viruses ...... 203 Drugs against herpesviruses...... 203 Nucleoside analogues...... 203 Guanosine analogues ...... 203 Acyclovir and valacyclovir...... 203 Mechanisms of resistance to acyclovir ...... 204 Penciclovir and famciclovir...... 205 Ganciclovir and valganciclovir...... 205 Other nucleoside analogues ...... 206 Brivudine...... 206 Kinase-independent antiherpesviral drugs...... 206 Foscarnet ...... 206 Cidofovir ...... 206 Adefovir ...... 207 Topically used antiherpesviral drugs...... 207 Idoxuridine ...... 207 Trifluridine ...... 207 Docosanol ...... 208 Fomivirsen ...... 208 Novel investigational antiherpesviral agents...... 208 Drugs against HBV...... 208 α-interferon ...... 209 Lamivudine ...... 209 Emtricitabine ...... 209 Adefovir, tenofovir ...... 209 Entecavir ...... 210 Penciclovir, famciclovir and ganciclovir ...... 210 Drugs under development ...... 210 Drugs against other DNA viruses...... 211 Drugs against adenoviruses...... 211 Drugs against papillomaviruses...... 211 Drugs against poxviruses ...... 211 Drugs against RNA viruses...... 212 Drugs against HCV...... 212 Ribavirin ...... 212 α-interferon ...... 212 New agents ...... 212 Drugs against picornaviruses ...... 212 Drugs against influenzaviruses...... 212 Adamantanes ...... 212 Neuraminidase inhibitors ...... 213
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Ribavirin ...... 213 Drugs against HIV ...... 214 Nucleoside-type reverse transcriptase inhibitors (NRTIs) ...... 214 Acyclic nucleoside phosphonates (ANPs) ...... 215 Non-nucleoside-type reverse transcriptase inhibitors (NNRTIs) ...... 215 Foscarnet ...... 215 Protease inhibitors (PIs) ...... 216 Fusion inhibitors ...... 217 Chemokine receptor antagonists ...... 217 Integrase inhibitors ...... 218 Other drugs against HIV ...... 218 Combination therapy against HIV...... 218 Other drugs against RNA viruses...... 218
ix
Sterilization and disinfection
Sterilization and disinfection
Killing of harmful microorganisms or decreasing of microbe numbers to an acceptable level is crucial for prevention of disease transmission in the clinical practice, for prevention of microbial contamination and the consequent spoilage of the products as well as for prevention of transmission of pathogenic microbes through pharmaceutical or food products. This can be achieved using physical and/or chemical disinfecting or sterilizing procedures. The aim of sterilization is to achieve a total germ-free status through killing or removing of all microbes. For this aim mostly physical methods (heat, ionizing radiation, filtration) are used. During disinfection the primary aim is to kill pathogenic microbes or to decrease their numbers significantly, below the infective limit; usually by means of chemical methods using antimicrobial compounds. Disinfectants are compounds with non-selective antimicrobial activity, which are exclusively used on non-living surfaces due to their toxicity. Antiseptics are chemical compounds with antimicrobial activity, which can be used on skin and mucosal surfaces due to their lower toxicity. Usually a diluted solution of a disinfactant is used as an antiseptic, which has negligible toxicity and not irritating due to the relatively low concentration. Preservatives are used in pharmaceutical products, food and cosmetics to inhibit the microbial contamination and replication of microbes to prevent spoilage and decomposition of these products and to prevent product-mediated spread of infections. By application of some physical (cold, aeration) and chemical (soaps) agents only a certain proportion of microbes can be eliminated; the microbial load decreases below an acceptable, safe level, but this decrease cannot be quantified accurately. This effect is called sanitation effect . Physical and chemical effects leading to inactivation of microbes are cidal effects, while static effects result only in inhibition of microbial growth and replication. Biocidal or germicidal are generalizing expressions used to designate killing of microbes (germs). If during disinfection the vegetative forms of microbes are not killed, only inhibited in growth, we have a bacteriostatic effect . Using bactericidal agents or procedures kills the vegetative forms of bacteria. Sporocidal effects kill even the highly resistant bacterial endospores produced by spore-forming bacteria ( Bacillus , Clostridium ) as well as fungal spores. In contrast, sporostatic effects inhibit germination of these spores, but do not lead to their inactivation. Agents and procedures leading to inactivation (loss of infectivity) of viruses are virucidal . Effects leading to killing or inhibition of the replication of fungi are fungicidal or fungistatic , respectively. Parasiticidal effect may be achieved using procedures or agents which kill protozoal vegetative forms and cysts as well as pathogenic helminths, their ova and larvae.
The kinetics of the destruction of microbes
Thorough knowledge on the kinetics of the destruction of microbes is required to understand and to correctly apply the principles of sterilization and disinfection. If a microbial population or community is exposed to a lethal effect, progressive decrease of the viable cell count occurs over time. The kinetics of the destruction of microbes is determined by the intensity of the lethal effect. Effects with sufficiently high intensity lead to exponential decrease in the viable cell count. If the logarithm of the number of surviving cells is plotted against the exposure time, the resulting curve is a line with a negative slope; this slope gives the death rate of the microbial population.
1 Sterilization and disinfection
500 7 450 6 400 350 5 300 4 250 200 3 surviving cells
150 2 Natural logarithm of the number of
Number of surviving cells (CFU/ml) 100 1 50
0 0 0 1 2 3 4 5 6 0 1 2 3 4 5 6
time (hours) time (hours) The kinetics of the destruction of microbes
Death rate (K) shows the proportion of the microbial population surviving the exposure after a given exposure time. The equation showing this relation is shown below. 1 N K = ln 0 , T N where N0 is the starting cell count of the microbe population, N is the cell count of the surviving microbes after exposure time T . The number of surviving microbes ( N) after exposure time T can be calculated as given below. = × −KT N N 0 e , where N0 is the starting cell count of the microbe population and K is the death rate. The actual value of the death rate depends on the procedure applied, on the composition and starting cell number of the microbe population and on the environmental conditions (composition of the medium, temperature, etc.). As mentioned above, this exponential curve occurs when the lethal effect has sufficient intensity. With decreasing intensity, the curve describing the correlation between the exposure time and the number of surviving microbes becomes increasingly sigmoid, i.e. killing is slowed down in an increasingly wide range of low and high microbe numbers compared to the exponential decrease. This also means that a lethal effect with sufficient intensity (appropriate concentration of the disinfectant, appropriate temperature or radiation dose, etc.) should be applied for optimal killing effect. Knowing the death rate of a given microbe population, the exposure time necessary for appropriate sterilization or disinfection using a lethal effect with a given intensity can be calculated by the rearrangement of the above equation. N − ln N T = 0 . K
2 Sterilization and disinfection
Factors influencing the efficacy of sterilization/disinfection
A crucial requirement for the antimicrobial activity is a direct contact between the microbe and the agent/effect, which is maintained for a sufficient time. The development and persistence of this direct contact, however, is influenced by a number of factors, which may modify the antimicrobial action of the physical effect or the disinfecting chemical agent markedly. 1. Properties of the material to be disinfected The quality of the material to be disinfected determines which disinfecting procedures can be applied, as only such a method should be used for disinfection, which does not damage the material. The efficacy of the disinfection is also affected by the surface characteristics, hydrophobicity and structure of the material. (Materials with continuous, smooth, non-porous surface can be disinfected more easily than rough, uneven, porous surfaces.) 2. Level of contamination on the material to be disinfected Contamination, even in very small amounts, may decrease or totally inhibit the disinfecting effect. Contamination may mechanically hinder the contact of the disinfecting agent and the microbes due to poor penetration of the disinfectant into or through the contamination; organic contamination (blood, serum, mucus, feces, urine, etc.) may surround the microorganisms and may provide a barrier protecting them from the disinfecting agent or effect. Organic contaminants may directly inactivate certain disinfectants (e.g. those containing chlorine or iodine). For this reason, it is important to remove all contamination from the materials or surfaces before disinfection. 3. Properties of the lethal agent In case of physical effects, their nature and intensity are important. (E.g. higher temperatures results in more intensive killing; moist heat has higher killing efficacy than dry heat.) In case of disinfectants, their chemical composition, stability, water-solubility and other characteristics will determine their antimicrobial spectrum, efficacy and utility. 4. Type, number and resistance of the microbes to be killed Different groups of microbes exhibit different resistance to the various disinfecting procedures. Intensively growing populations (communities) are more susceptible than slowly growing or non-replicating ones. Killing of higher number of microbes needs longer exposure times and higher intensity of the effect. The natural and acquired resistance (see below) of the microbes to physical effects and chemical agents determines ultimately the methods/procedures which can be selected. a. Resistance of microorganisms to physical effects Out of physical effects, resistance of microbes shows big differences in case of heat and ultraviolet radiation. The extremely high resistance of proteinaceous infectious particles (prions) to physical effects is notable. b. Resistance of microorganisms to chemical disinfectants The resistance of the microbes to chemical agents shows considerable diversity as compared to physical effects. It is also important, that using disinfectants in inappropriate concentrations or with inappropriate exposure times may lead to development of acquired resistance to disinfecting compounds. Mechanisms of resistance to disinfectants are discussed in detail below. 5. Temperature At higher temperatures (within a certain temperature range) killing is faster and higher temperatures are consequently more efficient. Above a certain temperature optimum,
3 Sterilization and disinfection
however, heat may inactivate the disinfectant or cause damage to the material to be disinfected. 6. Exposure time Sufficient exposure time is a prerequisite of the sufficient disinfecting/sterilizing effect. Suboptimal exposure times lead to incomplete killing; only a certain proportion of the microbes present in the material or on the surface to be disinfected is killed, consequently after cessation of the disinfecting activity, the surviving microbes resume replication. 7. Concentration In case of chemical disinfecting procedures, the concentration of the disinfectant is of paramount importance. Concentrated solutions need shorter, more diluted ones need longer exposure times for efficacy. Under a certain concentration level, the disinfectant cannot exert antimicrobial activity. For that reason, the prescribed optimal concentration should be used during chemical disinfection. (The active concentration is not always the same as the applied concentration, due to decreases caused by binding of disinfectant molecules to contamination.) The optimal concentration may also be influenced by the material to be disinfected. 8. pH of the environment The effect of the pH on the activity of disinfecting procedures is very complex, but it influences the efficacy of chemical agents much more than that of physical effects. Besides pH itself may influence the survival and growth rate of microbes, it also affects the efficacy of the chemical disinfectanting agents and their ability to bind to (cell) surfaces. Ionization status of acidic or alkalic disinfectant is directly influenced by the pH. Some disinfectants (e.g. phenolics, acetic acid, benzoic acid, etc.) are active in non-ionic form, and the alkalic pH favouring the ionized form decreases their activity. The antimicrobial activity of other agents (e.g. glutaraldehyde, cation-active detergents), conversely, increases with increasing pH; their activity is highest at alkalic pH. The binding of the agents to microbial cell surface is influenced by the electric charge of the cell surface, which, in turn, is ultimately influenced by the pH. With increasing pH, the negative charge of the cell surface increases, which leads to alteration of the capacity to bind disinfectants (e.g. chlorhexidine or cationic tensides) and to increase in the active concentration of the disinfectant on the cell surface enhancing the antimicrobial activity. 9. Bivalent cations These influence primarily the efficacy of chemical procedures. Bivalent cations (Mg 2+ , Ca 2+ ) present in hard water may interact with the microbial cell surface, leading to inhibition of binding of disinfectants (e.g. cationic detergents) and to consequent inhibition of the antimicrobial activity. They may also bind to and modify the activity of the disinfectants directly.
4 Sterilization and disinfection
Physical methods for sterilization and disinfection
High Temperature
High temperature is the most widely used method for sterilization and disinfection. Similarly to other methods, the number of surviving microbes decreases gradually and the kinetics of microbial death is exponential. The microbicidal activity of high temperatures can be described with the following parameters. The lowest temperature at which the given microbe population is killed after ten minutes of exposure is the thermal death point . The relationship between the time needed to reach total sterility and the exposure time is shown by the thermal death time , which equals to the minimum time span required to the death of all microbes at given environmental circumstances and at a given temperature. Even small alterations in the temperature used may lead to significant changes in the thermal death time. Decimal reduction time ( D) is the time during which 90% of the microbe population is killed.
Mechanisms of thermal injury
Moist heat leads to denaturation and coagulation of cellular proteins. Even relatively low temperatures lead to single-strand breakages in the DNA. High temperatures also lead to loss of membrane integrity, leading to loss of small molecules. Heat activates ribonucleases, which destroy ribosomes. Damage caused by dry heat is mostly due to water loss, cells dry out, cellular proteins are denatured; oxidative damage and damage due to increase in the electrolite concentration can be observed. Due to the loss of water, the number of the polar groups in the proteins decreases, consequently more energy is necessary to maintain stable structure.
Application of moist heat
Both moist and dry heat can be used for sterilization; however, the more widely used method is the application of moist heat (vapour) due to its better penetration and consequent higher efficacy. The vegetative forms of most bacteria are relatively susceptible to thermal injury; members of the genera Staphylococcus , Enterococcus , Pseudomonas and Mycobacterium show higher heat resistance. Most bacteria are killed after 30-60 minutes at 58-60°C, after 5-10 minutes at 70°C and within a few seconds at 100°C. Heat treatment of 80°C for 5-10 minutes kills the vegetative forms of most pathogenic bacteria and fungi; killing vegetative cells of mycobacteria requires at least 95 °C. Bacterial spores have markedly higher heat resistance, their killing requires treatment with high-pressure vapour at 121°C in an autoclave. E.g. the time necessary for inactivation of spores of Clostridium botulinum is 4 minutes at 121°C, while 5.5 hours at 100°C. Viruses are inactivated at 50-60°C, excepting the members of the Picornaviridae family (enteroviruses, polioviruses, hepatitis A virus, etc.), which are killed safely only by temperatures higher than 80°C. The heat resistance of the vegetative forms of fungi and protozoa is similar to that of the vegetative forms of bacteria, while the fungal spores and protozoal cysts show much higher heat resistance. This resistance, in case of certain species, reaches that of bacterial endospores. The heat resistance of helminths is low. Treatment with moist heat may be achieved using boiling or vapour flow (100°C, 30
5 Sterilization and disinfection minutes), or by high-pressure vapour in autoclave (121°C, 1 atm overpressure, 30 minutes). The most efficient of these is high-pressure vapour; at 1 atm overpressure the spores are inactivated after 15 minutes (the longer exposure time is used for safety reasons). With further increases in the pressure (2 atm overpressure) the sterilization temperature can be increased further (134°C); in this case 3 minutes is sufficient to reach total sterility. For heat treatment of heat-sensitive fluids and semi-fluid materials fractionated heat treatment ( tyndallization ) can be applied. This means that the material to be sterilized is heated to 80-100°C then cooled to 37°C in three consecutive days. During the 80-100°C heat treatment the vegetative forms are killed, while at 37°C the spores may germinate to give rise to heat-susceptible vegetative forms, which become susceptible to killing by the 80°C treatment next day. This way the heat-sensitive materials can be sterilized by heat-treating them at least three times. Pasteurization involves the heat treatment of fluids at 63-66°C for 30 minutes. This is suitable to kill the vegetative forms of most pathogenic bacteria, but it does not lead to killing of all bacteria (microbes) and spores are spared. This kind of heat treatment is used in case of milk and other foods (beer, yoghurt, fruit juice, etc.). To decrease the damage of food exerted by the long exposure to the high temperatures, fast pasteurization (72°C, 15 seconds or 95 °C 5 seconds) or ultrapasteurization (134-140°C, 1-3 seconds) is preferred. Pasteurization is not a sterilization method; total sterility cannot safely be achieved.
Application of dry heat
Sterilization using dry heat requires higher temperatures and longer exposure time, since dry heat penetrates less efficiently than moist heat. The longer exposure time to high temperature may, in turn, damage the materials to be sterilized. Therefore dry heat is applied mostly to sterilize heat-resistant materials, primarily glasswares, but metal tools, oils, powders or gels may also be sterilized in this manner. Heat treatment is performed in heat sterilizers , which have two types. The older, gravitational appliances heat the air in the inner space of the appliance, while the more modern appliances circulate the heated air. Consequently in the latter type the air reaches the working temperature faster, and the temperature is evenly high within the appliance. Heat treatment is performed at 140-180°C for 2-3 hours; equipments utilizing air circulation allow for shorter exposure times than gravitational systems. The other application of dry heat is the direct heat treatment . This method may be used for fast sterilization of metalwares, or to sterilize inoculator loops and glass slides during bacteriological work. Contaminated bandages, sample collectors and –containers as well as dead experimental animals may safely be eliminated by burning them.
The control of the efficacy of heat sterilization
Attainment of the optimal parameters of heat sterilization or the efficacy of the sterilization process may be controlled by mechanical, chemical or biological indicators. The mechanical control of the optimal parameters (temperature, exposure time, etc.) is performed by continuous monitoring of the control panels of the appliance. Chemical indicators also allow solely for controlling for the optimal sterilization parameters; appropriateness of the temperature and the exposure time is shown by a colour change in the indicators. Different indicators are used in autoclaves and in heat sterilizers. Both of these methods provide opportunity to indirect control only. Efficacy of the heat sterilization can directly be controlled using biological indicators
6 Sterilization and disinfection containing bacterial spores (Bacillus stearothermophilus or Bacillus subtilis var. niger ). The test spores undergo the sterilization process together with the materials to be sterilized; then the spores are inoculated into culture media to examine their viability. A successful sterilization process leads to loss of viability of the test spores, shown by lack of germination and growth. Autoclaves must be controlled using biological indicators every half a year.
Cold, freezing
Though low temperatures kill a number of different microbes, freezing is not a reliable sterilization method, since total sterility can never be achieved through freezing. Freezing leads to relative water loss due to crystallization of intracellular water, leading to electrolite disequilibrium and to denaturation of proteins. Freezing also damages the cell membrane, which leads to loss of intracellular material and ions; besides these effects, it activates ribonucleases and peptidases. Repeated freezing-melting cycles are much more harmful for the cells than prolonged freezing. If the cells are cooled during vacuum drying (are lyophilized), cell loss can be decreased substantially. Lyophilization and freezing are widely used for long-term storage of bacterium or fungus cultures and virus suspensions as well as a preservation method in food industry. The widely used preservative effect of cooling is based mainly on inhibition of the replication of microbes. At 4°C most pathogenic microorganisms are unable to grow and replicate; more sensitive microbes may even be inactivated.
Radiation
Ultraviolet (UV) radiation
UV radiation is a non-ionizing radiation, which possesses mutagenic and cell damaging effect. These activities are closely dependent upon the wavelength of the UV light. Best microbicidal effect is exhibited by radiations with wavelengths of 240-280 nm. The optimal wavelength is 260 nm, which corresponds to the absorption maximum of the DNA. The microbicidal activity is due to the UV absorption of the DNA and to the consequent DNA breakages. UV radiation leads to the formation of timine dimers, which results in breaking or structural distortion of the DNA chain, disturbing normal base pairing and DNA replication. If the bacterial cells are exposed to visible light of 300-400 nm wavelength immediately after UV exposure, the frequency of mutations and the bactericidal activity of the UV light is significantly decreased. This phenomenon of photoreactivation is due to the activity of hydrolytic enzymes induced by the visible light, which hydrolyse timine dimers. Another repair mechanism, excision repair , is also involved in repairing the UV-induced DNA damage. The enzymes of this repair function excise the dimers from the DNA chain and mend the damaged strand. High activity of this excision repair is thought to be responsible for the high UV resistance in some bacteria. Susceptibility to UV radiation is highest in case of viruses and Gram negative bacteria. Vegetative forms of Gram positive bacteria, due to their cell wall structure, are more resistant. UV resistance of bacterial and fungal spores as well as of protozoal cysts is prominent. Though germicidal effect of the UV light is unquestionable, it cannot be regarded as a perfect sterilization method, due to its low penetration and low energy. UV does not penetrate into solid objects and materials and penetrates poorly into fluids. For this reason it does not
7 Sterilization and disinfection affect microbes not directly exposed to the UV radiation, and it cannot reliably ensure total sterility. The most important application of UV light as a disinfecting method is disinfecting the air in closed rooms or in biological safety cabinets. It can also be used to sterilize smooth, level surfaces. It should be used with caution as it can also damage the skin and the eye. The significant natural germicidal activity of the solar radiation is caused by its ultraviolet component.
Ionizing radiation
Ionizing radiation is classified into two groups based on their physical properties; (i) corpuscular radiations ( α- and β-radiation), and (ii) high-energy electromagnetic radiations (X- and γ-radiation). Ionizing radiation has much more practical utility than UV, due to its markedly higher energy, better penetration and greater lethal effect. γ-radiation is used in most cases. The lethal effect of the ionizing radiation is mediated by direct and indirect activity on cellular macromolecules. The less important is the direct effect, which is exerted by direct energy transfer resulting in lysis of covalent chemical bonds, e.g. in double-strand breakages in the DNA. As biological systems contain high amount of water, the lethal effect of the ionizing radiation is mostly exerted indirectly, by the radiolysis of the water and the consequent effect of the released active radicals on the macromolecules. Water molecules are ionized by the radiation as follows. + − H2O → H 2O + e The produced water cation reacts with non-ionized water molecules inducing further lysis of water leading to production of free hydroxyl radicals. + + • H2O + H 2O → H 3O + OH The free electron produced by the ionizing radiation also reacts with non-ionized water molecules producing free hydrogen radicals and hydroxide ions. − − • e + H 2O → OH + H Hydroxyl radicals possess potent oxidizing activity, while hydrogen radicals are potent reducing agents. These free radicals are extremely reactive; they damage the macromolecules, e.g. lead to double-strand breakages in the DNA. Intracellular oxygen molecules also enhance the activity of the ionizing radiation. This enhancement is the result of the reaction between intracellular oxygen molecules and free radicals, giving rise to an autooxidative chain reaction. • • H + O 2 → HO 2 • 2 HO 2 → H 2O2 + O 2 Compounds containing sulfhydryl groups protect the cells from the damaging effect of the radiation by converting the absorbed energy through being oxidized to disulfides. Vegetative forms of most pathogenic microbes are susceptible to ionizing radiation, but bacterial and fungal spores as well as protozoal cysts and worm eggs are more resistant. At the end of the sterilization process using radiation, the extent of the microbial death may decrease significantly; for this reason it is important to use appropriate exposure time and to provide the necessary radiation intensity throughout the whole exposure time. Ionizing radiation is extensively used in medical practice to sterilize suture materials
8 Sterilization and disinfection and disposable utensils (syringes, needles, etc.).
Ultrasound
High-frequency resonance, in the upper part of the range of audible sound and in the range of ultrasound (20-40 kHz) are capable of disrupting the cells. High frequency soundwaves, due to continuous alteration of the pressure, induce cavitation in fluids (including the intracellular fluids); then these induced intracellular bubbles collapse leading to disintegration of the cell. In fluids containing soluble oxygen, ultrasound also induces H2O2 production and consequent oxidative damage. Vibration may also cause depolymerization of macromolecules and intramolecular reorganization of functional groups as well as to DNA breakages. Susceptibility of microorganisms to ultrasound is extremely variable. The most susceptible microbes are Gram negative rods, while staphylococci exhibit extremely high resistance. Since high-frequency sound treatment does not lead to total sterility, and its utilization is difficult, it is rarely used as a sterilizing method. It is used in dentistry practice to disinfect the root canal. It is a widely used method, however, in molecular laboratory practice to disrupt cells and/or to fragment macromolecules (sonication).
Filtration
The most widely used method to sterilize heat-sensitive fluids and solutions is filtration . Though the most important effect is the mechanical effect based on size exclusion, electrostatic charge, adsorption and the physical characteristics of the filter also influence the filtration effect. Many different types of filters are commercially available. The older types, i.e. porcelaine (Chamberland) filter, asbestos (Seitz) filter, diatome earth (Bekerfeld) filter and glass filters, share the disadvantage of uneven pore size and of the tendency to alter the chemical composition of the transmitted fluids through absorption of some components or through dissolution of filter components. Presently, membrane filters separating exclusively on the basis of pore size are the only filter type in use. They may be made of cellulose-acetate, cellulose-nitrate, polycarbonate, polyester, polypropylene or polysulfonate. The most widely used filter pore size is 0.22 m in diameter, which produces a bacterium-free filtrate. To filter viruses, filters with a pore size of 0.01 m are applicable. The fast flow-through of fluids is ensured using vacuum. To filter the air of enclosed spaces (sterile boxes or rooms) high-efficiency particulate air (HEPA) filters are used.
Gas sterilization, plasma sterilization
During gas sterilization and plasma sterilization the sterilization is performed using disinfectants in gas or plasma phase, respectively; i.e. both physical and chemical effects are involved in the sterilization effect. Gas sterilization utilizes ethylene-oxide , formaldehyde , glutaraldehyde or presently more frequently ortho-phtalaldehyde or β-propiolactone . Ethylene-oxide and aldehydes are broad spectrum alkylating agents, which denature proteins, nucleic acids and other macromolecules by alkylating them. Their activity is concentration dependent, but also influenced by temperature, exposure time and relative humidity. Gas sterilization is used to sterilize heat-sensitive devices, large appliances or rooms. The agents used are highly irritating and toxic (see in detail in the section ‘Chemical disinfecting procedures’); therefore
9 Sterilization and disinfection the residual agent must be removed by a thorough aeration. The efficacy of gas sterilization can be evaluated using chemical and biological indicators. A novel biological indicator allows for detection of the growth of the test Bacillus subtilis spores using a fluorescent detection system four hours after an improper sterilization process. The plasma-phase hydrogen peroxide or peroxi-acetic acid are much more active oxidizing agents than the liquid state compounds. Both disinfectants are used in plasma sterilization systems . Their most important advantage in contrast to compounds used for gas sterilization is their low toxicity, because they are degraded into substances harmless to the user and to the environment (water, oxygen, acetic acid). In plasma state, these broad spectrum sporocidal oxidizing agents show rapid action, their microbicidal activity is mediated by the free oxygen radicals produced. The sterilizing effect of plasma state compounds can be enhanced further using simultaneous UV radiation. Further advantage is that they are active at low temperatures (46±4°C); therefore plasma sterilization can be used to sterilize heat-sensitive materials. Their disadvantage is, however, that their penetration is poor and their utility is limited; they cannot be used to sterilize fluids, powders, materials in closed containers and linen- or cellulose-containing materials. Plasma sterilizing systems may be operated at normal atmospheric pressure or in vacuum; presently totally automated systems using vacuum are widespread. The process starts with producing the vacuum in the inner space of the plasma sterilizer followed by heating it. Then water and hydrogen peroxide or peroxi-acetic acid is injected into the sterilizing chamber, where the sterilizing agent evenly fills the chamber. In the next phase the agents are transformed into plasma state using electric or magnetic field, and the free radicals are produced. This sterilizing cycle is repeated 2-10 times (taking approximately an hour altogether), and total sterility is achieved. At the end of the process pressure is levelled off by injecting sterile filtered air into the chamber.
Spectrum of efficacy of physical methods
Bactericidal Sporocid al Fungicidal Virucidal Para siticidal Physical methods activity 1. high temperature 1.1. Boiling + - + ± ± 1.2. Vapour flow (100ºC, 30 min) + - + ± ± 1.3. Autoclave 121ºC, 30 min, 1 atm + + + + + 134ºC, 3 min, 2 atm + + + + + 1.4. Fractionated heat treatment + + + + + 1.5. Pasteurization 63-66ºC, 30 min + - + + + 72ºC, 15 min + - + + + 134-140ºC, 1-3 s + - + + + 1.6. Heat sterilizers 160 -180ºC, 2-3 h + + + + + 1.7. Direct heat treatment + + + + + 1.8. Burning + + + + + 2. low temperature, freezing - - - - - 3. radiation 3.1. UV radiation ± ± ± ± ± 3.2. Ionizing radiation + + + + + 4. ultrasound ± ± ± ± ± 5. filtration removes microbes selectively as determined by pore size Gas sterilization, plasma sterilization + + + + +
10 Sterilization and disinfection
Chemical methods for sterilization and disinfection
Chemical agents used against microorganisms for inactivation, killing, inhibition of replication or for decreasing their numbers can be classified into two groups on the basis of their usage. i) chemotherapeutics, antibiotics , which are agents with selective antimicrobial action exerted at a specific target in the microbe and are not or slightly toxic to the host; and ii) disinfectants , which are non-selective microbicidal agents. As most disinfectants damage all living cells, they are applicable to disinfect objects, devices and surfaces only. A distinct group of disinfectants that can be used to disinfect skin and mucosal surfaces is called antiseptics ; these are used mostly in the dentistry and dermatology practice or prior to invasive intervention. Another distinct group is the group of preservatives , which inhibit the microbial growth in pharmaceutical products, cosmetics and foods and thus inhibit their spoilage and protect against infections transmitted by these products. As preservatives are or at least may be introduced into the body, they must conform to special requirements (very low toxicity and irritating property, lack of accumulation in the body, etc.). This section discusses the criteria influencing the selection of disinfectants, the mechanism and spectrum of action of the different disinfectants and their fields of application. The criteria for the ideal disinfectant are the following. • It should be active against a wide range of microorganisms, i.e. should have a broad spectrum. • It should kill or inactivate microorganisms at relatively low concentrations and after a relatively short exposure time. • It should have good water-solubility, good penetrating characteristics; it should not damage the materials to be disinfected. • It should have as few disadvantageous properties (foul smell, toxicity, corrosive activity, flammability) as possible. • Its use should be cost-effective. • If combined, interactions between different disinfectants must be taken into consideration; combinations of agents antagonizing the activity of each other should be avoided.
Though the range of the available disinfectants is wide and is continuously widening, finding an ideal disinfectant meeting all requirements may be difficult. Always the agent or method most appropriate for the actual aim should be chosen.
Determination of the efficacy of the disinfectants
The classical method (developed at the 1930s and now rarely used) for investigating the efficacy of disinfectants is the determination of the phenol coefficient . Phenol coefficient shows how much more or how much less active is the studied disinfectant compared to phenol against the studied microorganisms under standard circumstances (at a given disinfectant dilution, exposure time, temperature, medium etc.) Phenol coefficient of a given disinfectant is usually given against three important pathogenic bacteria, Salmonella typhi, Pseudomonas aeruginosa and Staphylococcus aureus . The development and usage of newer disinfectant groups, the decrease in the usage of phenol as a disinfectant, the diversity of microbes and the need for quantitative evaluation of disinfectant efficacy under circumstances closer to the real application environment brought about the development of new methods for determination of efficacy. Besides, since the extent
11 Sterilization and disinfection of microbial death may be expressed in different manners (as a percentage of the initial number of microbes or the log 10 of the number of killed or surviving cells), the standardization and comparability of the different methods is problematic. Determination of an appropriate threshold of microbial death is also an important issue in evaluation of efficacy. During testing of novel disinfectants the efficacy is usually given in comparison to one or more well-known commercial disinfectant. Bactericidal activity of disinfectants is usually tested against Staphylococcus aureus , Escherichia coli and Pseudomonas aeruginosa. Presently the most widely used method to test the bactericidal activity of disinfecting agents is the suspension test . The test involves the mixing of different predetermined dilutions of the agent to be tested to a suspension containing standard number of microbes, albumine (to model ‘dirty environment’) and bivalent cations (Ca 2+ and Mg 2+ ; to model hard water), these test suspensions are incubated at a given temperature (frequently at ambient temperature), then the test suspensions are sampled at predetermined time points. The residual disinfectant is neutralized in these samples by diluting the agent (as in case of alcohols and phenol derivatives) or by means of a neutralizing agent (formaldehyde– NH 3; glutaraldehyde – glycine; phenol derivatives and quaternary ammonium compounds– Tween 80); and following the neutralization the number of surviving cells is determined for each sample. The applicability of the method is hindered by aggregation of bacterial cells in case of certain disinfectants (e.g. in case of some quaternary ammonium compounds), which leads to distortion of the results. This can be circumvented by adding compounds inhibiting aggregation (e.g. non-ionic tensides when testing quaternary ammonium compounds). The result of the suspension test (bactericidal activity of an agent at a given concentration, BA
12 Sterilization and disinfection
The use-dilution test or carrier test evaluates the bactericidal activity of the disinfectant against Salmonella choleraesuis , Staphylococcus aureus and Pseudomonas aeruginosa at the dilution that will be routinely used. Small metal cylinders are used for performing the test, which are immersed in broth cultures containing a predetermined CFU number of the abovementioned bacteria, then dried at 37°C. The inoculated cylinders are incubated for 10 minutes in the dilution of the disinfectant recommended by the manufacturer for routine use. After incubation, the residual disinfectant is rinsed off from the cylinders and they are placed into fresh broth medium to monitor bacterial growth. The efficacy of the tested agent is acceptable if no growth is detected in the test. During evaluation of the bactericidal activity of disinfectants, some bacteria may exhibit markedly higher resistance than the test bacteria. Therefore, the following should be taken into account. i) Making a homogeneous suspension out of mycobacteria, due to their highly hydrophobic cell wall, is difficult. Besides, the efficacy of disinfectants is tested on fast-growing mycobacteria (e.g. Mycobacterium terrae ), because of the slow-growing nature of M. tuberculosis . ii) Testing of the sporocidal activity of disinfectants is also difficult and cumbersome, because for assessing the final (residual) viable cell number, the time necessary for germination of the spores and growth of vegetative cells must be provided. Sporocidal activity is usually tested on Clostridium sporogenes or Bacillus subtilis spores. iii) The biofilm-producing activity of the microbes should be taken into account during testing for efficacy. The expectable efficacy of the disinfectants against microbial biofilms is tested on bacteria growing on suitable surfaces (glass, metal, microtitre plate, etc.). iv) In case of bacteria present intracellularly in other organisms (e.g. Legionella pneumophila in Acanthamoeba polyphaga free-living amebae) the disinfectant efficacy should be tested against suspensions containing both extracellular bacteria and host protozoa. The final (residual) viable cell number should be determined after lysis of the protozoal cells. These procedures can be used to test antifungal efficacy of disinfectants as well. However, it is crucial to correctly select the nature of the fungal inoculum (blastoconidia, hypal elements, spores) used in the test, as their susceptibility to the disinfectant may be profoundly different. Moreover, to correctly set the initial inoculum size is technically challenging. Fungicidal agents should be tested against Candida albicans blastoconidia and against Aspergillus niger spores and hyphae, the number of surviving cells should be performed at 20 °C using at least 48 hours incubation time. To evaluate the virucidal activity of disinfectants the suspension test and the use-dilution test are used most frequently. The disinfectant may be termed virucidal if it decreases the infectivity of the test virus by at least 99.99%. Testing of efficacy is technically hindered by the obligate intracellular replication of viruses. The cell-free filtrate of the supernatant of the cell culture infected with the virus, which contains virions, is treated with the disinfectant. After the exposure time, the disinfectant is neutralized and the residual infectivity of the virus is tested on appropriate cell cultures. Virucidal activity should be tested against polioviruses and adenoviruses (representing the non-enveloped viruses) and against herpes simplex virus (representing enveloped viruses). It is frequently desirable to test the efficacy against further viruses as rotavirus, human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV) as well. Virucidal activity against unculturable viruses or viruses difficult to culture (e.g. HCV) can be measured using methods demonstrating viral nucleic acid (e.g. polymerase chain reaction). Testing for parasiticidal activity (against protozoa and worms) is tedious; it is heavily influenced by the developmental form of the parasite (trophozoite or cyst; worm egg, larva or adult) and its culturability. During measurements of efficacy against prions, the infectious protein is contained in
13 Sterilization and disinfection a tissue homogenate. The residual infectivity of the test prions after incubation with different concentrations of the potentially effective disinfectant can be determined by means of inoculation of test animals. Testing of solid-phase disinfectants should include evaluation on solid carriers (surfaces) besides the abovementioned approaches. To avoid air-borne infections or contamination, aerosolized disinfectants may also be used. The efficacy of these air-disinfectants is evaluated by sampling the air, which can be performed by sedimentation or filtration onto solid medium or by bubbling the air through broth medium. Novel, rapid methods for testing of efficacy may yield results within a few hours, in contrast to traditional methods using at least 24 hours incubation time for evaluation of microbial growth. Such new methods are vital staining (e.g. viable cells stained with acridine orange emit green fluorescence, while dead cells are orange), detection of the oxygen utilization (decreasing oxygen concentration) or CO2 production either directly or using the resulting pH change, or fast detection of microbial nucleic acids using a molecular technique. In case of evaluation and control of efficacy of preservatives measuring the long-term efficacy represents an additional problem. Furthermore, in emulsion formulas the preservative is concentrated in one of the phases and its concentration is lower in the other phase; it may even be absent in some phases. Thus contaminating microbes may survive in the phase containing less preservative. Components in the formulas, cosmetics or foodstuff may also inactivate the preservative. Therefore evaluation of efficacy should involve testing in the formula/food besides testing in pure form. Preservatives are evaluated mostly using the simulated use test; bactericidal activity is usually tested against Staphylococcus aureus , Escherichia coli and Pseudomonas aeruginosa , and fungicidal activity against Candida albicans . Testing for biocidal activity is especially important for preservatives used in ophthalmic formulas and in ointments. Mechanism of action of disinfectants is complex, their biocidal activity, depending on the concentration, may be exerted on a specific target (in case of low concentration) or by damaging aspecifically several different structures and metabolic processes simultaneously (in case of higher concentration). The biocidal action of most disinfectants is the result of a number of different processes and not of inhibition or damage of one target (metabolic process, structural component or macromolecule). Biocidal agents may be classified into three main groups according to their main target and most important mechanism of action; i) agents damaging the cell membrane, ii) protein denaturing agents, and iii) agents modifying the functional groups of proteins and nucleic acids.
14 Sterilization and disinfection
Groups of disinfectants
Dis infectants 1. agents damaging the cell membrane 1.1. surface-active compounds 1.1.1. cationic tensides cetylpyridinium chloride, cetylpyridinium bromide, cetyl-trimethyl-ammonium bromide, benzalkonium chloride, tetradecyl-trimethyl-ammonium bromide, trimethyl-hexadecyl-ammonium chloride, lauroyl-dimethyl-benzylammonium chloride, alkyl-trimethyl-ammonium chloride, alkyl-dimethylbenzyl-ammonium chloride, alkyl-dimethyl-ethylbenzyl-ammonium chloride, didecyl-dimethyl-ammonium chloride 1.1.2. anionic tensides Na-lauroyl-sulfate, Na-n-dodecyl-benzene-sulfonate, Na-palmitate, triethanolamine oleate 1.1.3. non-ionic tensides pentaerythryl palmitate, long-chain alcohol ethers and fatty acid esters of glycerol and polyethylene glycol polyoxyethylene sorbitane monooleate (Tween 80) 1.1.4. amphoteric tensides alkyl-betains, alkyl-amidopropyl-betains 1.2. alcohols ethanol, isopropanol, chlorbuthanol 1.3. phenol derivatives 1.3.1. phenol and phenolics phenol, cresol 1.3.2. chlorinated biphenyls triclosane, hexachlorofene 1.4. biguanids chlorhexidine, alexidine, vantocyl, diamidines 2. protein denaturating agents 2.1. strong inorganic acids hydrochloric acid, sulphuric acid 2.2. organic acids acetic acid, lactic acid, citric acid, propionic acid, salicylic acid, benzoic acid 2.3. salts&esters of inorganic acids salicylates, sorbate 2.4. strong bases Na-hydroxide, K- hydroxide, Ca-hydroxide 3. agents modifying functional groups 3.1. heavy metal compounds thiomersal, phenylmercury-citrate (Hg), Ag-acetate 3.2. oxidizing agents 3.2.1. halogen compounds 3.2.1.1. iodine compounds iodine tincture, iodophors 3.2.1.2. chlorine and compounds chlorine, hypochlorites, chloramines (halazone) 3.2.2.és vegyületei peroxides 3.2.2.1. hydrogen-peroxide 3.2.2.2. peroxyacetic acid 3.3. dyes 3.3.1. aniline dyes malachite green, brilliant green, crystal violet 3.3.2. acridine dyes acriflavine 3.4. alkylating agents 3.4.1. aldehydes formaldehyde (taurolidine, noxythioline, polynoxyline) glut araldehyde , ortho -phtalaldehyde 3.4.2. ethylene oxide 3.4.3. β-propiolactone
15 Sterilization and disinfection
Disinfectants damaging the cell membrane
Structural and functional integrity of the cell membrane depends on the organization of the proteins and lipids it is comprised of. The agents damaging the cell membrane cause the desorganization of this structure, which lead to inhibition of membrane function resulting in disruption of active transport mechanisms, the electron transport chain and (in case of bacteria) the proton gradient dependent ATP synthesis. These, in turn, will lead to dysfunctional energy metabolism, loss of cellular electrolytes and macromolecules, to consequent osmotic disequilibrium, resulting in cell death.
Surface-active compounds
Surface-active agents (detergents or tensides) are comprised of hydrophobic, apolar hydrocarbon chain(s) and hydrophilic groups. The hydrophobic part of the molecule is usually a lipophilic, long fatty acid chain, while the hydrophilic part is polar (ionic or non-ionic). Based on the characteristics of the hydrophilic groups, the detergents are classified as cation-active, anion-active, non-ionic or amphoteric tensides.
Cation-active tensides
The most important surface-active agents with antimicrobial activity are the cation-active tensides, i.e. the quaternary ammonium compounds. They dissociate to a complex cation and its counter-ion; the antimicrobial activity is due to the cationic part. The polar (ionic) part binds to the phosphate group of phospholipids, while the apolar side chains penetrate to the hydrophobic inner layers of the membrane. This results in distortion of the membrane structure and in the consequent loss of the semipermeability of the membrane. High concentrations of the cation-active agents will denature the cellular proteins when penetrating into the cell. The spectrum of efficacy and the grade of activity of the compounds and their susceptibility to organic contamination are determined by the side chain substituting the quaternary nitrogen atom. They have excellent bactericidal activity; they are virucidal against enveloped viruses, and show parasiticidal activity against certain parasites. They lack sporocidal and fungicidal activity. The activity of cation-active detergents is highest at alkaline pH. Their toxicity is low when used on skin or mucosal surfaces. Their further favourable characteristics are that they are not corrosive or irritating, they do not have a bad smell and they are water-soluble. They are, however, easily inactivated by acids (below pH 3.5), organic contaminants, porous materials or by bivalents cations prevalent in water (Mg 2+ , Ca 2+ ), as well as by anion-active tensides. The cation-active detergents are usually combined with other disinfectants (e.g. biguanides or iodine compounds). The most widely applied active compounds are • cetylpyridinium-chloride , • cetylpyridinium-bromide, • benzalkonium-chloride , • cetyl-trimethyl-ammonium-bromide, • tetradecyl-trimethyl-ammonium-bromide, • trimethyl-hexadecyl-ammonium-chloride , • lauroyl-dimethyl-benzylammonium-chloride , • alkyl-trimethyl-ammonium-chloride ,
16 Sterilization and disinfection
• alkyl-dimethylbenzyl-ammonium-chloride , • alkyl-dimethyl-ethylbenzyl-ammonium-chloride , • didecyl-dimethyl-ammonium-chloride . The last three possess marked activity against HIV and HBV as well. Some of these compounds (benzalkonium-chloride, alkyl-trimethyl-ammonium-chloride) are also used as preservatives, mostly in cosmetics, eye drops or contact lens storage solutions.
+ Cl N
cetylpyridinium-chloride
CH3 + N benzalkonium-chloride Cl CH tetradecyl-dimethyl-benzyl-ammonium-chloride 3
Br CH3 + N CH3 tetradecyl-trimethyl-ammonium-bromide CH 3
CH3 + N CH3 Cl
lauroyl-dimethyl-benzyl-ammonium-chloride
CH 3 Cl + N
CH3 didecyl-dimethyl-ammonium-chloride
Anion-active tensides
The most well-known representatives of anion-active tensides are soaps and synthetic cleaning agents. These detergents are comprised of a long hydrocarbon chain, a hydrophilic anionic part (usually containing carboxyl- or sulphate group) and its counter-ion (usually a sodium ion). Their activity is provided by the negatively charged anionic part of the long-chain fatty acid released upon dissociation. They themselves do not possess disinfecting (microbicidal) activity, they only show cleaning effect, but they can enhance the microbicidal activity of other disinfectants. Their activity is best at weakly acidic pH. It must, however, be taken into account that they interfere with the activity of some disinfectants (e.g. cation-active tensides). Such compounds include sodium-n-dodecyl-benzene-sulfonate , sodium-palmitate , sodium-lauroyl-sulfate , triethanolamine-oleate .
O + O SO Na sodium-lauroyl-sulfate O O + O SO Na sodium-n-dodecyl-benzene-sulfonate O
17 Sterilization and disinfection
O + Na sodium-palmitate O
O C2H4OH + HN C2H4OH O C2H4OH triethanolamine-oleate
Non-ionic tensides
As their name suggest, they do not carry ionic groups. The polar part of the molecule contains alcohol-, ether- and/or ester group(s) (carbohydrates are also possible), the apolar part is usually a long-chain fatty acid. Their antimicrobial activity is negligible, but they have excellent cleaning efficacy. They are used mostly in washing gels. Active compounds include pentaerythryl-palmitate , fatty acid esters of glycerol and polyethylene glycol , ethers of glycerol formed with long-chain alcohols and polyethylene glycol , polyoxyethylene-sorbitane-monooleate (Tween 80).
O CH2OH pentaerythryl-palmitate O CH2 CH2OH CH OH 2 O H C O 2 glycerol-monostearate HC OH H C OH 2
CH2 polyethylene-glycol-ether (PEG-lauroyl-ether) HO CH2 O n
O polyethylene-glycol fatty acid esther (PEG-stearate) CH 2 HO CH2 O n
CH 2 CH2 HO CH O O CH2 OH 2 n O m CH 2 CH OH O 2 o O
O (CH2)14CH3
O
fatty acid esthers of PEG-sorbitane (Tween80, PEG sorbitane monostearate)
Amphoteric tensides CH O CH 3 O 3 O The molecules of amphoteric + + R N CH2 R N N CH2 tensides carry both basic and acidic groups; H O CH O CH consequently they may behave both as 3 3 cationic and anionic tensides depending on alkyl-betaine alkyl-aminopropyl-betaine the environmental pH. Their activity is
18 Sterilization and disinfection highest at neutral pH. Their disinfecting efficacy is similar to that of the cationic tensides; the spectra of the two groups are identical. Their advantage is that anionic detergents not at all, while organic contaminations only slightly decrease their activity. Such active compounds are alkyl-betains and alkyl-amidopropyl-betains (e.g. lauroyl-amidopropyl-betain)
Alcohols
H C Cl Alcohols exert their microbicidal activity by 3 H HO Cl dezorganization of the phospholipid bilayers, as well as H3C OH H C Cl by their dehydrating effect and by denaturation of 3 CH3 proteins. Increasing chain length is directly proportional chlorbuthanol isopropanol to microbicidal activity and, unfortunately, to toxicity. Penetration (water-solubility), however, is better in case of alcohols with short alkyl chains, therefore the latter CH2OH CH OH tend to have better antimicrobial efficacy when used. Cl 2 Their activity is primarily bactericidal and fungicidal, their virucidal activity is limited to enveloped viruses; they do not show sporocidal and Cl parasiticidal activities. Alcohol-based disinfectants may benzyl-alcohol contain (and transmit) bacterial and fungal spores, 2,4-dichlorobenzyl-alcohol therefore alcohols cannot be used to disinfect skin CH2CH2OH OCH CH OH surfaces, invasive diagnostic and therapeutic equipments 2 2 (e.g. endoscopes), nor to disinfect machinery used in the manufacturing of sterile products unless sterilized by filtration. Alcohols evaporate rapidly; they do not leave phenyl-ethanol phenoxy-ethanol residual disinfectant contamination, but the rapid evaporation also limits the available exposure time. Being highly flammable, they should be used with caution. As they penetrate poorly into organic matter, they should be used only on clean surfaces (free of organic contamination). Microbicidal activity is optimal at concentrations 60 to 90%. In practice, the 70% solution of ethyl-alcohol (ethanol, CH 3CH 2OH) and the 60-70% solution of isopropyl-alcohol (isopropanol, 2-propanol) are used, mostly to disinfect skin and artificial surfaces. When combined with other disinfectants (e.g. with iodine compounds), alcohols potentiate their microbicidal activity. Some alcohols ( chlorbuthanol , benzyl - and dichlorobenzyl-alcohol , phenyl - and phenoxy -ethanol , phenoxypropane -2-ol , 2-bromo -2-nitropropane -1,3 -diol ) are used as preservatives.
Phenol and phenolics OH
Phenol and derivatives, even at low concentrations, are very efficient bactericidal and parasiticidal compounds; they have virucidal activity against enveloped viruses, as well as fungistatic and sporostatic activity. Their activity phenol against non-enveloped viruses is variable. Besides disintegration of the cell wall and the cytoplasmic membrane, they irreversibly inhibit the membrane-associated oxidases and dehydrogenases, and at higher concentrations they are also capable of protein denaturation. Phenol was the first disinfectant used (Lister in the 1860s), but due to its toxicity and the availability of more potent and less toxic derivatives it is used no longer. Antimicrobial activity of alkylated phenol derivatives increases with the length of the alkyl chain (up to six carbon atoms) as well as the sensitivity to inactivation by organic
19 Sterilization and disinfection contamination, but water-solubility and toxicity OH decreases. Their water-solubility may be increased OH OH CH by means of combining them with emulgeating 3 agents (e.g. soaps). The potency of halogenated phenolics and biphenyls is multiple times higher as CH3 CH compared to the phenol itself. The simplest 3 alkylated phenolic compound is cresol , which is a ortho-cresol meta-cresol para-cresol corrosive fluid with a characteristic smell, containing ortho-, meta- and para-cresol. Its efficacy can be enhanced using higher temperatures during disinfection, and it preserves its efficacy even in the presence of organic contamination. The chlorinated as well as the isopropyl derivative of the meta-cresol are also used as preservatives. Halogenated, mainly chlorinated, biphenyl derivatives, Cl OH triclosan and hexachlorophene exhibit primarily antibacterial O activity. Triclosan has a marked antibacterial effect against Gram positive bacteria; its efficacy against Gram negatives and Cl Cl fungi is lower, but may efficiently be potentiated by EDTA. triclosan Certain Gram negative bacteria (e.g. pseudomonads) are capable of multiplication in triclosan. Its virucidal activity is limited to HO Cl enveloped viruses, lacks sporocidal and parasiticidal activity. OH Bivalent cations and fatty acids decrease the antimicrobial Cl efficacy. Besides its antimicrobial activity (which is utilized Cl mostly in toothpastes and mouthwashes), triclosan also has anti- Cl Cl inflammatory effect. It is rarely used due to its neurotoxicity, Cl hexachlorophene detected primarily in neonates.
Biguanides
Biguanides are compounds containing two guanidine groups. Their target is the cytoplasmic membrane of the microorganisms and the envelope of the enveloped viruses, but they cause protein denaturation if penetrating into the cell at sufficiently high concentrations. Their primary activity is antibacterial; they are more efficacious against HN NH Gram positives than against Gram negatives. The activity HN N N NH H H N N against the latter may be potentiated by ethanol or H H isopropanol. They are active against enveloped viruses; HN NH they have a weak antifungal activity. They are inactive against mycobacteria, bacterial spores and parasites as well as against non-enveloped viruses. The most frequently used member of this group is Cl chlorhexidine Cl chlorhexidine , as it is only slightly irritating. Chlorhexidine is used mostly in hand- and mouthwashes, but it is also used as a preservative. The molecule is poorly water-soluble, for that reason it is marketed as a water-soluble salt, gluconate or hydrochloride. The pH range for optimal activity is pH 7-8, where the molecule is in a dicationic form. Consequently, anionactive detergents (soaps) and bicarbonate, borate, carbonate, chloride, citrate and phosphate ions abundant in hard water decrease its activity markedly, by forming non-soluble salts with chlorhexidine. A further disadvantage is that its activity is highly concentration dependent, and that it is readily inactivated by organic contamination.
20 Sterilization and disinfection
Alexidine differs from chlorhexidine in the presence of ethyl-hexyl groups and consequently in the rapidity of bactericidal action. The disinfectant exerts its rapid H HN NH 2 H2 bactericidal activity by H C C 3 H HN N N NH H C CH 2 H H 2 3 phase separation and C C N N C C H H formation of lipid 2 C C N H N C C H2 H H H H H H domains in the microbial C 2 2 C H C H2 H2CH cytoplasmic membranes. 3 alexidine 3 Biguanide polymers, such as vantocyl , which is comprised of heterodisperse mixture of polyhexamethylene biguanides, is widely used in ophthalmic formulas and in the food industry. It is equally active against Gram positive and Gram negative bacteria, though Pseudomonas aeruginosa and members of the Proteus group are less susceptible. Vantocyl, similarly to alexidine, forms lipid domains in the membranes leading to permeability changes and to disturbances in the function of certain membrane-associated enzymes. In case of Gram negative bacteria, vantocyl interferes with the function of the outer membrane as well. Diamidines are biguanide derivatives with a two-ring system structure. Propamidine and its bromide derivative dibromopropamidine are used as antibacterial disinfectants, mostly for wound disinfection and in eye drops. Their main target is the bacterial cytoplasma membrane, but their mechanism of action is not exactly known. Their antimicrobial activity decreases at acidic pH and in the presence of organic contamination.
HN NH O O R=H: propamidine R=Br: dibromopropamidine H2N NH2 R R Certain diamidines, as pentamidine, are used as antiprotozoal drugs as well as against Pneumocystis jiroveczii (see below). Otherwise their spectrum is similar to other biguanide derivatives.
Protein denaturing agents
Native proteins possess a characteristic conformation, which is necessary for activity and proper function. Some chemical agents, due to their denaturing activity, alter the conformation of the polypeptide chains, leading to loss of function. (When penetrating to the cell, agents damaging the membranes, surface-active agents, phenol and phenolics, alcohols, are also capable of protein denaturation, but in the activity of these agents this mechanism is of secondary importance.)
Acids and alkali compounds
These agents have bactericidal, virucidal and sporocidal activity. Their effect leads to dimerization of protein side chains and to distortions of the secondary and tertiary structure of the proteins, and consequently to loss of function. The disinfecting potency of strong inorganic acids (HCl, H 2SO 4) and alkalis (NaOH) is determined by their dissociation (generation of free H +- and OH −-ions), as their activity is based on alteration of the environmental pH around the microorganisms. In case of some alkalis, the toxicity of the metal ion released upon dissociation also contributes to the disinfecting activity. Strong acids and alkalis are more active against Gram negative bacteria
21 Sterilization and disinfection and against viruses than against Gram positive bacteria, fungi or protozoa; the resistance of mycobacteria to strong acids and alkalis is extraordinary. Out of the inorganic acids sulphuric acid-dichromate is used to sterilize glassware, while out of the alkalis sodium hydroxide (NaOH) is the most widely used. Due to their strongly corrosive nature they must be used with caution. The 2N (2M) solution of NaOH is capable of inactivating prions. Organic acids dissociate weakly, their antimicrobial activity is exerted in a non-dissociated state. They penetrate the cell, where they accumulate and act by inducing an osmotic disequilibrium in the cytosol directly. (Their salts are converted to non-dissociated organic acids and exert their activity as such.) Polyvalent organic acids (e.g. citric acid) frequently have metal ion chelator activity as well, which may contribute to their microbicidal activity. Their spectrum is bactericidal; they lack mycobactericidal, sporocidal, fungicidal, virucidal and parasiticidal activity. Organic acids ( acetic acid , lactic acid , citric acid , propionic acid , benzoic and hydroxybenzoic acid as well as their salts and esters; salicylic acid and its salts, sorbate and its salts, etc.) are widely used in food and cosmetic industry as preservatives.
CH OH O 3 HO OH OH HO H3C OH HO OH CH3 OH OH O O O O O O O O H acetic acid propionic acid lactic acid citric acid benzoic acid salicylic acid
Agents modifying the functional groups of proteins and nucleic acids
The catalytic site of the enzymes contains specific functional groups, which are actively participating in, and are crucial for, the catalyzed processes. Damage or modification of any of these functional groups leads to loss of the enzyme function. Similarly, cell wall, cytoplasmic membrane, nucleic acids and other cellular molecules have functional groups crucial to normal function. Arsenic and mercury compounds react to sulfhydryl groups, formaldehyde and acidic dyes to amino- and imidazolyl groups, while basic dyes bind to hydroxyl- and phosphate groups. The common disadvantage of these agents is that organic and inorganic contamination containing free reactive groups significantly decreases their disinfecting efficacy.
Heavy metal compounds
Water-soluble salts of mercury react with the sulfhydryl side chains of proteins forming mercaptans. The initial step in the inactivation of the sulfhydryl groups is reversible, if the microbial cell has access to glutathione or sodium-thiosulfate, the sulfhydryl group is restored and the cell survives. The salts of silver and other heavy metals take effect in a similar manner. The heavy metal salts are primarily bactericidal and fungicidal; Gram positive bacteria are more susceptible than Gram negatives or mycobacteria. The compounds are virucidal only against enveloped viruses, the parasiticidal acitivity depends on the parasite targeted; all lack sporocidal activity. Mercury compounds bind to carboxyl- and phosphate groups as well as to amines besides sulfhydryl groups; at higher concentrations they denature cytoplasmic proteins. Many mercury compounds have been and are used for disinfection. Mercury(II)-chloride (HgCl 2) was a widely used disinfectant, but it is no longer used due to toxicity. Organic mercury compounds as thiomersal , phenylmercury-citrate and -acetate are less toxic; they are used
22 Sterilization and disinfection as antiseptics for skin disinfection and as O ONa preservatives in different pharmaceutical O products (e.g. in eye drops or vaccines). S O Hg CH However, due to their potential toxicity and 3 Hg irritating property, even the use of these newer compounds is decreasing. thiomersal phenylmercury-acetate Silver compounds are widely used antiseptics, used as salt solutions or in colloidal form. Inorganic silver salts are efficient antibacterial agents, but their use is less and less common due to their irritating property. The most frequently used compound was silver-nitrate (AgNO 3), which has excellent activity against Neisseria gonorrhoeae , therefore it was used to prevent ophthalmia neonatorum , a gonococcal eye infection of the neonates. In the present days it is replaced by 1% silver-acetate solution. In colloidal silver compounds the silver ion is bound to proteins, therefore its release is slower and the disinfecting effect is prolonged. Silver compounds are used chiefly in ophthalmology and, for the treatment of burn patients, in dermatology.
Oxidizing agents
The most widely used disinfectants, halogen derivatives, hydrogen-peroxide and peroxy-acetic acid, belong to the group of oxidizing agents. Their primary target is the sulfhydryl group of proteins, which are oxidized to form disulfide cross-links. Some compounds (e.g. chlorine compounds) may oxidize amino- and indolyl groups, as well as the hydroxyl group of the phenol side chain of tyrosine.
Halogen derivatives
Derivatives of chlorine and iodine are used as disinfectants since the 19 th century. Iodine compounds are mainly used in dermatology, while chlorine and its derivatives are frequently used for water disinfection. Iodine derivatives are active against both Gram positive and Gram negative bacteria, they have fungicidal and virucidal activity; when used for longer exposure times, they are sporocidal and parasiticidal. The microbicidal activity of iodine is best below pH 6.0 in the form of elemental iodine (I 2), the microbicidal activity is decreasing with increasing pH. The − − antimicrobial effect of iodide ions (I and I 3 ) formed in aqueous solutions is negligible. Iodine tincture is an alcohol-based solution of 2% iodine and 2% potassium-iodide (KI), which is used as skin antiseptic, however, due to its strong irritating and discolouring properties, its use is decreasing.
Iodofors (e.g. Betadine®, I Povidone®) are stable complexes of 3 H O N iodine formed with surface-active N O N O agents. Their water-solubility is CH CH CH CH3 CH2 CH2 excellent; they are not corrosive and n m not irritating. They may be used as iodofors single-phase disinfectants, as besides their disinfecting effect, their cleaning activity also takes effect. Their disinfecting activity is due to the elemental iodine gradually released from the complex; upon decreasing iodine content, their microbicidal effect is also decreased. The depletion of the iodine content is marked by a change of colour. Their disadvantage is that light and temperatures above 35°C rapidly decrease the active iodine content; only the freshly prepared solution should be used
23 Sterilization and disinfection for disinfection. Their further disadvantages include rapid inactivation by organic compounds; they may also provoke allergic symptoms in persons sensitive to iodine. Chlorine is a yellowish-green, irritating, suffocating and highly toxic gas. Elemental chlorine is solely used for water disinfection, but even in this field it is gradually replaced by its less toxic derivatives. The different chlorine compounds, hypochlorites and chloramines are widely used cheap disinfectants with rapid activity. The exact mechanism of their microbicidal activity is unknown; they act most probably through chlorination of different macromolecules. (Chlorination of the amino groups of the DNA-stabilizer polyamines may be one of the main targets.) In case of hypochlorites, the released oxygen also contributes to microbicidal activity. The efficacy of chlorine compounds can be enhanced by increased temperatures. They exhibit bactericidal, fungicidal and virucidal activities; some derivatives also possess sporocidal and, against certain parasites, parasiticidal activity at higher concentrations. They are highly sensitive to changes in the pH and their activity is concentration dependent, they are irritating, toxic and prone to damage the material to be disinfected. Besides these, their antimicrobial activity is significantly lower in the presence of organic contamination, as the active chlorine first binds to the contamination surrounding the microbes, forming a poorly soluble precipitate around the microbe, which protects it from the disinfectant. They are unstable compounds, prone to spontaneous decomposition and inactivation, for that reason only freshly prepared solution should be used for disinfection. Hypochlorites are the oldest chlorine compounds used for disinfection. They are cheap disinfectants, which can be stored as a ready-to-use solution; they can be combined with cationic or anionic detergents. Hypochlorites are available in powder or solution forms, the most widely used compounds are sodium hypochlorite (NaOCl) and calcium hypochlorite (Ca(OCl) 2). The antimicrobial activity of the hypochlorites depends upon the environmental pH; at weakly acidic and neural pH the non-dissociated hypochloric acid (HOCl) is dominant, while at basic pH the 100-fold less active hypochlorite ion (OCl −). With decreasing pH the maximum of activity is reached at pH 5, further decreasing the pH will also decrease the stability of the solution as well. The stability is provided by adding NaOH for long-term storage. The organic chlorine derivatives used for disinfection are the chloramines, including chloramin-B, dichloramin-B and halazone, the latter is applied mainly for water disinfection. Their advantage over hypochlorites is that they are more stable, less prone to inactivation by organic contamination and they are less corrosive.
O O O Cl O Cl S N S N S N Cl Cl O Cl O HO O
chloramin B dichloramin B halazone
Ozone
Ozone (O 3) is an unstable molecule containing three oxygen atoms, its oxidizing activity arises form the release of reactive oxygen radicals (e.g. superoxide) formed upon decomposition. It is a toxic and irritating gas, but its advantage is that it does not form toxic decomposition products. It kills all microbes. Its sole use is water disinfection.
24 Sterilization and disinfection
Peroxides
Hydrogen peroxide (H 2O2) is a colourless, transparent fluid; it is marketed as 3-90% aqueous solution. Hydrogen peroxide is non-toxic; it is decomposed to biologically harmless materials, oxygen and water. It is rapidly decomposed in living tissue due to the activity of the catalase enzymes, for this reason its germicidal activity is of short duration. It exhibits a wide microbicidal spectrum; it is bactericidal, fungicidal, virucidal and parasiticidal. At higher concentrations (35%) and at higher temperatures it also has sporocidal activity. Gram positive bacteria are more susceptible than Gram negatives, but most bacteria are capable of inactivating hydrogen peroxide by means of catalase and peroxidase enzymes. Though the mechanism of the antimicrobial action is primarily the oxidizing activity, toxic hydroxyl free radicals (formed in iron ion dependent reactions) also contribute in some cases. Under aerobic conditions hydrogen peroxide may also cause DNA breakage directly, but these are repaired by the DNA repair mechanisms. Hydrogen peroxide is used primarily as an antiseptic (at a concentration of 3%) to disinfect skin or wounds, in contact lens storage solutions and for plasma sterilization (see above). Peroxyacetic acid has much more potent microbicidal activity compared to hydrogen peroxide; it is bactericidal, fungicidal, virucidal, parasiticidal and sporocidal even at concentrations as low as 0.3%. It may also be used in plasma sterilizers (see above). Its main advantage, similarly to hydrogen peroxide, is that it is environment-friendly, because it is decomposed to oxygen and acetic acid. In contrast to hydrogen peroxide, it is not inactivated by catalases and peroxidases, and its activity is not impaired by organic contamination. However, it is highly corrosive and irritating. Its main application is sterilization of heat-sensitive medical devices (e.g. haemodialysis equipment), but it is also used to disinfect surfaces. Peroxyacetic acid, similarly to hydrogen peroxide, denatures cellular proteins, increases the permeability of the cell wall by oxidative lysis of the disulfide bonds, and inactivates enzymes and other proteins through oxidization of sulfhydryl groups. Peroxyacetic acid and hydrogen peroxide synergistically enhance the activity of one another.
Dyes
The utility of aniline and acridine dyes is not confined to staining of microbes, but they also exhibit antimicrobial activity even at low concentrations. Their activity is mostly bacteriostatic (rarely bactericidal), fungistatic and parasiticidal against certain parasites. They uniformly lack sporocidal and virucidal activity. They react to the acidic phosphate groups of nucleic acids and proteins. They are easily inactivated by serum and by other proteins; their usage is also hindered by their colour and potential carcinogenicity. Dyes are used primarily in dermatology, and they may serve as selective components in different growth media used for bacteriological diagnostics.
25 Sterilization and disinfection
Aniline dyes
Aniline dyes are the derivatives of the triphenyl-methane, their most frequently used representatives are malachite green , brilliant green and crystal violet . They are highly selective; they inhibit Gram positive but not Gram negative bacteria efficiently. Their putative mechanism of action is damaging membrane proteins. Crystal violet is thought to interfere with the biosynthesis of the cell wall peptidoglycane as well, by inhibition of the conversion of UDP-acetyl-muramic acid to UDP-acetyl-muramic peptide. Lipopolysaccharide of the Gram negative cell wall inhibits the uptake of dyes, explaining their lack of activity against Gram negative bacteria.
CH C H 3 2 5 CH H C N 3 3 C2H5 - H3C N HSO4 Cl CH C H + 3 + 2 5 CH N N + 3 C H N CH3 2 5 CH3
Cl brillant green malachit green H C N 3 CH3 crystal violet
Acridine dyes
Acridine dyes or flavins exhibit euflavine bacteriostatic, sometimes bactericidal, activity against a number of Gram positive and Gram + negative bacteria. Acridine dyes are heterocyclic H2N N NH2 molecules, which intercalate into the DNA helix, CH3 acriflavine causing DNA breakage. In clinical practice Cl acriflavin is used as wound disinfectant, which is the mixture of proflavin and euflavin. Only H2N N NH2 euflavin has antimicrobial activity. proflavine
Alkylating agents
The germicidal activity of aldehydes, ethylene-oxide and β-propiolactone is mediated by their ability to alkylate protein side chains. This activity is irreversible, leading to modification of enzymes and loss of enzyme activity. Their importance arises from the broad spectrum of their activity; they readily kill not only the vegetative forms of microbes (they are bactericidal, mycobactericidal, fungicidal, virucidal and parasiticidal), but they also show potent sporocidal activity. Besides the oxidizing agents, alkylating agents are the widest spectrum disinfectants.
26 Sterilization and disinfection
Aldehydes
A number of aldehydes exhibit microbicidal activity. Formaldehyde (HCHO), regarding its targets, is one of the least selective antimicrobial agents; it reacts to carboxyl-, hydroxyl- and sulfhydryl groups of proteins, and inhibit the microbial DNA synthesis by formation of protein-DNA crosslinks. Formaldehyde is available as 34-38% aqueous formalin solution or as solid paraformaldehyde polymer with 91-99% active material content. Formaldehyde gas should be released by heating (formalin solution) or by potassium-permanganate treatment with added water (paraformaldehyde) prior to usage. It kills all microbes when used at a sufficiently high concentration at temperatures 20 °C or higher in an environment with a relative humidity of 60-80%. A 1:10 diluted formalin solution (4% solution) can be used for surface disinfection. Is usage is hindered, besides its significant toxicity, by its irritating property, poor penetration and by its rapid inactivation by organic contaminants as well as by its liability to polymerization at room temperature. It is used to disinfect buildings, rooms, equipment and to sterilize heat-sensitive materials. As it is highly toxic and potentially carcinogenic, thorough aeration is necessary after formalin disinfection. Formalin is also used to inactivate viruses or bacteria as well as to convert toxins to toxoids during vaccine production, as it does not alter antigenicity. To decrease the irritating property of formaldehyde with S preserved activity and spectrum, formaldehyde concentrates were HO C N NH developed, which release formaldehyde gas gradually. Noxythioline H2 H CH3 (N-hydroxymethyl-N-methylthiourea) powder releases formaldehyde noxythioline and N-methylthiourea when treated with water. This agent is also used for disinfection of body cavities and to treat peritonitis. Polynoxyline (poly[methylene-di(hydroxymethyl)urea] is a polymeric condensate of carbamide and formaldehyde, with an effect similar to that of noxythioline, marketed as gel or as pastilles. Taurolidine is a condensate of two taurine and three O H H N N O formaldehyde molecules. Its activity surpasses that of the pure S S O O formaldehyde; its area of use is identical to that of noxythioline, but N N the aqueous solution of taurolidine is more stable. The urine decontaminating action of methenamine is also based on release of taurolidine formaldehyde (see below). Glutaraldehyde is the most widely used dialdehyde, which, H2 H2 similarly to formaldehyde, exhibit bactericidal, fungicidal, virucidal, H C C O C C C sporocidal and parasiticidal activity. It is tenfold more active than H formaldehyde and less toxic at the same time, its additional advantage O 2 H is that its microbicidal activity is not impaired by organic glutaraldehyde contamination. Its antimicrobial action is mediated mostly by inactivation of amino- and sulfyhydryl groups. Its activity is best at mildly alkalic pH, as this pH exposes more binding site for the disinfectant on the cell surface, but above pH 8 stability is rapidly decreasing due to polymerization. The compound is highly stable in acidic solutions, but its activity is much poorer. Activity of acidic solution can be increased by increasing the temperature, which leads to depolymerization and release O of the dialdehyde. In practice, glutaraldehyde is marketed as a stable, 2% or H more concentrated acidic solution; prior to usage it should be activated by H alkalizing to reach the optimal activity. This activated solution has a short half-life; it preserves its activity for up to two weeks only. Glutaraldehyde O is used mainly for cold sterilization of heat-sensitive medical (surgical) ortho-phtalaldehyde
27 Sterilization and disinfection materials or equipments (e.g. endoscopes). Ortho-phtalaldehyde is an aromatic compound containing two aldehyde groups. Its antimicrobial spectrum is similar to that of glutaraldehyde, but its efficacy is much higher. Its advantage over glutaraldehyde that it is less irritating, does not have a foul odour, it is stable in a wide pH range (pH 3-9) and therefore does not need activation. Its disadvantage is that it stains grey protein-containing materials including the skin; therefore its usage needs increased attention. It is primarily used in the clinical practice to disinfect endoscopes.
Ethylene-oxide
Ethylene-oxide is a fluid below 11°C, at higher temperatures it is a O rapidly evaporating compound. For sterilization it is used in a gaseous H H form, its application requires special equipments and circumstances H H (certain temperature and humidity). As the pure ethylene-oxide is highly ethylene-oxide explosive, irritating and corrosive, it is used mixed with 10-90% CO 2, Freon or other gases. It is one of the widest spectrum disinfectants; it kills all bacteria including mycobacteria and the spores, though its killing is slow. It also exhibits fungicidal, virucidal and parasiticidal activity. The ring of the ethylene-oxide opens up in the presence of the labile hydrogen of carboxyl-, amino-, sulfhydryl-, hydroxyl- or phenolic groups, and • replaces the hydrogen atom with the radical formed ( CH 2CH 2OH), leading to impaired function. Moreover, it also reacts to DNA and RNA, probably by binding to the nitrogen atoms of the purine and pyrimidine bases as well as by esterification of the phosphate groups. It penetrates excellently and, as it is highly active even at low temperatures, can be used to sterilize heat-sensitive materials. Its disadvantages include severe toxicity, mutagenicity and carcinogenicity; due to these properties its usage demands special attention. Due to its toxicity its usage is decreasing, being replaced by β-propiolactone possessing more favourable characteristics.
β-propiolactone O O The compound is a fluid with sweetish, irritating odour at room temperature. As it is unstable at room temperature, it should be stored at beta-propiolactone 4°C. β-propiolactone is not flammableor explosive, and its activity is many times higher than that of the ethylene-oxide, in spite of its poorer penetration. Its spectrum is similar to the spectrum of ethylene-oxide, it kills practically all microorganisms. To reach optimal potency, β-propiolactone requires high relative humidity (75-80%) and a temperature around 25°C. The agent is chiefly used to replace formaldehyde, in the sterilization of surfaces or large spaces. Its antimicrobial action is faster than that of formaldehyde, and traces of β-propiolactone can be removed faster from the sterilized spaces.
Disinfectant combinations and disinfectant systems
Unfortunately, all available chemical agents used for disinfection has limitations arising from their spectrum, sensitivity to organic contamination, unfavourable stability, interactions with other disinfectants or from their toxicity, irritating property and corrosiveness. To circumvent these limitations, a potential solution can be to form and use disinfectant combinations. E.g. ethanol or isopropanol combined with quaternary ammonium compounds, sodium-hypochlorite or iodine compounds may yield a markedly more effective mixture. Combining cationic tensides or phenol derivatives with aldehydes may result in
28 Sterilization and disinfection decreased toxicity and irritating property. In a number of combinations, disinfectants may synergistically enhance each other’s efficacy; this can be observed with the combinations of hydrogen peroxide and other peroxides or iodine compounds and cationic tensides. Highly sporocidal combination (peroxi-formic acid) can be prepared by mixing hydrogen peroxide and formic acid. Development of antimicrobial agents that can be incorporated into the material of surfaces or medical equipment for on-site antimicrobial effect is a field of intensive research. Such bioactive surfaces can be prepared by using silver salts, biguanides and triclosane, which decreases the microbial adhesion and biofilm formation on the surface. Enhancing the activity of disinfectants is also possible by physical effects. An excellent example is plasma sterilization. Besides this, ultrasound increases the activity of aldehydes and biguanides, while ultraviolet light shows synergy with hydrogen peroxide. Combination of ozone- (or superoxide-) treated water and oxidizing agents yields a cheap and highly potent disinfectant system.
Spectrum of activity of disinfectants
Bactericidal Sporocidal Fungicidal Virucidal Parasiticidal Chemical disinfecting procedures Gram+ Gram- Mycob. activity 1. agents acting on the cell membrane 1.1. surface-active agents 1.1.1. cationic tensides + + + - - ± ± 1.1.2. anionic tensides ------1.1.3. non-ionic tensides ------1.1.4. amphoteric tensides + + + - - ± ± 1.2. alcohols + + + - + ± - 1.3. phenol derivatives 1.3.1. phenol and compounds + + + -1 -1 ± + 1.3.2. chlorinated biphenyls + ± - - ± ± - 1.4. biguanides + ± - - ± ± - 2. protein denaturing agents 2.1. inorganic acids and bases + + - + - ± - 2.2. organic acids&derivatives + + - - - - - 3. agents targeting functional groups 3.1. heavy metal compounds + ± ± - + ± ± 3.2. oxidizing agents 3.2.1. halogen compounds 3.2.1.1. iodine compounds + + + +2 + + +2 3.2.1.2. chlorine compounds + + + +2 + + +2 3.2.2. ozone + + + + + + + 3.2.3. peroxides 3.2.3.1. hydrogen-peroxide + + + ± + + + 3.2.3.2. peroxyacetic acid + + + + + + + 3.3. dyes 3.3.1. aniline dyes -1 - - - -1 - ± 3.3.2. acridine dyes -1 -1 - - -1 - ± 3.4. alkylating agents 3.4.1. aldehydes + + + + + + + 3.4.2. ethylene oxide + + + + + + + 3.4.3. β-propiolactone + + + + + + + 1 the effect is only static 2 only after prolonged exposure time
29 Sterilization and disinfection
Microbial contamination and spoilage of pharmaceuticals. Preservation of pharmaceuticals.
During manufacturing of pharmaceutical products strict microbiological requirements must be adhered to; even in case of non-sterile products only minimal microbiological contamination is tolerated. In spite of the strict regulations and close quality control, however, products may become contaminated, which cannot be marketed due to contamination with a potentially pathogenic organism, to multiplication of the contaminating microbes or to high levels of original contamination. The microbial contamination may lead to spoilage of the pharmaceutical, which results in chemical and physicochemical degradation of the components of the formula and consequently in its unsuitability for further use. Besides, a formula contaminated with a (potentially) pathogenic microorganism may serve, and has served, as a source of infection or intoxication. The economical and prestige loss due to product reclaims for microbiological contamination may also be significant for the manufacturer. Materials used in formulae may serve as nutrients for different microbes. The ability of different microorganisms to utilize and degrade pharmaceutical formulae is highly diverse. While the degradation capacity of simple, monospecific communities or communities comprised of a few species is generally moderate, mixed populations with numerous involved species show potent degrading ability; by cooperation of different microbial species even very complex substrates may be degraded and utilized as nutrients. Under suitable selection pressure, novel degradation pathways may be evolved, enabling microbes to degrade newly developed synthetic components as well. The time required for degradation of different compounds is highly variable, they may be degraded within hours (e.g. phenol), within months (certain detergents), but also within years or decades only (e.g. halogenated pesticides). The speed of the degradation of a certain drug or chemical compound is influenced by a number of factors. i) The chemical structure and ii) physicochemical properties of the molecule, iii) type and number of microorganisms present, and iv) the usability of the compound as a precursor or as an energy source during the biosynthetic processes of the microbes present. To assess the liability of the formula for degradation it is important to determine the proportion of the highly degradable component (natural products or fully degradable synthetic molecules, e.g. detergents). Naturally, degradation is not exclusively microbial; the formula may be degraded by physical (high temperatures, sun exposure) or chemical (oxidative degradation, autodegradation) effects as well, but these issues are not discussed further.
Sensitivity of formula components to microbial degradation
Therapeutic agents (drugs)
Components of the formulas with therapeutic activity may be degraded to less active or inactive derivatives by microbial spoilage. Many microbes were shown to be capable of metabolizing a wide array of drugs in the laboratory setting. Different alkaloids (morphine, strychnine, atropine), painkillers (Aspirin, paracetamole), thalidomide, barbiturates, steroid esters are easily metabolized and may serve as an energy source for the growth of microbes. Fortunately the degradation of the active drug component is relatively rare in the pharmaceutical practice, but in case of certain formulae it should be taken into account (e.g.
30 Sterilization and disinfection degradation of atropine in eye drops, gradual decreasing of the concentration of the active component in steroid-containing unguents due to activity of moulds, hydrolysis of Aspirin by esterase-producing bacteria or inactivation of chloramphenicol by bacteria producing chloramphenicol acetyl-transferase).
Surface-active agents
Anionic detergents, due to their slightly alkalic nature, are relatively stable to microbial degradation, though may be totally degraded when diluted. Stability is increased with increasing length and with increasing complexity of the branching of the alkyl chain. Some non-ionic detergents, e.g. alkyl-polyoxyethylene-alcohols (PEG-lauroyl-ethers) emulgeating agents, can easily be degraded by a number of different microbes. Similarly to anionic detergents, liability to microbial metabolization is decreasing with increased length and with increasing complexity of the branching of the alkyl chain. Other non-ionic tensides, e.g. alkylphenol-polyoxyethylene-alcohols are markedly more resistant to microbial spoilage. Amphoteric tensides, e.g. betains, are also susceptible to biodegradation. Cationic tensides are degraded after longer times and only in high dilutions; however, Pseudomonas species, important as nosocomial pathogens, can grow and multiply in cationic detergents; such contaminated solutions may serve as a source of nosocomial outbreaks.
Organic polymers
Most organic polymers used in pharmaceutical formulae as suspending agents (pectin, cellulose, dextran, etc.) can easily be depolymerised by a wide array of microbes using extracellular microbial enzymes (amylases, pectinases, cellulases, dextranases or proteases). Polymers with lower molecular weight (e.g. polyethylene-glycol) may be degraded totally by oxidization of the hydrocarbon chain. Synthetic polymers used as packaging material (nylon, polystyrene, polyester), are extremely resistant to microbial degradation.
Moisturizing agents
Small molecular weight compounds used to inhibit water loss in certain formulae, as glycerine or sorbitol, can be metabolized totally. These are very prone to microbial degradation; and they can resist degradation only when used in high concentrations.
Fats and oils
These hydrophobic materials are most susceptible to degradation in the form of oil-in-water emulsions, as this formulation increases the solubility of oxygen in the oils. Water drops within the oil phase or the moist film condensed on the surface of the oil may be colonized by fungi.
Sweeteners, aromas and colouring agents
Most sugars and sweeteners used in pharmaceutical formulas can be utilized as nutrient by microorganisms, but the used concentration may be high enough to inhibit microbial growth due to decreased water activity. Aromas and colouring agent used to be stored as stock solutions, but colonization with Pseudomonas species occurred frequently. For this reason, presently these stock solutions are
31 Sterilization and disinfection either stored containing preservatives or are freshly diluted from their alcohol-based solutions, as alcohol-based solutions are less susceptible for microbial degradation.
Preservatives and disinfectants
Many Gram negative bacteria are capable of metabolization of preservatives and disinfectants, especially when used or stored in suboptimal concentrations. Multiplication of Pseudomonas species in stock solutions of quaternary ammonium compounds led to nosocomial outbreaks multiple times. Pseudomonads are capable of metabolization of 4-hydroxybenzoate ester used as preservative in eye drops as well as preservatives used in different solutions or oral suspensions.
Visible signs of microbial degradation in pharmaceutical formulae
Even the first signs of microbial degradations are easily noticed using examinations by sensory organs, due to formation of metabolites with unpleasant smell and taste (‘sour’ fatty acids, amines with fishy smell, other materials with bitter or nauseating smell and taste). The formulae may also be discoloured by microbial pigment production. Depolymerization of suspending agents (e.g. carboxymethyl-cellulose) leads to loss of viscosity and sedimentation of the suspended materials. On the other hand, microbial polymerization of sugars and surface-active agents may lead to formation of viscous mass in syrups, shampoos and creams. Fungal colonization leads to formation of granules in the creams. In viscous formulas bubbles of gas-phase metabolites may be formed. The pH alteration of the product depends on the nature and further degradation of the metabolites formed. The pH changes during the spoilage will influence the further growth of the contaminating microbes. Spoilage of oil-in-water emulsions is characterized by a progressive degradation. Degradation of surface-active agents decreases the stability of the formula. Lipolytic release of fatty acids from the oils leads to pH decrease, to separation of phases and to the collapse of the emulsion. Ketonic oxidation of fatty acids results in development of sour taste and unpleasant smell.
Factors influencing the microbial spoilage
Type and number of colonizing microorganisms
Low numbers of contaminating microbes, if incapable of multiplication in the product, will not necessarily cause noticeable spoilage in the formula. Such a low level contamination may originate from contamination of raw materials, from inadequacy or violation of the cleaning protocols of manufacturing equipment or from inadequate use of the product. Degrading potential is not reliably predicted by the number of contaminating microorganisms. The highly aggressive pseudomonads, even when present in low numbers, present a greater danger regarding degradation or for the user than larger volume contamination of tablets with bacterial or fungal spores. In case of contamination, the signs of spoilage may appear after an ‘incubation time’ of variable length. As between manufacturing and usage of the products long periods may pass, the growth of the contaminant microbes and the consequent spoilage may become significant during this time. Isolating a certain contaminant microbe from a spoiled product does not necessarily indicate that this microbe initiated the spoilage. This isolated microorganism can be a
32 Sterilization and disinfection secondary colonizer overgrowing the primary contaminants; in this case the degrading activity of the primary contaminants created a physicochemical environment favourable for the growth of the isolated secondary contaminant. Contamination with obligate pathogenic microorganisms is unfavourable primarily not because of the spoilage caused. In this case the main risk posed is that these pathogens remain viable in the product, which may serve as a source of infection in this manner. Contaminating opportunistic pathogens (e.g. Aspergillus spores) may represent a similar danger for immunocompromised patients.
Nutrients
Microorganisms responsible for microbial spoilage of pharmaceutical products are frequently non-fastidious with simple nutrient requirements and high metabolic adaptability; these may utilize many components of the pharmaceutical formulae as substrates for growth and biosynthetic processes. Plant- and animal-derived raw materials represent natural, easily utilizable nutrient sources. Even ion-exchanged water may contain enough nutrients to enable a slow growth of Pseudomonas species, frequently associated with water-borne infections. Survival and growth of contaminant is limited primarily not by depletion of nutrients, but by accumulation of toxic microbial metabolites and physicochemical properties of the product unfavourable for microbial growth.
Moist content – water activity
For growth of microorganisms directly accessible free water is crucial. The water activity (A w) of the product shows the proportion of this directly available water, which is calculated as the ratio of the steam pressure of the formula and the steam pressure of pure water measured under the same circumstances. Higher concetration of dissolved material is associated with decreased water activity. Most microorganisms grow faster in more diluted solutions (at higher A w); by increasing the concentration of the dissolved material (with decreasing A w) the growth rate gradually decreases until reaching the minimum (growth) inhibitory water activity . This value is 0.95 for most Gram negative bacteria, 0.9 for staphylococci, micrococci and lactobacilli, 0.88 for most yeasts. Osmotolerant yeasts capable of fermenting syrups may degrade formulas with water activity as low as 0.73, while some extremely xerophilic (drought tolerant) moulds (e.g. certain aspergilli) can grow at 0.61 water activity. Degradation susceptibility of watery formulas can be improved by adding high concentration sugars or polyethylene-glycol, though usage of BP syrup (67 m% saccharose, Aw = 0.86) does not fully inhibit the osmotolerant yeast, and further preservative approaches may be necessary. (The usage of saccharose is decreasing in the pharmaceutical formulae; it is replaced by fructose and sorbitol, which are less cariogenic.) Decreasing the water activity of a formula is also possible by drying, but in this case to prevent water absorption and the consequent microbial growth, these dry and frequently hygroscopic products (tablets, capsules, powders) need special, waterproof packaging. Coating of the film tablets can substantially decrease the possibility of absorption of moisture without hindering the water-solubility of the tablet. This formulation increases the microbial stability of the product even under moist conditions during storage, but also increases the production costs markedly. In moist places with unstable temperature a layer of condensed water may be accumulated on the surface of the originally dry products (e.g. tablets), leading to high local
33 Sterilization and disinfection water activity which may enable the growth of fungi. Similar condensation and consequent fungal growth may develop on the surface of viscous materials and hydrogels (e.g. syrups or cream) as well.
Redox potential
Microbial growth is ultimately determined by the oxido-reduction status of their environment together with the presence of a terminal electron acceptor sufficient for their respiration. The redox potential is normally high even in highly viscous emulsions, as oxygen is well-soluble in oils and fats.
Storage temperature
Microbial spoilage of pharmaceutical products may take place in a wide temperature range (-20 - +60ºC). The storage temperature of the products will fundamentally determine the range of potential contaminants. The long-term storage of pharmaceutical raw materials and even the short-term storage of solutions used for total parenteral nutrition needs a temperature of -20ºC or lower. Refrigeration (4-8ºC) is advisable for multiple-dose topical formulae (eye drops, syrups) to inhibit or slow the growth of microorganisms contaminating the formula during usage. In contrast, water used for injections should be stored at +80˚C between the distilling and packaging steps to prevent regrowth of and endotoxin release from Gram negative bacteria. pH
Providing extreme pH values will prevent microbial contamination and spoilage. Bacterial spoilage is most probable at pH values close to neutral; heavy growth of Gram negative bacteria may occur in antacids, mouthwashes, and even in distilled or demineralised water. At alkalic pH (above pH 8), e.g. in soap-based emulsions, spoilage is uncommon. In products with low pH (pH 3-4; e.g. in syrups with fruit flavouring) the most common contaminants are fungi. Yeast, through degradation of organic acids and by consequent increasing of the pH, may provide opportunity for secondary colonization and growth of bacteria. Though using low pH to prevent microbial growth and spoilage is widely used in the food industry, its application in pharmaceutics is usually not possible.
Packaging
The packaging will fundamentally influence the microbial stability during storage and usage in case of most pharmaceutical products through keeping out of contaminating microbes. Damaged packaging is always associated with increased risk of contamination. The protection of parenteral formulations is of paramount importance, as their parenteral usage carries a high risk of infection. Wide containers for creams are replaced by containers with small-diameter dosing tubes. If low water activity is important in prevention of spoilage of the products, the packaging must be waterproof. Under moist conditions even the paper used for packaging can serve as a nutrient for the microorganisms, therefore preservatives are frequently applied to preserve the packaging itself.
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Resistance of microorganisms in pharmaceutical products
Survival of microorganisms under special environmental circumstances may be influenced by non-living material. Different polymers (e.g. starch or gelatine) may increase the resistance of microbes to heat and to drying. Similarly, adsorption to small-particle materials may aid the microorganisms in survival under certain circumstances.
Health risk arising from microbial contamination
It is now unquestionable that application of contaminated pharmaceutical products carries certain risk for the patients. Although drug-related infections have been documented since the beginning of the 20 th century, the importance of microbial contamination of pharmaceutical products was fully revealed by the 1960-70s. Healthcare was first notified of pharmaceutical-related health risk when Salmonella serovariants were isolated from different tablets, pancreas- and thyroid extracts and salmonellosis was reported from patients using these medications. Similarly, isolation of non-fastidious, saprophytic organisms, which are practically non-pathogenic for healthy people, but may cause life-threatening infections in immunocompromised patients drew attention to the adverse consequences of microbial contamination of pharmaceuticals. Non-fastidious Gram negative bacteria (Pseudomonas , Serratia , Klebsiella ) capable of rapid multiplication in aqueous solutions were many times found responsible for medication-transmitted infections. Improperly sterilized ophthalmic solution contaminated with Pseudomonas aeruginosa caused eye infections even resulting in loss of sight. Similarly, pseudomonads were responsible for contamination of antiseptics used for skin decontamination of burned patients, leading to rejection of the transplanted skin graft and to sepsis. Skin and airway infection of neonates were reported to be caused by Gram negative bacteria or fungi contaminating lotions and balms used for skin care of the babies. Gram negative bacteria multiplying in antacids and in oral formulas, caused severe infections in immunocompromised (tumour) patients. Gram negative bacteria contaminating washing fluids used for dialysis and parenteral nutrition solutions prepared in hospital pharmacy caused severe urinary tract infections and even fatal sepsis in small children. Similar fatal septic consequence of contamination of intravenous infusions during aliquoting with multiresistant Gram negative enterobacteria even reached the media in Hungary recently. Contaminated human tissues and human-derived components of pharmaceutical products may transmit fatal viral infections, e.g. rabies. The initial spread of HIV among haemophiliacs was due to VIII factor preparations derived from collected human blood not tested properly. Prion infections (Creutzfeld-Jacob disease) were also transmitted by growth hormone preparations extracted from human hypophysis. Transfused blood has transmitted hepatitis B and C virus as well as cytomegalovirus infections, even transfusion-transmitted syphilis was reported. In endemic regions malaria or Trypanosoma infections may also be transmitted by contaminated blood or blood products. Susceptibility of different pharmaceutical formulations to contamination may differ significantly. Disinfectants, antiseptics, powders, tablets provide environments unfavourable for microbial growth. In contrast, other products, like creams or body lotions, contain high amounts of nutrients utilizable by microbes (e.g. carbohydrates, amino acids, vitamins) together with substantial amounts of water. The consequences of contamination are ultimately determined by the type and amount of contaminants, as well as the manner of administration. Contaminated injection or infusion preparations lead to the most severe consequences; the resulting sepsis may lead to fatal outcome. Different wounds or skin lesions may also be colonized by microbes contaminating pharmaceuticals; these infections lengthen the hospital stay and consequently increase the healthcare costs. It must be emphasized, however, that in most cases of pharmaceutical-transmitted infections the role of the pharmaceutical product remains unnoticed. Microbial contamination may cause problems even if viable microbes are not present in the product. Lipopolysaccharide (endotoxin) component of the Gram negative cell wall, which is released after cell death may remain active after heat sterilization of sterilized products (injections, infusions and haemodialysis solutions). This endotoxin will induce a number of unfavourable physiological reactions through cytokine activation when entering the bloodstream, causing fever in milder cases, but in severe cases leading to shock, which sometimes may be fatal. Microbial exotoxins are fortunately rarely found as contaminants of pharmaceutical products, but aflatoxin-producing aspergilli were found in plant-derived raw materials.
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Potential sources and prevention of contamination
Contamination during the manufacturing process
Regardless that the pharmaceutical product is manufactured in (hospital) pharmacies or in the pharmaceutical industry, the microbiological quality of the product is determined by the quality of the raw materials used; the manufacturing environment, and the manufacturing process itself. This means that the microbiological quality of the product is influenced by each step of the manufacturing process. The high microbiological quality of the raw materials, especially the quality of water and natural products is very important. The regular maintenance of the manufacturing equipment as well as their thorough cleansing after use must always be meticulously performed to avoid cross-contamination between batches. The choice of appropriate cleaning appliances and their perfect performance is similarly important. The appropriate buildings and manufacturing environment as well as filtered air of appropriate quality must be provided. Besides, the continuous training of employees in proper personal and manufacturing hygiene is required, together with the regular screening of the health of the employees, including screening for asymptomatic carriage of pathogens. The product should be packaged into appropriate, contamination-free packaging, which protects it from contamination during storage and shipping.
Production in the hospital pharmacy – magistral formulations
Preparation of formulas in the hospital pharmacy involves special problems in avoidance of microbiological contamination.
Water quality
Pharmaceutical manufacturing requires high-quality water, which should be subjected to further treatments (distillation, reverse osmosis, ion exchange treatment), depending on the mode of usage. As these treatments require continuous control and monitoring, in parallel with these the control of the microbiological quality of the water can also take place. Storage of water needs special precautions, as certain Gram negative bacteria may survive in water containing only traces of organic material and may start growing when the water is warmed to room temperature. To avoid survival of Gram negative bacteria, the water used for pharmaceutical production should be stored at 80˚C, with continuous circulation (1-2 m/s) to prevent biofilm formation.
Manufacturing environment
The microbial flora of the environment of the hospital pharmacy mirrors the microbiological status of the hospital environment. Free-living opportunistic microorganisms, e.g. Pseudomonas aeruginosa , are present in moist places (water tubes, basins, taps) throughout the hospital. By contaminated cleaning equipment (mops, buckets, cleaning machines) or by humans these microbes can be transferred easily to the area of the pharmacy. The cleaning equipment, if stored wet, provides an environment suitable for bacterial growth leading to high-rate contamination of these equipments. Microbial contamination of the manufacturing environment can be minimized by adherence to the proper manufacturing practice, using special washbasins and containers,
36 Sterilization and disinfection proper cleansing and maintenance of manufacturing equipment, including cleaning equipment as well. Moreover, cleaning of the manufacturing units should be performed according to the manufacturing guidelines.
Packaging
Packaging in sacks, papers, or with corks is not appropriate for packaging pharmaceutics, as these may be contaminated with bacterial or fungal spores. These materials are replaced by plastics resistant to biodegradation. In the past packaging of pharmaceutical products was reused for economic reasons. End-users sent back high number of containers and tubes to the pharmacies, unfortunately together with the microbial contamination it suffered during use. Presently the reusable containers should be thoroughly cleaned and dried then sterilized prior to reuse. Serious problems may arise from improper use of disinfectant solutions, i.e. when the containers are refilled with freshly prepared solution without discarding of the remainder of the used solution and without cleaning of the container. In this manner, the contamination remains in the container and the contaminated disinfectant is used. Another improper and dangerous, but widely used, practice is the repackaging of products and tools bought in large batches to smaller batches, not infrequently in a non-sterile manner. This increases the risk of microbial contamination significantly, and therefore should be avoided, or the repackaging should be performed in a sterile manner.
Contamination during use
The contamination occurring during use is primarily a problem encountered when using multiple-dose formulation. Fortunately, the frequency of such contaminations is decreasing, mainly due to improvements in packaging techniques and in patient care.
Contamination of human origin
During using pharmaceuticals, patients may contaminate the formulations with their own microbial flora, which may result in autoinfection during further use. Topically used formulas are exposed to a markedly higher risk of contamination, as these are usually administered directly to the area of application using the hands, thus can easily be contaminated with the normal flora of the skin (staphylococci, Micrococcus species, diphtheroids), but the transiently carried Pseudomonas may also appear in the formula. The risk of contamination can be decreased significantly by using disposable dosing devices. Contamination of multiple-dose formulations in the hospital may serve as a source of cross-contamination and cross-infection between patients. E.g. zinc-containig ointments used for care of pressure sores are easily contaminated with Pseudomonas aeruginosa or with Staphylococcus aureus . If the formula does not contain preservatives, these contaminants start growing, especially in the presence of water (oil-in-water emulsion, fluid films, condense water). The next use of the formula may transmit the contamination to another patient and in this manner may be the source for a nosocomial outbreak. Personnel involved in patient care and responsible for dosing and application of pharmaceutics may also serve as a source of contamination. During patient care the hands of the nurses are contaminated with the pathogens present in the hospital environment. Though these contaminations can be efficiently removed by proper hand hygiene, when handwashing and hand disinfection is improper, e.g. due to high number of patients or crowding, they can
37 Sterilization and disinfection find their way into the formulas. Hand-protecting creams used by the nurses may also become contaminated, especially if their containers are kept uncapped at the washbasin. For the above reasons, the proper hand hygiene (handwashing and hand disinfection) of the hospital personnel is paramount for prevention of nosocomial cross-infections. The hand creams used for skin care of the employees should be appropriately preserved and it is advisable to use disposable applicators. The best approach for patient care is using a newly opened formula for each patient and to avoid touching the patients during application of pharmaceutics.
Environmental sources
From the air a few microorganisms will contaminate the pharmaceutics left open. High number of contaminants will find a way to the topical formulas if left open next to the washbasin or if used with wet hands. As these contaminating microbes are not fastidious, usually they replicate quickly. This problem is even worse if the formulas are kept in the warm patient room or in the warm and moist bathroom. Body washes used in the patient care can be contaminated with opportunistic pathogens (e.g. Pseudomonas ) rapidly, and microbes can replicate quickly in these products. The risk of cross-infection is increased further if multiple patients (in shared bathrooms) share the same shower gel for prolonged periods. The microbial population of homes and hospitals differ markedly. Pathogenic microorganisms are found more frequently in the hospital environment, and consequently can be isolated more frequently from pharmaceuticals used in hospitals. In the home of the patients the risk of contamination is substantially lower as the medication used is comprised of a smaller amount of pharmaceuticals and patients rarely share formulae.
Contamination originating from devices and equipment
For preparation and dosing of pharmaceutics a number of different tools (spatulas, sponges, brushes) are used by the patients or by the healthcare personnel, which may serve as a contamination source if used multiple times. Moreover, if reused on a new batch they may transmit contamination to the newly opened sterile products. Contamination originating from these issues may be prevented by using disposable dosing devices. Apparatuses used in hospital patient care (moisturizing devices, incubators, ventillators, etc.) need proper maintenance and proper disinfection after use. The decontamination of the equipment is performed by disinfectants, which themselves may ironically be contaminated with opportunistic microbes, especially if used improperly, and in this manner the ‘disinfected’ devices may become a source of infection. To prevent disease transmission by disinfectant, these should always be used as prescribed (see above).
Factors influencing the outcome of pharmaceutical-transmitted infections
Contaminated pharmaceuticals contribute to spread of cross-infections in hospitals, and these nosocomial infections cause excess morbidity and mortality, increase the length of hospital stay and consequently lead to increased healthcare costs. Clinical manifestations range from mild local symptoms (wound infection caused by contaminated ointments) through gastrointestinal symptoms (caused by using contaminated oral formulations) to fatal septic consequence (contaminated intravenous infusions). The most severe cross-infections arise from direct inoculation of the contaminated pharmaceutical to the bloodstream; the group with the highest risk is the groups of immunocompromised patients and patients requiring intensive care.
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The outcome of the cross-infections caused by contaminated pharmaceuticals or equipment is determined basically by three factors; i) type and extent of microbial contamination, ii) mode of administration, and iii) patient resistance to infection.
Type and extent of microbial contamination
Microorganisms contaminating pharmaceuticals may be obligate or opportunistic pathogens. Fortunately, obligate pathogens, e.g. Clostridium species or Salmonella serovariants are rarely found in pharmaceuticals, but their presence may have dire consequences. Wound infections with occasional fatalities were caused by powder contaminated with Clostridium tetani spores in neonates. Contaminated thyroid- and pancreas extract was the source of a number of salmonellosis outbreaks. In contrast, opportunistic pathogens, e.g. Pseudomonas aeruginosa , Klebsiella , Serratia species, are isolated from pharmaceuticals with higher frequency. These normally do not cause infection in immunocompetent individuals, but may induce life-threatening infection in patients with impaired immunity, e.g. in the elderly, in transplant recipients or in patients with malignant disease. The virulence of microorganisms and the related minimum infective dose differs by species and even within a certain species by strain. A number of exogeneous factors (wounds, foreign bodies, drugs causing local vasoconstriction, etc.) may significantly decrease the infective dose of the microbe.
Mode of administration
The most severe consequences arise from direct inoculation of contaminated pharmaceuticals into the bloodstream or into normally sterile body sites (cerebrospinal fluid, eye, peritoneal cavity, etc.). In medial practice, therefore, epidural injections are given using a bacterium filter. Solutions for injections and for eye drops are easily contaminated by Gram negative bacteria, and endotoxins released upon death of Gram negative cells also increase the risk of adverse effect. Solutions for total parenteral nutrition are also good media for microbial growth. Pseudomonas aeruginosa contaminating eye drops or contact lens solutions may infect the conjunctiva and the cornea, which may lead to blinding eye infections. In case of per os formulations, the consequences of contamination are influenced by the acidic pH of the stomach (decreased in patients taking antacids), the acid tolerance of the microorganism and the amount of stomach content. The risk posed by contamination of topical formulations is highest when applied on injured skin (traumatic wounds, burns, pressure sores), which are rapidly colonized by contaminating microbes; application on the intact skin carries lower risk. Local steroid treatment increases the risk of nosocomial skin infections.
Resistance of the patients to infection
The susceptibility of the host to infection will ultimately determine the outcome of pharmaceutical-transmitted infections. Hospitalized patients are more exposed and more susceptible to infections than patients cared for at home. The immune response of neonates, elderly people, diabetic and traumatized patients, patients with malignancies or patients treated with immunosuppressive drugs is impaired, making them especially susceptible to infections. For this reason, pharmaceuticals should be applied with special care in case of these patients to prevent nosocomial infection.
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Preservation of pharmaceutical products
To prevent microbial spoilage of pharmaceutical products and to eliminate small-scale contamination possibly occurring during storage or usage, preservatives with antimicrobial effect are used. As microbial growth and biodegradation is inhibited when water is lacking, preservatives are used only in case of formulae containing water (solutions, suspensions, creams). In case of products with low water activity (tablets, capsules) preservatives are not necessary. It is forbidden to use preservatives to compensate for the effects of a poor manufacturing environment. Ideal preservatives i) have broad spectrum of activity, ii) react to the contaminating microorganisms only, and not with formula components, iii) non-toxic and not irritating, and iv) remains stable and active until the use of the formula. Unfortunately, most antimicrobial agents are unselective; frequently reacts to compounds in the formulae and many are toxic or irritating. Excluding toxic and irritating agents, the efficiency of other agents that may be used as preservatives is not sufficient. Among the presently used agents there are no such preservatives that can be used in formulae where toxicity is a very important issue (injections administered to the eye or to the central nervous system). Many preservatives used in cosmetics may cause contact dermatitis, making them ineligible for using in pharmaceutical creams and ointments as well. Though it is a prerequisite for a preservative to show the fastest killing possible, this rapid killing is provided only in relatively simple aqueous solutions (eye drops, injections). In physicochemically complex systems (emulsions, creams, etc.) the killing effect is of significantly shorter duration and rather the microbistatic activity is the rule. In formulae with low water activity (tablets, capsules) preservative effect is not at all possible, as the water phase, the medium for the antimicrobial activity, is not present.
Factors affecting the efficacy of preservatives
Factors affecting the efficacy of preservatives are the very same to those affecting the efficacy of disinfectants. Besides these, the characteristics of the formula to be preserved and even packaging influence the availability (effective concentration) of the preservative and consequently the efficacy of preservation. Molecules of the preservative interact with the microorganisms, with the components of the formula as well as with the contaminating compounds present. These interactions lead to decreasing effective concentration of the preservative, and consequently decreased activity of the remaining preservative. In multiple-phase formulas the preservative is distributed in an unstable equilibrium between the different phases, as i) between the water and the oil phase, ii) between the water phase and the micelles of the surface-active agents, iii) between the water phase and the phase of polymeric suspending agents, iv) between the water phase and the phase adsorbed to the wall of the container or to the surface of granules and v) if microorganisms are present, between the different phases of the formula and the surface of the microorganisms. The full efficacy of the preservative usually depends on the concentration of free preservative molecules in the water phase. Availability of the preservative can be decreased markedly by interaction with the packaging materials. Phenolics may diffuse through rubber corks, dispenser tubes of multiple-dose injections or eye drops, and even may react to the nylon tubes of creams. The availability of the quaternary ammonium compounds may be decreased by adsorption to plastic or glass surfaces of containers. The concentration of volatile preservatives decreases
40 Sterilization and disinfection significantly during regular opening and closing of containers during usage, therefore, though their efficacy is well-preserved during storage in containers closed in an airtight manner, it will rapidly and significantly decrease after opening of the container. Consequently, the type and concentration of the preservative to be used in a formula should be determined for each formula individually during the formula design, based on the components of the formula and on the contamination expected during usage. Capacity of the preservatives is the contamination level tolerated by the given formula containing the given preservative without degradation or loss of activity. This capacity varies from formula to formula; and is determined by the characteristics of the preservative and the formula and is also influenced by the type and number of the contaminating microbes as well as by their capability to degrade formula components and the preservative(s). Capacity can be measured using two principles. Some laboratory tests use cultures with a relatively high CFU number of different predefined microbes for modelling contamination and the efficacy of the preservative is determined by the examination of the number of surviving microbes after a predetermined time period. Other tests monitor the efficacy and inactivation of the preservative by repeated inoculations until the total expiration of the preservative efficacy. The latter technique measures the capacity in the given system more exactly, but the test is expensive and time-consuming.
Control of the microbiological quality of the products
Quality assurance includes all control processes, which ensure with high probability that pharmaceuticals continuously comply with the special quality requirements. The quality assurance process involves formula design and development, the good manufacturing practice (GMP), the quality control processes and the post-marketing follow-up of the product. As many microorganisms may represent a potential risk for the patients or may cause spoilage of the formula, it is necessary to assess the risk of contamination for all formulas. This risk assessment should follow up the product throughout its whole life cycle, from raw materials to the product to be marketed and to the end user.
Quality assurance during formula design and development
Pharmaceutical-related infections and spoilage of the product caused by microbial contamination during manufacturing, storage or usage may be prevented by production of the pharmaceuticals in sterile single-dose formulas. Though the single-dose formulas are more expensive, its usage significantly decreases the risk of infections transmitted by contaminated pharmaceuticals. The high infection risk and more adverse outcome expected in case of parenteral formulations, together with high toxicity of systemically inoculated preservatives necessitate the development of sterile single-dose formulas. In case of formulas with lower risk of infection, prevention measures with lower efficacy and less cost are also acceptable. In case of eye drops used at home, as the infection risk is lower, these are marketed as sterile multiple-dose formulas protected with preservatives. In case of oral and topical formulas, the risk of infection arising from microbial contamination is relatively low; the most important problem is to avoid chemical and physicochemical (non-microbial) spoilage and preservation of the quality of the product. For this reason, multiple-dose formulas are more cost-effective in case of these products.
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Good pharmaceutical manufacturing practice
Good pharmaceutical manufacturing practice (GPMP) is a collection of standards to be applied during manufacturing pharmaceutical products, including guidelines on the planning and construction of factories, on quality control of raw materials, on validation of processes during manufacture, on the production process and on the control of contamination. Quality control (QC) is the part of GPMP involved in examination of the adherence to the specific standards and of the related documentation. These issues are discussed in detail in the course of Pharmaceutical technology.
Quality control processes
The first critical point during microbiological quality control is the timing of sampling and choosing appropriate (representative) samples. For instance, uneven distribution of contaminating microorganisms in viscous formulas represents a serious problem for proper sampling. The number of viable cells that can be demonstrated from a given formula or other sample is ultimately determined by the type of the medium used (even different batches of the same medium may be different), and by the conditions of the isolation and incubation. The European Pharmacopoeia prescribes qualitative and quantitative standards for microbiological purity of pharmaceuticals; depending on the mode of usage, there are different threshold total cell numbers and different contaminants to be excluded. Sterile formulas ( microbiological purity class 1 ) must not contain any contaminating microorganisms. In formulas used topically or in the airways ( microbiological purity class 2 ) the maximum level of contamination is 10 2 microbes (aerobic bacteria and fungi) per one gram or millilitre of the formula, presence of Pseudomonas aeruginosa and Staphylococcus aureus is not tolerated. In formulas for oral or rectal use (microbiological purity class 3 ) the tolerable level of contamination is 10 3 aerobic bacteria and 10 2 fungi per one gram or millilitre of the formula, Escherichia coli must not be present. A higher level is tolerated if the formula contains raw materials from natural sources (10 4 bacteria, 10 2 fungi and 10 2 enterobacteria or other Gram negative bacterium), but presence of Salmonella , E. coli and S. aureus is not tolerated. In case of medicinal herb products (microbiological purity class 4 ) the maximum tolerated level of microbial contamination is 10 7 bacteria and 10 5 fungi, including a maximum of 10 2 E. coli per gram or millilitre for products prepared for usage by boiling water. For herbs not prepared by boiling water (prepared without heat treatment) the levels are 10 5 bacteria and 10 4 fungal cells; enterobacteria may be present in cell numbers not higher than 10 2, E. coli and Salmonella are not tolerated. Most manufacturers regularly test their products for the total microbial cell numbers and for the specific pathogens to be excluded. Altered or oscillating total cell count or appearance of a specific or new microorganism indicates problems of the manufacturing process or arising of special problems. Parenteral formulas must not contain endotoxin (pyrogenic compounds), to prevent endotoxin-mediated adverse effects. Previously the presence of endotoxin was tested using animal (rabbit) inoculation, when fever of the animal was the indicator of the presence of endotoxin. Presently the test is performed by the Limulus lysate test. This test utilizes the lysate of the amoebocytes of the horseshoe crab (Limulus polyphemus ), which reacting to the bacterial lipopolysaccharide (LPS) forms an opaque gel-like precipitate even with very small concentration of LPS. In a modified version of the test the endpoint is indicated by colour change of a chromogenic substrate, this test is evaluated by means of a spectrophotometer. In a yet experimental version of the test cell cultures are used to evaluate not only the presence of
42 Sterilization and disinfection the endotoxin, but also its activity by measuring the induced cytokine release. Food industry has developed and uses a wealth of analytical procedures to demonstrate microbial toxins from plant-derived foodstuffs. These methods can also be used to test natural raw materials (medicinal herbs, plant oils). Though the examination of physicochemical and chemical spoilage of pharmaceuticals is not part of the regular quality control process, these tests may occasionally be necessary during formula development or to track an unexpected manufacturing error. To detect and identify metabolites produced during the degradation process, chemical analytical procedures (gas- and liquid chromatography, mass spectrometry, etc.) can be used.
Resistance against disinfectants
Resistance of different microbes to biocidal agents is highly variable; it is determined by the (cellular) structure and the physiological properties of the microorganism. In general, the highest resistance is exhibited by oocysts of cryptosporidia, bacterial endospores, mycobacteria and protozoal cysts, intermediate resistance is seen in case of Gram negative bacteria and Gram positives show the highest susceptibility. However, this classification is not absolute; susceptibilities may vary between species and even between different strains of the same species. For example, spores of Clostridium difficile are markedly more susceptible to disinfectants than spores of Bacillus subtilis or even than vegetative cells of Mycobacterium avium-intracellulare . Within the group of Gram negative bacteria, members of the Pseudomonas , Proteus and Providencia genera show disinfectant resistance above the Gram negative average. Disinfectant resistance of vancomycin resistant enterococci (VRE) significantly exceeds the resistance of vancomycin susceptible strains, but both resistant and susceptible strains are more resistant to disinfectants than antibiotic-susceptible and antibiotic-resistant Staphylococcus Prions (CJD, BSE) aureus strains. The figure shows the groups of microorganisms in Certain protozoa (Cryptosporidium) order of decreasing disinfectant resistance. This order is roughly the same regarding resistance to physical effects as Bacterial and fungal spores well. The order is in general sense, in case of certain Mycobacteria disinfectants or physical effects it may be different. (M. tuberculosis, M. avium) The resistance of microbes to disinfectants (and to Protozoal cysts physical effects as well) derives from natural physiological (Giardia) characteristics or processes (sporulation, adaptation ability, Small non-enveloped viruses biofilm production) of microorganisms; resistance may be (polioviruses) regarded as decreased susceptibility or tolerance rather than Protozoal trophozoites true resistance. (Acanthamoeba) In the development of secondary (acquired) resistance Gram negative bacteria or tolerance the improper use of disinfectants and improper (Pseudomona, Providencia) infection control have a crucial role. Decreased susceptibility Vegetative cells of fungi to disinfectants, besides the resulting decreased efficacy of (Candida, Aspergillus) disinfectants and consequent survival of microbial Larger non-enveloped viruses (Adenoviruses) population, may provide cross-resistance to certain antibiotics; some genes providing acquired resistance to Gram positive bacteria (S. aureus, Enterococcus) disinfectants may also provide resistance to certain antibiotics. In this manner, misuse or inappropriate use of Enveloped viruses disinfectants may select for resistance not only to the (HIV, influenzavirus)
43 Sterilization and disinfection disinfectant, but also to antibiotics. At the same time, certain mechanisms of antibiotic resistance may lead to decreased susceptibility to certain or all disinfectants or physical microbicidal effects as well.
Bacterial resistance to disinfectants
Resistance of bacteria to disinfectants may originate from the natural physiological properties of the bacteria (intrinsic or natural resistance) or may be acquired by the bacteria. The acquired resistance may be mutational or may be developed by uptake and expression of genes on mobile genetic elements (plasmids, transpozons or integrons). Natural (generic) resistance is commonly seen in Gram negative bacteria, in bacterial spores, in mycobacteria and under certain circumstances in staphylococci. This natural resistance is mediated by the cellular structure, poor permeability of the cell wall (lipoid outer layer in mycobacteria), spore wall (thick multilayer wall), outer membrane (in Gram negative bacteria) or glycocalix, or by biofilm production of the bacterium. Acquired resistance is most frequently found against heavy metal compounds, primarily against mercury-based disinfectants. Spreading of the resistance to multiple disinfectants is observed in recent times, especially among staphylococci.
Natural resistance of bacteria
Natural or intrinsic (generic) resistance is the net result of properties which provide means for the bacteria to evade disinfectant action and are uniformly characteristic to (almost) all strains of a given species. These properties are not developed specifically against the disinfectant effect driven by selection pressure exerted by the disinfectant; resistance is the ‘side effect’ of physiological mechanisms with functions important for survival. The cell envelope provides a permeability barrier that may hinder or even totally inhibit the uptake of certain compounds, thus fundamentally determines the susceptibility of the cell to disinfectants. Intracellullar accumulation of disinfectants may also be decreased by active efflux of the agents. Cells may constitutively produce enzymes, which are capable of degrading and inactivating disinfectants. Bacteria growing in biofilms show special properties necessary for biofilm growth (biofilm phenotype), due to these properties organisms growing in biofilms are more resistant to harmful effects (physical effects, disinfectants and antibiotics); on the other hand, disinfectants (as well as antimicrobials) penetrate poorly into the biofilms. Generally, Gram negative bacteria are more resistant to disinfectants than Gram positives.
Intrinsic resistance of Gram positive bacteria
Cell wall of Gram positive bacteria is constructed from peptidoglycane and teichoic acid. Disinfectants, even larger molecules, penetrate easily through this structure; the cell wall structure of Gram positive bacteria, therefore, provides explanation for their relatively high susceptibility to disinfectants. In case of Gram positives producing glycocalix, the susceptibility is decreased; partly because the glycocalix serves as a permeability barrier and partly because by reacting to or by adsorbing disinfectant molecules, it decreases the effective concentration of the agent. The disinfectant resistance of enterococci is especially high.
44 Sterilization and disinfection
Intrinsic resistance of Gram negative bacteria
The resistance to disinfectants of Gram negative bacteria is higher than that of Gram positives. This is definitely due to the barrier function of the outer membrane, primarily of its lipopolysaccharide (LPS) layer. The uptake of solutions and hydrophilic molecules is regulated by the hydrophilic porin channels of the outer membrane. The barrier function, and consequently the disinfectant resistance may be decreased significantly by EDTA; in contrast, Mg 2+ ions increase the stability of the cell envelope. The intrinsically decreased susceptibility of Gram negative bacteria to certain disinfectants (e.g. triclosane) may also be mediated by active efflux. Active efflux pumps of E. coli and P. aeruginosa provide resistance not to triclosane only, but also to certain antibiotics; such as fluoroquinolones, tetracyclines and ampicillin in case of E. coli or ciprofloxacin in case of P. aeruginosa . In E. coli the efflux pump AcrAB is part of a multidrug efflux system providing resistance to a number of different antibiotics and disinfectants. The expression of the gene coding for the Acr AB pump is regulated by an activator providing multiple antibiotic resistance (multiple antibiotic resistance activator, MarA). To certain environmental stimuli (e.g. in the presence of triclosane or pine oil) the expression of the MarA is increased, leading to hyperproduction of the Acr AB efflux pump and to increased resistance to disinfectants as well as to certain antibiotics, e.g. tetracyclines.
Intrinsic resistance of mycobacteria
The explanation for the extreme resistance of mycobacteria to disinfectants and to other harmful effects is, similarly to the case of bacterial spores, the presence of a complex cell envelope, which acts as a permeability barrier inhibiting uptake of disinfectants. The highly hydrophobic cell wall containing peptidoglycane, mycolic acids and arabinogalactane efficiently inhibits most disinfectants, especially hydrophilic molecules, in reaching their cellular target. Only certain disinfectants have mycobactericidal activity, including some phenol derivatives, peroxides, alcohols and glutaraldehyde. Biguanides and quaternary ammonium compounds are only mycobacteriostatic, even at high concentrations; but may be modified (e.g. alkylated) to become mycobactericidal.
Intrinsic resistance of bacterial spores
The resistance of bacterial endospores (produced by members of the Bacillus and the Clostridium genera) is excellent. Most disinfectants (including alcohols, phenol derivatives, cationic detergents, biguanides and organic mercury compounds) do not exhibit sporocidal activity even at high concentrations, the action is only sporostatic. Disinfectants with sporocidal activity (aldehydes, iodine- and chlorine compounds, hydrogen-peroxide and peroxyacetic acid, ethylene oxide, β-propiolactone) kill the vegetative cells of the sporogenic genera rapidly even at low concentrations, but to kill the endospores, high concentration of the disinfectant and longer exposure times are necessary. The extreme resistance of bacterial spores is the consequence of the presence and chemical structure of the layers surrounding the core of the spore, i.e. the cortex and the coat. The high disinfectant resistance of the spores develops gradually during sporulation, at phase IV to VII (during development of the cortex and the coat and the release of the mature spore). While resistance to biguanides (and high temperatures) appears at phase IV of the sporulation, the susceptibility to glutaraldehyde decreases significantly only in the last phase of the sporulation. Low molecular weight basic proteins present in the core of the spore in 10-20% and small acid-soluble spore proteins play a role in providing resistance to ultraviolet
45 Sterilization and disinfection light and to peroxides. These small protein molecules bind to DNA and protect it from the activity of free radicals harmful for the hereditary material. After germination, the susceptibility of the cell to biocidal effects returns to properties characteristic to the vegetative cell.
Biofilm production as a mechanism of the intrinsic resistance
Biofilms are microbial communities associated with solid (living or non-living) surfaces, consisting of microbial cells and an exopolysaccharide matrix produced by the biofilm microorganisms. Biofilms play important role in biocorrosion, may decrease water quality and may contaminate disinfectants or cosmetics. They may be formed on catheters, prostheses or medical devices serving as reservoir for microbes and increasing the risk of infection and reinfection. Biofilms may be formed by a single bacterial species, (sometimes it contains strains of a single species with different phenotypes), but may also be comprised of a number of different species, including fungi and protozoa as well. In the different regions of the biofilm, microbes live under diverse environmental conditions, which affect their physiological properties as well. For instance, for bacteria living in deeper layers of the biofilm availability of nutrients is limited, leading to slower growth, which, in turn, will result in altered susceptibility to antimicrobial agents. Biofilms contribute to decreased susceptibility to different agents with antimicrobial activity, including disinfectants, in various ways, including 1. poorer penetration of disinfectants into the central region of the biofilm; 2. biofilm material may interact with the disinfectant leading to its inactivation; 3. the biofilm provide a special microenvironment for microbes, which affects the composition of the cell envelope, their physiological properties and processes as well as their growth and replication, consequently affecting the susceptibility of the cells to antimicrobials; cells with ‘biofilm phenotype’ show increased resistance to disinfectants (and antibiotics) several orders of magnitude higher than cells not growing in a biofilm; 4. some microbes in the biofilm may produce and secrete enzymes neutralizing or degrading disinfectants, or enzymes capable of repairing damages caused by the disinfectants, and these enzymes may also protect other species originally susceptible to the biocidal agent; 5. and the biofilm provides good opportunity to exchange genetic elements coding for disinfectant (or antibiotic) resistance, thus facilitates spread of resistance. The cells showing biofilm resistance usually regain their susceptibility if removed from the biofilm.
Acquired resistance of bacteria to disinfectants
Similarly to resistance to antibiotics, resistance to disinfectants may also be developed by mutation or by acquisition of resistance genes encoded on mobile genetic elements (plasmids, transpozons or integrons) and it is also driven by selection pressure exerted by exposure to disinfectants. The mutation may affect the target of the disinfectant or may lead to hyperproduction of the target by overexpression of the coding gene, while plasmid-mediated transferable mechanisms more frequently involve increasing of the active efflux or enzymatic inactivation of the disinfectants.
46 Sterilization and disinfection
Resistance of bacteria transferred by mobile genetic elements
Plasmids may mediate the resistance to silver- and organic mercury compounds, to some membraneactive agents (biguanides, quaternary ammonium compounds, diamidines), to some dyes and to formaldehyde. Resistance may be mediated by decreased uptake (silver compounds) or active efflux of the disinfectant (biguanides, cationic detergents, dyes) as well as by enzymatic inactivation and degradation of the disinfectant (chlorhexidine, formaldehyde, mercury compounds). Plasmid-mediated resistance was reported in case of staphylococci, and in case of several species of the Enterobacteriaceae and Pseudomonadaceae families. Integron-transmitted resistance to quaternary ammonium compounds is frequently found among Gram negative bacteria. In case of methicillin-resistant Staphylococcus aureus (MRSA) strains the gene products of the qac gene family ( qac A-D) provide resistance to several membrane-active disinfectants and to some dyes, by decreasing the intracellular concentration of the agents through active efflux. The proton-dependent exporter proteins encoded by the qac A and qac B genes providing multidrug resistance show high level of homology to efflux pumps providing tetracycline resistance. A wealth of data is available on the plasmid-mediated resistance of Enterobacteriaceae to mercury compounds. Plasmids transmitting narrow spectrum resistance provide protection against Hg 2+ salts and a few organic mercury derivatives, while plasmids coding for broad spectrum resistance protects the cells from practically all mercury compounds. Narrow spectrum resistance is mediated by inactivation of the compounds by enzymatic reduction, while the mechanism of broad spectrum resistance is enzymatic hydrolysis of the disinfectants. In Staphylococcus species the efflux pumps providing resistance to quaternary ammonium compounds is very frequently found together with β-lactamase-mediated resistance. The reason for this is that the qac A/B providing disinfectant resistance and the β-lactamase gene is encoded by the very same plasmid, thus selection with either of the agents will coselect resistance to the other agent as well (coresistance).
Resistance of bacteria due to mutations
Resistance to disinfectants may develop through mutations in the bacterial genome. The driving force for this phenomenon is the selection pressure exerted by the usage of disinfectants. Development of mutational resistance is enhanced by using disinfectants in suboptimal concentrations. The biochemical basis of the resistance to membrane-active disinfectans (quaternary ammonium compounds and biguanides) is the decreased permeability of the outer membrane. In case of E. coli , the main target for triclosane is the enoyl-acyl-protein reductase, a key enzyme in fatty acid synthesis; mutational alteration of this enzyme provides resistance to triclosane. Triclosane resistance in P. aeruginosa is caused by a mutation in the nfx B regulatory gene, leading to hyperproduction of the MexCD-OprJ efflux pump. Resistance to acridine dyes may also develop by mutation of proteins involved in the membrane transport system or by active efflux (as in case of E. coli ). Mutations in the gene coding for the repressor of MarA resistance gene providing intrinsic resistance to some agents (multiple antibiotic repressor, MarR) lead to hyperproduction of the efflux pump providing intrinsic resistance and consequently to cross-resistance to a number of disinfectant families (as well as to some antibiotic families). As disinfectant damage microbial cells at more than one target simultaneously, many different mutations are necessary to efficiently provide resistance. For this reason, in the development of disinfectant resistance, mutational resistance is less important.
Phenotypic adaptation as a part of acquired resistance
Changes in the fatty acid and protein content of the microenvironment of bacteria is mirrored in alterations in the cell surface hydrophobicity and ultrastructure of bacteria, which may lead to decreased susceptibility to disinfectants. The composition of the cell envelope is continuously changing depending on the environmental conditions and nutrient availability,
47 Sterilization and disinfection which may result in changes in susceptibility. For instance, Staphylococcus aureus cultured in a lipid-rich environment will form a cell wall with higher lipid content; these cells are less susceptible to phenolics than normal cells. In this manner, Staphylococcus aureus may be adapted to disinfectants by culturing in lipid-rich media. Bacterial growth may be slowed down or even arrested by lack of nutrients, which may also lead to inefficacy of certain disinfectants. Stress response given to environmental stressor stimuli may also lead to decreased susceptibility to disinfectants. Oxidative stress and adaptation to it leads to decreased susceptibility to peroxides, as in case of E. coli , for example.
Fungal resistance to disinfectants
In contrast to bacteria, data available on disinfectant resistance in fungi is scant. Generally, moulds are more resistant than yeasts. Though the resistance of the vegetative fungal cells exceeds the resistance of most Gram positive and Gram negative vegetative bacterial cells, the susceptibility of both the vegetative fungal cells and the fungal spores is lower than that of bacterial endospores. To kill vegetative fungal cells significantly higher disinfectant concentration is necessary than to cause growth arrest (the fungicidal concentrations are markedly higher than fungistatic concentrations), moreover, death of fungal cells also require longer exposure times. The intrinsic resistance of fungal cells, similarly to the case of bacteria, arises and is determined by the barrier effect of the fungal cell wall; and the level of this resistance differs in different phases of growth. This differential resistance is based on differences in the quality of the cross-links between cell wall subunits and thus on the porosity and barrier function of the cell wall. Their susceptibility is highest in the logarithmic phase, while it is lowest in the stationary phase. The microenvironment of the fungal cells also affects the susceptibility to disinfectants; the fatty acid concentration in the environment will influence cell membrane fluidity, which, in turn, determines the susceptibility to membrane-active disinfectants, e.g. susceptibility to alcohols in yeasts. Some Saccharomyces cerevisiae strains were reported to be capable of enzymatic degradation of certain microbicidal agents. The toxic effect of heavy metal compounds is counteracted by production of hydrogen-sulfide, which precipitates the heavy metal compounds as water-insoluble sulfides. The enzyme formaldehyde dehydrogenase was demonstrated in some Penicillium species. Disinfectant resistance based on active efflux or on development of acquired resistance due to mutations or to uptake of resistance genes (mobile genetic elements) from foreign cells have not yet been reported in fungi.
Viral resistance to disinfectants
The resistance of viruses to disinfectants is highly variable. The resistance is fundamentally determined by the presence of the lipid envelope; non-enveloped viruses are significantly more resistant than enveloped viruses. In case of enveloped viruses, the primary target for disinfectants is the envelope, while in case of non-enveloped viruses the target is the capsid, but disinfectants may also damage the viral nucleic acid. Enveloped viruses (HSV, HIV, rabies virus, influenzavirus) are easily inactivated by membrane-active disinfectants (quaternary ammonium compounds, biguanides, isopropanol), while these agents are inactive against non-enveloped viruses (picornaviruses, parvoviruses, adenoviruses, reoviruses). Some picornaviruses, rota- and adenoviruses exhibit outstanding
48 Sterilization and disinfection chemoresistance. Another mechanism of resistance of viruses to disinfectants is the formation of virus aggregates, into which the penetration of the disinfectants is poorer, similarly to the case of bacterial biofilms. Acquired disinfectant resistance has never been reported in viruses.
Protozoal resistance to disinfectants
Intestinal protozoa, as Cryptosporidium spp. , Entamoeba histolytica and Giardia intestinalis have a cyst form serving as the infective form, which is highly resistant to disinfectants. The level of resistance is similar to that seen in case of bacterial endospores; this high resistance is based on the barrier function of the cyst wall. The resistance of Acanthamoeba castellani , a free-living amoeba causing severe eye infection, especially in contact lens wearers, to disinfectants is well studied. The resistance is based on the biofilm formation of the vegetative form and on the resistance of the cysts. Disinfectants with reliable activity against protozoa are found in the groups of oxidizing and alkylating agents. Acquired disinfectant resistance has not been reported.
Resistance of prions to disinfectants
Prions are highly resistant to most physical effects and chemical agents; they survive acid treatment, are resistant to high temperatures, ionizing radiation, to most disinfectants and to proteolytic enzymes as well. Formaldehyde, acidic glutaraldehyde and ethylene oxid decreases the infectivity of prions only slightly. Out of the chemical approaches, boiling in 2M sodium-hydroxide for at least an hour or treatment with concentrated (20 000 ppm active chlorine content) sodium hypochlorite may be used for inactivation of the abnormal (prion) protein, but safe inactivation necessitates the combination of sodium hydroxide with autoclaving, to exploit their synergistic effect. The full explanation for this extreme resistance of the prion proteins is not yet available.
Disinfectant policy
The aim of the disinfectant policy (institutional disinfection programme) is the development and continuous application of a disinfection system, which complies with the infection control programme of the institution and provides sufficient long-term microbial purity. To achieve this aim, first the disinfecting methods to be applied including the range of disinfectants and antiseptics and the rules for application should be determined; guidelines should be detailed for all the different applications. A similarly important activity is the regular control of the efficacy of the disinfecting methods and of the disinfectant solutions as well as the monitoring of the meticulous adherence to the rules. Disinfectant policy 1. prescribes the range of the disinfectants to be used; 2. regulates the acquisition and storage of the disinfectants in detail; 3. determines to rules for the preparation of the working solutions of disinfectants; 4. prescribes the correct practice of disinfection; 5. regulates the safety rules during the process of disinfection; 6. prescribes the rules for the documentation related to disinfection; 7. includes the regular control and monitoring of the adherence to the rules of microbial safety and disinfection.
49 Sterilization and disinfection
The range of disinfectants to be applied is determined by the properties of the working process (institution) and the level of microbial safety to be achieved, and may also be limited by the site of application. During production of pharmaceuticals and foodstuffs, obviously a great deal of attention should be given to choose disinfectants with remainders of low toxicity, which can easily be removed as well (e.g. heavy metal compounds are generally not allowed). In the healthcare setting this issue is less important. In case of industrial application sites, but also in case of healthcare-related equipment, it is also important that equipment and appliances should not suffer corrosion during or due to disinfection (e.g. application of disinfectants with chlorine content or producing oxygen are not fortunate). Since disinfection may be very expensive, cost-effectiveness is also an important issue. By prescribing and full adherence to rules for acquisition and storage, the availability of disinfectants in the proper quality and quantity can be ensured and the loss of activity during storage can be prevented. This is very important, since potential loss of activity may remain hidden for a long time until a severe contamination with serious consequences occurs. By the guidelines on working solutions the continuous and reliable efficacy of the disinfectant solutions may be ensured, and the risk of microbial contamination of the disinfectant solutions and the consequent spread of microbes by contaminated disinfectant solutions may be prevented. The policy prescribes, individually for each procedure and process, the timing of disinfection (during work, after work, at the end of the day, weekly, etc.). It provides rules for the technology of the disinfection, on the choosing, proper concentration and exposure times of disinfectants, individually for each material or surface to be disinfected. At this stage it is useful to state the combinations to be used and to be avoided as well. The criteria for selection of the disinfection rules and disinfectants to be applied vary with the risk of infection caused by microbial contamination of the devices, equipment or environment to the product, patients or users. Devices used in direct contact with the product, open wounds or damaged mucosal surfaces or those used in physiologically sterile body sites carry the highest risk, therefore should be sterile. This category includes isolators, most pharmaceutical raw materials, gloves, needle, syringes as well as invasive devices (catheters, surgical devices and materials). For sterilization of these, if physical methods cannot be applied, only fluid disinfectants should be use; if possible, disposable devices should be used. Obviously, this group also includes the sampling devices and media used for monitoring of microbial purity, as contamination of these devices and media will lead to false results in the sterility tests. Devices and products with medium risk include those contacting the intact skin or intact mucosal surfaces, i.e. pharmaceuticals used on the intact skin or those for oral administration, as well as endoscopes, respirators, equipment used for anaesthesiology, washbasins and hospital bed linings. For eradication of contamination from these devices disinfectants are used; the criterion of microbial purity is a tolerably low total cell count and absence of certain pathogens ( Staphylococcus aureus , indicators for fecal contamination, etc.) Low-risk areas are those which are never in contact directly with the products or patients, i.e. walls and floors of hospital rooms or rooms not directly related to production. These surfaces may be decontaminated by proper cleaning and washing with disinfectant solutions. A more detailed discussion of these rules is not provided; the reader is referred to pharmaceutical technology for further information. To limit the number of procedures and disinfectants used within a given time period is a good approach. Lower number of procedures and agents, by decreasing the risk of human mistakes, provides better average efficacy than meticulous regulation of all the small parts of the procedures. Naturally, simplification can never lead to decreased efficacy of sterilization
50 Sterilization and disinfection and disinfection. On the other hand, to prevent the development of resistance, it is necessary to alternate the agents (and sometimes the protocols as well), to use agents in a rotating manner. This means that a technology relying on the usage of a given disinfectant (or combination of disinfectants) is used for a predetermined period, then a novel protocol (preferably using agents with different targets or mechanisms of action) is applied. After a certain time lapse, the latter protocol is exchanged for a third one; later the original first protocol may be resumed. It is important, that the duration of rotations should be long enough for the protocols to become a routine (all participants should be well-practiced), but short enough not to cause a selection pressure leading to resistance development. With the above approaches a continuously and reliably high efficacy of disinfection may be achieved and maintained; and proper documentation allows for rapid detection and elimination of the occurring mistakes. The development of and adherence to the proper practice is also extremely important in terms of cost-efficiency. Beyond disinfection, the practice of cleaning should also be regulated, as proper cleaning is a fundamental prerequisite for effective disinfection. The frequency of cleaning should be determined differentially for the rooms with different functions; the range of cleaning agents should also be prescribed. The appropriateness and efficacy of the cleaning should be supervised prior to disinfection. The training of involved personnel, of course, should also be an integral part of the disinfectant policy. Disinfectant policy also serves the aims of working safety; the protection of the personnel involved in disinfection can also be provided by adherence to the rules. The actions to be done in case of accidents occurring during disinfection (spillage, contamination of the skin, mouth or eyes, etc.) should be regulated. The toxicity of the disinfectants and the harmful substances potentially released from disinfectant combinations should be stated. The safe storage, application and disposal of concentrated and working disinfectant solutions should be regulated separately. The control measures should be extended to regular monitoring of microbiological purity. This should be performed by regular sampling of surfaces (as well as of air, if necessary), and the microbiological culture of the samples should be performed. If the nature of the potential contaminating microbes necessitate, other techniques (ELISA, molecular biology techniques) may also be necessary. The other important element of control is the regular monitoring of the adherence to the regulations on disinfectant usage and the related documentation. In the view of the abovementioned, in all activities where disinfection has a role, it is necessary to employ a person responsible for the reliability of disinfection. This person should prepare the regulation of disinfection, trains the employees performing the disinfection, performs and supervises the related work and documentation and is involved in the regular control as well.
51 Antimicrobial chemotherapy
Antimicrobial chemotherapy
Despite the continuous progress of medicine, infections remain a major cause of death even in developed countries. With the increasing number and better survival of patients more susceptible to infections (leukemic patients, transplant patients, patients with cystic fibrosis, etc.), the importance of different pathogenic microbes also has increased. Besides long-known pathogens, opportunistic microbes with lower virulence have emerged, a number of which are multiresistant or untreatable with presently known antimicrobial agents. On the other hand, the increasing number of hospitalized patients and the spread of invasive diagnostic and therapeutic applications lead to increasing frequency of nosocomial infections. Most nosocomial pathogens are multiresistant posing a serious therapeutic challenge. As a consequence, a significant proportion of healthcare costs are related to prevention and treatment of infections. Demonstrating and identification of pathogens has become a minimum requirement in clinical microbiology, and susceptibility testing is indispensable in most infections. The best studied group of pathogens is the bacteria; we have the widest choice out of antibacterial agents. However, with the emergence of opportunistic pathogens, antifungal, antiviral and antiprotozoal agents as well as susceptibility testing are becoming equally important. The primary requirement of antimicrobials is selective toxicity . This means that the agent should be highly toxic to the targeted microorganism, but should exhibit mild or no toxicity to the host. Its measure is the therapeutic index , which (calculated similarly as in case of other therapeutic agents) is the quotient of the dose producing therapeutic effect and the toxic dose. There are drugs with wide (even high doses has low toxicity) and narrow therapeutic range (the therapeutic dose is close to the toxic dose). In case of drugs with narrow therapeutic range, monitoring of serum levels may be necessary for timely detection of toxicity.
Classification of antimicrobial agents
Antimicrobials can be classified in several ways. 1. according to target organisms a. antibacterial agents b. antifungal agents c. antiprotozoal agents d. anthelminthics e. drugs against ectoparasites f. antiviral agents 2. according to spectrum of efficacy a. broad spectrum (active against most microbes within the target group) b. narrow spectrum (active against only certain microbes within the target group) 3. according to exerted effect a. cidal agents (killing the microbes) b. static agents (inhibiting the growth and replication of microbes) 4. according to mechanism of action (biochemical target) 5. according to origin a. natural products b. semisynthetic drugs (chemically modified derivatives of natural products) c. synthetic drugs
52 Antimicrobial chemotherapy
Originally, the antibiotics were natural products with antimicrobial (or anticancer) activity, and synthetic antimicrobials were called chemotherapeutics . Today both concepts are used as a collective name for antimicrobials regardless of their origin, however antibiotics mostly refer to antibacterial agents and drugs against other microorganisms are frequently named chemotherapeutics. The first group of antimicrobials coming into use was the group of antibacterial drugs, and these are the most frequently used agents as well. The explanation is twofold. i) Bacterial infections are frequent and most are relatively easy to diagnose. ii) Due to their prokaryotic cell structure there are a number of targets which can be inhibited selectively. As fungi, protozoa and helminths are eukaryotes similarly to their host, targets that can be inhibited selectively are harder to find. The situation is even worse in case of viruses as these use the host’s biosynthetic apparatus for replication; therefore targets for selective therapy are scant. For these reasons the number of available antifungal, antiprotozoal and antiviral agents is smaller. Though we do not know any antimicrobial agent which is clinically effective both against bacteria and fungi, the principles and targets of antibacterial and antifungal therapy are similar. Resistance to antibacterial and antifungal agents is also frequently analogous functionally. There are certain similarities in case of antiprotozoal drugs as well. Anthelminthics and antivirals are totally different, however; due to the multicellular organization of worms and the peculiar mode of virus replication the principles of anthelminthic and antiviral therapy and the targets of drugs used are significantly different (see below).
Principles of antibacterial and antifungal therapy
Antibacterial and antifungal therapy can be initiated according to susceptibility results (targeted therapy ), without that knowledge, based on choosing a drug most probably effective against all expectable pathogens ( empirical therapy ) or with the aim of prevention of infection ( chemoprophylaxis ). If the condition of the patients allows, it is frequently practical to wait until susceptibility results become available and administer a targeted therapy. If the situation demands immediate initiation of antimicrobial therapy, a drug or a drug combination effective against all probable pathogens should be chosen. Consequently, broad spectrum antibiotics or combinations with broad coverage are to be chosen for empirical therapy. Similar reasons drive the choice in chemoprophylaxis. The choice of antibiotics in empirical therapy and in chemoprophylaxis is aided by several guidelines, which are specified according to the indication (site and characteristics of infection). Choosing the appropriate antibiotics may be crucial for the patient, decreases the risk of resistance development and is cost-effective. The required input information is the expectable pathogens in the particular disease, the range of antibiotics effective against them and their local resistance characteristics. Recognition of local pathogen distribution and resistance characteristics (and consequently to plan an effective and cost-effective empirical therapy) necessitates regular monitoring activities ( surveillance ; see in detail in the chapter ’Antibiotic policy’). Surveillance means that besides regular statistical evaluation of clinical isolates and their resistance patterns we perform an active prospective survey in order to gather information on distribution of resistance in isolates from patients, staff, environment and healthy individuals. Based on surveillance results, an effective empirical therapy can be predicted with better probability, leading to increased quality of healthcare as well as to
53 Antimicrobial chemotherapy decrease in direct and indirect healthcare costs. This saving on healthcare cost is much higher than the (relatively high) cost of surveillance activity. For the same reason, microbiological examination and susceptibility testing, even subsequently, is important in all serious and/or therapy-resistant infections, since the data gathered this way will aid in choosing an appropriate empirical therapy (and occasionally prophylaxis). Besides efficacy, prevention of resistance development and spread becomes an increasingly important goal. In practice, this is generally unreachable; increasing of resistance is frequently unavoidable to some extent. However, slowing this process is the strategically most important task of clinical microbiology at present (see in detail in the chapter ’Antibiotic policy’). Antibiotics may be administered as monotherapy (alone) or in combination with other antibiotics. When combined, the antibiotics may interact and influence one another’s efficacy. These interactions may be
synergy 1,20E+06 1,00E+06 A 1. synergistic interaction B 8,00E+05 combination (the efficacy of the combination is 6,00E+05
better than the efficacy of the drugs CFU/ml 4,00E+05 alone) 2,00E+05 e.g.: β-lactam+aminoglycoside 0,00E+00 0 4 8 16 24 36 48 time (hours)
2. additive (indifferent) interaction 1,20E+06 additive effect A (the efficacy of the combination is 1,00E+06 B similar to the efficacy of the drugs 8,00E+05 combination alone; some authors distinguish additive effect, which is the net sum 6,00E+05 CFU/ml of the efficacies of the two drugs, 4,00E+05 from indifferent interaction 2,00E+05 corresponding roughly to the efficacy 0,00E+00 of one of the drugs) 0 4 8 16 24 36 48 e.g.: β-lactam+fluoroquinolone time (hours)
1,20E+06 antagonism
1,00E+06
8,00E+05 3. antagonism (the efficacy of the combination is 6,00E+05 CFU/ml 4,00E+05 lower than the efficacy of the drugs A alone) 2,00E+05 B β combination e.g.: -lactam+bacteriostatic agent or 0,00E+00 macrolide+clindamycin 0 4 8 16 24 36 48 time (hours)
54 Antimicrobial chemotherapy
Principles of choosing an appropriate antibiotic are the following. 1. therapy should be administered using the appropriate dose for the appropriate duration 2. therapeutic concentration of the drug(s) should be reached at the site of infection 3. antagonistic drugs should not be combined 4. targeted therapy should be administered, if possible 5. a cidal agent should be chosen, if possible 6. broad and narrow spectrum agents should be used for empirical and targeted therapy, respectively.
Resistance to antibiotics
Failure of the antibiotic therapy (when the condition of the patient does not improve) means clinical resistance ( therapeutic failure ). Clinical resistance can be caused by 1. poor absorption of the drug, 2. poor penetration of the drug to the site of infection, 3. dosing mistake, 4. drug interaction, 5. inactivation of the drug by non-pathogenic bacteria (e.g. the normal flora), 6. tolerance of the pathogen to the drug, 7. resistance of the pathogen to the drug (microbiological resistance).
A frequent cause of the therapeutic failure is the microbiological resistance of the pathogen to the antibiotic. This may derive from physiological causes (phenotypical) or may be genetically encoded. Phenotypic resistance does not require genetic changes, it is a result of the actual phenotype of the microbe. This generally means that removing the organism from that particular environment will lead to disappearance of resistance, i.e. the descendants are not resistant. Phenotypic resistance may be caused by 1. slow growth rate or non-replication of the population, 2. phenotypic variability (resistance is a by-product of the expressed phenotype), 3. biofilm growth (antibiotics penetrate poorly into biofilms, the environmental conditions are varied within different parts of the biofilm leading to a wealth of different phenotypes, growth is slowed down in biofilms, biofilm growth induces a stress reaction leading to overexpression of repair mechanisms; there is a certain biofilm phenotype).
Genetically encoded resistance always involves a change at gene or gene expression level, does not depend on physiological (environmental) factors, and is passed to descendants. Further on, by resistance we refer to this genetically encoded resistance. The degree of resistance may be variable among and even within populations. Some strains or species are more resistant (higher level resistance), while in case of others the resistance is less pronounced (lower level resistance). In some cases the resistance can be demonstrated, but has no immediate clinical consequence. This is determined primarily by nature of the underlying resistance mechanisms and/or the level of their expression.
Classification of resistance
From the viewpoint of antibiotic therapy the resistance can be primary resistance , when the pathogen was resistant before the initiation of the antimicrobial therapy, or
55 Antimicrobial chemotherapy secondary resistance , when resistance develops during therapy. Primary resistance may be caused by intrinsic ( natural , generic ) resistance , which is an inherent characteristic of the given species (or higher taxon); or occurs when the infection is caused by an organism with previously acquired (secondary) resistance . (The expression ’acquired resistance’ is sometimes used in a narrower sense meaning only resistance developing through acquisition of foreign genetic material coding for resistance, in contrast to mutational resistance acquired through new mutation(s) leading to resistance.) Secondary resistance may also cover situations when superinfection with a resistant organism leads to clinical resistance. If a particular strain is resistant to at least two important antibiotic family traditionally used against that species, the strain is multiresistant . If only one or two antibiotics remain active, the strain is panresistant . Cross-resistance occurs if a resistance mechanism (natural or acquired) conferring resistance to a certain agent also produces resistance to another agent. Cross-resistance may be complete , when shown by virtually all species (as in case of ampicillin and amoxicillin); or partial , when present only in certain cases (as in case of erythromycin and clarithromycin). Cross-resistance frequently occurs between drugs from the same drug class (drugs with the same target). Coresistance arises from the coupled expression of different resistance mechanisms conferring resistance to different antibiotic classes, e.g. due to the co-presence on the same plasmid or integron. In contrast to cross-resistance, two different mechanisms and genes exist, which are expressed and inherited in a connected manner. Heteroresistance is a characteristic of resistance mechanisms which lead to a certain disadvantage to the cell expressing it. In this case, all cells of the population carries the resistance gene (the population is genetically homologous), but it is expressed only in a small minority of cells in the absence of the drug, most cells have susceptible phenotypes (even though all cells harbour the resistance gene). Therefore, in the absence of the drug most cells in the population show the normal growth rate (similar to that of the susceptible strains). However, there is a small minority of cells in reserve expressing the resistant phenotype, and upon exposure to the drug, these cells maintain the population (are not killed or inhibited), even if they show decreased growth rate compared to the susceptible phenotype. When the drug is withdrawn, the original high proportions of susceptible to resistant phenotype is restored, allowing for optimal growth rate both in drug-free and drug-exposed conditions. A typical example of heteroresistance is methicillin resistant Staphylococcus aureus . Knowledge of natural resistances is fundamental for choosing an appropriate empirical therapy, while being acquainted with cross-resistances helps in the interpretation of the antibiogram. The most important natural resistances are listed at the end of the chapter.
Development and spread of resistance
Besides the desired effect, antibiotic treatment also has adverse consequences. Similarly to other drugs, the treated patient may experience side effects or allergic reactions of different severity, but in contrast to other drugs antibiotics have vast ecological effect not directly connected to the patient treated. This effect may have impact on the microbe targeted by the antibiotic through selection for resistance, but may adversely influence the environmental microbe communities as well. These effects not directly connected with the patient treated are called collateral damage . Selective pressure exerted by the antibiotic leads to development of acquired resistance. In all microbe populations there are mutants appearing randomly that resist the
56 Antimicrobial chemotherapy antibiotic effect and consequently have selective advantage in the presence of the antibiotic. These mutants are selected upon exposure to an antibiotic. The speed of resistance development depends on the extent of the necessary genetic alteration; the smaller the necessary change, the faster the resistance is developed. It develops extremely quickly, for example, if one point mutation is sufficient for high-level resistance (one mutation confers resistance of a level high enough for survival of therapeutic doses and the gene to be mutated is present in one copy in the genome). Selection for resistance, of course, not only occurs in the microbe responsible for the targeted infection, but in all pathogenic and non-pathogenic strains exposed to the antibiotic, including the patient’s normal flora as well as potentially pathogenic microbes currently colonizing the patient (collateral damage). The risk of resistance development may be decreased by judicious selection of the antibiotic and adequate therapy, underlining the importance of appropriate antibiotic treatment (see above). Exposure to antibiotics provokes resistance in the normal flora of the patient as well as in the pathogens present simultaneously in the body. (It was proven in case of Helicobacter pylori that clarithromycin resistant strains are significantly more frequent in patients previously treated with clarithromycin for airway infection.) The latter effect is unavoidable even with the most prudent use of antibiotics. Resistance mechanisms developing in the normal flora may persist for extended periods, serve as reservoirs for resistance and may be transferred back to pathogens. Collateral damage affects not only pathogens, but the environmental (soil, water, etc.) microflora as well. Antibiotics released to the environment (e.g. with hospital waste water or waste water from pharmaceutical companies) lead to spread of resistance in environmental microorganisms. Resistance mechanisms selected are preserved indefinitely in the environment, serve as reservoirs of resistance, and eventually may get back to pathogens or to members of the human normal flora. Besides, the contaminating antibiotics alter the composition of the environmental microbial communities due to selection of more resistant species. The ecological consequences are not even predictable at this time. Besides provoking resistance, antibiotic treatment affects the composition of the patient’s normal flora, may kill it off or suppress it, allowing for colonization with multiresistant nosocomial pathogens. This colonization may be derived from the hospital flora or the patient may harbour resistant bacteria in small numbers (e.g. in the gut), which are normally suppressed by the normal flora, but can rapidly grow and may cause disease without its inhibitory effect. (A characteristic example is the post-antibiotic colitis caused by Clostridium difficile .) These harboured resistant organisms may originate from the nosocomial flora acquired during a previous admission to another (or to the same) ward, from the environment, and from animals. The latter may be acquired through direct animal contact (pets have the greatest importance in this regard) or from animal-derived food contaminated with resistant organisms. Colonization of human beings will result in better survival of these strains, as eradication from patients is more difficult than removal from the environment by appropriate disinfection. On the other hand, colonization may lead to infection; in this case the choice of effective antibiotics is automatically limited, due to the resistance of the causative agent; therefore the treatment necessitates broad-spectrum agents. With the spreading of resistance, doctors are forced to use drugs with broader and broader spectra; as a consequence, resistance will be developed and spread to these broader spectrum agents as well, leading to routine use of the broadest spectrum agents available. This self-sustaining process will ultimately result (and was shown to have resulted in case of Klebsiella pneumoniae ) in selection of strains resistant to all potentially useful antimicrobials.
57 Antimicrobial chemotherapy
This process is called the resistance spiral . Acquired resistance may develop not only in the pathogen exposed to the antibiotic, but may arise from horizontal gene transfer (conjugation, transformation), i.e. by transmission of genes coding for resistance from resistant to susceptible microbes, where they cause resistance if expressed properly. The source of these genes may be
1. organisms possessing generic resistance, sometimes the antibiotic producers, 2. human or animal pathogenic bacteria, which acquired resistance during antibiotic therapy, 3. members of human or animal normal flora, which acquired resistance during antibiotic therapy, 4. members of animal normal flora, which acquired resistance to veterinary antibiotics or antibiotic analogues used for production enhancement or treatment and are cross- resistant to human antibiotics belonging to the same drug family or exerting activity on the same cellular target, 5. environmental bacteria, acquiring resistance through selection pressure exerted by ’polluting’ antibiotics released to the environment.
Genetic background of resistance
Resistance is usually mediated by one gene and gene product; less frequently requires concerted expression of more genes. The gene(s) coding for the resistance mechanism may be located on the bacterial chromosome, on a plasmid, as well as on mobile genetic elements, i.e. on transpozons or integrons. Transpozon s are DNA fragments, which may be translocated from their chromosomal location to another part of the chromosome or to another chromosome during conjugation or from chromosome to plasmid. Two types of transpozons are known, type 1 transpozons contain insertion sequences (IS elements) on both ends, these are transferable alone or together with the gene(s) (frequently resistance genes) in the transpozon (between the two terminal IS elements). Translocation of insertion sequences may lead to expression of silent (promoterless) resistance genes through providing a promoter for the gene (e.g. expression of the cfiA gene coding for the carbapenemase of Bacteroides spp . is activated in this manner). Type 2 transpozons code for a tranpozase enzyme, which is responsible for transfer; and contain one or more genes (frequently resistance genes), which are translocated and disseminated together with the transpozase gene. Integrons are genetic elements with an own integrase enzyme, which may be integrated at specific sites into the genome by means of site-specific recombination. Besides the integrase, they contain recombination sites allowing for capturing different genes, mostly coding for resistance to different antibiotics and disinfectants or for virulence properties. These genes are located on so-called gene cassettes, which do not have a promoter, but possess a recombination site. They are inserted into the integron using this site; the integron, in turn, provides the promoter necessary to expression of the gene. Thus the promoter of the integron will mediate the expression of genes of all carried gene cassettes frequently leading to coresistance. The closer a gene is located to the promoter in the integron, the more effective is its expression. Gene cassette sequence within the integron is easily rearranged through the recombination sites, enabling the gene providing the greatest advantage to the bacterium at the moment to get closest to the promoter to reach the highest level of expression. It was revealed from a number of resistance genes that it is encoded by a gene cassette carried on an integron. Since transpozons and integrons may contain more than one resistance gene simultaneously, they have a role in multiresistance as well. Chromosomally coded resistance is not directly transferable, while genes encoded by plasmids or other mobile genetic elements can be transferred during conjugation. Therefore, these mobile elements have paramount importance in the spread of resistance within and between bacterial species. Resistance genes encoded by the chromosome may, though rarely,
58 Antimicrobial chemotherapy be translocated to plasmids or to other mobile elements. These genes originating form organisms with generic resistance become ancestors of resistance genes encoded on plasmids, transpozons or integrons, which are capable of quick spread. These genes frequently undergo a number of genetic changes, and in forms significantly different from the ancestral gene provide high level resistance and serious selection advantage. Resistance may be expressed constitutively , i.e. independently of the presence of the antibiotic (substrate) in question, or in an inducible way , when the resistance phenotype is expressed only in the presence of the antibiotic. In case of inducible resistance, the efficacy of the antibiotic depends not only on the ability of the mechanism to provide resistance, but also on the efficiency of the antibiotic as an inducer. Weak inducers will induce the production of only small amount or no resistance gene product at all; consequently the weak inducer antibiotic may retain clinically sufficient activity by not inducing the mechanism sufficiently for preventing the antibiotic effect. Inducible resistance may cause discrepancy between in vitro and in vivo susceptibility results. This discrepancy may frequently lead to a false susceptible result, if the in vitro induction is poor leading to in vitro susceptibility, but in vivo the inducing efficacy is enough to compromise the efficacy of the antibiotic.
Possible mechanisms of resistance
Mechanisms of antibiotic resistance may involve i) the alteration of the chemical structure of the drug, ii) decrease of intracellular concentration of the drug or iii) decreasing the efficacy of the interaction between the target and the antibiotic through various changes involving the targeted molecule or biochemical process. In case of antibiotics needing activation, iv) the loss or alteration of the activator mechanism may also cause resistance. The efficiency of different mechanisms is variable, certain mechanisms are alone capable of producing a resistant phenotype (these provide high level resistance ), while others lead to a certain decrease in the efficacy of the drug (increase the minimum inhibitory concentration, to be defined later), but this decrease alone is not sufficient to provide clinically relevant resistance (these provide low level resistance ). The importance of mechanisms conferring low level resistance lies in their ability, by slightly increasing the chance of survival of bacteria exposed to the antibiotic (e.g. between two doses when the serum concentration is low), to provide opportunity for development more efficient resistance mechanisms. It occurs frequently that the resistant phenotype is produced by not one resistance mechanism alone, but is a result of several independent mechanisms with different mode of action and efficacy. For instance, a pattern of resistance to β-lactam antibiotics may arise from the action of more than one lactamases and/or efflux pumps may also play a role besides lactamases.
Alteration of the chemical structure of the drug
Cleavage
Resistance is caused by an enzyme which cleaves a chemical bond crucial for antibiotic action in the drug. These enzymes may be encoded by the chromosome, but the majority of problems are caused by quickly spreading variants encoded on plasmids or on other mobile genetic elements. They may provide resistance of different levels. These mechanisms are highly specific, they usually provide resistance to members of only one drug family, and do not lead to cross-resistance between drug families. The most important
59 Antimicrobial chemotherapy examples are β-lactamases. There are lactamases involved in intrinsic resistance, e.g. in β- lactam resistance of mycobacteria or in ampicillin resistance of klebsiellae.
Enzymatic modification
During modification a chemical substitution of a functional group on the antibiotic molecule takes place. Modifiable functional groups may be hydroxyl- or amino- (more rarely carboxyl-) groups; to these groups an acetyl- or less frequently a phosphate- or a nucleotidyl group is bound. Chromosomally coded enzymatic modification is typical in antibiotic producers, in other bacteria it is more frequently encoded on plasmids or on other mobile genetic elements. They frequently provide high level resistance. Modification is highly specific, provides resistance to only members of one drug class; extremely rarely leads to cross-resistance between different drug families. Modification is the most important mechanism of aminoglycoside resistance.
Decrease of intracellular concentration of the drug
Low permeability/permeability decrease
Membranes effectively hinder the uptake of hydrophilic molecules (sugars, aminoacids, etc.), these are internalized by means of different transport systems or transport proteins (porins). These porins are transmembrane proteins that bridge the lipid bilayer forming a hydrophilic channel (pore) with a narrow opening. Permeability to a given hydrophilic drug is limited by its size; above a certain size limit penetration is extremely slow. Besides molecule size, other factors determining uptake speed is hydrophobicity (hydrophobic molecules penetrate slower) and charge (negatively charged molecules are slower to penetrate). Obviously, the number of active channels influences the speed of uptake fundamentally. Permeability of the cell membrane has a key role in entry of antibiotics into the cell or in inhibition of antibiotic uptake. This is especially important in case of Gram negative bacteria, where besides the cytoplasmic membrane the outer membrane of the cell wall should also be crossed by the antibiotic. A similar barrier is formed by mycolic acids in the cell wall of mycobacteria. In case of these groups, consequently, many antibiotics can penetrate the barrier (outer membrane or layer of mycolic acids) only through porins or can penetrate not at all. For this reason, low permeability has a key role in intrinsic resistance to many drug classes, and explains the extensive generic resistance of Gram negatives and mycobacteria (to lincosamides, glycopeptides, etc.). If porins are necessary for antibiotic uptake, these may serve as a target for development of acquired resistance through mutation or decreased expression of the porins involved. Mutation in the porin protein gene may alter the structure of the porin channel (pore size, surface charge, hydrophobicity), and consequently the range of molecules the channel will allow through. Decrease in expression of the porin gene takes effect by decreasing the number (density) of the porin channels. Both of these mechanisms will lead to decrease of the intracellular drug concentration. As efflux of the antibiotic will further decrease the concentration, this mechanism is frequently coupled with efflux-based resistance mechanisms (see below). Loss or modification of porin function, of course, rarely results in total blockade of penetration, thus leads rarely to so efficient inhibition of penetration as impermeability
60 Antimicrobial chemotherapy causing intrinsic resistance. Though porin loss or modification slows down antibiotic uptake significantly, cannot altogether prevent it. (The cell cannot lose all porins; some of them must be maintained to take up hydrophilic nutrients, etc.) Accordingly, this mechanism will decrease the intracellular drug concentration, but uptake of a certain amount of drug is inevitable. Consequently the level of the provided resistance is rarely high, but lower intracellular drug concentration facilitates development and selection of other resistance mechanisms. As a consequence of the abovementioned facts, this mechanism is important primarily in Gram negative bacteria and mycobacteria, though it occurs in Gram positives as well. Acquired decrease in permeability may produce cross-resistance between different drug classes. All types of permeability-related resistance are coded on the chromosome.
Active efflux
This type of resistance is caused by the activity of the pumps naturally involved in detoxification of the cell. It is a prominently important mechanism in case of eukaryotic organisms as well (see for instance the chloroquine resistance of Plasmodium falciparum ). Efflux pumps may be divided into two large groups, primary active transporters use an energy source (usually ATP) to cover the energy demand of the process, while secondary active transporters use a transmembrane ion gradient (usually the proton gradient) as an energy source (the latter are antiport, or less frequently synport proteins). Primary transporters are characteristic to eukaryotes, while in prokaryotes secondary transporters have the greater importance. Bacterial primary transporters belong to the ABC (ATP-binding cassette) transporter family, while secondary transporters may belong to several protein families (major facilitator superfamily, MFS; resistance-nodulation-division, RND; small multidrug resistance, SMR, etc.). Bacterial secondary transporters utilize the transmembrane proton gradient and act as a proton-drug molecule antiport proteins. Some efflux pumps are coupled with porins through a membrane fusion protein, this way the porin activity and efflux may act synergistically in decreasing intracellular drug concentration both in case of natural and acquired resistance. Efflux pumps are generally coded by the chromosome, but plasmids coding for efflux pumps were also described. Development of efflux-based acquired resistance involves acquisition and expression of new efflux pump genes or overproduction of the efflux apparatus. The latter may arise from mutation(s) in the promoter or the repressor of the gene coding for an element of the efflux apparatus, from altered activity of another regulator, and may be caused by an insertion sequence (IS) element, which increases the expression level of the pump gene. In general, the level of resistance is high only in case of intrinsic resistance; efflux- based acquired resistance is usually of low or intermediate level. However, supplementing the effect of other resistance mechanisms (cleavage, modification, target mutation, etc.) efflux may contribute to development of high level, clinically important resistance. Furthermore, the low level resistance provided by efflux may create favourable situation for selection of other resistance mechanism frequently leading to high level resistance. Another important aspect is that different antibiotics from different drug classes with different targets may serve as a substrate to a certain efflux pump, leading to cross-resistance between drugs with totally different mechanisms of action. Thus efflux may contribute to selection for multiresistance even through overuse of only one antibiotic class. Efflux-based resistance has examples in case of most antibiotic classes, different efflux pumps are important in the resistance to macrolide antibiotics. It is frequently found in the background of intrinsic resistance, mostly in case of Gram negative bacteria. E.g. intrinsic resistance of Pseudomonas aeruginosa to a number of different antibiotics may be explained with the produced efflux pumps besides its porins with narrow specificity.
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Changes connected with the target
Target modification
The target of the drug may be altered by mutational events (commonly by a point mutation), leading to a target with lower affinity to bind the antibiotic and to decrease or, less frequently, to total cessation of inhibition. This mechanism must be coded for on the chromosome, as it is mediated by a mutation in the chromosomal gene. It frequently provides high level resistance, but it is not rare that multiple stepwise mutations are necessary for development of high level resistance. It is not uncommon that the mutations leading to resistance simultaneously cause decreased virulence, which may be compensated by further mutations. This mechanism is highly specific, provides resistance against antibiotics acting on the target involved. Fluoroquinolone resistance is the most obvious example. Naturally, this mechanism cannot play a role in intrinsic resistance (though a small genetic difference of the targets in two related species may result in a large difference in susceptibility).
Overproduction of the target
Due to increased gene expression, the amount of the target may increase in the cell. Inhibition of higher amounts of the target needs higher intracellular concentration of the antibiotic. This mechanism usually provides low or intermediate level of resistance, which may be reversible by increasing the amount (dose) of the drug. Similarly to the former, this mechanism is usually chromosomally coded, being caused by mutations in the promoter of the chromosomal gene coding for the target (but rarely may be mediated by insertion elements providing more efficient promoter for the target gene). The mechanism is highly specific, provides resistance against antibiotics acting on the target involved. Target overproduction is exemplified by trimethoprim resistance due to overproduction of the target dihydrofolate reductase. The mechanism has no role in intrinsic resistance, but may be a source for differences in intrinsic resistance between species.
Production of a new target
In this case resistance is provided by a new target produced simultaneously with the original target, which performs the function of the original target, but is not sensitive to the inhibitory effect of the drug. It is usually encoded by plasmids or other mobile genetic elements, and provides high level resistance. The mechanism is specific, providing resistance to the drugs acting on the target replaced. It is not involved in intrinsic resistance, but a similar mechanism may exist, i.e. a target with altered biochemical properties as compared to the drug-sensitive one, though in these cases sensitive target never existed (as in the background of cephalosporin resistance of enterococci). A very important example of acquisition of a new target is the methicillin resistance of staphylococci.
Development of a metabolic bypass
A metabolic bypass means that the function of the target is overtaken by another metabolic process, i.e. a new metabolic pathway develops or gains ground, permitting the normal proceeding of the metabolic process in question, even in the presence of the drug. In
62 Antimicrobial chemotherapy this manner the drug exerts its inhibitory activity on the target, but this does not lead to disfunction of the cell. This mechanism frequently involves the modulation of the expression of or alteration of more than one gene, thus in most cases chromosomally coded, but sometimes a whole operon devoted to resistance may be coded by a plasmid. It usually provides high level of resistance, but may also lead to decrease of virulence. The mechanism is specific, provides resistance only to drugs acting on the target. It is frequently involved in intrinsic resistance, in the manner that the target is not present on the intrinsically resistant cell, e.g. the resistance of mycoplasmas to cell wall active agents originates in the lack of cell wall.
Protection of the target
Resistance can be mediated by production of a new protein, which protects the target molecule from the effect of the antibiotic (prevents the binding of the drug to the target). Two mechanisms are possible, either the target binds the protector molecule, which in turn hinders the binding of the drug, or the protector molecule enzymatically modifies the target to convert it to a form not binding the drug. This mechanism is usually coded by mobile genetic elements, most frequently on plasmids. Depending upon the efficiency of the protection provided, they may result in low to high level of resistance. They are specific mechanisms; provide resistance only to drugs acting on the target. It has no role in intrinsic resistance. This mechanism is exemplified by ribosomal protection in case of macrolide or aminoglycoside resistance.
Decreased activation
If the antibiotic needs activation by the target microorganism, resistance may possibly be mediated by the decrease or loss of this activation. This decrease or loss may be caused by decrease or loss of the activity of the activating enzyme, or by enhancement of a bypass metabolic process leading to decreased expression of the activator. As the activator activity is frequently a process important for growth of the organism, decrease in activation is frequently connected with decreased ability to survive in the host, i.e. decreased virulence. This decrease in virulence may be compensated by further genetic changes and/or by a metabolic bypass, leading to a resistant strain which retains its virulence as well. This compensation may even lead to a resistant strain with increased virulence. These activator mechanisms are invariably chromosomal; therefore this mechanism is always coded by the chromosome. The level of resistance provided depends on how efficient is the blocking of activation, varying from low to high level resistance. The examples among bacteria are confined to metronidazole and to some antituberculotic drugs.
Tolerance to antibiotics
Besides antibiotic resistance therapeutic failure may also be caused by antibiotic tolerance . Tolerance is a phenomenon when the expected bactericidal action of a bactericidal agent fails to develop, but a bacteriostatic action takes place instead. Thus tolerance may occur in case of bactericidal agents only. In case of infection with tolerant isolates antibiotic treatment provides significant clinical improvement, but a relapse occurs after the end of treatment, the antibiotic is frequently unable to eradicate the pathogen. Importantly, antibiotic
63 Antimicrobial chemotherapy resistance is not demonstrable, isolates prove to be susceptible in routine susceptibility testing (e.g. MIC determination); therefore demonstration of antibiotic tolerance in vitro is difficult (unless suspected and specifically sought for). Similarly to resistance, tolerance may be phenotypic or genetically coded. The background of phenotypic tolerance is similar to that of phenotypic resistance, i.e. it is a frequent characteristic of non-replicating or slowly growing cells, but tolerance may also be induced by biofilm growth. Phenotypic tolerance has key importance in infections caused by the Mycobacterium tuberculosis complex . Genetically encoded tolerance has so far been described mostly against antibiotics acting on the cell wall synthesis, β-lactams and glycopeptides. The explanation is the alteration of the regulation of autolytic processes, which take part in the normal cell wall synthesis and contribute to the cell death induced by antibiotics acting on the cell wall synthesis (e.g. in case of Streptococcus pneumoniae ).
Antibiotic susceptibility testing
One of the most important tasks of the clinical microbiology laboratory is to determine the antibiotic susceptibility of the pathogenic bacteria and the interpretation of results. The aim of antibiotic susceptibility testing is to predict the efficacy of the tested antibiotics in the infection involved. For this prediction to be made we should know the extent of activity of a particular drug against the pathogen (which drug concentration is necessary to kill the pathogen or to inhibit its growth), and the expectable amount of antibiotic at the site of infection (what concentration can be attained with a particular therapeutic dosage using a particular route of administration). In vitro susceptibility tests measure the activity of the antibiotic against the given pathogen under standardized conditions, consequently, do not predict clinical efficacy directly. The standardized conditions applied in vitro vary greatly in vivo . A characteristic example is meningitis, when, besides antimicrobial activity, clinical efficacy is fundamentally influenced by the ability of the drug to penetrate to the cerebrospinal fluid. It is thus not always true that out of two antibiotics the more effective is that which is active at lower concentrations. For this reason, the correct evaluation of results and their interpretation to the clinician is important. In practice, this means categorizing the quantitative results into categories ’susceptible’, ’intermediate’ and ’resistant’. It is extremely important to choose an appropriate method to examine the susceptibility of a certain pathogen. Some methods may be unsuitable for testing of certain pathogens (e.g. disc diffusion for testing of anaerobic bacteria), or some bacteria may require specific procedures (e.g. for testing of Mycobacterium tuberculosis complex proportion methods were developed). There are pathogens in case of which susceptibility testing in the narrow sense is impossible (e.g. Mycobacterium leprae ). The density of the initial inoculum used in the test fundamentally influences the results. Resistance to most antibiotics is not independent of inoculum size, tests with lower inoculum yield false susceptible results, while in case of large inocula (usually resembling the in vivo situation more closely) the same isolate proves to be more resistant ( inoculum-effect ). Sometimes prediction of the in vivo efficacy is possible in vitro only under special circumstances. To demonstrate heteroresistance, the cells with resistant phenotypes must be selected. This can be achieved using specialized media or culture conditions (detection of MRSA in agar medium with high salt concentration, at 30 °C), or in media containing antibiotics. If the mechanism of resistance is inducible, it is possible that the antibiotic to be tested does not induce the resistance in vitro (but induces it in vivo ) leading to a false susceptible result. Such types of resistance are tested with an antibiotic, which shows total cross-resistance with the drug to
64 Antimicrobial chemotherapy be tested, but is a good inducer (e.g. demonstration of clindamycin resistance of staphylococci using the good inducer lincomycin). Regular quality control of the tests is crucial. This is performed by testing reference strains with known susceptibilities.
Antibiotic susceptibility testing methods
Susceptibility testing can be performed quantitatively, by measuring the concentration killing or efficiently inhibiting bacteria, or with semiquantitative methods which use the same principle, but are less accurate. The most frequently determined susceptibility indicator is the Minimum Inhibitory Concentration (MIC). The MIC is the lowest drug concentration, which efficiently inhibits the growth and proliferation of bacteria. As MICs of an antibiotic may vary widely even within a species, to characterize the susceptibility of a certain taxonomic category (usually a species) we use population markers computed from MICs of individual strains. 1. MIC range The range between the lowest and the highest MIC encountered 2. MIC 50 The concentration inhibiting 50% of the strains examined 3. MIC 90 The concentration inhibiting 90% of the strains examined. The lower and closer to each other these values are, the more susceptible is the taxon to the antibiotic in question. These population markers have significant importance in interpretation of MIC values. For interpretation of MIC values, i.e. to translate the quantitative results to the categories susceptible-intermediate-resistant, which are easier to use clinically, the breakpoints that determine the categories had to be developed. These breakpoints should correspond to the clinical efficacy of the drug against the particular pathogen; therefore they are frequently determined based on the MIC distribution of the pathogen and the pharmacokinetics of the drug ( pharmacological approach ). Another, less frequently used approach is when the category determination is based on the discrepancy between the MIC of the strain in question and the population or species average ( biological approach ). Presently laboratories use the US (Clinical Laboratory Standard Institute, CLSI) or the European (European Committee on Antimicrobial Susceptibility Testing, EUCAST) guidelines and standard protocols. Both are based on the pharmacological approach, but EUCAST also takes the biological approach into account. Based on the breakpoints, an isolate is susceptible to a given drug if the MIC of the drug equals to or is below the lower breakpoint, and resistant if the MIC equals to or is above the higher breakpoint. Intermediate susceptibility is characterized by MIC between the two breakpoints. (Some drugs have only one breakpoint against certain pathogens, in this case intermediate susceptibility does not occur, an isolate is either susceptible or resistant unequivocally.) In case of susceptible isolates the rate of clinical success with the antibiotic (when used appropriately and if it can be used to treat that particular infection) is higher than 95%, while in case of resistant isolates the chance of clinical failure is 95%. For intermediate isolates the therapeutic response is unpredictable. The Minimum Bactericidal Concentration ( MBC ) or (in case of fungi ) Minimum Fungicidal Concentration ( MFC ) can also be determined. This is the lowest concentration, which kills the vast majority (99.9%) of the individual cells of the pathogen. (In contrast to MIC, in this case we examine not only the inhibition of growth, but also whether the cells
65 Antimicrobial chemotherapy remain viable or die.) If the MBC or MFC is close to the MIC, we regard the drug as cidal, while if the difference is significant (at least five twofold dilution steps), we regard the drug as bacteriostatic or fungistatic. (Even static drugs may kill the microbes in extremely high concentrations, but these are usually not achievable or toxic in vivo .) The phenomenon when the MBC of a primarily cidal drug differs significantly from the MIC, is called antibiotic tolerance (see above). The most direct method to assess the expected efficacy of a given drug in a given infection of a given patient is to determine serum bactericidal titer ( SBT ). This is the highest dilution of the serum (or liquor or any other body liquid) of the patient receiving antibiotic treatment (which, of course, contains the antibiotic), which shows microbicidal action against the tested pathogen (isolated from the same patient). SBT is determined similarly to MFC determinations. The most suitable method to demonstrate cidal activity and the kinetics of killing are the determination of time-kill curves . These are determined using a dilution series of the antibiotic inoculated with equal amounts of the isolate. Quantitative cultures are performed regularly at predetermined time points (e.g. at hours 0, 6, 12, 24, 36, 48; the time points are selected according to the purpose of the tests). The curve is drawn by plotting viable cell numbers depending on time. Conclusions on killing activity of the drug are drawn by comparing curves obtained at different drug concentrations to the drug-free control. The method is suitable for examination of the interaction of two drugs as well. For this aim, the curves with the two drugs alone are drawn and compared to the curves obtained with the combination. The combination is synergistic if the viable cell number is decreased with further two orders of magnitude compared to the sole effect of the more effective drug, while increase of the cell number with at least two orders of magnitude indicates antagonism. In all other cases, the interaction should be interpreted as additive.
Quantitative methods
Methods to determine MIC
Agar dilution method
Agar dilution is the reference method for MIC determination. (This method approximates best the proliferation environment in vivo for pathogenic bacteria and fungi.) The test is performed using plates with the appropriate nutrient agar medium, where the consecutive plates contain a dilution series of the antibiotic incorporated in the medium. All plates are inoculated with a liquid culture of standard CFU numbers. The MIC is the concentration of the drug in the first plate (the lowest concentration) where no growth is detected. The test should not be read and should be repeated if the drug-free control plate does not show appropriate growth, or if there is growth on plates containing higher concentrations, but not on those containing lower concentration. The advantage of the method is accuracy; MIC of certain antibiotics against certain bacteria can be determined only using agar dilution with acceptable accuracy. It is also advantageous that the use of solid medium aids in detection of contamination. The drawbacks are that it is expensive and labour-intensive.
66 Antimicrobial chemotherapy
Broth dilution method
It is similar to agar dilution, but in this case the test is performed not in solid, but in broth medium. This is the standard method of MIC determination. Broth micro- and macrodilution methods are known, depending on the volume of medium used. Microdilution can be performed on a microplate in small (100-200 l) volumes, while macrodilution uses 1 ml volumes necessitating the use of test tubes. All test should include positive (drug-free or growth) control (+) as well as a negative (organism-free or purity) control (-). Microdilution uses smaller amounts of test MIC=1 mg/l material including the drug, microplates are easy to MIC=1 mg/l inoculate and this can be automated easily, while evaluation of macrodilution MIC>64 mg/l is easier, and as uses a higher absolute amount of inoculum MIC ≤0,12 mg/l it is slightly more accurate. Both methods can be used
64 32 16 8 4 2 1 0,5 0,25 0,12 + – for MBC determination. MIC is the lowest concentration,
which suppresses the growth significantly as compared to the drug-free control. (In practice the inhibition of growth by approximately 80% is regarded as the endpoint, which roughly corresponds to the limit of growth detection by the naked eye. Thus MIC is usually the lowest concentration where no growth can be seen with the naked eye.) The test should not discontinuous be read and should be growth repeated if there is growth More than one in the purity control (-), if dilution difference there is no growth in the b/w parellel tests drug-free control (+), if Growth in the there is no growth at lower negative control concentrations, but at higher concentrations No growth in the positive control growth is detected (discontinuous growth), or 64 32 16 8 4 2 1 0,5 0,25 0,12 + – if the parallel tests differ in Drug concentration mg/l more than one dilution.
67 Antimicrobial chemotherapy
E-test®
The E-test method is performed using a plastic strip impregnated with a concentration gradient of the antibiotic by means of a gel-like substance and provided with a scale of drug concentration. This strip should be placed onto an agar plate inoculated with a culture of the test organism at a standard inoculum density. The drug diffuses out of the strip into the agar, reproducing the concentration gradient of the strip within the medium. The drug will inhibit the organism in a manner directly proportional to the drug concentration in the agar, resulting in an ellipsoid inhibition zone. MIC is read where the border of this inhibition zone intersects the strip. In case of antifungal susceptibility testing using fungistatic agents, the whole surface of the plate contains fungal colonies, but the size of the colonies is markedly smaller where there is inhibition (microcolonies). For reading the MIC value we use the ellipsoid shaped border of the zone of macrocolonies and microcolonies. As internal (growth and purity) controls cannot be included, it is crucial to ascertain that the growth seen on the plate is derived solely from the isolate to be tested. The method may be used for assessment of drug interaction, by removal of the strip with the first drug (A) after twenty minutes of incubation and replacing it with the strip with the second drug (B) positioned exactly where the first strip has been. On another plate this is performed in a reverse order of the drugs (the strip containing drug B is placed first followed by the strip with drug A). From the MICs of the two drugs alone and from MICs read from these plates the Fractional Inhibitory Concentration (FIC, see in detail in the section ’Checkerboard dilution’) can be calculated. This method is inaccurate, but may be used to assess interaction; for accurate measurement of the FIC a checkerboard dilution should be performed. The greatest advantage of the method is its simplicity, but the strips are relatively expensive.
Determination of MBC/MFC
For measuring of the killing activity of the drug we perform the macrodilution (rarely the microdilution) method first, then we plate an aliquot from the tubes containing drug concentrations above the MIC (no growth is observed) to drug-free medium. The number of colonies grown is compared to the inoculum size used to inoculate the test tubes in the macrodilution method. MBC/MFC is the concentration where the viable cell number of the original inoculum is decreased thousandfold (the 99.9% of the inoculum has perished). Usually two parallel tests are run for each isolate. When there is more than one dilution difference between the parallel tests after 48 hours of incubation, the test should be repeated.
Determination of SBT
This method provides direct information on the effect of the unknown drug concentration present in the serum (or rarely in other body fluid) of the patient. We prepare a dilution series of the serum collected from the patient during antimicrobial therapy (it contains the drug as well at the concentration it is present in the patient). The greatest dilution (titer) still exhibiting bactericidal effect is the SBT. The test technically closely resembles MBC determination. Besides being labour-intensive, the greatest disadvantage is that it cannot be
68 Antimicrobial chemotherapy standardized. Nevertheless, in case of some serious and hard-to-treat infections (e.g. infectious endocarditis) monitoring of SBT can be valuable.
Time-kill tests (curves)
These curves demonstrate the time dependence of the killing activity of the tested drug against the organism studied. The test is performed in a series of tubes containing a dilution series of the drug inoculated with a predetermined inoculum. Viable cell numbers are determined (usually using quantitative culture) in samples taken from these tubes at certain time-points, including immediately after inoculation. The observed changes in viable cell number plotted against time yields the time-kill curve. The definition for killing activity is again a thousandfold decrease in viable cell number. Besides demonstration or exclusion of killing activity, these curves provide information on the kinetics of the drug effect (e.g. how quickly the drug kills the test organism). If we compare curves of two different antibiotics and that of their combination, the nature of the drug interaction can be determined. Due to its being time-consuming and expensive, the method is not used in routine diagnostics.
Checkerboard dilution
This method is used to examine the interaction of two antimicrobial agents. The principle is a dilution matrix of the two drugs. It is prepared on a 96-well plate, the rows containing drug A in a dilution series while its concentration is the same throughout the columns; concentration of drug B is constant in the rows, but it is added as a dilution series to columns. The wells of the plate then contain (excepting the control columns) the two drugs in a unique combination of drug concentrations. This plate is inoculated and incubated, and at the end-point MICs of both drugs are determined (drug A from the rows, drug B from the columns). These MICs are compared to MICs of the two drugs tested separately. The nature of the interaction is shown by the Fractional Inhibitory Concentration (FIC) index calculated using the formula:
FIC index = FIC A + FIC B, where FIC A= lowest MIC of drug A in the combination divided by MIC of drug A alone and FIC B= lowest MIC of drug B in the combination divided by MIC of drug B alone. The combination is regarded synergistic if FIC index is equal to 0.5 or lower, antagonistic if FIC index is 4 or higher, and indifferent if FIC index is between 0.5 and 4. The advantage of the method over other methods assessing drug interaction that this interaction is tested at various combination of drug concentrations, not only in one or a few predetermined combinations. In this manner, checkerboard dilution is the most accurate method available to test drug interaction; it is possible that other methods do not detect an interaction when it depends strongly on the proportion of the two drugs. The drawback is that it can only be used if the microdilution method should be performed in the same way for the two drugs (frequently unfulfilled in case of fungi). Using the matrix dilutions for determination of MBCs, the Fractional Bactericidal Concentration (FBC) can also be calculated.
69 Antimicrobial chemotherapy
Drug B MIC A – + 0 MIC A=16 mg/l MIC B=8 mg/l 0,25 MIC A in the matrix: 0,5 MIC Am =8 mg/l Indifferent effect 1 MIC B in the (FIC index=1) matrix: 2 MIC Bm =4 mg/ 4 FIC A=8/16=0,5 8 FIC B=4/8=0,5
FIC index=1 16
64 32 16 8 4 2 1 0,5 0,25 0 Drug A MIC B
Drug B MIC A – + 0 MIC A=16 mg/l MIC B=8 mg/l 0,25 MIC A in the matrix: 0,5 MIC Am =4 mg/l 1 Synergy MIC B in the matrix: (FIC index=0,5) 2 MIC Bm =2 mg/ 4 FIC A=4/16=0,25 FIC B=2/8=0,25 8 FIC index=0,5 16
64 32 16 8 4 2 1 0,5 0,25 0 Drug A MIC B
Drug B MIC A – + 0 MIC A=16 mg/l MIC B=8 mg/l 0,25 MIC A in the matrix: 0, 5 MIC Am=64 mg/l
1 Antagonism MIC B in the matrix: (FIC index=5) 2 MIC Bm =8 mg/
4 FIC A=64/16=4 FIC B=8/8=1 8 FIC index=5 16
64 32 16 8 4 2 1 0,5 0,25 0 Drug A MIC B
70 Antimicrobial chemotherapy
Semiquantitative methods
Breakpoint determination
The method is a simplification of the dilution method, we test the drug only at two concentrations, namely at the breakpoint concentrations determining the susceptibility categories. These two concentrations can be tested in either agar or broth media, usually broth medium is used. If the tested isolate does not grow at either concentration, it is considered susceptible. If it grows at the lower but not at the higher concentration, it is intermediate; if it grows in both it is resistant. If there is growth at higher but not at lower concentration, the results are not valid and the test should be repeated. The test is also to be repeated if the drug-free growth control does not show growth or there is growth in the purity (sterile) control. The method is simple, it has low cost and low workforce demand, but is unsuitable for testing anaerobes.
Resistance screening
The method is similar to breakpoint determination. Screening, however, uses only one agar plate containing the drug to be tested at the concentration corresponding to the higher breakpoint (that which determines resistance). Isolates growing on this screening plate are suspected to be resistant to the drug. This resistance should be confirmed by a quantitative method. The method is useful in screening large quantities of samples for certain important resistances (glycopeptide and high-level aminoglycoside resistance in enterococci; the latter is important as high level resistance to aminoglycosides disrupts their synergy with agents inhibiting cell wall synthesis) as well as for detection of heteroresistance (methicillin resistance in Staphylococcus aureus ).
Disc-diffusion (Kirby-Bauer) method
This technique is the most frequently used method for routine susceptibility testing, suitable for testing of fast-growing aerobic and facultative anaerobic bacteria. In case of slow- growing bacteria, e.g. obligate anaerobes, it does not give reliable results, therefore in case of these bacteria it is used only for diagnostic purposes (demonstration of primary resistance). In case of fungi it is presently used only in case of azole antifungals. (Similarly to E-test, microcolonies are observable, which should be ignored during evaluation). The principle of the method is that agar plates are inoculated with a standard bacterium suspension, and then paper discs containing antibiotics are placed on the surface of the agar. The drug in the discs will diffuse into the agar, and will inhibit the growth of bacteria in proportion to its susceptibility, leading to development of inhibition zone s around the disc. The diameter of this zones is proportional to the drug concentration in the disc (how much drug is available), to the efficacy of the drug against the test isolate (which concentration is necessary to inhibit the given isolate, i.e. what is the MIC) and the diffusibility of the drug (how far from the disc can the drug travel). Using appropriate standardization and interpreting the observed zone diameters according to a standard, the susceptibility of the isolate can be measured. The main drawback is that the method gives only semiquantitative results, and that it is unsuitable for testing anaerobes and other slow-growing fastidious bacteria. Nevertheless it is
71 Antimicrobial chemotherapy used widely, being easy to perform and cheap, thus it is very convenient for testing of large number of isolates routinely. In case of fast-diffusing drugs (e.g. β-lactams), zone diameters can be used to calculate MIC by means of a predetermined calibration curve. This calibration curve is drawn by determination and plotting of zone diameters and corresponding MICs for a large collection of strains of the species to be tested. If a straight can be fitted onto the points, MIC of the drug in question can be extrapolated from the zone diameters for the particular species (regression analysis). This calibration should be performed for each drug in case of each species. If the relation is not linear, zone diameters do not predict MIC with tolerable reliability. Regression analysis is utilized mostly in automated disc-diffusion susceptibility testing.
Semiautomated and automated susceptibility testing
Out of the methods described above, broth microdilution, agar dilution and the semiquantitative methods can be automated. Endpoint detection and reading of results is performed by measuring optical density (in case of broth microdilution) or using computerized video systems (in case of plated agar media). The results yielded may be MICs (directly or through computerized regression analysis) or the results of the breakpoint determination. The main advantage is that the method is highly standardized, but may be less accurate than the corresponding non-automated method; may be unsuitable for reliable detection of certain resistance mechanisms.
Comparison of susceptibility testing methods
As the reference (agar dilution) and the standard (broth microdilution) methods are labour-intensive, expensive and time-consuming, it is necessary to use simpler methods in routine susceptibility testing. Accuracy and reliability of these methods should be determined against the standard or the reference method. (Hypothetically, comparison with clinical efficacy is better, but it is impossible to perform for all new methods to be introduced. As the standard and the reference methods have been compared to clinical success rates, it is simpler to compare the performance of a new method to one of these.) For quantitative data we simply compare the results of the method tested with those of the standard method using a sufficiently large collection of strains (it is important that these should be truly unrelated strains, not isolates); and we calculate the percentage of concordant results. The new method is acceptable if this percentage is above 90%. During comparison we consider results differing in only ± one dilution as the same, because a one-dilution difference may also occur in repetitions of the standard method. Comparison of interpretative categories is equally important. (In case of semiquantitative methods only categories can be compared.) We consider the results of the standard method as valid and we count the errors. Different errors may be found. A minor error is when a susceptible isolate is found intermediate or an intermediate isolate is categorized as susceptible, or on the other hand a resistant isolate is found intermediate or an intermediate isolate is categorized as resistant. Major error occurs when a susceptible isolate is classified as resistant and very major error is encountered if a resistant isolate is categorized as susceptible.
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Direct demonstration of resistance mechanisms
Demonstration of proteins responsible for resistance
Presently direct demonstration of certain β-lactamases, extended spectrum β-lactamases (ESBLs, see below), metallo-β-lactamases (MBLs, see below) and the alternative penicillin binding protein (PBP2a) of Staphylococcus aureus responsible for methicillin resistance (see below) is commercially available. Demonstration of the chloramphenicol-acetyltransferase and certain aminoglycoside modifying enzymes is also possible, but these, being less reliable than susceptibility testing, are not used in routine diagnostics.
Demonstration of βββ-lactamases
Direct demonstration of β-lactamases is important when provides more reliable results than the cultural susceptibility testing (e.g. in case of Neisseria gonorrhoeae or Haemophilus influenzae ). Detection of no β-lactamase activity does not mean that the isolate is susceptible, as other resistance mechanisms may be present. Therefore, direct β-lactamase detection does not replace cultural susceptibility testing. This is the main disadvantage of the method. Several different methods exist. 1. Nitrocefin -test Nitrocefin is a cephalosporin derivative, where the hydrolysis of the β-lactam ring results in a colour product. The majority of β-lactamases hydrolyses nitrocefin, thus this colour reaction is suitable for demonstration of most β- lactamases. Suitable for detection of β-lactamase production of haemophili, neisseriae, staphylococci and enterococci. In case of Moraxella catarrhalis this is the only reliable method (even cultural susceptibility testing is unreliable). Its drawback is that nitrocefin is not hydrolysed by all β-lactamases, therefore it cannot demonstrate all of them. 2. Iodometric test In case of penicillins and cephalosporins, lysis of the β-lactam ring produces a reducing group in the molecule, which reduces iodine to iodide. This reduction may be demonstrated using starch. It is recommended primarily in case of Neisseria gonorrhoeae . 3. Acidimetric test This method is based on demonstration of the decrease in pH caused by the acidic carboxylic group originating from the lysis of the β-lactam ring using an appropriate indicator. It can be reliable in case of haemophili, neisseriae and staphylococci.
Demonstration of ESBLs
Demonstration of ESBLs capable of hydrolysing 3 rd generation cephalosporins is based on their characteristic of being inhibited by β-lactamase inhibitors (e.g. clavulanic acid, see below). Therefore, the isolates are resistant to 3 rd generation cephalosporins, but these regain efficacy in vitro in the presence of β-lactamase inhibitors. 1. Double disc method We place the disc containing the inhibitor in the proximity of the cephalosporin
73 Antimicrobial chemotherapy
disc(s). In a positive test, the inhibition zone of the cephalosporin disc is elongated towards the disc containing clavulanic acid. We may use ceftazidime, cefotaxime or cefpodoxime as a 3 rd generation cephalosporin, but using all three gives the best results. 2. ESBL discs These discs contain a 3 rd generation cephalosporin (ceftazidime, cefotaxime or cefpodoxime) plus clavulanic acid. The diameter of the inhibition zones are compared to that of the disc containing the corresponding cephalosporin alone. A test is positive if the disc with the inhibitor exhibits a zone at least 5 mm larger than the disc containing only the cephalosporin. Positive results even with one cephalosporin is enough for the diagnosis of ESBL. 3. ESBL E-test This test is performed using a special Etest strip containing a cephalosporin alone in one end and the cephalosporin plus clavulanic acid on the other. The test is positive if the clavulanic acid lowers the apparent MIC with at least four scales. All of these methods share a disadvantage, i.e. they may yield false positive results with ampC-type β-lactamases (see below), or may yield confusing results when an ampC enzyme is produced simultaneously with the ESBL. As different ESBLs have different substrate preferences, using only one cephalosporin may lead to false negative results.
Demonstration of MBLs
The principle of the test is that metallo-β-lactamases can be inhibited by EDTA (ethylenediamine-tetra-acetic acid). It is performed similarly to ESBL detection, but uses a carbapenem as a test drug and EDTA as the inhibitor. MBL Etest is the most widely used. The main disadvantage of the test is that it may give false positive result due to the inhibitory activity of EDTA against the test bacteria.
Demonstration of PBP2a responsible for βββ-lactam resistance of MRSA
This protein can be demonstrated using commercial tests based on a monoclonal antibody (e.g. latex agglutination). The drawback is that this gene is frequently present in coagulase-negative staphylococci, therefore correct identification is crucial.
Direct demonstration of resistance genes
Resistance can be detected by demonstration of known resistance genes; usually by means of polymerase chain reaction. Naturally, only demonstration of a few genes resulting in clinically important resistance is used in routine diagnostics. The method is fast and can be very high-throughput. It is also suitable for assessing the susceptibility of non-culturable organisms. It detects only known resistance mechanisms with clear genetic background, therefore using only direct gene detection, isolates with new or rare resistance mechanisms remain undetected, if we use only this method.
Interpretation of in vitro susceptibility
Susceptibility testing is performed in vitro using highly standardized methodologies. Conditions in vivo are different, and may be significantly different at the site of infection,
74 Antimicrobial chemotherapy therefore the in vitro results should always be evaluated against the knowledge on the microbe and the drug. False resistant results may arise from methodological faults, e.g. using inappropriate culture medium for susceptibility testing (using medium containing para-aminobenzoic acid for testing of sulfonamide susceptibility, or when too long a time is spent between inoculation and application of antibiotic discs allowing for growth of bacteria before antibiotic inhibition). False resistant results may be obvious in case of some species, e.g. penicillin resistant isolate has never been found among β-haemolytic streptococci, similarly to carbapenem or 3 rd generation cephalosporin resistant ones among Haemophilus isolates. False resistant results lead to increased costs and increased spread of resistance due to the unnecessary use of more expensive higher spectrum antibiotics. False susceptible results may also arise from methodological problems (e.g. inoculum effect, if the inoculum used for susceptibility testing is too low, or if the drug used in the test is partially or totally degraded); but most frequently caused by the marked difference between the characteristics of the pathogen in vitro and in vivo . MRSA is a characteristic example, which may frequently appear susceptible to carbapenems and/or 1 st and 2 nd generation cephalosporins in vitro , but these drugs are invariably ineffective in vivo . False susceptible results are more frequent when using semiquantitative methods (primarily with disc diffusion); these methods do not reliably detect certain resistance mechanisms (e.g. methicillin resistance in staphylococci, glycopeptide resistance in staphylococci and enterococci, penicillin resistance of Streptococcus pneumoniae , ampicillin resistance of haemophili or resistance of ESBL-producing bacteria to 3 rd generation cephalosporins). In these cases a quantitative susceptibility testing method should be used, or the mechanism of resistance should be directly demonstrated. A false susceptible result bears a direct risk of therapeutic failure, and consequently in some cases jeopardizes the life of the patient. Therefore it is crucial to avoid these mistakes or to discover them in time. Thorough knowledge of natural resistances aids in recognizing such mistakes, as in this case (if the identification is correct) we have prior knowledge (without or even in spite of the susceptibility results) on to which antibiotics must the tested isolate be resistant. In these cases the clinician should be notified not of the results read in in vitro testing, but the real resistance pattern. (The view that what is seen should be reported is definitely incorrect; the results should be critically evaluated and interpreted to the clinician.) Both false susceptible and false resistant results can be minimized through good knowledge on the natural resistances and a sufficient quality control system.
Using antibiotic susceptibility testing for diagnostic purposes
Known generic resistances can be used for identification as well as in providing selectivity when added to media. For these purposes antibiotics not used in the therapy are also applied. 1. All Gram negative bacteria are glycopeptide resistant. 2. All Gram positive bacteria are resistant to polymyxin and colistine. 3. Enterococci are resistant to clindamycin. 4. Staphylococcus saprophiticus is identified based on its novobiocin resistance.
Mathematical description of the antibiotic effect
Modelling of in vivo behaviour of the antibiotics can be approached in two ways. The studies can concentrate on the changes of in vivo concentration of the drug (pharmacokinetics) or on the in vivo efficacy as a function of concentration (pharmacodynamics). As the general principles of pharmacokinetics of antibiotics are the same as for other drugs,
75 Antimicrobial chemotherapy pharmacokinetics is not discussed further. Pharmacodynamics of antibiotic drugs, somewhat simplifying the case, is concerned with the relationship between drug concentration and the expected (antimicrobial) effect. This problem has many different points compared to other, non-antimicrobial drugs, since in this case not only the characteristics of the treated individual have influence, but the properties of the infecting microbe (the pathogen to be treated) also have key importance. This means that pharmacodynamics of a certain drug can be totally different in regard of different pathogens, i.e. these data would ideally be necessary to be collected in case of each pathogen-drug system. It is true, however, that related pathogens tend to behave similarly in the presence of several different drugs, thus extrapolation of behaviour of a common pathogen to that of a rare one may be possible in many cases. Pharmacodynamic data show the extent and the nature of the antimicrobial effect (cidal or static effect); the degree of dependence of the effect on the number of cells (inoculum effect) and on the actual phenotypic traits of the infecting microbe population (growth rate, biofilm formation, etc.); how pharmacokinetic processes in the host (tissue penetration, distribution, protein binding, etc.) influence antimicrobial activity; and the degree of the risk of resistance development. Data collection on pharmacodynamics can be performed using pharmacodynamic models and model systems, animal experiments and clinical studies. Data collected in this manner are used even during the drug development process. The data aid in planning, optimization and rationalization of antimicrobial chemotherapy, and may contribute vastly to prevention of resistance development. Unfortunately, specific pharmacodynamic data are available almost exclusively in case of the most frequently used antibacterial agents and in case of the most important pathogens.
Pharmacodynamic parameters
Efficacy of an antibiotic against a given pathogen, besides the properties of the drug, depends primarily on two factors, concentration and length of exposure. In other words, how drug concentration changes over time at the site of infection, i.e. what is the highest attainable concentration ( Cmax ), and how quickly it increases and decreases (how long it is maintained). The properties of the drug and the target microorganism together determine which is the more important, concentration or exposure time. In certain drug-pathogen systems antimicrobial efficacy is directly proportional to the concentration of the drug. This is a concentration dependent effect (e.g. in case of aminoglycosides, fluoroquinolones). In other systems, in contrast, after the drug reached a certain threshold concentration, antimicrobial activity remains more or less the same, antimicrobial activity depends on the duration the microorganism is exposed to drug concentrations above the threshold. This describes the concentration independent or time dependent effect (e.g. in case of β-lactam antibiotics, glycopeptides, macrolides, oxazolidinones). Members of a drug class tend to behave according to one or the other effect, but against some pathogens they may behave differently. Besides the abovementioned, antimicrobial agents may possess postantibiotic (postantifungal ) effect . This means that antimicrobial activity is maintained for a certain period (even for hours) after the drug concentration at the site of infection has dropped below the MIC. The explanation of this effect is that the drug damages the surviving microbes, and they need some time to recover and to become capable of replication. This timed needed to regain the ability of growth and division is the time period of postantibiotic effect.
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Concievably, postantibiotic effect may contribute significantly to the overall antimicrobial activity of the drug. To predict therapeutic success or failure it is necessary to know the antimicrobial activity, to be measured quantitatively using different in vitro parameters (MIC, MBC, etc., see above), and pharmacokinetic parameters of the therapy (drug or combination of drugs). The effect of the antimicrobial can be described using three main parameters calculated from the in vitro activity and pharmacokinetic parameters. 1. proportion of the peak concentration at the site of infection and the MIC ( Cmax /MIC ), 2. the time the drug concentration is above the MIC at the site of infection (T>MIC ), 3. proportion of the area under the 24-hour concentration curve and the MIC (24-h AUC/MIC ) or area under inhibition curve ( AUIC ).
Peak concentration (C max )
AUIC serum concentration
MIC
T>MIC
Time of the first time 24 hours dose
These parameters do not correlate equally with clinical success or failure. In case of antibiotics with primarily concentration dependent effect (aminoglycosides, daptomycin, quinolones, ketolides), the proportion of the peak concentration at the site of infection and the MIC (Cmax/MIC) shows the closest correlation, while in case of concentration independent (time dependent) drugs the time the drug concentration is above the MIC at the site of infection (T>MIC) and the area under inhibition curve (AUIC) are better predictors of outcome. With increasing significance of postantibiotic effect, AUIC becomes the more important parameter; in case of drugs with negligible postantibiotic effect ( β-lactams, erythromycin, linezolide) T>MIC shows the best correlation with clinical outcome, while in case of drugs with measurable postantibiotic effect (azithromycin, clindamycin, tetracyclines, glycopeptides) AUIC is the better predictor. These data can be used for optimization of antibiotic dosage and administration schedule. For antibiotics with time dependent (concentration independent) effect frequent smaller doses or continuous infusion is the best way of administration, while in case of drugs
77 Antimicrobial chemotherapy with concentration dependent effect high doses given infrequently (one or two times daily) provides the best efficacy.
Pharmacodynamic model systems
The above parameters can be determined using clinical data (analyzing serum concentrations of the drug against clinical outcome), in animal models or by means of in vitro model systems.
In vitro models
In vitro models are constructed using a culture of the test organism challenged with the antimicrobial, which is continuously washed with a drug free buffer to model drug elimination. Different elimination rates are modelled with different speed of buffer flow. The effect on the microbe is measured by sampling the model regularly and determination of the viable cell count in the samples. Advantages of the in vitro models are the excellent reproducibility and relatively low cost, and they can be used for investigating pathogens when the amount of clinical data available does not allow statistical evaluation (e.g. in case of rare infections) or when ethical issues prevent clinical studies (e.g. in case of infections with high lethality). In vitro models allow for measuring a great deal more parameters compared to in vivo (animal or clinical) studies.
Animal models
In animal models the human infection is reproduced artificially in a suitable model animal, and outcome is compared in differently treated groups (treated using various doses and administration routes). It is a better model of human infection than the in vitro model systems, but both the course of infection and drug pharmacokinetics may differ from the human situation. Further drawback is that these tests are expensive. Presently animal models are an important step of drug development (see below).
Using clinical data
Pharmacokinetic characteristics are usually determined in healthy volunteers. The other possibility is to test the drug on a patient group fulfilling well-defined criteria. In the latter case both the outcome and the side effects can be measured well, but there may be several confounding factors (underlying illness, co-infection, etc.) present, which may interfere with the evaluation of data. In clinical studies only a few doses and administration routes can be tested. Costs are extremely high and ethical issues must be observed continuously.
Pharmacodynamics of resistance development
Pharmacodynamic studies can, besides directing the drug development process and predicting outcome of therapy, predict resistance development occurring under the selection pressure exerted by the drug. During therapy secondary de novo resistance can only develop if the few mutant cells with decreased susceptibility, which unavoidably evolve under drug pressure, can survive. Prevention of resistance development in this case has to ensure that these mutant cells die during the therapy (either through the microbicidal effect of the therapy or by killing mechanisms of the host immune system). These selected mutants with decreased susceptibility can be killed if the drug concentration at the site of infection surpasses not only the MIC of the wild-type cells, but that of the less susceptible first-step mutants (which carry one mutation resulting in an increase of the MIC) as well. This concentration (inhibiting the first-step mutants) is called mutant prevention concentration (MPC), because if the drug concentration exceeds MPC, the primary (first-step) mutants cannot survive, and consequently the development of mutants with further mutations and even higher MICs is avoided. However, if drug concentration remains lower, between MIC and MPC, the survival of the first-step mutants becomes
78 Antimicrobial chemotherapy possible, and the surviving first-step mutants can develop further mutations or acquire another resistance mechanism leading to development of higher level, clinically relevant resistance. Therefore the concentration range between MIC and MPC is referred to as mutant selection window .
Peak concentration (C max )
AUIC
MPC mutant selection serum concentration window MIC
T>MIC
time of the first time dose 24 hours
The risk of selection, naturally, is present and much higher if the drug concentration remains below MIC, but in this case the expectable outcome is therapeutic failure, and the drug should not be used. Concentrations corresponding to the mutant selection window may appear efficacious, but there is a danger of late therapeutic failure (or recurrence of infection) due to development of resistance during the therapy course. This is not always apparent during treatment of one patient. In the infecting microbe population the number (proportion) of resistant individual cells (mutants) grow slowly, and serial passage in more than one patient (and continuous increase in the proportion of mutant cells in the population) may be required to reach a critical rate of mutants that lead to therapeutic failure. As presently used breakpoints were established on the basis of MICs, routine susceptibility testing is not capable of predicting the risk of resistance development. Professional discussions are in progress to modify the breakpoints with regard to MPC and mutant selection window. Existence of the mutant selection window provides explanation to the advantage arising from production of resistance mechanisms which, though increasing the MIC slightly, do not provide clinically relevant resistance (confer low level resistance). In these strains the increase in the MIC is paralleled by an increase in the MPC as well, leading to widening of the mutant selection window. This, in turn, provides opportunity to selection and survival of mutants with more potent resistance mechanisms. MPC may be approximated by agar dilution performed with extremely high (10 10
79 Antimicrobial chemotherapy
CFU) inoculum. Such a large inoculum certainly contains a few primary (first-step) mutant cells with decreased susceptibility, which may grow at the drug concentration corresponding to the MIC against the wild-type cells. (Besides, more cells are extremely rarely present in vivo at the site of the infection.) These mutants will grow at plates containing drug concentrations between the MIC and MPC (corresponding to the mutant selection window). The lowest concentration inhibiting even these cells with decreased susceptibility is the MPC. Naturally, in cases when the high level resistance is developed in one step only (is due to only one mutation) or when resistance is acquired through horizontal gene transfer, MPC is higher than the peak concentration attainable during therapy. Therefore, the model presented above, though is capable of predicting resistance development, does not provide a solution in these cases. During development of newer antibiotics, risk of development of resistance due to mutations can be predicted. This provides opportunity to develop drugs with low risk of mutational resistance development.
Development of antimicrobial agents
Sources of compounds with antimicrobial activity
Chemical compounds with antimicrobial properties originate from two main sources; they may be of natural origin or may be produced by chemical synthesis. In the past, compounds produced by different microbes were the most important source of antimicrobial agents, and most antibiotic families used presently come from this source. Active research is in progress to discover and isolate natural products with antimicrobial properties from soil or marine microorganisms. First the soil extract or seawater is tested for antimicrobial activity, then the compound(s) responsible and/or the producing organism is isolated and characterized. The screening for antimicrobial activity can be performed directly by assessment of activity of the soil extract or seawater (e.g. demonstrated by agar dilution), but recently more sensitive methods specific to a certain drug class are also in use. (In case of β-lactams a β-lactamase induction or a D-D-carboxypeptidase test is used, while in case of aminoglycosides there is a competitive ELISA method.) Screening for genes involved in antibiotic production is also utilized. For isolation of the compound, different chromatographic methods (gel exclusion, ion exchange or adsorption chromatography, etc.) are applied; analysis of chemical structure is performed by means of NMR and/or X-ray crystallography. Researchers try to acquire new natural compounds by genetic modification of antibiotic producer organisms (usually by transformation or transfection with genes involved in drug biosynthesis) or by altering the natural drug production process by adding substrates during fermentation. In the latter case knock-out mutants engineered so that some genes involved in the drug biosynthesis are inactivated are used, making it easier to direct the biosynthesis to the required direction. Drugs from medicinal herbs used in traditional medicine may serve as source for new drugs, especially in China. For further information on research on natural antimicrobial products we refer to the material of pharmaceutical technology. The role of organic chemistry syntheses is presently more important. Though sometimes drugs produced by chemical synthesis for another purpose show antimicrobial properties, the new antimicrobials arise mainly from chemical modification of known structures to develop drugs with more favourable characteristics. As in this case a known
80 Antimicrobial chemotherapy molecule is modified, the risk of failure is lower. The basis for modification is the discovery of structure-activity relationship. It should be determined, which are the key elements of the molecular structure indispensable for activity, which structural elements influence pharmacokinetic properties, and which are the target points for resistance mechanisms modifying or degrading the drug. Utilizing this knowledge, derivatives of the original molecules can be produced, which may be more active, may have more favourable pharmacokinetics or may be insensitive to resistance provided by enzymatic modification or degradation. A new field is the rational drug design. Designing a new molecule with antimicrobial properties require a thorough knowledge of the physiology and virulence mechanisms of the target microbe, allowing for identification of biochemical processes that may be efficiently and selectively inhibited (potential targets). This is followed by the rational design of molecules inhibiting the potential targets identified earlier by means of molecular modelling, then the synthesis of the molecules is performed and they are tested for antimicrobial activity.
The drug development process
Antimicrobial activity of a new compound is first tested in vitro . During in vitro testing its antimicrobial spectrum, the degree and nature of its activity (cidal or static) as well as its mechanism of action is determined. If the drug candidate proves to possess good in vitro activity, then it may enter the development process proper. In the initial phase of the development process, the tolerability, the tissue penetration, metabolism and other important pharmacokinetic parameters are determined in animal experiments. Next step is testing the in vivo activity in different model infections. After the determination of the dose of the pathogen causing 100% lethality (LD 100 ), groups of animals challenged with pathogen doses above the LD 100 are treated with different doses of the drug candidate (the routes of administration and the duration of the treatment may differ according to the drug formulation planned to be marketed). The 50% protective dose ( PD 50 , the dose leading to survival of 50% of the challenged test animals) of the drug candidate is determined in the different groups. These tests are performed with different important pathogens, and thus the in vivo efficacy and spectrum of the drug is characterized. This is the phase where it can be discovered if the predictions suggested by the in vitro susceptibility results will expectedly differ markedly from the in vivo situation in humans. This is followed by investigations of efficacy in different organ-specific infection models (endocarditis, meningitis, pneumonia, abscess, osteomyelitis, etc.), and similar tests on immunocompromised animals. During these tests, the formulation and possible doses of the candidate drug are also determined. Promising candidates then enter the preclinical safety test phase, which is performed exactly as for other drug types (acute and chronic toxicity tests, exclusion of carcinogenic, mutagenic, allergizing effects as well as potential damage to embryogenesis, organogenesis and spermatogenesis), therefore it is not discussed in detail. Data collected on drug candidates proving efficacious and safe in preclinical test should be presented to the licensing authority for evaluation. Clinical tests can only be started with the consent of the authority. Parallel to the clinical phase, long-term toxicity is tested in appropriate animal models. Clinical testing has four phases. In phase I initially the safety and human pharmacokinetic parameters (oral absorption, attainable serum concentration, degree of protein binding, elimination half-life, recovery from the urine) of the drug is tested on a small group of healthy volunteers (20-80 individuals) under medical supervision. Later a wider-range study with more than one dose is performed still on healthy volunteers to
81 Antimicrobial chemotherapy determine the expectable bioavailability. In phase I the dose to be administered is determined, and a rough assessment of the activity becomes possible (see in the section ’Mathematical description of the antibiotic effect’.) Moreover, the more common adverse effects are recognized. In phase II the activity of the candidate drug is tested on a predetermined patient group (100-300 patients) in comparison with a licensed antibiotic used frequently for the same indication. Both clinical and microbiological improvement (pathogen eradication) as well as adverse effects are monitored. Phase III is similar to phase II, but involve more (1000-3000) patients. For this reason it is frequently a multicenter study. The most frequent study type is randomized comparative study, when it is determined randomly whether the patients receives the test drug or the comparator (proven to be effective), and neither the doctor nor the patient knows which drug is administered. This phase serves primarily to assess whether the new drug is more active than older ones, besides this, the side effects are also discovered. At the end of phase III the data collected should be analized and presented again to the licensing authority. After licensing the drug candidate becomes a licensed drug and the marketing can be started. Clinical phase IV is not an evaluation phase, the drug is available on the market, but the data on the new drug should be continuously collected, mostly on cases of rare but dangerous side effects. If such adverse events are encountered, the drug has to be withdrawn from the market. Phase IV is also the phase for further data collection, during this phase data are collected on efficacy in special patient groups or in rare infections.
Problems with development of new antibiotics
Due to the profit-oriented nature of drug development and marketing, financial issues play a key role in decision-making related to development of antimicrobial drugs. Compared to other drug groups, antimicrobials are at a disadvantage, since a number of drugs (mainly antiprotozoal and anthelminthic drugs) are used to treat infections which occur mostly in tropical, developing countries. In these countries neither the government nor the inhabitants can finance to buy the drugs; consequently not only development of new drugs is unprofitable, but, due to lack of profit, even production and marketing of older drugs has sometimes been stopped. Though antibiotics, antifungal and antiviral agents are widely used in developed countries with high healthcare expenditures, several factors decrease the profit expectable from marketing anti-infectives. In contrast to many other drugs, antimicrobial agents are taken only for relatively short periods, and this period not only cannot be lengthened, but needs to be shortened to slow down resistance development. Due to the low (and preferably decreasing) consumption the recovery time for the money invested into the drug development is longer than in case of other drugs, resulting in a significantly lower expectable profit than that promised by other, not antimicrobial drugs. Between 1960 and 1980 a lot of antibiotics have reached the market, therefore considerable profit can only be expected if the new antibiotics are prominently better than older drugs. Furthermore, for business-oriented drug development only broad-spectrum agents are profitable, which can be used to treat a wide variety of infections, as more may be sold of these drugs; but from clinical viewpoint narrow-spectrum agents are more desirable, as these bear less risk for spreading of resistance. For this reason the licensing authorities restrict the diagnoses in case of which the new drug is licensed. A further problem is that resistance development is unavoidable on the long run, which, sooner or later, will limit the utility of the anti-infective, therefore it is to be expected that sales rate is going to fall drastically. Besides the lower expectable profits, development of antimicrobial agents is more
82 Antimicrobial chemotherapy difficult and more expensive than development of other drugs types. As an antimicrobial possess different activity to different microbes, and even its mechanism of action may differ between different target organisms, efficacy studies should be performed in case of a number of pathogens. Consequently, more in vitro and in vivo studies are necessary than in case of other drugs. In summary, besides similar or rather higher development cost, the profit to be expected from marketing an antimicrobial is lower, for this reason the investment hardly or not at all yields the profit to be expected from other drugs during the ten years of the patent period. These facts give rise to two unfavourable consequences. On one hand, in developing countries, where the health level of the inhabitants tend to be poor, only the cheap drugs are available, which, partly because of lack of alternative drugs, partly because of lack of microbiological laboratory services, are seriously overused. This leads to high proportion of resistance, and these frequently found resistant strains are spread through international travel and tourism to developed countries, where travel has recently been recognized as an independent risk factor for acquisition of resistant microbes. The other unfavourable consequence, concerning developed countries more directly, that those pharmaceutical companies where the developing potential is concentrated and the founding for drug development is available, invest these financial, infrastructural and human resources into development of more profitable formulations taken for long periods continuously (anxiolytics, lipid metabolism regulators, or the recently marketed Viagra, etc.) instead into development of antimicrobials. (In the past 25 years only one new antibiotic class with a unique mechanism of action has been discovered; the oxazolidinones.) As a consequence, the number of newer antimicrobials with unique targets, which are expectedly unaffected by the presently known resistance mechanisms, is very small. For this reason it is crucial that efficacy of the known drugs should be preserved to the highest possible extent, and as long and as possible (see in the next section).
Antibiotic policy (antibiotic stewardship)
The spread of antibiotic resistance is the most serious problem of infection control. The cause of this spread is the overuse and misuse of antibiotics, which, besides being the fuel for spread of resistance, generates enormous expenditure as well. The aim of antibiotic policy is the rationalization of the use of antimicrobial agents (avoidance of overuse and misuse), for a better quality healthcare, for the decrease of unnecessary expenses and for prevention of development and spread of resistance. Antibiotic policy can be operated at different levels, may be operated at a hospital ward or hospital, can be regional, national and, theoretically, international. Antibiotic policy is most effective at the ward or hospital level, since it is developed taking the local peculiarities into consideration, but it is important to conform with regional and higher level regulations. Antibiotic policy is and has to be an integral part of the infection control; they should be developed and operated in accordance. As similar problems are expectable in case of chemotherapy against other microbe groups, especially in case of antifungal and antiviral agents, similar regulations would be desirable for these antimicrobials as well. Similarly to the antibiotic policy, the use of disinfectants should also be regulated. Disinfectant policy dealing with these issues is discussed above. Antibiotic policy demands the concerted action of clinicians, pharmacists, infectologists, clinical microbiologists (microbiology laboratory) and the whole infection control team. Furthermore, support from decisions makers as well as the cooperation of patients is also indispensable. (Antibiotic policy needs financial support, and patients should be familiar with antibiotic policy and accept it.) In order to develop, operate and continuously
83 Antimicrobial chemotherapy supervise a local antibiotic policy, all healthcare institutions should create a working group out of the abovementioned professionals. The main executive force of antibiotic policy are doctors working in patient care, as they are the prescribers of antibiotics and they have a direct influence on the prescribing practice. The therapeutic decision is influenced by three main factors, i.e. the knowledge of the prescriber about the infections and the available drugs, the real or imagined expectations of the patients and the affordable resources. The role of the other participants of antibiotic policy is to aid prescribers to choose an appropriate therapy through efficient providing and communication of the necessary information, giving advice and to ensure continuous availability of the appropriate antibiotics. Consequently, the main role of pharmacists is the organization of antibiotic supply and monitoring of antibiotic consumption. Clinical microbiologists perform the laboratory examinations necessary for targeted therapy and monitor antibiotic resistance. Infection control specialists organize the preventive as well as the outbreak-related countermeasures including disinfection. Decision-makers and financial staff should provide the necessary financial and human resources. Pharmacists and clinical microbiologists are sometimes involved in consultative tasks as well. For efficient antibiotic policy, the actual problems in regard to consumption (which antibiotics are overused and by whom) as well as concerning resistance (in case of which pathogens or infections is resistance problematic and against which drugs) should be discovered and the aims should be clearly defined at the ward, institute or regional level. Recommendations and regulations should be prepared, made accepted and strictly observed, and the efficiency of the antibiotic policy (reaching of goals) should be monitored continuously. The costs necessary to cover these expenses may be high, but it was shown in different healthcare settings that the savings arising from the decreased number of nosocomial infections and from decreased resistance always far exceed the invested money; antibiotic policy, besides improving the quality of patient care, is cost-effective on the long run. The tools for this approach are regular data collection, regulation of antibiotic prescribing, effective eradication in case of an outbreak (see in more detail in the section ’Sterilization and disinfection’), and continuous training of healthcare workers and patients.
Data collection
Data collection should be performed in two directions; antibiotic consumption as well as antibiotic resistance should be monitored. To monitor consumption, prescribing habits and the factors influencing therapeutic decisions should be investigated. According to surveys, prescription of inadequate or unnecessary therapy is driven by the lack of information and training, by the fear of the failure of empiric therapy or by the desire to conform to the patients’ requirements. Knowledge on these issues is necessary for surveying the extent of misuse and the drugs involved as well as for analysis of the relationship between consumption and resistance. On the other had, this knowledge will highlight the centre(s) of gravity for training. Monitoring of resistance should be extended both to nosocomial and to community acquired infections. Relative frequency and distribution of pathogens in different infectious diseases should be discovered and followed up, preferentially separately for special patients groups (children, the elderly, the immunocompromised, patients with diabetes, etc.). These data are indispensable for the prediction of the pathogens to be expected, consequently for prescribing appropriate empiric therapy. The resistance of the more frequent pathogens should also be monitored. These two activities form the microbiological surveillance. Data provided
84 Antimicrobial chemotherapy by the surveillance, besides aiding the choice of empirical therapy directly, are necessary for the studying the effect of the selection pressure exerted by the antibiotics, and are basic data for determination of medium- and long-term goals of antibiotic policy. For maximum efficiency, regional, national and international antibiotic policies would need monitoring of antibiotic consumption in veterinary practice (both in large-scale animal husbandry and in pet healthcare), since certain mechanisms of antibiotic resistance were developed in and spread to humans from bacteria in the animal normal flora. A characteristic example is the use of avoparcin as a growth promoter. Avoparcin is an antibiotic with a mechanism of action analogous to that of glycopeptides. Avoparcin use led to development of resistance to avoparcin in enterococci of the poultry normal flora, which proved to provide full cross-resistance to glycopeptides of the human medicine. These resistant enterococci were transferred to and established in the human normal flora, finally leading to emergence of glycopeptide resistant enterococci as human pathogens. (Banning avoparcin led to a significant decrease in prevalence of vancomycin resistant enterococci.) For this reason, involvement of professionals in veterinary medicine in the development and operation of antibiotic policies above ward/institution level would be desirable.
Measuring antibiotic consumption
Monitoring of antibiotic consumption is an important task of pharmacists in the frame of antibiotic policy. The major goal of monitoring consumption is to assess the actual selection pressure. Consumption data collected, of course, can be used in analyses with different aims (e.g. in pharmacoeconomy studies). (Obviously, the issues described below refer not exclusively to antibiotics, measuring of consumption of other drug types can be performed in an analogous manner.) Measuring requires variables that measure consumption objectively, and allows for comparison of data from different wards, institutions or even regions. Consumption may be measured at a collective level, based on sales rates in a region, or by purchasing rates in a ward or institution level, or at patient level using prescription (or drug purchase) rates or using patient documentation. In some countries national registers exist for that purpose. For practical reasons, drugs belonging to a class with the same mechanism of action are treated as one group. In case of antibiotics, those are practical to be treated as one group, which provoke resistance mechanisms in a similar manner (e.g. all 1st generation cephalosporins). This grouping is likely to result in groups still too large to be worked with, necessitating further contractions to be executed again on the basis of nature and extent of resistance provoked. Drug purchase costs, number of consumed cartons or amount of consumed drug are not suitable indices, as differences between prices, doses per cartons and therapeutic doses of different drugs will bias the data and compromise comparability. To ensure that data will be comparable even between different countries, standardized indices should be used, such as defined daily dose ( DDD ) and prescribed daily dose ( PDD ). DDD and PDD are technical indices allowing for standardized assessment of consumption. DDD is the daily drug dose for an average (70 kg) human, while PDD means the daily drug dose prescribed for one patient for treatment of the main indication of the drug in the given institution. (Consequently, the latter can be used only in case of a given institution or ward as main indications and thus prescribed doses may vary widely among different institutions.) These indices allow for circumvention of bias arising from the difference in the necessary doses. The number of DDDs, calculated as a quotient of the consumed amount of drug (grams, IUs, etc.) and the DDD, is a parameter independent of drug price and of the amount of
85 Antimicrobial chemotherapy the agent in a carton, a daily treatment weighs approximately the same. This allows for unbiased comparison of different drugs or application fields. Drawbacks of DDD include that it is not applicable in pediatric wards (daily doses vary according to patient age); does not reflect the number of patients exposed to the drug (combination therapy is possible); and does not always correspond to the daily dose administered in reality (lower doses are used for prophylaxis or in renal failure, higher doses are used in serious infections). For these reasons it is more suitable for large-scale comparisons (e.g. between countries), while PDD, which corresponds better to the applied dose, yields more accurate results for temporal or within-ward comparisons. The number of PDDs is calculated analogously to that of DDDs. By determination of the number of DDDs or PDDs consumed, the antibiotic usage during the study period of the studied unit (ward, clinic, region) can be characterized, and drug consumption of different units can be compared, or changes in consumption over time can be tracked. In case of antibiotics, however, a further index should be measured to accurately assess the selection pressure exerted, i.e. the number of patients exposed to the antibiotic. In other words, consumption should be correlated with patient turnover. In inpatient care number of patient-days (equal to the sum of the daily patient numbers during the examined period, but can be approximated by the number of bed-days , calculated as ’number of beds × bed occupancy × number of days’) is the most suitable index to measure patient turnover, while in outpatient care the best index is the number of inhabitant-days (calculated as ’number of inhabitants in the studied area × number of days’). (The latter is not suitable to describe inpatient flow.) To assess selection pressure in inpatient care, the number of DDDs or PDDs per 100 patient-days (100 bed-days) is suitable, while for outpatient care the number of DDDs per 100 inhabitant-days is an appropriate approach. Exposure of the patients can be expressed as the percentage of patients receiving antibiotics. These measurements, naturally, cannot include drug consumption through non-registered (self-medication) or illegal (through unlicenced drug sources) usage.
Regulation of antibiotic usage
Antibiotic policy intervenes to antibiotic usage directly through regulation of drug choice. This intervention needs accurate planning, the basis for which should be the consumption and resistance data. Both choosing empirical therapy and choosing form the antibiogram can (and should) be regulated. Different strategies exist.
1. Guidelines. These are the least effective of the strategies, but they represent a useful approach in situations where other tools cannot be applied (e.g. in case of independent general practitioners, or when understaffing and lack of funding prevents more complicated approaches). 2. Restriction of usage. For this intervention antibiotics are classified into three groups. There are unrestricted (freely usable) drugs (1 st group), antibiotics with low risk of resistance induction and used more or less rationally. Some antibiotics may be used only in selected cases (2 nd group). Different restrictions are possible, e.g. prescribed only by specialists, for a predefined set of diagnoses or only at certain wards (e.g. intensive care units), etc. Antibiotics with prominent efficacy or extremely high cost are put into the reserve (3 rd group). These can only be used if other drugs cannot be used, frequently only with the consent of the representative of the infection control team or the antibiotic policy
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group. It is also necessary to regulate the antibiotic prophylaxis in parallel. Naturally, the viewpoints driving the selection of the circle of restricted antibiotics are different in outpatient care and in clinical usage, according to the different indications (prophylaxis or treatment) and severity of infections. This means that these regulations should be individually prepared in case of each ward, or even each disease. It is possible that the microbiology laboratory reports only certain drugs as a part of restriction. Advantage of restriction is that it provides direct control on consumption, but may increase drastically the consumption of unrestricted drugs. Thus, though preserving the efficacy of certain drugs for a time, may lead to a situation when only restricted drugs remain effective, leading to their increased use and eventually to resistance against them. For this reason, restriction is not always enough to prevent spread of resistance in the long run. An appropriately chosen restriction, however, may prevent the selection and spread of multiresistant organisms (e.g. restriction of use of 3 rd generation cephalosporins led to significant decrease in the prevalence of ESBL production among nosocomial enterobacteria). A further drawback is that prescribers may be annoyed by restricting their right to independent therapeutic decision, thus it may be hard to have this approach accepted. 3. Rotation. Rotation or cyclineg means that all patients receive the same antibiotic protocol if possible, i.e. as an empirical therapy and if it is effective according to the antibiogram. This therapy protocol is changed to another after a predetermined period, possibly followed by a third one, after which the first protocol is returned to and the rotation is started again. Efficiency in preventing resistance depends mainly upon the length of the cyclineg periods. If these periods are sufficiently short, resistance spread can be controlled well, but using too long periods, though slows the spread of resistance, cannot slow it enough to have favourable impact. Problems with the rotation are the following. The efficacy of the different protocols should be roughly equal; the number of protocols necessary may be unmanageably high for wards with wide patient care profile (e.g. internal medicine wards); and in case of frequent patient exchange between wards, the protocols should be harmonized, creating an unbearable workload to the personnel in charge for the regulations. 4. Diversification. During diversification the aim is to use as many drug protocols simultaneously as possible, i.e. individualize therapy as much as possible. It is even possible that all patients receive different antibiotic regimens. This minimizes the selection pressure exerted by a certain overused antibiotic class, as practically no drugs are used excessively. This may be very efficient in prevention of resistance, but requires extremely high level of competence of the antibiotic policy group and, even more from the part of prescribers and of professionals involved in organizing antibiotic supply. (Many different drugs should be cost-effectively supplied continuously, and the microflora of the ward and the expected patient population as well as its resistance characteristics should be known intimately and in an up-to-date manner.) Besides, requiring intensive surveillance work to continuously provide the necessary data, a fast-reacting microbiology service and infection control team is also indispensable. For these reasons, this approach needs the most investment.
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5. Introduction of new antibiotics. This possibility is almost totally theoretical, because the speed of drug development cannot keep pace with the spread of resistance (see in more detail in the section ’Problems with development of new antibiotics’).
Training
As misuse of antibiotics often arise form lack of knowledge, training is an important part of antibiotic policy. Training has two main target groups; personnel involved directly (doctors, nurses) or indirectly (pharmacists, laboratory personnel, etc.) in patient care and patients. Training should involve the appropriate infection control measures (e.g. good hand hygiene drastically decreases the risk of transmission of nosocomial infections) both for healthcare personnel and patients. A very important issue is to transfer knowledge on antibiotics, antibiotic resistance and on prudent use of antibiotics to healthcare personnel, especially to prescriber doctors and pharmacists involved in antibiotic supply. Through patient training, prescribing on patient’s demand can be avoided. (Many patients do not know that the most common causes of sore throat are viruses, against which antibiotics are ineffective, and consequently needless.) Training in connection with antibiotic policy can be divided into three main parts. Knowledge on infections, on antimicrobials and on antimicrobial use should be communicated thoroughly during graduate and postgraduate training of healthcare personnel. Similarly important is the regular and up-to-date reporting of data collected during execution of antibiotic policy to a wide range of healthcare personnel, especially to those directly involved in patient care. Finally, problems should be communicated to patients (and to patients-to-be, i.e. to the general public) continuously in order to facilitate acceptance of regulations.
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Antibacterial agents
Drugs targeting cell wall synthesis
Drugs targeting cell wall synthesis 1. β-lactam antibiotics 1.1. Penicillins 1.1.1. Early penicillins penicillin-G, penicillin-V 1.1.2. β-lactamase-stable penicillins oxacillin 1.1.3. Broad spectrum penicillins 1.1.3.1. Aminopenicillins ampicillin,amoxicillin 1.1.3.2. Carboxypenicillins carbenicillin, ticarcillin 1.1.3.3. Ureidopenicillins piperacillin, mezlocillin, azlocillin 1.1.4. Temocillin 1.1.5. Mecillinam (amdinocillin) amoxicillin+clavulanic acid, 1.1.6. Penicillins protected with a β-lactamase piperacillin+tazobactam, inhibitor ampicillin+sulbactam 1.2. Cephalosporins 1.2.1. 1st generation cefalothin, cefalexin, cefazolin 1.2.2. 2nd generation cefuroxime, cefaclor, cefprozil cefixime, ceftibuten, cefotaxime, 1.2.3. 3rd generation ceftriaxone, ceftazidime, cefoperazone, cefsulodine, moxalactam 1.2.4. 4th generation cefepime, cefpiromee 1.2.5. „5th generation” ceftobiprole cephalosporins proper cefazolin e, cefuroxim e, cefixim e, cefepim e oxacephems moxalactam carbacephems loracarbef cefamycins cefoxitin, cefetamet, moxalactam 1.3. Carbapenem s imipenem, meropenem, e rtapenem 1.4. Monobactams aztreonam 2. Glycopeptides vancomycin, teicoplanin 3. Lipoglycopeptides telavancin, dalbavancin, oritavancin 4. Daptomycin daptomycin 5. Fosfomycin (fosfonomycin) fosfomycin 6. Lipoglycodepsipeptides ramoplanin
βββ-lactam antibiotics
β-lactams are drugs with rapid bactericidal action, they exert their activity by inhibiting cell wall synthesis. Their target molecules are the penicillin binding proteins (PBPs), which are key enzymes of the cell wall synthesis responsible for transpeptidation. This inhibition leads to a shift towards autolytic processes within the cell wall; the ultimate cause of cell death is autolysis. Primarily they act on actively growing and replicating cells, consequently bacteriostatic drugs antagonistically interfere with the activity of β-lactams. With aminoglycosides they potentiate the activity of each other (synergy). Due to their lacking of cell wall, β-lactams do not have activity on mycoplasmas. β-lactams are also ineffective against legionellas, chlamydiae and rickettsiae.
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Penicillins
Basic structure of penicillins is the 6-amino-penicilloic acid, which contains a β-lactam ring condensed with a thiazolidine ring. Semisynthetic derivatives differ in the substituents on the amino group at the 6 th position. With the exception of the natural penicillin G, all are semisynthetic antibiotics. Different subgroups exist, as H2N 6 S 1. Early penicillins CH3
2. β-lactamase-stable penicillins N 3 CH3 O 3. Broad spectrum penicillins COOH 1. Aminopenicillins 2. Carboxypenicillins 6-amino-penicilloic acid 3. Ureidopenicillins 4. Penicillins protected with β-lactamase inhibitor
Early penicillins
This group contains, among others, the acid-labile penicillin-G (not to be used per os ) and the acid-stable penicillin-V.
H H N N S CH S 3 O CH3 O N O N CH3 CH3 O O COOH COOH benzylpenicillin phenoxymethylpenicillin (penicillin G) (penicillin V)
They are active against most Gram positive bacteria including both aerobic and strictly anaerobic species, but only bacteriostatic against enterococci. They are also active against Neisseria spp. , pasteurellae and strictly anaerobic Gram negatives as well as against spirochaetes and spirillums.
β-lactamase- (penicillinase-) stable penicillins
This group include methicillin (no longer used due to severe side effects), oxacillin , cloxacillin and nafcillin .
O CH3 O CH3 H N H N S N S CH3 CH3 O O N O N CH3 CH3 O O CH3 COOH COOH methicillin oxacillin
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CH3
O H N S CH3 O N CH3 O COOH nafcillin They are resistant to lysis by staphylococcal penicillinase, but have narrower spectrum than early penicillins. The majority of penicillin-susceptible streptococci and coryneforms are resistant to oxacillin, as all Gram negative aerobic and anaerobic species. They are used against β-lactamase producing staphylococci exclusively.
Broad spectrum penicillins NH 2 H N S Aminopenicillins CH3 O N 3 CH3 R O Ampicillin and amoxicillin belongs here, and COOH their derivatives esterified on the 3-carboxylic group R=H: ampicillin (bacampicillin , pivampicillin , talampicillin ). R=OH: amoxicillin Cross-resistance and cross-suceptibility is total between all aminopenicillins, but there may be slight differences in activity in case of certain species (amoxicillin is slightly more active against Streptococcus pneumoniae than ampicillin, however, the activity of penicillin G is superior to both against susceptible isolates). Their spectrum is broader against Gram negative bacteria as compared to early penicillins. They are active against all bacteria susceptible to early penicillins, but also have potent activity against Haemophilus spp. , Escherichia coli, Proteus mirabilis , salmonellae and shigellae as well as against Helicobacter pylori .
Carboxypenicillins
Carbenicillin and ticarcillin belongs here. Their activity is broadened with an antipseudomonas action. They are no longer available in many countries.
COOH COOH N S N H CH S CH 3 S H 3 O N O N CH3 CH3 O O carbenicillin COOH ticarcillin COOH
Ureidopenicillins
This group contains mezlocillin , azlocillin and piperacillin . These have the broadest spectrum out of unprotected penicillin derivatives; they may remain effective against bacteria resistant to other penicillin derivatives (e.g. Pseudomonas aeruginosa , strictly anaerobic bacteria).
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O O O O O
H3C S N N HN N O NH NH N S N S H CH3 H CH3 O N O N CH3 CH3 O O mezlocillin COOH azlocillin COOH
O O O
H3C N N NH N S H CH3 O N CH3 O piperacillin COOH
Temocillin
Temocillin is the 6-methoxy derivative of ticarcillin (a COOH CH carboxypenicillin, see above). This 6-methoxy group, similarly to O 3 cefamycins substituted analogously, lend excellent lactamase stability to N S CH the molecule, but decreases the activity against Gram positives, strict S H 3 O N anaerobes and Pseudomonas . Therefore, temocillin is used primarily CH3 against ESBL producing enterobacteria, an increasingly important usage O temocillin COOH is treatment of Burkholderia cepacia infections of patients with cystic fibrosis. It has no activity against Gram positives and strictly anaerobes.
Mecillinam (amdinocillin)
N N S The difference between mecillinam and other penicillin CH3 th derivatives is that the substituent in the 6 position is connected through N 3 CH3 an imino- instead of an amino group. Its orally bioavailable derivative is O pivmecillinam , which carries a pivaloyl-oxymethyl group on the 3rd mecillinam COOH position carboxylic group. Its main cellular target is PBP2, in contrast to other penicillins (where the main target is PBP1A, PBP1B and PBP3). Its spectrum is similar to that of temocillin; it is active mainly against Gram negative enterobacteria. It is inactive against Gram positives, the majority of strict anaerobes and against pseudomonads. It is rarely used.
Penicillins protected with β-lactamase inhibitor
β-lactamase inhibitors are compounds containing a β-lactam ring, which are poor inhibitors of transpeptidation (clinically ineffective when used alone), but due to the structural relationship, they are capable of irreversibly inhibit the β-lactamase enzymes which inactivate β-lactam antibiotics. β-lactamase inhibition leads to preservation of the other drug (a β-lactam antibiotic) in the combination, which, in turn, remains capable of antibacterial action. Such combinations include amoxicillin+clavulanic acid (Augmentin), ampicillin+sulbactam (Unasyn), and piperacillin+tazobactam (Tazocin). In the USA, ticarcillin+clavulanic acid is also used frequently. Their common drawback is that the β-lactamase inhibitor component cannot penetrate into the cerebrospinal fluid.
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CH OH N 2 N O O O O O S CH3 S N
CH CH3 N N 3 N O COOH O COOH O COOH clavulanic acid sulbactam tazobactam
β-lactamase inhibitors differ in their inhibitory efficacy against different β-lactamase enzymes. TEM- and SHV-type β-lactamases are inhibited more effectively by clavulanic acid and tazobactam than by sulbactam. Tazobactam can inhibit ampC-type lactamases, while the other two inhibitors are much less efficient. All three inhibit staphylococcal penicillinase and chromosomal lactamases of Gram negative strictly anaerobic bacteria (see below a detailed description of different types of β-lactamases). Besides the properties of the inhibitors, inhibition may be pH dependent in case of certain β-lactamases. Rarely the β-lactamase inhibitor may also show antibacterial activity (e.g. the activity sulbactam in Unasyn against Acinetobacter spp .)
Cephalosporins H H N S Their basic structure is the 7-amino-cephalosporanic acid 2 7 (7-ACA). Its semisynthetic derivatives differ in substituents at N 3 O the 3 rd and 7 th carbon atom. All cephalosporins in clinical use are COOH semisynthetic molecules. 7-amino-cephalosporic acid Cephalosporins are classified into generations on the basis of the year of clinical introduction. Different countries may classify cephalosporins slightly differently according to the tradition in the national literature; the classification below is based on the Hungarian tradition. Members of later generations show increased β-lactamase stability and consequently higher activity against Gram negative bacteria, but in parallel their activity decrease against Gram positives. The only exceptions to this rule are the truly broad spectrum 4 th generation and the derivatives not yet licensed (‘5 th generation’). Besides new, yet unlicensed drugs, all cephalosporins are inactive against enterococci and Listeria monocytogenes (these bacteria possess PBPs which cannot bind cephalosporins). They are also uniformly inactive against the majority of clinically important Gram negative strictly anaerobic bacteria ( Bacteroides , Prevotella , Porphyromonas ) due to production of a chromosomal cefalosposinase ( β-lactamase, see below). Some drug groups with strong structural relationship are also classified with cephalosporins such as carbacephems (loracarbef) and oxacephems (moxalactam). These contain carbon or oxygen atoms, respectively, in the six-membered ring of the basic structure instead of the sulphur atom of the cephalosporins proper. These structural differences have small influence on the antibacterial spectrum of the compounds; therefore these related compounds are usually included in the classification based on generations. This summary also follows this practice. Similarly, structurally related cefamycins (containing an extra methoxy group at the 7 th position lacking in cephalosporins proper) are also included into this classification scheme usually. However, as this methoxy group provides excellent stability to β-lactamases (as seen in case of temocillin), spectrum of cefamycins differs from that of cephalosporins proper. For this reason, besides including them in the generational classification, they are discussed in a subsection on their own.
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1st generation (drugs licensed before 1978)
This generation includes cefalothin , H H S N S cefaloridine, cefalexin , cefadroxil and cefazolin . + These drugs can cross the placenta, the cavities lined O N N with serous membranes, but cannot penetrate the O blood-brain barrier. Their activity is confined to cefaloridine COOH Gram positives and certain enterobacteria (their H H spectrum is similar to that of aminopenicillins), but S N S possess weaker activity against Gram positives than O N O CH penicillins. They have weak activity (and are O 3 clinically inefficacious) against Haemophilus spp ., COOH O Moraxella (Branhamella) catarrhalis , Enterobacter cefalothin spp . and Klebsiella spp . They show no efficacy against most non-fermentative Gram negative rods including Pseudomonas aeruginosa, Acinetobacter baumanni , Burkholderia cepacia and Stenotrophomonas maltophilia . Though some differences may exist between activities against different bacterial species, cross-resistance and cross-susceptibility is practically total between members of the 1 st generation. NH 2 H H N S
H H O N N S O CH N N 3 COOH NN S cefalexin O N S CH3 O N NH 2 H H cefazolin COOH NN N S
O N HO O CH 3 cefadroxil COOH 2nd generation (drugs licensed between 1978-1981)
Cefuroxime, cefamandole, cefprozil , O CH N 3 cefaclor , and the carbacephem loracarbef belong H H O N to the 2 nd generation. Two cefamycins, cefoxitin S and cefetamet are also included (see also in the O N O NH2 subsection ‘Cefamycins’). O st These drugs, similarly to the 1 generation, cefuroxime COOH O do not reach therapeutic concentrations in the OH H H cerebrospinal fluid. They are active against all N S CH bacteria included in the spectrum of the former 3 generation, but also have clinically useful activity O N S N O N against Haemophilus spp . Due to increased NN (acquired) β-lactamase activity, they are inactive cefamandole COOH against most Moraxella (Branhamella) catarrhalis and Enterobacter strains. They also remained inactive against non-fermenting Gram negative rods. Cross-resistance and cross-susceptibility is practically total between members of this
94 Antimicrobial chemotherapy: antibacterial agents generation, but slight differences may exist in NH activity against different species (e.g. cefaclor 2 H H has slightly better activity than cefuroxime N S against Streptococcus pneumoniae ). Cefamycins O N HO O CH classified into this generation are the only 3 exceptions to this rule. cefprozil COOH
NH NH 2 H H 2 H H N S N
O N O N O Cl O Cl COOH COOH cefaclor loracarbef
3rd generation (drugs licensed after 1981)
The third generation (the most important members sometimes collectively referred to as oxyimino-cephalosporins) is characterized by a β-lactamase stability superior even to the 2nd generation, which is partially based on their being poor inducers of β-lactamase production. With the broadening Gram negative spectrum, the activity against Gram positives decreased further. These drugs are generally inactive against staphylococci (variable in vitro activity is accompanied with poor clinical efficacy), though, with some notable exceptions, remain active against streptococci (but not against enterococci, see above). Members of the third generation differ considerably in their spectrum and pharmacokinetic properties; the cross-resistance observed in the former two generations does not apply. The Gram negative spectrum of the orally bioavailable cefixim and ceftibuten does not differ significantly from that of the 2 nd generation, but their β-lactamase stability increased further, therefore may remain active against bacteria which are resistant to the 1 st and the 2 nd generation through production of a β-lactamase.
O COOH O COOH N N H H H H N S N S S S N O N CH N O N O 2 O H2N H2N COOH COOH cefixim ceftibuten
The drugs cefotaxime and ceftriaxone penetrate excellently into the cerebrospinal fluid, and are the drugs of first choice for empirical therapy of bacterial meningitis. Besides, they have excellent activity against enterobacteria and some Acinetobacter strains. For this reason these drugs are among the most frequently used antibiotics (and consequently are very frequently overused and misused). They are inactive against Pseudomonas aeruginosa , Stenotrophomonas maltophilia and Burkholderia cepacia . Between the two drugs the cross-resistance and the cross-susceptibility is total. Some less frequently used drugs with similar spectrum are the cefdinir , the cefpodoxime and the ceftizoxime .
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O CH N 3 H H O CH3 N S N S CH H H 3 N S N O N S N S O H N N N 2 O N O CH3 COOH N O ceftriaxone O H2N cefotaxime COOH O O
OH O CH N N 3 H H H H N S N S S S N O N CH N O N O 2 H N O 2 H2N COOH cefdinir ceftizoxime COOH
O CH N 3 H H O CH N S cefpodoxime 3 S N H H N O N N S O S O CH3 H2N + CH3 O CH3 N O N N O H N H C O O O O 2 3 ceftazidime COOH
The member of the 3 rd generation with the most favourable β-lactamase stability is ceftazidime . Furthermore, compared to the spectrum of the cefotaxime-ceftriaxone group its spectrum of activity is broadened with activity against Pseudomonas aeruginosa és Burkholderia cepacia , but is inactive against all Gram positive bacteria including streptococci susceptible to cefotaxime. It is also inactive against Stenotrophomonas maltophilia . There are 3 rd generation cephalosporins with a chemical structure differing from the oxyimino-cephalosporin structure, characteristic to most members of the generation. The narrow-spectrum cefsulodin is active exclusively against non-fermeting Gram O H H negative rods ( Pseudomonas , Acinetobacter ), O N S S NH2 and is inactive both against Gram negative HO + O O N N enterobacteria and against Gram positives. O
Cefoperazone has a spectrum similar to that of cefsulodin COOH cefotaxime, penetrates well into and is excreted through the bile (as opposed to the majority of β-lactams metabolized and OH excreted by the kidneys); cefoperazone is also marketed in combination with lactamase inhibitor (sulbactam) in some O O H H countries. The cefamycin moxalactam O N S N N CH3 is an oxacephem-based structure (its H H C N O N S N spectrum and its chemical structure can 3 O N be found in the section ‘Cefamycins’). COOH NN cefoperazone
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4th generation
This generation includes cefepime and cefpiromee. These have very broad spectrum including both Gram negative and Gram positive bacteria. They are active against all bacteria against which a member of the 3 rd generation is active, they may have better activity against Pseudomonas aeruginosa and Acinetobacter baumannii than ceftazidime, and they have excellent activity against most Gram positive genera. These drugs, however, are also inactive against enterococci, Listeria monocytogenes and cephalosporin-resistant strictly Gram negative anaerobic bacteria. They have weaker activity than ceftazidime against Burkholderia cepacia and are totally inactive against Stenotrophomonas maltophilia .
O CH O CH N 3 N 3 H H H H N S N N S S S + + N O N N N O N N O O H N H2N 2 CH3 cefepime COOH cefpirome COOH
‘5 th generation’
Several molecules in the experimental drug development phase belong to the theoretical 5 th generation, none of these molecules has been licensed, and therefore they are not classified into a generation. Such a compound is ceftobiprole , which binds PBPs more strongly than former β-lactams, it may even bind to alternative PBPs providing resistance to other β-lactams (e.g. PBP2a of MRSA, see below). Consequently they are active against MRSA, Listeria and enterococci (including the β-lactam resistant Enterococcus faecium ). Otherwise their spectrum is similar to that of the 4 th generation, ESBLs hydrolyse, and consequently provides resistance to them, and they remain inactive against Gram negative strictly anaerobic rods as well. They have weak activity against certain non-fermenters including Acinetobacter baumannii , Burkholderia cepacia and Stenotrophomonas maltophilia , most probably due to constitutive production of β-lactamases.
OH N H H N N S S O N O N N O O O N O H2N ceftobiprole COOH O O H C 3
Cefamycins
Cephalosporin derivatives containing an extra methoxy substituent group at the 7 th position are called cefamycins. This structural property, similarly to temocillin, provides excellent β-lactamase stability, therefore they may remain active against bacteria producing β-lactamases, e.g. the chromosomal cephalosporinase of Gram negative strictly anaerobic rods does not always provide resistance to cefamycins. However, they still do not have activity against species resistant to cephalosporins due to PBPs with low affinity to cephalosporins (e.g. in case of enterococci and Listeria monocytogenes ). Cefamycins are frequently classified into one of the cephalosporin generations (see above). Cefamycins include cefoxitin and cefotetan (2 nd generation) and moxalactam (3 rd generation). They are used primarily against anaerobes and ESBL-producing bacteria.
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CH 3 CH3 H O H H O H S N S HOOC S N S CH3 H2N S N O N O NH2 O N S O O O N COOH O COOH NN cefoxitin cefotetan
COOH CH3 H O H N O CH3 O N S N HO O N COOH NN moxalactam
Carbapenems
This group includes imipenem , meropenem , the newer doripenem and the orally bioavailable ertapenem , as well as a drug before being licensed, the orally administrable faropenem . All members are semisynthetic. They have significant postantibiotic effect, in contrast to other β-lactams. They have a broad spectrum, and are resistant to hydrolysis by most β-lactamases, therefore they are the broadest spectrum β-lactams currently available.
OH H H OH H H H3C CH H S 3 N H3C N NH N CH3 S O N COOH N O O H COOH imipenem meropenem
OH H H COOH H3C S H N N O COOH N O H ertapenem
They are active against most Gram positive and Gram negative aerobic and anaerobic bacteria. Exceptions are Stenotrophomonas maltophilia , Clostridium difficile , some Gram positive coryneform rods (e.g. Corynebacterium jeikeium ). They are also inactive, similarly to other β-lactams, against legionellae, chlamydiae, mycoplasmas, rickettsias and bartonellas. Imipenem, but not meropenem, is also inactive against Burkholderia cepacia . Meropenem has better activity against Pseudomonas , but has weaker activity against enterococci, and is applicable in case of meninigitis (in contrast to imipenem), as penetrates well into the cerebrospinal fluid. Ertapenem has weaker activity than imipenem and meropenem against enterococci and non-fermenting Gram negative bacteria ( Pseudomonas , Acinetobacter ); therefore it can be used only against Gram negative enterobacteria. (As the risk of misuse and overuse is higher due to its oral formulation and it favours the development of carbapenem resistant isolates due to its weaker activity, ertapenem should be used with caution.)
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Monobactams
H3C CH3 The only licensed member of this group is the N O COOH semisynthetic aztreonam . It is inactive against Gram N H2N H positive bacteria and against the Bacteroides group, but has N CH S 3 excellent β-lactamase stability, and is consequently active O against most β-lactamase producing Gram negative N O bacteria. aztreonam O S O OH
Mechanism of resistance to β-lactam antibiotics
Resistance due to production of βββ-lactamases
This is the most important mechanism of active compound inactive compound resistance to β-lactams; it is more common in Gram penicillin (CH3)2 S (CH3)2 H 6 S negatives than in Gram 6 R1 N R1 N H2O positives. β-lactamases are H N O N enzymes hydrolysing the COOH O COOH HO H β-lactam bond in the antibiotic, which is crucial for antibacterial ß-lactam bond activity, i.e. they inactivate the to be hydrolyzed antibiotic. A very high number cephalosporin of different β-lactamases has 7 S H S R1 N 7 H O R1 N been described. The following H 2 O N 3 R2 O O N 3 R2 parameters characterize the O HO H β-lactamases. COOH COOH • ß-lactam bond substrate profile (which to be hydrolyzed β-lactam antibiotics can be carbapenem inactivated) R2 R2 • inducibility (whether it is H O produced constitutively, i.e. S R1 2 S R1 always; or produced only N O N O when the substrate, i.e. the COOH HO H COOH β-lactam antibiotic is ß-lactam bond present) to be hydrolyzed monobactam • sensitivity to β-lactamase H inhibitors R1 N R1 N H H O 2 O O N SO H • cellular localization O N 3 O SO H (intracellular or secreted) 3 HO H • the nature of the DNA ß-lactam bond coding for the enzyme to be hydrolyzed (chromosomal, plasmid or transpozon DNA) Substrate profile determines which β-lactam antibiotics may be degraded by the β-lactamase. The substrates are not degraded with equal speed; there are substrates to which the enzyme has high affinity (these are degraded fast even by relatively small amounts of the
99 Antimicrobial chemotherapy: antibacterial agents enzyme) and there are ones to which the enzyme has low affinity and are degraded slowly and inefficiently, necessitating high enzyme concentrations to be degraded. Substrate profile alone does not show the resistance provided, as hydrolysing activity also depends on the amount of enzyme produced. On the other hand, some drugs may be substrates, but do not induce β-lactamase production, therefore, as the β-lactamase is not produced, they are preserved (see below). The enzymes may be produced constitutively, i.e. independently from the presence of substrate; in case of these enzymes substrate profile and enzyme amount determines whether resistance is provided to a certain drug. The enzyme production may be inducible, i.e. the enzyme is not produced until a substrate (drug) with an inducer capacity is encountered by the cell. Sensitivity to β-lactamase inhibitors shows whether the effect of the β-lactamase can be countered by an inhibitor, which inhibitor(s) are effective and how efficient they are (what amount of the enzyme remains active in the presence of a certain amount of inhibitor. This means that bacteria can counter the effect of a β-lactamase inhibitor using two stategies; they may produce an inhibitor resistant β-lactamase or they may hyperproduce the β-lactamase, so that therapeutic inhibitor concentrations could not decrease the amount of active β-lactamase sufficiently, and the β-lactamase activity remains enough for drug degradation. The cellular localization is different in case of Gram positive and Gram negative bacteria; β-lactamases of Gram positives are secreted to the environment, while Gram negative β-lactamases are located in the periplasmic space. Secreted enzymes protect the whole population, and even other bacterial species (therefore it is possible that co-infecting bacteria or bacteria in the normal flora degrade the drug targeting the pathogen). In contrast, periplasmic β-lactamases protect only the cell which produced them. Cellular localization is an inherent characteristic of the enzyme and is not determined by the producing organism. This means that if an enzyme originating from a Gram positive organism is transferred to a Gram negative species, it still preserves the property of being secreted (e.g. BRO enzyme in Moraxella catarrhalis , see below). Substrate profile ultimately determines the range of drug against which it may provide resistance, but other factors play an important role as well. The amount of the enzyme produced is crucial, as some substrates may require higher enzyme concentration to be degraded efficiently. This is even more important in case of secreted β-lactamases, which degrade the drug before it reaches the cell. Such resistance always shows strong inoculum effect. If the β-lactamase produced is inducible, resistance provided is heavily influenced by the inducer capacity of the drug, since the amount of the β-lactamase depends primarily on this capacity. Consequently a poor inducer substrate usually remains effective, as it does not induce production of the β-lactamase in amounts sufficient for drug degradation. (This inducer is not necessarily the same as the substrate to be degraded. E.g. 3 rd generation cephalosporins are generally poor inducers, and therefore remain active against bacteria with inducible β-lactamases, e.g. Enterobacter spp ., but this activity is lost if another drug, e.g. ampicillin, induces β-lactamase production.) A considerable number of β-lactamases is known, and large differences exist in substrate specificity and in other properties, necessitating classification. Two approaches exist; Ambler’s system is based on genetic relatedness of β-lactamase genes and classifies β-lactamases into four groups (A, B, C and D). Groups A, C and D include enzymes with a serin crucial for activity in the active centre, while group B contains the metallo-β-lactamases, which have a zinc atom at the active centre and a different hydrolytic mechanism. Ambler group A contain enzymes with penicillinase activity, regardless of which other drug types may also be degraded;
100 Antimicrobial chemotherapy: antibacterial agents group C includes enzymes with cephalosporinase activity, while enzymes with capacity to degrade oxacillin belong to group D. This phylogenetic grouping is not based on properties important from practical point of view; there are significant differences within the groups regarding the range of antibiotics against which resistance is provided. The other classification is functional, based primarily on substrate specificity and inhibitor resistance. The presently accepted functional classification is the Bush-Jacoby-Medeiros scheme. This has three large groups (1 to 3), but group 2 has numerous subgroups (2a to 2f). The scheme is presented in the table below.
Bush- Jacoby- Ambler substrate(s) inhibitor resistance example Medeiros group group clavulanic acid EDTA enzyme bacterium 1 C cephalosporins - - AmpC Enterobacter 2a A penicillins + - lactamases of Staphylococcus Gram positives 2b A penicillins, 1st gen. + - TEM1, SHV1 E. coli cephalosporins 2be A penicillins, + - TEM and SHV E. coli , Klebsiella cephalosporins , derived ESBLs, monobactams CTX-M ESBLs 2br A penicillins ± - inhibitor-resistant E. coli , Klebsiella TEM 2c A penicillins + - PSE P. aeruginosa 2d D penicillins, oxacillin ± - OXA P. aeruginosa 2e A cephalosporins + - CUM Proteus vulgaris 2f A penicillins, + - IMI, Enterobacter , cephalosporins, MNC-A, Serratia carbapenems SME-1 3 B almost all - + L1 Stenotrophomonas β-lactams maltophilia
Besides the examples mentioned in the table, a number of other β-lactamases exist. The clinically most important enzymes are presented in the following section.
Important β-lactamases by functional groups
1. AmpC enzymes (Bush-Jacoby-Medeiros group 1). These are enzymes originally encoded chromosomally, with predominantly cephalosporinase activity. They are resistant to β-lactamase inhibitors (excepting tazobactam). Their interesting, but clinically not useful property is that cloxacillin functions as an inhibitor. Inducible AmpC enzymes are poorly induced by cephalosporins, consequently in spite of their potent cephalosporinase activity, they do not always provide resistance against cephalosporins, but if cephalosporins are administered together with good inducer antibiotics (e.g. aminopenicillins), they are capable of inactivating cephalosporins as well. Most enterobacteria produce a certain amount of chromosomal AmpC enzyme, with the exception of salmonellae, klebsiellae and Proteus mirabilis . In case of E. coli and Shigella spp . the AmpC β-lactamase is produced in extremely small amounts and is not inducible, therefore these species are originally susceptible to aminopenicillins, but the proportion of aminopenicillin resistant strains is increasing. This secondary resistance is mediated by other β-lactamases (e.g. TEM, see below). Isolates belonging to the genera Enterobacter , Citrobacter (excepting most C. diversus strains), Serratia , Providencia and Proteus (excepting P. mirabilis ) produce an inducible variety of AmpC. These strains possess natural aminopenicillin resistance. Due to production of high amount of the enzyme, most Enterobacter spp . is naturally resistant to aminopenicillins protected with β-lactamase inhibitor, in addition to 1 st and sometimes even to 2 nd generation cephalosporins as well. A small minority of Enterobacter and Citrobacter freundii strains are characterized by constitutive AmpC production and natural resistance similar to that of E. coli . Using poor inducer substrate β-lactams (most characteristically 3 rd generation cephalosporins), may
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provoke the development of derepressed mutants, most frequently among Enterobacter spp . and, rarely, in other species producing chromosomal AmpC β-lactamases. Derepression means that the normally inducible enzyme loses inducibility and is produced permanently (as if the presence of substrate had ceased repression and had induced enzyme production; mimicking permanent presence of inducer substrates). These strains are resistant to amino-, carboxy- and ureidopenicillins and to their β-lactamase-inhibitor protected derivatives, excepting piperacillin+tazobactam, as well as to 1 st , 2 nd and 3 rd generation cephalosporins including cefamycins, but fourth generation cephalosporins and carbapenems preserve their activity. Pseudomonas aeruginosa and Acinetobacter baumannii also produce inducible AmpC enzymes, leading to their intrinsic resistance to aminopenicillins and their β-lactamase-protected derivatives, and to 1 st and 2 nd generation cephalosporins. In case of Pseudomonas aeruginosa , this enzyme has a clinically irrelevant but detectable imipenemase activity, which does not lead to imipenem resistance nor does it act on other carbapenems. Derepressed mutants may also develop in case of Pseudomonas aeruginosa , the amount of the enzyme produced vary widely, leading to major differences in β-lactam susceptibility. Even partial derepression (low enzyme amounts) provides ureidopenicillin resistance, while ceftazidime, carboxypenicillin or 4 th generation cephalosporin resistance requires total derepression (high amounts of the enzyme). Chromosomal AmpC genes were translocated to plasmids (coding for enzymes MIR, FOX, MOL, LAT, MOX, ACT and CMY, etc.). These genes are usually expressed constitutively (not in an inducible manner like most chromosomal AmpC-type genes), providing a resistance pattern similar to that of the permanently derepressed mutants; strains carrying plasmid-borne AmpC enzymes are resistant to all penicillins excepting sometimes piperacillin+tazobactam (MIC of piperacillin+tazobactam are higher than in wild-type strains, but in the upper range of the susceptible category), to all 1 st , 2 nd and 3 rd cephalosporins including cefamycins. Most enzymes hydrolyse and inactivate aztreonam as well. Susceptibility to 4 th generation cephalosporins and to carbapenems is preserved. 2. Staphylococcal penicillinase (Bush-Jacoby-Medeiros group 2a) Extracellular enzymes (with strong inoculum effect) encoded on plasmids, they are inhibitor sensitive. These enzymes inactivate all penicillin derivatives with the exception of penicillinase-stable penicillins (i.e. early penicillins, amino-, carboxy- and ureidopenicillins), but cannot hydrolyse cephalosporins or carbapenems. Hyperproduction of the enzyme leads to borderline methicillin resistance. This resistance type, in contrast to real methicillin resistance, do not lead to cross-resistance to all β-lactams; these strains are resistant to all penicillins including penicillinase-stable penicillins, but remains susceptible to cephalosporins, carbapenems and β-lactamase inhibitor combinations. (Even more rarely this borderline methicillin resistance is mediated by a methicillin-hydrolysing β-lactamase.) Borderline methicillin resistance is hard to detect in the laboratory and its clinical importance is also largely unknown. 3. ROB and BRO enzymes (Bush-Jacoby-Medeiros group 2b) Plasmid-borne, extracellular, inhibitor sensitive enzymes. The enzyme ROB is characteristic to Haemophilus spp ., while BRO is produced by Moraxella (Branhamella) catarrhalis . Both enzymes originate from an unknown Gram positive organism. They provide resistance to aminopenicillins and 1 st and 2nd generation cephalosporins. 4. TEM and SHV enzymes (Bush-Jacoby-Medeiros groups 2b, 2be and 2br) The two gene families code for genetically different, but functionally similar enzymes. Gene family TEM include at least 133, while gene family SHV at least 54 different enzymes. All are plasmid-borne, constituvely produced enzymes. They are generally inhibitor sensitive, but sulbactam is a much weaker inhibitor than clavulanic acid or tazobactam. Both enzyme families are most probably derivatives of chromosomal β-lactamases of Klebsiella spp . translocated to plasmid. The most frequently found varieties (TEM1, TEM2, SHV1) provide a certain level of resistance to all penicillin derivatives excepting temocillin as well as to 1 st and 2 nd generation cephalosporins (Bush-Jacoby-Medeiros groups 2b). The resistance pattern depends on the amount of enzyme produced. Even extremely low enzyme amounts lead to aminopenicillin, ureidopenicillin and 1 st generation cephalosporin resistance, though in case of the latter two groups the isolates may appear falsely susceptible in vitro . Susceptibility to β-lactamase inhibitor combinations and to 2 nd generation cephalosporins depends highly upon enzyme amounts; resistance to piperacillin+tazobactam requires very high amounts of enzyme, which is rarely encountered. The enzymes are found in many Gram negative genera, are primarily characteristic to enterobacteria, but have also been described in P. aeruginosa . Aminopenicillin resistance of Haemophilus spp . and Neisseria gonorrhoeae is also frequently mediated by these enzymes. Both gene families contain inhibitor resistant mutants (Bush-Jacoby-Medeiros groups 2br), but these provide resistance only to aminopenicillins, 1 st and 2 nd generation cephalosporins and their combinations with β-lactamase inhibitors.
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The clinically most important TEM and SHV mutants are the TEM- and SHV-type extended spectrum β-lactamases (ESBLs). (Naturally, ESBLs not belonging to TEM and SHV families also exist, see below.) These enzymes hydrolize all β-lactams excepting carbapenems and cefamycins (Bush-Jacoby-Medeiros groups 2be). In spite of their sensitivity to β-lactamase inhibitors, due to the high amounts of enzyme produced these are also inactive. As ESBLs are inducible enzymes, their in vitro efficiency heavily depends on the inducer ability of the substrate to be inactivated, thus in many cases not all drugs appear inactive, frequently only the MIC of the excellent inducer ceftazidime is increased significantly. However, in vivo only carbapenems and cefamycins remain reliably active, therefore ESBL production must be demonstrated, and positive isolates should be regarded as resistant to all other β-lactams, regardless of the in vitro susceptibility results. (Piperacillin+tazobactam and 4 th generation cephalosporins may retain some activity, but in vitro assessment of this activity is totally unreliable, therefore the safety of the patients demand that these should also be regarded as inactive and excluded from therapeutic choice.) ESBL production is primarily detected in nosocomial Klebsiella pneumoniae and less frequently in nosocomial E. coli , but infrequently may be found in most Gram negative nosocomial pathogens ( Citrobacter , Serratia , Enterobacter , P. aeruginosa , etc .) 5. K1, KOX enzymes (Bush-Jacoby-Medeiros group 2be) These enzymes are characteristic to Klebsiella spp . They are chromosomally coded, and are genetically similar to the plasmid-borne SHV enzymes; these enzymes, being translocated to plasmids, are the hypothetical ancestors of SHV enzymes (see above). They provide resistance to amino-, carboxy- and ureidopenicillins and to 1 st generation cephalosporins, though MICs of drugs in the last two groups may remain in the upper range of the susceptible category. However, these drugs are clinically ineffective with the exception of urinary tract infections where extremely high drug levels can be achieved. Hyperproduction of these enzymes leads to resistance to all penicillins except temocillin (including β-lactamase-protected formulations and to all 1 st , 2 nd and 3 rd generation cephalosporins except ceftazidime (which is not a substrate of these enzymes). Hyperproducer strains still preserve their susceptibility to temocillin, cefamycins, ceftazidim, 4 th generation cephalosporins, monobactams and carbapenems. 6. CTX-M enzymes (Bush -Jacoby-Medeiros group 2be) These plasmid-borne ESBL enzymes were recently discovered, but became very widespread among Enterobacteriaceae . They originate most probably from a chromosomal β-lactamase of a member of the Gram negative genus Kluyvera . They are sensitive to β-lactamase inhibitors; tazobactam is a significantly more efficient inhibitor than sulbactam or clavulanic acid. However, the levels of the enzyme produced are practically always enough for conferring resistance to inhibitor-protected drug formulations as well including piperacillin+tazobactam. Thus, the phenotype provided is an ESBL phenotype highly similar to that conferred by TEM and SHV ESBLs, excepting that cefotaxim is a better substrate than ceftazidime and 4th generation cephalosporins are inactivated more readily than in case of TEM and SHV ESBLs. Susceptibility to carbapenems and cefamycins is retained. These enzymes are characteristic to Enterobacteriaceae , and, importantly, they also appeared in Salmonella Typhimurium. 7. Other ESBL-type enzymes (Bush-Jacoby-Medeiros group 2be and 2f) These are rare plasmid-borne enzymes, which provide an ESBL phenotype. The enzymes belonging here are the ones frequent in Turkey (PER-1), South-America (PER-2) and the Far East (VEB-1), but very rare in other regions (these are related to the chromosomal cephalosporinase of Gram negative strict anaerobes) and some genetically similar enzymes (TLA-1, CME-1) together with some unrelated and also extremely rarely found Serratia - (SFO-1) and P. aeruginosa -derived (GES-1) enzymes. 8. PSE enzymes (Bush-Jacoby-Medeiros group 2c) These are the chromosomal β-lactamases of P. aeruginosa . They are inhibitor sensitive and they inactivate amino- and carboxypenicillins. 9. OXA enzymes (Bush-Jacoby-Medeiros group 2d) These are plasmid-borne β-lactamases characteristic to P. aeruginosa and A. baumannii. They are sensitive to β-lactamase inhibitors, but clavulanic acid and sulbactam are weak inhibitors, their combinations are not more active than the β-lactam component alone, but piperacillin+tazobactam is more active than piperacillin alone. Their (clinically irrelevant) characteristic is that they are capable of hydrolysing oxacillin, which is resistant to hydrolytic activity to other β-lactamases. Their original varieties provide aminopenicillin and low level 1 st generation cephalosporin resistance, but a high number (at least 57) of mutant enzymes are known. These include OXA-type ESBLs, conferring a resistance phenotype similar to that provided by other ESBLs, but have better hydrolysing activity against 4 th generation cephalosporins. OXA-type carbapenemases are broad spectrum enzymes with the ability to inactivate carbapenems; these may hydrolyse and inactivate practically all β-lactam drugs.
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10. cepA and cfxA (Bush-Jacoby-Medeiros group 2e) These are chromosomally coded, or exceptionally plasmid-borne, constituvely produced inhibitor-sensitive enzymes, mostly with cephalosporinase activity. They are found characteristically in Gram negative strictly anaerobic bacteria, primarily in species of the Bacteroides fragilis group, but similar enzymes have also been described in Prevotella and Porphyromonas species as well. The enzyme cepA provides resistance against all cephalosporins excepting cefamycins even when produced in low amounts; higher enzyme levels confer resistance to unprotected penicillins as well. The enzyme cfxA provides resistance to both cephalosporins and penicillins, together with a low level cefamycin resistance. Neither enzyme influences the efficacy of inhibitor combinations and carbapenems. 11. CUM (cefuroximases of P. vulgaris and P. penneri ) (Bush-Jacoby-Medeiros group 2e) Inducible, inhibitor sensitive chromosomal enzymes, hydrolysing amino-, carboxy- and ureidopenicillins and 1 st , 2 nd and 3 rd generation cephalosporins. As ureidopenicillins and 3 rd generation cephalosporins are poor inducers, therefore they remain active. These strains also remain susceptible to cefamycins, carbapenems, monobactams and β-lactam+ β-lactamase inhibitor combinations. Similar enzymes are produced by the majority of Citrobacter diversus strains. Extremely rarely hyperproducers are encountered, these lose their susceptibility to the poor inducer drugs (ureidopenicillins and 3 rd generation cephalosporins) and remain susceptible only to cefamycins, carbapenems, monobactams and β-lactam+lactamase inhibitor combinations. 12. PenA (penicillinase of Burkholderia cepacia ) (Bush-Jacoby-Medeiros group 2e) Chromosomally coded, inducible enzyme. It has a substrate specificity similar to that of the CUM enzyme, hydrolyses amino- and carboxypenicillins and 1 st and 2 nd generation cephalosporins; it is inhibitor sensitive. 13. L2 enzyme of Stenotrophomonas maltophilia (Bush-Jacoby-Medeiros group 2e) Chromosomal, inhibitor sensitive, inducible enzyme, the inducer mechanism is shared by the chromosomal metallo-β-lactamase (L1, see below) of the species, inducers of either enzyme induces the production of both β-lactamases, i.e. these two enzymes are almost always present and determine the susceptibility profile together. The enzyme L2 alone provides resistance to cephalosporins and monobactams, but together with the L1 enzyme results in resistance to all β-lactams. 14. Weak carbapenemases of Enterobacter and Serratia spp . (Bush-Jacoby-Medeiros group 2f) Very rare, chromosomal, inhibitor sensitive enzymes with a weak carbapenemase activity (NMC-A, IMI, SME-1). Besides carbapenems, they inactivate penicillin derivatives and monobactams. They confer only low level carbapenem resistance. 15. Carbapenemase ESBLs (Bush-Jacoby-Medeiros group 2f) Rare but emerging enzymes, coded for by plasmids or integrons (KPC, GES-2). They are inhibitor sensitive. They inactivate practically all β-lactams, piperacillin+tazobactam may possibly remain active in vitro , but its in vivo efficacy is questionable. They are found primarily in Klebsiella and Enterobacter strains (KPC) and Pseudomonas aeruginosa (GES-2), but may occur in any species belonging to the Enterobacteriaceae family. Recently KPC is emerging in Europe, causing panresistance in Klebsiella pneumoniae . 16. Metallo-β-lactamases (Bush-Jacoby-Medeiros group 3) Metallo-β-lactamses hydrolyse β-lactams with a unique mechanism differing from the mechanism of other β-lactamases. They need zinc for activity. They are not inhibited by β-lactamase inhibitors, but efficiently inhibited by EDTA. They have different substrate specificity, some enzymes hydrolyse carbapenems only, e.g. CphA enzyme of Aeromonas spp .; others hydrolyse all β-lactams excepting monobactams, e.g. L1 chromosomal enzyme of Stenotrophomonas maltophilia , CcrA (or cfiA) enzyme of Bacteroides fragilis , as well as plasmid- or integron-borne enzymes (VIM and IMP) found mostly in P. aeruginosa and A. baumannii . Enzymes encoded on plasmids and integrons cause problems mainly in Japan and in other Asian countries. In Europe these are emerging, but problems also arise from chromosomal metallo-β-lactamases, mainly that of S. maltophilia . This enzyme shares the inducer mechanism with the other chromosomal enzyme of Stenotrophomonas (see above), and hydrolyses all β-lactams except monobactams. Together with monobactamase activity of the other, concertedly produced L2 enzyme, confer resistance to all β-lactam antibiotics. (Hypothetically, aztreonam+ β-lactamase inhibitor combinations remain active, as L1 does not hydrolyse aztreonam and L2 is inhibited by the inhibitor, but this combination is not in use.) Some susceptibility standard systems (e.g. CLSI) consider some β-lactams (e.g. piperacillin+tazobactam or ceftazidime) possibly effective (explaning in vitro susceptibility to these drugs with isolates producing low levels of one or both enzymes), but as all β-lactams are targets for L1 or L2, clinical efficacy of any β-lactams is questionable, as stated by e.g. the European standard (EUCAST).
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Phylogenetic classification of the clinically most important β-lactamases
β-lactamases can be classified into four large phylogenetic groups (Ambler groups, see above). The first group (Ambler group A) include the majority of β-lactamases, i.e. all members of the 2 nd functional group excepting OXA enzymes (K1, TEM, SHV, CTX-M, PSE, CUM, L2 enzymes, staphylococcal penicillinase, chromosomal weak carbapenemases of enterobacteria and chromosomal cephalosporinases of strict anaerobes), the second (Ambler group B) consist of the metallo-β-lactamases, the third (Ambler group C) comprises the AmpC type enzymes, while the fourth (Ambler group D) the OXA-type β-lactamases and the enzyme PenA.
Clinically important β-lactamases by groups of bacteria
1. β-lactamases of Gram positive bacteria These β-lactamases are secreted into the environment and can exert their activity before the contact of the drug and the cell. The most important of the Gram positive-derived β-lactamases is the staphylococcal penicillinase. Out of the other Gram positive species Bacillus spp . frequently produce chromosomal β-lactamases providing intrinsic resistance to early- and aminopenicillins, but B. anthracis remain susceptible to penicillin and to other β-lactams. In acid-fast rods (mycobacteria and Nocardia spp .) β-lactamase production also occurs frequently. Some opportunistic Clostridia also have chromosomal β-lactamases. In Gram positive genera apart from the abovementioned, resistance to β-lactams is not mediated by production of β-lactamases, though acquisition of staphylococcal penicillinase was observed in enterococci in rare cases. Resistance is due to modification of PBPs or alternative cell wall synthesis (production of a new PBP). 2. β-lactamases of Gram positive rods This group include many enzymes of different origin and activity. These act in the periplasmic space of the cell; they inactivate only drug molecules that have entered the cell. They may be encoded by the chromosome, by plasmids, transpozons or integrons. Transmission of β-lactamase genes is common; nosocomial, and frequently interspecies, gene transfer cause serious problems. β-lactamase production is the most important mechanism of resistance to β-lactam drugs in Gram negative rods. Due to diversity of β-lactamases and the different phenotypes conferred in different species or in different in vitro circumstances (depending on differences in induction efficiency and the levels of enzyme production) by the same β-lactamase gene, it is difficult to determine which particular gene is in the background of a given phenotype. Producing more than one β-lactamase and possessing other mechanism of resistance to β-lactams simultaneously also encountered frequently and complicate the picture further. a. Chromosomal β-lactamases These are primarily found in Enterobacteriaceae and in non-fermenters; being encoded on the chromosome, they lead to natural resistance, which is not easily transmitted to other species. • AmpC enzymes ( Enterobacter , Serratia , Citrobacter , Providencia , Proteus morganii , Pseudomonas aeruginosa ) • K1 and KOX enzymes ( Klebsiella ) • PSE enzymes ( P. aeruginosa ) • L1 and L2 enzymes ( Stenotrophomonas maltophilia ) • CUM ( Proteus vulgaris ) • cepA ( Bacteroides fragilis ) • PenA ( Burkholderia cepacia ) • CcrA ( B. fragilis ) • Different chromosomal carbapenemases ( Enterobacter , Serratia ) b. Plasmid- or integron-borne β-lactamases Widely distributed, easily acquired enzymes, giving rise to serious clinical problems. • TEM and SHV enzymes ( E. coli , Klebsiella , Haemophilus , Neisseria , Branhamella , numerous other Gram negative genera) • TEM and SHV ESBL ( E. coli , Klebsiella , less frequently other enterobacteria) • CTX-M ESBL ( E. coli , Klebsiella , less frequently other enterobacteria) • Other ESBLs (different enterobacteria) • OXA enzymes ( P. aeruginosa , A. baumannii) • OXA ESBL ( P. aeruginosa , A. baumannii) • Carbapenemase ESBL ( Klebsiella , Enterobacter , other enterobacteria) • IMP, VIM metallo-β-lactamases ( P. aeruginosa , A. baumannii)
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3. β-lactamases of Gram negative cocci and coccobacilli Plasmid-borne TEM-1 enzymes were demonstrated in case of both Haemophilus spp . and Neisseria gonorrhoeae (but rarely in case of N. meningitidis ). These confer resistance to early penicillins, aminopenicillins and 1 st generation cephalosporins, but the level of resistance is significantly lower than in case of enterobacteria, as the permeability of the cells is higher to the drugs and lower enzyme levels are produced. These isolates remain susceptible to 2 nd and higher generation cephalosporins, to inhibitor-protected penicillins, to monobactams and to carbapenems. In case of Haemophilus spp . an enzyme ROB-1 (Ambler group A, functional group 2b; possibly of Gram positive origin) may also play a role in resistance. This enzyme is frequently found in North-America, but rare in other regions. It produces a resistance pattern similar to that of TEM-1, but, in contrast, it inactivates 2nd generation cephalosporins as well. More than 90% of Moraxella ( Branhamella ) catarrhalis strains produce a BRO-type enzyme (Ambler group A, functional group 2b), which is plasmid-borne and inhibitor sensitive. This enzyme was shown to originate from a Gram positive bacterium; it is secreted into the environment. They confer resistance to amino- and carboxypenicillins and to 1 st and 2 nd generation cephalosporins. Inhibitor-protected combinations remain active. In this group production of β-lactamases should be demonstrated not by traditional susceptibility testing, but by direct demonstration of the β-lactamase activity. This may be achieved by means of a chromogenic test, e.g. using nitrocefin (see above). 4. β-lactamases of Gram negative strictly anaerobic bacteria The majority of Bacteroides spp . produces a chromosomally coded β-lactamase with primarily a cephalosporinase activity (cepA), which is capable of inactivation of all cephalosporins except cefamycins, and confer low level resistance to penicillin and its derivatives as well. Inhibitor-protected drugs, cefamycins and carbapenems retain their activity. The most susceptible species is B. fragilis , other species, particularly B. thetaiotaomicron and B. vulgatus produce higher enzyme levels, and thus may become resistant even to β-lactamase inhibitor combinations. By secondary acquisition of different insertion sequences enhancing gene expression, cepA may be produced in higher amounts by all species, leading to resistance similar to that of B. thetaiotaomicron and B. vulgatus , i.e. resistance to all cephalosporins and unprotected penicillins with the possibility of resistance also to inhibitor-protected combinations. Cefamycins and carbapenems invariably remain active. Inhibitor resistant cepA varieties have also been found. The enzyme family cfxA provides resistance similar to high level cepA production; these enzymes were described from Bacteroides , Prevotella and Porphyromonas spp . Unlike cepA, these enzymes also confer low level cefamycin resisance. Rarely, Bacteroides spp . may produce a (zinc-dependent, inhibitor resistant) metallo-β-lactamase (ccrA) capable of degrading all β-lactams. These isolates are resistant to all β-lactam derivatives. Fusobacteria produce mainly penicillinase-type enzymes (Ambler group D, functional group 2d) similarly to β-lactamase producing clostridia ( C. butyricum , C. clostridioforme ). These strains are resistant to all penicillins except inhibitor combinations; but retain their cephalosporin and carbapenem susceptibility. Other strict anaerobes do not produce β-lactamases.
Resistance conferred by alteration of PBPs
The mechanism of the alteration of PBPs is usually transformation. The transforming DNA undergoes recombination with one of the PBP genes yielding a mosaic gene structure, which, in turn, results in production of a modified PBP. If this mosaic protein is capable of performing its function and at the same time binds β-lactams with lower affinity, a certain level of resistance to β-lactams is developed. In case of bacteria not producing β-lactamases, this is the main mechanism of β-lactam resistance. This mechanism is the basis of penicillin resistance of Streptococcus pneumoniae and other α-haemolytic streptococci, where the extent of alteration determines the resistance pattern. First (in case of smaller alterations) susceptibility to early penicillins is impaired, followed by decreased susceptibility to aminopenicillins, then 3rd generation cephalosporins. Rarely carbapenem resistance may also develop. Even small decreases in susceptibility to early penicillins (increasing of the penicillin MIC to the intermediate category) lengthen the
106 Antimicrobial chemotherapy: antibacterial agents treatment duration necessary for eradication significantly, and mortality rates are higher in case of invasive infections. This mechanism is in the background of some cases or borderline methicillin resistance in case of staphylococci (it may also be derived from hyperproduction of the staphylococcal penicillinase, see above). This mechanism of aminopenicillin resistance is recently emerging in Haemophilus influenzae ; these strains, unlikely β-lactamase producers, are resistant not only to aminopenicillins but, naturally, to their inhibitor-protected varieties, and 2 nd generation cephalosporins as well. Amoxicillin resistance of Helicobacter pylori and Campylobacter spp . is also conferred by such a mechanism. This mechanism may also be found rarely in case of Clostridium perfringens , Neisseria spp. , Pseudomonas aeruginosa , Acinetobacter spp. and Bacteroides spp.
Resistance caused by alternative cell wall synthesis
This mechanism is based on production of a totally novel PBP in addition to the normal original PBPs. Though cell wall synthesis is a concerted action of several different PBPs with slightly different functions, this novel PBP can alone sufficiently perform the cell wall synthesis. Resistance is caused by its very low affinity to β-lactam antibiotics; therefore the drugs cannot inhibit its function. This mechanism provides resistance invariably to all marketed β-lactam antibiotics. This mechanism is in the background of classical methicillin resistance of staphylococci (a novel PBP, PBP2a is produced). As PBP2a is less efficient than the normal PBP arsenal of the bacteria, strains utilizing PBP2a grow slower than wild-type strains. For this reason, if the drug is absent, only a small minority of cell in a population expresses the resistant phenotype at a certain time (heteroresistance), necessitating selection for these cells in the course of in vitro susceptibility testing (as these will be selected in vivo during β-lactam therapy). These isolates frequently possess a multiresistant phenotype, e.g. almost invariably show co-resistance to macrolides and lincosamides. The same mechanism explains the intrinsic resistance of almost all Enterococcus faecium strains to β-lactam antibiotics (a PBP5 is produced). This confers relatively low level resistance to all β-lactams, but it is frequently transformed to high level resistance by different secondary changes.
Resistance based on decreased permeability (slower drug uptake)
Generally it produces a low level, clinically less important resistance, but is frequently coupled with other resistance mechanisms (production of β-lactamases, active efflux) and contributes to high level resistance. Sometimes decreased permeability provides clinically important resistance alone, e.g. this is the mechanism of the sole carbapenem resistance of some Pseudomonas aeruginosa and Enterobacter strains or of the cefoxitin resistance of ESBL-producing Klebsiella pneumoniae .
Efflux-based resistance
This mechanism usually provides low or intermediate level of resistance. It is frequently associated with decreased permeability, some efflux pumps exert their activity connected with a porin protein (see above). The resistance provided is not always confined to β-lactams, efflux-based resistance (even the same pump system) may lead to cross-resistance to different other drug families as well, moreover, may also be involved in resistance to disinfectants, possibly leading to cross-resistance between antibiotics and biocides. This mechanism is most frequently found in nonfermenters ( Pseudomonas aeruginosa , Acinetobacter baumannii ).
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Glycopeptides
These glycosilated CH oligopeptides exert their bactericidal 3 OH activity by inhibiting cell wall O OH synthesis; they bind to the D-Ala - NH HO O 2 D-Ala end of the pentapeptide CH3 involved in transpeptidation, leading to sterical hindrance of the process. HO O O Cl This cidal activity is slower than that O O β of -lactams. Aminoglycosides HO OH Cl synergistically enhance their efficacy. O O They do not penetrate well to the H H O N N cerebrospinal fluid and to the bile. N N NH H H H Vancomycin and teicoplanin HN O O N CH O NH O 3 belongs to this group; the latter is a HOOC 2 H C mixture of compounds differing in 3 the monosaccharides bound to the OH vancomycin CH3 peptide backbone. Teicoplanin A2 is HO OH the most important component, which contains an 8-10 carbon atom lipoic acid (marked with R in the figure) bound to one of the monosaccharide side chains via an amide bond. Teicoplanin A1 do not contain a carbohydrate component, and teicoplanin A3, also present in significant amount in the antibiotic, contain less monosaccharide substituents and lacks the lipid side chain as well. Their spectrum is narrow, they are active solely on Gram positive bacteria; they are only bacteriostatic against OH enterococci. All Gram negative HO NHR bacteria possess natural resistance, HO HO though a small minority of HO O O Cl Neisseria gonorrhoeae strains is O O O susceptible in vitro . Glycopeptides HO are inactive against obligate O HN Cl intracellular pathogens. There are O O O H H some intrinsically resistant strains O N N H3C N N NH (in genera Leuconostoc , H H HN O O NH Lactobacillus , Erysipelothrix , O 2 Nocardia ) among the Gram HOOC positives as well. These Gram HO O OH positives contain a pentapeptide HO O ending in D-Ala - D-lactate instead OH teicoplanin A2 OH of D-Ala - D-Ala, which binds O glycopeptides less efficiently, OH leading to their inactivity. (The OH OH alternative pentapeptide provides normal cell wall synthesis.)
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Mechanisms of glycopeptide resistance
1. Target modification This mechanism is based on modification of the pentapeptide involved in the transpeptidation by changing the terminal peptide to D-lactate or to D-serine instead of the normal D-alanine. This mechanism of resistance is very similar in biochemistry to the mechanism of natural resistance of Gram positives, but genetically distinct. The genetic element providing resistance codes for three enzymes and their regulatory genes. One of the enzymes is responsible for synthesis of the low-affinity precursor, another incorporate this precursor into the pentapeptide, and the third eliminates the normal, high-affinity precursor. Sufficient activity of all three enzymes is necessary for the resistance phenotype. Several genetically unrelated types has been described, these differ in the level of the resistance provided, in the affected drugs as well as in inducibility and in the genetic element on which they are encoded.
Binding of vancomycin to the normal (D-Ala - D- Ala) pentapeptide. The arrow points to the hydrogen bond critical for binding.
CH2OH HO O HO H C H C3 3 O O O Cl HO O O H3C HO H CH3 Cl + H H O OH N O NH2 H O N N H H N CH N H O H 3 H N H O H H O H O H
CH3 O O NH2
OH HO OH
O H H O CH N 3 R N O CH3 H O H
Lack of -NH- group critical for hydrogen bond formation prevents Sterical inhibition by the larger side chain of D-Ser leads to binding of vancomycin to pentapeptides with D-Ala - D-lactate end. weakness of the hydrogen bond critical for vancomycin binding.
CH OH CH2OH 2 HO O HO O HO HO H C H3C H C3 H C O O Cl 3 O O O Cl 3 O HO HO O O O O H C H3C 3 H HO H HO CH CH 3 Cl 3 Cl + H + H H O OH H O OH NH N O NH2 H N O 2 H O N O N N N H H N H H N CH N H CH N H O H 3 O H 3 H N H O H H H N H O H H O H O H O H O H CH C CH O 3 O 3 O O NH NH2 2 HO OH OH HO OH HO OH CH2 O H O O H H O CH O 3 N R N O R N O CH3 CH H O H H 3 O H
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1. vanA type Inducible mechanism encoded on plasmid or transpozon. The three enzymes necessary for resistance are D-Ala - D-Ala peptidase (cleaving the dipeptide conferring glycopeptide susceptibility), D-2-ketoacid-reductase (synthesizing D-lactate) and D-Ala - D-lactate-ligase (forming the dipeptide conferring resistance). This mechanism provides high-level resistance to both vancomycin and teicoplanin. It was found in Enterococcus faecalis and E. faecium , in staphylococci and, very rarely, in other Gram positive genera. 2. vanB type Inducible mechanism encoded on plasmid or transpozon. Biochemical mechanism is identical to that of vanA, but vanB is genetically distinct. It was described in Enterococcus faecalis and E. faecium . Provides extremely high level of resistance to vancomycin. As teicoplanin is a poor inducer, the mechanism does not provide teicoplanin resistance, but prior vancomycin exposure may render it also ineffective. According to newer results, the normal anaerobic flora ( Clostridium and other genera) in the gut may serve as a reservoir for vanB-type resistance. 3. vanC type Chromosomally coded mechanism responsible for natural resistance of Enterococcus gallinarum and E. casseliflavus . Provides constitutive low level resistance (MIC values around the breakpoint signifying the susceptible category). Contrasting vanA and vanB types, D-serine is incorporated into the altered pentapeptide instead of D-lactate, and the enzyme activities necessary for resistance differ accordingly. Isolates are resistant to vancomycin only, teicoplanin retains activity. 4. vanD type Rare, chromosomally coded mechanism, the biochemical background is similar to that of vanA and vanB. Provides medium level of resistance, vancomycin is inactive, but teicoplanin may remain efficacious. 5. vanE and vanG types Chromosomally coded, inducible, rare mechanisms with a biochemical background similar to vanC. Type vanG is transmissible, while vanE is not. Both provide low level resistance; vancomycin susceptibility is decreased, teicoplanin activity is spared.
There are glycopeptide dependent Enterococcus strains. In these strains the normal cell wall synthesis is damaged and they cannot synthesize the normal D-Ala - D-Ala dipeptide, but contain resistance determinants of vanA or vanB type. These allow for synthesis of D-Ala - D-lactate dipeptide, but as both vanA- and vanB-type resistance are inducible, this replacement dipeptide synthesis is expressed and works only in the presence of glycopeptides. Without glycopeptides, the strain loses its ability to synthesize cell wall and becomes nonviable.
2. Modification of the cell wall synthesis This mechanism involves a more rapid cell wall synthesis, leading to thicker cell wall. This results in higher number of glycopeptide binding sites, consequently, the drugs are sequestered in the cell wall and significantly more glycopeptide molecules are required to block all pentapeptide sites. This mechanism provides low level resistance to all glycopeptides, and also decreases daptomycin susceptibility. It was described only in case of methicillin resistant MRSA strains with intermediate susceptibility to glycopeptides (glycopeptid intermediate Staphylococcus aureus , GISA). Despite that it confers low to intermediate resistance as measured in vitro , the risk of therapeutic failure of glycopeptides against these strains is substantial and the drugs are not recommended.
Lipoglycopeptides
These are glycopeptide derivatives containing a long hydrophobic side chain. Their target and mechanism of action is identical to those of glycopeptides; some authors claim that
110 Antimicrobial chemotherapy: antibacterial agents the hydrophobic side chain increase the efficiency of binding to the cell wall. However, presence of the hydrophobic side chain results in another target, which is more important in determination of their activity, i.e. the cytoplasma membrane. For this reason, they are discussed in detail in the section ‘Drugs targeting the cell membrane’.
Daptomycin
See in detail in the section ‘Drugs targeting the cell membrane’. Daptomycin has activity on two targets, the cell wall synthesis and the cytoplasma membrane. Inhibition of the synthesis of the cell wall component lipoteichoic acid plays a relatively minor role in its activity.
Fosfomycin (fosfonomycin) O HO P CH3 Fosfomycin is a methylated and phosphonated derivative of O OH ethylene oxide. Its target is the cell wall synthesis through inhibiting the enzyme synthesizing UDP-N-acetyl-muramic acid. Though penetrates fosfomycin well into different tissues including the cerebrospinal fluid, its primary field of application is urinary tract infections. Its spectrum is broad, but a high number of species with intrinsic resistance is known both among Gram positives and Gram negatives. It has a good activity against Staphylococcus aureus and S. epidermidis , but has variable or no activity against other staphylococci. It is active against streptococci and enterococci, with the exception of S. agalactiae . Out of enterobacteria it is active against E. coli , Shigella , Salmonella , Klebsiella pneumoniae , Serratia marcescens , Citrobacter spp ., Yersinia enterocolitica , Proteus vulgaris and P. mirabilis , all other enterobacterial species (e.g. Enterobacter spp ., Klebsiella oxytoca , Proteus morganii , etc.) possess some degree of intrinsic resistance. It is also active against Aeromonas and Campylobacter jejuni , as well as against Gram positive strictly anaerobic species (including Actinomyces spp. ), Veilonella and Fusobacterium . It is less active or inactive against non-fermenting Gram negatives, against Bacteroides , Prevotella , Porphyromonas , against obligate intracellular pathogens, spirochaetes, Vibrio , Brucella , Bordetella , Legionella , against corynebacteria, mycobacteria and Nocardia .
Mechanisms of fosfomycin resistance
1. enzymatic modification This mechanism involves conjugation of the drugs with glutathione together with opening of the epoxide ring. The mechanism may be coded by the chromosome or on plasmids, but in case of Gram negative bacteria, chromosomally coded resistance leads to decreased virulence. 2. resistance due to decreased permeability This mechanism is based on losing the expression of transporter systems (glycerol-phosphate and glucose-6-phosphate systems) involved in drug uptake. Rare mechanism of low clinical significance. 3. enzymatic cleavage It is mediated by cleavage of the C-P bond. Rare mechanism, with low clinical significance.
Lipoglycodepsipeptides
Cyclic oligopeptides inhibiting the cell wall synthesis by interfering with synthesis of the undecaprenyl structure of lipid II, which serves as a platform for synthesis of peptidoglycane elements. They are not absorbed from the gut, may be used for selective bowel decontamination. These are presently under development and not yet marketed. Ramoplanin belongs to this group. They have a narrow, Gram positive spectrum; they are active against most Gram positive aerobic and anaerobic pathogens including Clostridium difficile . They are applicable for eradication of glycopeptide resistant enterococci from the gut. They are inactive against all Gram negative bacteria. No data are available on lipoglycodepsipeptide resistance.
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Drugs targeting the cell membrane
Drugs targeting the cell membrane 1. Polymyxins polymyxin B, colistin 2. Daptomycin daptomycin 3. Lipoglycopeptides telavancin, dalbavancin, oritavancin
Polymyxins
Polymyxines are cationic cyclic peptides. They exert their bactericidal activity by damaging the cytoplasmic membrane by pore formation disrupting the barrier function and increasing permeability. They have primarily been used for topical treatment due to their marked toxicity in systemic applications, but with the spread of multiresistant Gram negative bacteria, their systemic use is on the rise presently. Polymyxin B and colistin belongs to this drug class.
CH3 R=CH3 colistin A H2N O R=H colistin B CH3
N CH N H 3 H H C 3 OH O NH O H CH3 CH3 O O O N H H R N N N N O HN NH H H 2 H O O O N N H
NH2 NH2 O HO CH NH 3 2
NH2 NH2
O O O O H H H H N N N H N CH3 R N N N H H HO CH CH3 O O NH2 O 3 H C HN O H C OH 3 3 H N N H N NH2 R=CH3 polymyxin B1 H O R=H polymyxin B2 O O
NH 2
Their spectrum is narrow; they are active against certain Gram negative bacteria, including Pseudomonas aeruginosa , Stenotrophomonas maltophilia , acinetobacters and enterobacteria excepting the Proteus group. They are inactive against Gram positive bacteria. Resistance is mediated by alteration of the electric charge of the membrane. Recently in case of panresistant Klebsiella pneumoniae strains, decreased uptake through increased capsule production and capsule thickness has emerged as a clinically important mechanism.
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Daptomycin
Daptomycin is a cyclic peptide with a lipoic acid side chain. It has two cellular targets. It forms a pore in the membrane, depolarizing it and causing increased permeability and arrest of ATP synthesis. It also inhibits synthesis of lipoteichoic acid and consequently normal cell wall synthesis. The clinically more important target is the cell membrane. Its activity is bactericidal, with a marked postantibiotic effect. It penetrates well into tissues, including the cerebrospinal fluid. Its activity is inhibited by the pulmonary surfactant; therefore it is ineffective in pneumonia.
H N COOH 2 CH NH COOH O 3 O O CH O O H 3 H H H N N N N H N H N O N N H H H O O O H O O O O COOH O O NH H 2 N N O N N H N COOH H H H O O CH NH 3 daptomycin 2
CH 3
Its spectrum is narrow; it is active only against Gram positives. Its primary field of application is against multiresistant Gram positive bacteria (MRSA, vancomycin resistant enterococci). Acquired resistance has been described but the mechanism of resistance is yet unknown. Accelerated cell wall synthesis found in glycopeptide intermediate staphylococci (GISA) seem to provide cross-resistance to daptomycin.
Lipoglycopeptides Cl HN H C These are structurally 3 OH related to glycopeptides, but possess an additional lipophilic O CH3 OH side chain; oritavancin and oritavancin telavancin was synthesized from O OH vancomycin, while dalbavancin OH OH H N CH O from teicoplanin. They retained 2 3 O Cl H C the activity of the glycopeptides 3 O O O on the cell wall synthesis, but due O OH to the lipophilic side chain they Cl O O gained activity on the cell H H O N N membrane, which is damaged by N N NH H H H pore formation. Consequently in HN O O N O O CH contrast to the slow bactericidal HOOC 3 NH activity of glycopeptides, the 2 H3C bactericidal action of OH CH3 lipoglycopeptides is rapid, and HO OH
113 Antimicrobial chemotherapy: antibacterial agents they are active against glycopeptide resistant strains. The lipophilic side chain also improved the pharmacokinetic properties, they penetrate better than glycopeptides, reaching therapeutic concentrations in the cerebrospinal fluid and gaining the ability of intracellular accumulation. All three drugs are before marketing. Their spectrum is identical to that of glycopeptides, i.e. they are active against most Gram positive bacteria, with activity against isolates with acquired glycopeptide resistance (VRE, GISA, GRSA), with the exception of dalbavancin, which proved to be inactive against strains carrying vanA-type resistance. Data on lipoglycopeptide resistance is lacking.
H N CH3 HN H C 3 OH
O CH3 OH O OH telavancin
OH O O Cl O O
HO OH Cl O O H H O N N N N NH H H H HN O O N O O CH HOOC 3 NH2 H3C OH CH HO OH 3 OH N P H O OH
CH OH 3 H N OH H3C COOH O O O Cl O O
dalbavancin OH O O H H N H N O HN N N H CH O Cl O NH H 3 O N N N CH H C H 3 3 OH O O OH HO O O OH OH HO OH OH
114 Antimicrobial chemotherapy: antibacterial agents
Drugs targeting the ribosome
Drugs targeting the ribosome 1. Drugs targeting the 30S subunit 1.1. Aminoglycosides 1.1.1. Molecules containing streptidine streptomycin 1.1.2. Molecules containing 4,6 disubstituted kanamycin, gentamicin, tobramycin, netilmicin, deoxystreptamine amikacin, isepamicin, sisomicin, arbekacin, etc. 1.1.3. Molecules containing 4,5 disubstituted neomycin, paromomycin deoxystreptamine 1.1.4. Aminocyclitoles spectinomycin 1.2. Tetracyclines oxytetracycline, minocycline, doxycycline 1.3. Glycylcyclines tigecycline 2. Drugs targeting the 50S subunit 2.1. Chloramphenicol 2.2. Macrolides 2.2.1. macrolides in the narrow sense 2.2.1.1. molecules with 14-membered rings erythromycin, clarithromycin, roxithromycin 2.2.1.2. molecules with 16-membered rings spiramycin, josamycin 2.2.2. azalides (15-membered ring) azithromycin 2.2.3. ketolides (14-membered ring) telithromycin 2.3. Lincosamides clindamycin 2.4. Streptogramins 2.4.1. Streptogramin A type compounds dalfopristin 2.4.2. Streptogramin B type compounds quinupristin 2.5. Oxazolidinones linezolide 2.6. Fusidic acid 2.7. Pleuromutilines retapamulin
Aminoglycosides and aminocyclitoles
They are composed of aminated monosaccharides bound with glycosidic bonds, while aminocyclitoles contain only one aminated monosaccharide molecule, and do not contain glycosidic bonds. They have a rapid bactericidal action with marked postantibiotic effect. They target the 30S subunit of the prokaryote-type ribosome and cause misreading of the genetic code by the ribosome, leading to formation of proteins incapable of normal function. They exhibit synergy with cell-wall active antibiotics ( β-lactams and glycopeptides). Their uptake into the cell requires energy (ATP) and divalent cations (calcium- or magnesium ions); high salt concentration, acidic pH and anaerobic milieu prevent penetration. They do not penetrate into the cerebrospinal fluid, abscesses, bone; reach only low concentration in the bile and in the lungs (but penetrate well into the pleural cavity). They may damage the kidneys and the acoustic nerve.
115 Antimicrobial chemotherapy: antibacterial agents
streptomycin neomycin B paromomycin NH 2 NH 4' 2 H N HO 4' 2 O HO O NH 3' HO 3' H N HO HN 2 3 H N OH 2' O NH 2 3 HO 2 2' O NH OH 6 2 HN OH H2N OH O O O HO NH2 1 O HO NH2 NH OH O OH CHO OH O NH 2 O OH HO O O O NHCH OH OH HO 3 H N OH 2 OH H N 3" 2 OH
kanamycin gentamicin C1a tobramycin H C 6' NH 6' 3 NH2 NH2 4 ' 2 6' 4' HO O O HO O 3' HO H N H N H N 2 3 2' 2 3 2' 2 3 2' O NH2 O NH2 O NH2
HO HO HO NH NH O NH2 O 2 O 2
O OH O O OH 2" 2" 2" HO HO HO OH HN CH3 OH NH2 NH2 CH3 OH dibekacin amikacin arbekacin
NH 2 NH NH 6' 2 6' 2 6' O 4' HO O O 3' HO H2N 3 2' HO H2N O NH2 O NH2 2' O NH2 HO O O NH HO H HO O 2 N NH N NH O 2 O H 2 O OH OH OH O OH O OH 2" HO OH 4" HO HO NH2 OH OH NH2 NH2
netilmicin isepamicin sisomicin NH 6' 2 NH NH 4' 6' 2 2 HO O 6' 3' O O HO H N 2 H N H N O NH 2 3 2 3 2 2 ' O 2' O NH2 O NH2 HO N NH HO HO O H 2 NH N OH O 2 O H O O O HO HN CH 2" 3 HO CH HO HN 3 HN CH3 CH OH 3 CH OH CH OH 3 3
spectinomycin
9 OH
H3C H H H N O O CH3
HO O H OH HN O CH 3
116 Antimicrobial chemotherapy: antibacterial agents
They are classified according to their chemical structure as follows. 1. Drugs containing streptidine, the natural drugs streptomycin and dihydrostreptomycin belongs here. 2. Drugs containing 4,6-disubstituted deoxystreptamine, this group includes most clinically significant aminoglycosides, i.e. - the clinically frequently used gentamicin (a natural mixture of different highly similar molecules), tobramycin (natural), and the semisynthetic netilmicin and amikacin , - the natural antituberculotic kanamycin (see below), - arbekacin (semisynthetic), sisomicin (natural) and isepamicin (semisynthetic). Netilmicin and sisomicin contain a monosaccharide molecule with a 4-5 double bond, and consequently without hydroxyl- or amino- functional groups. 3. Drugs containing 4,5- disubstituted deoxystreptamine; this group includes the natural neomycin used topically or for selective bowel decontamination and paromomycin (natural) used primarily as an antiprotozoal drug. 4. Aminocyclitoles, where the only clinically used member is spectinomycin (natural), which is used almost exclusively for treatment of penicillin resistant Neisseria gonorrhoeae infection.
Their spectrum is broad, they are active against most aerobic and facultative anaerobic bacteria, including some (but not all) obligate intracellular pathogens ( Bartonella quintana , Ehrlichia ). They have activity against Leptospira and Nocardia . Amikacin, kanamycin and streptomycin are also used as antituberculotics (see below). Their activity against streptococci and enterococci is weak, administered alone they are only bacteriostatic against these bacteria. In combination with cell wall active antibiotics, the activity is bactericidal. They are inactive against obligate anaerobic bacteria and in an anaerobic environment. They are inactive against Burkholderia cepacia , and weakly active against Serratia spp. and Stenotrophomonas maltophilia , as well as against haemophili and Mycoplasma spp .
Mechanisms of aminoglycoside resistance
1. Enzymatic modification This is the most important mechanism in case of Enterobacteriaceae , non-fermenting Gram negative rods and Gram positive bacteria. The genes coding for these enzymes are mostly plasmid-borne, and easily transmissible between different species. However, there are modifying enzymes coded on the chromosome of certain species ( Serratia spp. , Stenotrophomonas maltophilia ), accounting for the intrinsic resistance of these species. The mechanism usually provides high level resistance, but may lead to disruption of the synergy with cell wall active agents even in case of relatively low level of resistance. Drug modification and inactivation may occur by virtue of acetylating of amino groups ( aac enzymes) or by phosphorilation or nucleotidylation of hydroxyl groups ( aph and ant enzymes). The enzymes usually are capable of modifying one certain functional group; the provided resistance pattern is the function of the presence of the group. The site of modification (modified functional group) is marked by the number of the group in the name of the enzyme in parenthesis; genetically unrelated but functionally similar enzymes are designated by roman numerals, e.g. AAC(6’)-I; different genes are marked by lower-case letters, e.g. aac(6’)-If . (In the figure the modification sites are marked by the appropriate numbers; unnumbered groups mean that enzymes modifying that particular group has not yet been found in case of that particular drug.) One modifying enzyme is
117 Antimicrobial chemotherapy: antibacterial agents
normally capable of modifying one functional group only, but in this manner may be capable of inactivating more than one drug provided they contain that particular functional group. Consequently, cross-resistance between drugs belonging to the same aminoglycoside subgroup is not infrequent. In contrast, cross-resistance due to modifying enzymes between drugs belonging to different subgroups is rare. One modifiable functional group may be target for several different enzymes with different substrate specificity (different spectrum of inactivated drugs). Moreover, there are bifunctional enzymes with the ability to modify two functional groups simultaneously; these provide resistance to many different aminoglycosides. An important example is the bifunctional enzyme produced by enterococci and staphylococci AAC(6’)-APH(2”), providing extremely high level of resistance to all clinically important aminoglycosides (excepting streptomycin), leading to elimination of the aminoglycoside synergy with cell wall active drugs. Some strains produce more than one enzyme simultaneously. Recently an acetylating enzyme capable of modifying certain fluoroquinolones besides aminoglycoside substrates has been described in Gram negative species (see below).
Clinically important aminoglycoside modifyig enzymes and their spectra
AAC(6'): ANT(4'): -I: A, T, N, D, S -I: A, T, I, K, D -II: G, T, N, D, S -II: A, T, I, K kanamycin -III: extremely weak activity APH(3'): 6 ' -AAC(6')-APH(2") bifunctional enzyme: -I: K, G(B), N, P 4 ' NH2 A, G, T, N, D, I, K, S -II: K, G(B), N, P HO O 3 ' -III: K, G(B), A, I, N, P HO AAC(3): -IV: K, N, P H2N 3 -I: G, S O NH -V: N, P 2 ' 2 -II: G, T, N, D, S -VI: K, G(B), A, I, N, P, -III: G, T, K, D, S, Neo, P -VII: K, N HO 1 -IV: G, T, N, D, S O NH2 A: amikacin -VI: G, S AAC(2'): (several other varieties) D: dibekacin O OH G: gentamicin -I: G, T, D, N, Neo 2" G(B): gentamicinB HO OH NH I: isepamicin 2 K: kanamycin ANT (2"): AAC(1): N: netilmicin -I: K, G, T, D, S APH(2"): -I: P Neo: neomycin -I: K, G, T, S, D P: paromomycin (part of the bifunctional enzyme, S: sisomycin also possess AAC(6') activity) T: tobramycin
2. Resistance based on modification of the ribosome This mechanism is always chromosomal, mostly caused by point mutation of a rRNA or a ribosomal protein gene. This is the mechanism in case of the aminoglycoside resistance of mycobacteria and Campylobacter spp ., as well as of the spectinomycin resistance of Neisseria gonorrhoeae . 3. Resistance due to decreased permeability Mostly found in Pseudomonas aeruginosa , but may be in the background of aminoglycoside resistance of other Gram negatives and staphylococci as well. 4. Resistance due to protection of the ribosome This mechanism is coded by tranpozons, most probably originates from aminoglycoside producer soil bacteria. It is mediated by enzymatic methylation of the 16S rRNA in the ribosome; provides very high level resistance to all drugs containing 4,6-disubstituted deoxystreptamine (gentamicin, tobramycin, netilmicin, amikacin, etc.), i.e. to most aminoglycosides used as antibacterial agents in the clinical setting. Other aminoglycoside subgroups including aminocyclitoles retain their activity. This mechanism is spreading rapidly among members of the Enterobacteriaceae family, and has also been described in Pseudomonas aeruginosa .
Interestingly, in some aminoglycoside resistant strains aminoglycoside dependence may develop. This arises from compensation of lethal mutation(s) by the misreading of the genetic code caused by the aminoglycoside drugs.
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CH Tetracyclines 3 R1 N(CH ) HO H H 3 2 OH Their structure is based on a ring system consisting of four condensed rings. Their cellular target is the 30S NH2 subunit of the bacterial ribosome; they interfere with OH OH O OH O O aminoacyl-tRNA binding. They are bacteriostatic. ATP and magnesium ions are required for their entry into the cell. R1=H tetracycline R1=OH oxytetracycline They are lipid-soluble, penetrate well into different tissues, N(CH ) N(CH ) are concentrated in the intracellular space and in the bile, 3 2 H H 3 2 but cannot reach therapeutic concentration in the OH cerebrospinal fluid and in the urine. Their most important NH2 side effect is that they cause dental and bone development OH disorders in children, besides this, they cause sensitivity to OH O OH O O light. The most important members of the group are the minocycline CH natural oxytetracycline , and the semisynthetic doxycycline 3 HO N(CH ) H H 3 2 and minocycline . OH Their spectrum is broad; they are active against most Gram positive and Gram negative aerobic and NH2 facultative anaerobic species, as well as against OH mycoplasmas, spirochaetes and obligate intracellular OH O OH O O bacteria. Their activity against anaerobic bacteria is weak, doxycycline with the exception of actinomycetes. They are the drugs of first choice against obligate intracellular pathogens ( Chlamydia , Ehrlichia , Rickettsia ) and against certain spirochaetes (Leptospira ), in brucellosis (in this case they exhibit synergy and are combined with aminoglycosides), in plague and against multiresistant Burkholderia cepacia . Members of the Proteus group and Stenotrophomonas maltophilia possess generic resistance to tetracyclines.
Mechanisms of tetracycline resistance
1. Resistance due to protection of the ribosome This plasmid- or transpozon-borne mechanism is based on production of a new protein structurally related to elongation factors. This new protein protects the ribosome by inhibiting the destabilization of the aminoacyl-tRNA binding exerted by the tetracyclines. 2. Changes in permeability This mechanism is coded by the chromosome. It is found mainly in Gram negative bacteria, a porin protein in the outer membrane is lost or modified. Normally provides low level resistance, but may cause cross-resistance to a variety of other drugs including β-lactams and fluoroquinolones, as these drugs also use porins for the entry into the cell. Due to this cross-resistance, tetracycline overuse facilitates not only the spread of tetracycline resistance, but may play a role in the development of multiresistance as well. 3. Active efflux Efflux may be based on multidrug resistance (MDR) pumps capable of exporting many substrates, or on tetracycline-specific pumps. MDR pumps provide resistance to various antibiotics (e.g. fluoroquinolones) and biocides, while tetracycline-specific pumps eliminate only tetracyclines. Tetracycline-specific pumps are inducible, plasmid- or transpozon-borne, and are the most common mechanism of tetracycline resistance. 4. Target mutation Mutations of the rRNA is a mechanism suspected in the background of tetracycline resistance of Mycoplasma species, as no other mechanism has been found. However, this mechanism remains unproved.
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Glycylcyclines N(CH ) N(CH ) tigecycline 3 2 H H 3 2 OH O Glycylcyclines are structurally H related to tetracyclines; they have a H3C N NH2 N longer additional side chain containing a H C CH H OH 3 3 OH O OH O O substituted glycine. The only marketed member is tigecycline . Its target and mechanism of action are identical to those of tetracyclines, but has higher affinity and binds stronger to the ribosome. It is generally bacteriostatic, but is bactericidal against Streptococcus pneumoniae , Haemophilus influenzae , Moraxella (Branhamella) catarrhalis and Neisseria spp . In contrast to tetracyclines, glycylcyclines have marked postantibiotic effect. Spectrum of glycylcyclines is slightly broader than the spectrum of tetracyclines; it has good activity against strict anaerobes, though its activity is weak against the Bacteroides fragilis group. Intrinsic resistance of the Proteus group to tetracyclines also confers generic glycylcycline resistance; moreover, Pseudomonas aeruginosa also possess natural glycylcycline resistance. Activity against other non-fermenting Gram negative bacteria (e.g. Acinetobacter spp .) is controversial. Due to stronger ribosome binding, tetracycline resistance based on protection of the ribosome does not provide glycylcycline resistance, and glycylcyclines are poor substrates for many tetracycline efflux pumps, therefore, tigecycline may retain activity against tetracycline resistant bacteria. Data on glycylcycline resistance is scarce. Some authors suggested that some efflux pumps may provide low level tigecycline resistance; these were implicated in the background of tigecycline resistance of some Acinetobacter baumannii and Staphylococcus aureus isolates.
Chloramphenicol
Chloramphenicol is a natural substitutionized nitrobenzene derivative, but its large-scale production is carried out purely with chemical synthesis. Its target is the 50S subunit of the bacterial ribosome, where it inhibits the translocation of the polypeptide chain being synthesized during the protein elongation, similarly to macrolides, lincosamides and type B streptogramins. Due to the common ribosomal target, chloramphenicol and the mentioned drugs antagonize each other. Its action is bacteriostatic. Its tissue penetration is excellent; it reaches therapeutic concentrations in the cerebrospinal fluid, in the eye and in abscesses as well. Highly toxic, may result in irreversible bone marrow damage, therefore it has been withdrawn from the market in many countries including Hungary. It is still licensed for topical use (eye or ear drops, ointments, etc.) It has a broad spectrum; it is active against both HO CH OH aerobic and anaerobic bacteria. It is highly active 2 O N C C N CHCl against rickettsias, bartonellas and Borrelia recurrentis , 2 H H H 2 but Chlamydia infections may recur after O chloramphenicol therapy. Pseudomonas aeruginosa chloramphenicol exhibits natural resistance.
Mechanisms of chloramphenicol resistance
1. Enzymatic modification The molecule is modified by an acetyltransferase enzyme. The acetyl derivative does not bind to the ribosome and has no antibacterial activity. Many different acetylating enzymes
120 Antimicrobial chemotherapy: antibacterial agents
are known, some are coded by the chromosome, but many of them are plasmid- or integron-borne. 2. Active efflux Resistance may be mediated by either drug-specific or multidrug efflux pumps. 3. Protection of the ribosome This mechanism is based on the methylation of the 23S RNA of the ribosome; a plasmid-borne specific enzyme ( cfr ) is responsible for the ribosome methylation. This enzyme is not related to the erm methylase important in macrolide-lincosamide-streptogramin B resistance (see below). The mechanism is plasmid-borne, easily transferable; it was demonstrated in animal-derived staphylococci. This mechanism is most probably has been selected by the extensive use of chloramphenicol and other phenicols (e.g florphenicol) in the veterinary medicine; it has not yet been demonstrated in human isolates. Its importance may lie in its ability to provide cross-resistance to pleuromutilines, lincosamides, streptogramin A derivatives and oxazolidinones.
Macrolide antibiotics
Their chemical structure consists of a 14, 15 or 16-membered macrolide lactone ring and different monosaccharide molecules bound to the ring. Their target is the 50S subunit of the bacterial ribosome, where they inhibit the translocation of the synthesized polypeptide chain, similarly to chloramphenicol, lincosamides and streptogramin B type antibiotics. Consequently these drugs and the macrolides antagonize each other. Normally they are bacteriostatic, but may become bactericidal at appropriately high concentrations against certain pathogens (e.g. Corynebacterium diphtheriae , Bordetella pertussis , Streptococcus spp ). Macrolides exhibit intracellular accumulation, and have excellent activity against intracellular pathogens. They do not penetrate into the cerebrospinal fluid and reach only subtherapeutic concentrations in the urine. They have significant immunomodulating activity. Their spectrum is narrow; they are active primarily against Gram positives. They are active against several atypical mycobacteria, but the Mycobacterium tuberculosis complex ( M. tuberculosis , M. bovis and M. africanum ) exhibit natural resistance. Out of Gram negative bacteria, macrolides inhibit neisseriae, haemophili (including H. ducreyi ), Bordetella spp ., Moraxella ( Branhamella ) catarrhalis , Helicobacter pylori , Campylobacter jejuni and C. coli , as well as against Bartonella spp . and against Treponema pallidum . Macrolides are the drugs of first choice in infections caused by Legionella , Chlamydia and Mycoplasma . Moreover, macrolides possess antiprotozoal activity (see below). Macrolides are always inactive against Enterobacteriaceae , pseudomonads and acinetobacters as well as against other non-fermenting Gram negative rods; their antianaerobic activity is weak except against Actinomycetes (excluding telithromycin, see below). They can be classified according to their chemical structure as follows.
Macrolides in the narrow sense
This group includes natural antibiotics as erythromycin (14-membered ring) or spiramycin and josamycin (16-membered ring), as well as semisynthetic derivatives, as clarithromycin and roxithromycin (14-membered ring). Their spectrum does not differ from the general macrolide spectrum described above. Semisynthetic derivatives are slightly more active than natural products, but cross-resistance is practically total. Clarithromycin is more active than any other members of this subgroup against Helicobacter pylori and atypical mycobacteria. An important difference between spectra of 14- and 16-membered macrolides that Mycoplasma hominis exhibits intrinsic resistance to 14-membered rings, but not against
121 Antimicrobial chemotherapy: antibacterial agents
16-membered rings. This resistance is due to a characteristic single nucleotide alteration (compared to M. pneumoniae ) in the 23S rDNA.
O CH3
H3C CH3 H C CH H3C CH3 3 9 3 N CH3 N H C 9 CH N 10 8 3 3 OH OR 11 11 HO 8 7 OH OR HO 12 H C OH CH 7 3 12 6 3 H C OH CH 13 H C 5 3 13 6 3 3 CH 4 O 3 14 H C 5 CH CH CH O 14 O 3 O 3 3 2 4 O 15 CH3CH2 O 1 3 2 CH3 1 3 O O 2 CH O O 3 CH CH 3 3 CH CH3 3 O R=H: erythromycin OH azithromycin O OH R=CH 3: clarithromycin CH 3 N CH3
N
H3C O O N O N H3C CH3 H C CH N O 3 9 3 10 O H C CH 8 CH 3 3 OH OH HO 9 3 11 N CH N 7 10 8 3 H C OH CH O 3 12 6 3 HO O CH3 7 13 H C 5 CH 11 3 O 3 CH 4 12 6 3 CH CH O 14 O H C 3 2 3 13 5 H3C 4 CH 1 3 CH CH CH O 14 O O 3 2 3 3 2 O O 1 3 CH3 2 O O CH3 O telithromycin roxithromycin CH3 OH
CH3 CH3 CH3 OH H3C N CH O 10 9 3 8 11 7 O O 12 CH 3 6 CH 10 9 3 13 5 O 8 14 O 4 O 11 O CH 7 O 15 3 12 3 CH3 16 1 2 6 H C O O 3 13 O 5 O O 14 4 O O CH H C 3 3 O 15 3 N CH 16 1 2 3 H C O O O 3 HO CH 3 O
josamycin H3C O H3C CH3 N CH3 O O CH HO CH 3 spiramycin 3
H3C CH3
O CH3 OH
Azalides
These differ from the former subgroup in containing a 15-membered ring possessing a nitrogen atom in the ring. Azalides are semisynthetic molecules. The only marketed member is the azithromycin . Its main advantage is the very slow elimination resulting in very long periods with high serum and tissue drug levels, allowing for once-daily administration. However, some authors suggest that slow elimination leads to presence of subtherapeutic
122 Antimicrobial chemotherapy: antibacterial agents azalide concentration for relatively long time after cessation of the therapy, possibly facilitating resistance development. Spectrum of azithromycin is similar to that described above, but it has better activity against Haemophilus influenzae , Helicobacter pylori , Campylobacter jejuni and atypical mycobacteria. Excepting these species, cross-resistance with 14-membered macrolides can be regarded as total. Its utility as an antiprotozoal drug is increasingly exploited.
Ketolides
This semisynthetic macrolide group was devised by exchanging a monosaccharide substituent of the original 14-membered macrolide ring to a keto-group and by further modification of molecule. The first marketed member is telithromycin . The spectrum of this group is also similar to that of other macrolides, but ketolides have better antianaerobic activity. Its antiprotozoal activity (mostly tested against Toxoplasma gondii ) is sufficiently good, but it has weaker activity than clarithromycin against atypical mycobacteria. The most important advantage of ketolides compared to other macrolides is that they may remain active against isolates possessing certain mechanisms of macrolide resistance (active efflux and methylation of the ribosome) due to its higher affinity to the ribosomal binding site and to its poor ability to induce expression of the resistant phenotype. However, ketolides are inactive against isolates resistant through ribosomal mutations, including the intrinsically resistant Mycoplasma hominis .
Mechanisms of macrolide resistance
1. Target modification via enzymatic methylation of the ribosome (ribosomal protection) This mechanism is based on production of a methylase enzyme ( erm ), which is capable of methylating of a specific adenine in the 16S rRNA of the ribosome. Methylated ribosome cannot bind the antibiotic. This enzyme may be encoded by the chromosome, plasmids or transpozons, gene expression may be constitutive or inducible. Constitutively produced forms of the methylase provide high level resistance to all macrolides including ketolides, and total cross-resistance to lincosamides and streptogramin B derivatives (quinupristin), but not to chloramphenicol. This mechanism does not influence binding of streptogramin A derivatives (dalfopristin), these retain their activity, therefore the quinupristin+dalfopristin combination remains active, but shows only bacteriostatic effect (see below). In case of the inducible form of methylase production, some or all of the poor inducer 16-membered macrolides, ketolides and streptogramin B derivatives may remain active as they do not always induce methylase production sufficiently to confer clinical resistance. 14- and 15-membered macrolides and lincosamides are affected by inducible methylases just as by constitutive enzymes. This mechanism is the most important mechanism of macrolide resistance in Gram positive bacteria, but has no significant role in other bacterial groups. 2. Mutational alteration of the target Mutations in the rDNA or in ribosomal protein genes result in weak binding of the drug by the ribosome; the mechanism leads to high level resistance. Cross-resistance between all macrolides (including ketolides) and to lincosamides is always total; certain mutations provide crossresistance to streptogramin B derivatives (quinupristin) as well. Mutations of the 16S rDNA are characteristic to Campylobacter spp ., Helicobacter pylori and to the Mycobacterium avium-intracellulare complex , and have recently emerged to be
123 Antimicrobial chemotherapy: antibacterial agents
the third most important mechanism of macrolide resistance in Streptococcus pneumoniae . Mutational alteration of one of the ribosomal proteins may also confer resistance; such resistance has been found in S. pneumoniae , Staphylococcus aureus and Haemophilus influenzae . Macrolide resistance of Mycoplasma spp . is also based on multiple mutations in the rRNA and ribosomal protein genes. 3. Active efflux This mechanism provides low level resistance to different macrolides (to some or all of the 14-, 15- and 16-membered rings) and to streptogramin B derivatives (quinupristin). Ketolides are less vulnerable to efflux; they remain active against isolates with efflux-based macrolide resistance. Cross-resistance between different macrolides and the streptogramin B derivatives is usually only partial, depending upon the specificity of the pump produced. Susceptibility to lincosamides and streptogramin A derivatives (dalfopristin) remains unaltered. Efflux pumps may be classified according to their specificity as follows. 3.1. Partial macrolide (PM) type efflux Resistance is provided only to 14- (erythromycin) and 15-membered rings (azithromycin), susceptibility to 16-membered rings, ketolides, lincosamides and streptogramins is preserved. 3.2. Macrolide (M) type efflux This provides resistance to all macrolides excepting ketolides regardless of ring size, but does not confer resistance to lincosamides or streptogramins. 3.3. Partial macrolide and streptogramin (PMS B) type efflux Resistance is provided to 14- (erythromycin) and 15-membered rings (azithromycin), susceptibility to 16-membered rings is unaltered, but this mechanism also provides resistance to streptogramin B derivatives (quinupristin). It does not provide resistance to streptogramin A derivatives (dalfopristin) and lincosamides. 3.4. Macrolide and streptogramin (MS B) type efflux This type confers resistance to all macrolides, except ketolides, and to streptogramin B derivatives (quinupristin). Lincosamide and streptogramin A (dalfopristin) susceptibility is unaffected. Efflux is frequently found in staphylococci and streptococci, but has also been described in campylobacters as well. 4. Enzymatic degradation of the antibiotic This mechanism involves the enzymatic hydrolysis of the macrolide ring. This is a rare mechanism, several different enzymes are known. These are selective according to the number of atoms in the rings, i.e. some enzymes hydrolyze 14-, while others hydrolyze 16-membered rings. Enzymes with activity against both 14- and 16-membered rings have also been found; however, hydrolysis of a 15-membered ring has not yet been described. This mechanism does not provide cross-resistance to lincosamides or streptogramins. 5. Enzymatic modification of the drug This mechanism is based on inactivation of the drug via phosphorylation. This mechanism is also a rare one and the different enzymes are differentiated according to the preferred ring size of the substrates. This mechanism does not provide cross-resistance to lincosamides or streptogramins either.
Isolates carrying a plasmid coding for more than one macrolide resistance mechanism has been characterized (e.g. a Staphylococcus aureus strain carrying a methylase, an efflux pump and a phosphotransferase gene on the same plasmid).
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Lincosamides
R CH3 CH O CH Drugs of this antibiotic family are comprised 3 3 N O S of an amino acid and an eight carbon atom N H monosaccharide. Members of this family include H C HO OH the natural lincomycin and its semisynthetic 3 derivative clindamycin . Only clindamycin is used R=OH: lincomycin OH in human medicine. R=Cl: clindamycin Its target is identical to that of macrolides, streptogramin B derivatives and chloramphenicol; it inhibits the translocation of the polypeptide chain synthesized in the 50S subunit of the prokaryotic ribosome. Due to the common target, lincosamides exhibit antagonism towards the abovementioned drugs, and should not be combined with them. Their action is mostly bacteriostatic, but may rarely be bactericidal against certain bacteria at higher concentrations. It is accumulated in the intracellular space, crosses the placenta and penetrates excellently into the bone, but does not reach sufficiently high concentration in the cerebrospinal fluid. It is a narrow spectrum antibiotic, it is active mostly against Gram positives. It is the drug of first choice against Gram positive strict anaerobes, and is active against most Gram negative strict anaerobes as well. It is also active against Actinomycetes and campylobacters. However, it does not inhibit Clostridium difficile , enterococci, Chlamydia spp ., Mycoplasma spp ., Bartonella spp ., spirochaetes and is inactive against all Gram negative aerobes and facultative anaerobes (including species susceptible to macrolides). It is also used as an antiprotozoal drug.
Mechanisms of lincosamide resistance
1. Target modification via enzymatic methylation of the ribosome (ribosomal protection) Target modification may be realized through activity of two different methylases. Resistance may be based on production of the methylase described at macrolide resistance (erm ), which methylates 16S rRNA. This mechanism results in total cross-resistance between macrolides and lincosamides and partial cross-resistance with streptogramin B derivatives (see above in detail). The other methylase conferring lincosamide resistance is the ( cfr ) methylase methylating the 23S rRNA described at chloramphenicol resistance. This latter mechanism, in turn, provides resistance to chloramphenicol, streptogramin A derivatives (dalfopristinnel), oxazolidinones and pleuromutilines. It is a mechanism found in veterinary pathogenic bacteria, and has not yet been demonstrated in human isolates. 2. Mutational alteration of the target This mechanism is identical to that described at macrolide resistance both in its genetics and in the phenotype it provides; and similarly to the erm methylase, it also causes a total cross-resistance between linconsamides and macrolides. Certain mutations provide cross-resistance to streptogramin B derivatives (quinupristin) as well. 3. Enzymatic modification of the drug This mechanism is characteristic to staphylococci. This mechanism is based on enzymatic transfer of a nucleotidyl group to the lincosamide molecule leading to loss of antibiotic effect. Susceptibility to members of other drug classes is preserved, cross-resistance is never provided. Production of the nucleotidyl tranferase enzyme is inducible; as clindamycin is a poor inducer, this resistance mechanism should be demonstrated using the better inducer lincomycin during in vitro susceptibility testing.
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Streptogramins
Two subgroups are known, streptogramin A compounds (polydesaturated macrocyclic lactones) and streptogramin B derivatives (polydesaturated cyclic peptides). The target of streptogramin B derivatives is identical to the target of macrolides, lincosamides and chloramphenicol; they act on the 50S subunit of the prokaryotic ribosome. The common target results in antagonism between the abovementioned drug classes and streptogramin B derivatives, precluding their usage together in a combinational therapy. The target of streptogramin A derivatives is also on the 50S ribosomal subunit, but distinct from that of streptogramin B derivatives (as well as from that of macrolides, lincosamides and chloramphenicol). Both streptogramin subgroups inhibit polypeptide chain translocation, but with different biochemical mechanism, therefore cross-resistance does not exist between the two subgroups, and they synergistically enhance the activity of each other. (Streptogramin A derivatives also facilitate ribosomal binding of streptogramin B derivatives.) The only presently marketed drug is a combination of two streptogramins, dalfopristin (streptogramin A derivative) and quinupristin (streptogramin B derivative). The combination is bactericidal (except against enterococci, against which it is bacteriostatic), and possess an immunmodulatory effect, similarly to macrolides. It has a marked postantibiotic effect.
O O CH CH 3 OH 3 OH N N H H H C H3C 3 O O O O O O N H C N H C 3 O N 3 O N O CH O CH3 3 O S streptogramin A O dalfopristin
H3C N CH3
H C CH H C CH 3 3 3 N 3 N
CH3 CH3 N N N N N O O H H CH3 CH3 O O S N O O N O O NH NH N N O H O O H O O O H C H C 3 O 3 O HN O HN O
OH OH N N quinupristin streptogramin B
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Spectrum of the streptogramins (and thus that of the combination) is primarily Gram positive. Excepting Enterococcus faecalis , which possess an efflux-based intrinsic resistance, it is active against all Gram positive aerobic and anaerobic bacteria, including methicillin resistant Staphylococcus aureus , vancomycin resistant Enterococcus faecium (against E. faecium it is only bacteriostatic, it should be combined with a cell wall active drug for bactericidal effect) and Clostridium difficile . It is active against Mycoplasma pneumoniae , but has a weak activity against Moraxella ( Branhamella) catarrhalis , against Neisseria spp ., Haemophilus spp . and Legionella spp . as well as against Leptospira and Borrelia . It is inactive against Gram negative strict anaerobes, excepting a weak activity against Bacteroides fragilis ; against Enterobacteriaceae , pseudomonads and Acinetobacter spp . It was shown to possess antiprotozoal activity, e.g it is active against Toxoplasma gondii .
Mechanisms of streptogramin resistance
1. Enzymatic modification of dalfopristin A dalfopristin-acetyltransferase enzyme may inactivate the drug. This mechanism does not provide cross- resistance; the isolates remain susceptible to macrolides, lincosamides and streptogramin B derivatives (quinupristin). Unfortunately, the resulting loss of synergy leads to significant loss of activity; despite quinupristin remains active, therapeutic failure of the combination is not uncommon in case of dalfopristin resistant isolates. 2. Active efflux of dalfopristin This mechanism provides the natural resistance of Enterococcus faecalis . Similar mechanism may be found in a minority of Staphylococcus epidermidis strains, but in S. epidermidis it only provides low level resistance and the combination remains effective. 3. Enzymatic modification of the target of dalfopristin (protection of the ribosome) The biochemical background for this mechanism is the methylation of the 23S rRNA in the ribosome, resistance is provided by the methylase cfr described at chloramphenicol resistance. This mechanism provides cross-resistance to chloramphenicol, lincosamides, oxazolidinones and pleuromutilines. It has not yet been found in human clinical isolates.
4. Target modification via enzymatic methylation of the ribosome (ribosomal protection) This mechanism is based on the production of the erm methylase described at macrolide resistance. Provides cross-resistance to all macrolides, lincosamides and streptogramin B derivatives (quinupristin), but does not affect the susceptibility to dalfopristin. As a result, the quinupristin-dalfopristin combination loses its bactericidal activity and becomes bacteriostatic through the activity of dalfopristin alone. 5. Active efflux of quinupristin Pumps affecting quinupristin may be PMS B or MS B types as described above at macrolide resistance. 6. Enzymatic degradation of quinupristin Quinupristin may be degraded by a specific quinupristin hydrolase. This mechanism does not provide cross-resistance, the isolate remain susceptible to macrolides, lincosamides and dalfopristin.
7. Disruption of the dalfopristin-quinupristin synergism This mechanism is based on mutational alteration of a ribosomal protein. It has been described in Staphylococcus aureus and Streptococcus pneumoniae ; it led to resistance to the combination in these strains.
It is possible though rare that one isolate carries more than one resistance mechanism. E.g. it may produce an erm methylase, leading to quinupristin resistance, simultaneously to a dalfopristin acetyltransferase, which inactivates dalfopristin. Such combinations of resistance mechanisms may also lead to total loss of activity of the quinupristin/dalfopristin combination, but it is extremely rare.
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Oxazolidinones O
These drugs are oxazole derivatives. Their O N NO O target is the bacterial ribosome; by binding to the CH3 F N 50S subunit, they inhibit the association of the two H (30S and 50S) ribosomal subunits, consequently, linezolid the initiation of translation. They are generally bacteriostatic, but may be bactericidal against some species ( Streptococcus pyogenes , Streptococcus pneumoniae , Bacteroides fragilis , Clostridium perfringens ). Their only marketed member is the linezolid . The spectrum of linezolid is mainly Gram positive; it is active against all Gram positive pathogens, and against all strict anaerobes including Gram negative strict anaerobes. It has a weak antituberculotic activity, it is active against certain atypical mycobacteria. It is inactive against Gram negative aerobic and facultative anaerobic species, but has a demonstrable weak activity against Haemophilus , Neisseria , Bordetella , Pasteurella and Legionella . It is also inactive against Mycoplasma spp .
Mechanisms of oxazolidinone resistance
1. Mutational alteration of the target The mutation leading to resistance takes place in the 23S rRNA gene. Most Gram positive bacteria have multiple copies of this gene, and resistance requires mutational alteration of all these genes, consequently, development of high level resistance is slow. In case of organisms carrying only one or two copies of the 23S rRNA gene, development of resistance may be faster. This type of resistance has been described in some Enterococcus strains and in one MRSA strain. 2. Enzymatic modification of the target (protection of the ribosome) This mechanism is mediated by the cfr methylase described at chloramphenicol resistance. This mechanism provides cross-resistance to chloramphenicol, lincosamides, streptogramin A derivatives (dalfopristin) and pleuromutilines. It has not yet been found in human isolates.
Fusidic acid
Fusidic acid is a triterpen derivative. It is a bacteriostatic H C COOH antibiotic inhibiting the function of one of the ribosomal elongation 3 factors, leading to inhibition of the translocation of the polypeptide OH H H C chain being synthesized. It penetrates well due to its lipophilic 3 CH CH O nature; it reaches a markedly high concentration in the bone. 3 3 CH It has a narrow spectrum; it is active only against 3 H CH staphylococci and Gram positive anaerobes. Its activity against 3 O streptococci and enterococci is weak, as well as against Gram HO H negative anaerobes. It may be effective against Gram positive rods, CH3 fusidic acid Nocardia spp ., and against Neisseria spp ., Moraxella ( Branhamella ) catarrhalis and Bordetella spp . It is inactive against Gram negative aerobes and facultative anaerobes. Resistance to fusidic acid may be mediated by the mutational alteration of the target elongation factor encoded on the chromosome, but plasmid-borne mechanisms have also been implicated.
Pleuromutilines
Pleuromutilines consist of a basic structure of multiple ring system and a side chain connected through a thiol bond. The mechanism of their bacteriostatic action is the direct inhibition of the peptid bond formation in the ribosome. These drugs has been in use for a long time in veterinary medicine (tiamulin, valnemulin), but they are not yet used in human medicine. A semisynthetic topical pleuromutilin, retapamulin is to be marketed soon. (Retapamulin is planned to replace mupirocin, which is compromised by spread of resistance.)
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Their spectrum is narrow, they are active primarily against Gram positives, and has also good activity against CH2 Haemophilus (and other Gram negative coccobacilli), against OH strict anaerobes, Mycoplasma spp . and spirochaetes; CH3 H C 3 CH nevertheless, these data are derived mostly from the veterinary N H 3 O experience. Data on human pathogens are available only in case H of staphylococci ( S. aureus and different coagulase-negative S O species), streptococci ( S. pyogenes and S. pneumoniae ), H H3C Haemophilus influenzae and Moraxella ( Branhamella ) retapamulin O catarrhalis ; against these species retapamulin showed good H3C activity. Pleuromutilins are inactive against enterococci, Gram negative aerobes and Gram negative facultative anaerobes. Pleuromutilin resistance is mediated by enzymatic methylation of the 23S rRNA ( cfr ) described at chloramphenicol resistance. This mechanism provides cross-resistance to chloramphenicol, lincosamides, streptogramin A derivatives and oxazolidinones.
Inhibitors of nucleic acid metabolism
Inhibitors of nucleic acid metabolism 1. quinolones, fluoroquinolones 1.1. 1 st generation (does not contain a fluor atom) nalidixic acid, oxolinic acid 1.2. Norfloxacin 1.3.2nd generation ofloxacin, pefloxacin, ciprofloxacin 1.4. 3 rd generation levofloxacin 1.5. 4th generation moxifloxacin 1.4. „5th ” generation garenoxacin, prulifloxacin 2. Rifamycins rifampin, rifabutin, rifapentin
Quinolones, fluoroquinolones
These are bactericidal antibiotics with a marked postantibiotic effect. Their target is the bacterial DNA metabolism. Two target enzymes has been identified, the DNA-gyrase, responsible for decoiling of the supercoiled DNA; and topoisomerase IV, which mediates the separation of the two DNA copies during replication. Quinolones stabilize the DNA-enzyme active complex, blocking DNA replication and transcription. The two target enzymes has different affinity to the drugs; in Gram negatives generally the gyrase, while in Gram positives topoisomerase IV is the more susceptible, i.e. the primary target, but this may be variable among species. Quinolones are classified into different generations, similarly to cephalosporins. In case of later generations, the differences in affinity to one of the target enzymes decrease, later generation quinolones bind to both target enzymes with similar affinity. O O st 1 generation O COOH COOH
O Nalidixic acid and oxolinic acid N N H3C N N belongs to this group. These drugs do H C oxolinic acid H C nalidixic acid not yet contain a fluor atom. The drugs 3 3 have poor tissue penetration, but are accumulated in the urine; therefore they are used solely to treat urinary tract infections. Their spectrum is narrow; they are active only against members of the Enterobacteriaceae family, excepting the Proteus group, which possess natural resistance. They are inactive against all other Gram negatives as well as against all Gram positives.
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Norfloxacin O Classification of norfloxacin is controversial; some F COOH authors classify it to the 1 st generation, according to others it is a member of the second generation. This controversy arises N N N from the facts that norfloxacin contains a fluor atom, but still HN norfloxacin has poor tissue penetration and is useful only in urinary tract H3C infections. Its spectrum is slightly broader than that of the 1 st generation, it is active against the Proteus group, against staphylococci and enterococci; it also has a weak antipseudomonas activity. Enterobacteria susceptible to the 1 st generation are susceptible to norfloxacin.
2nd generation
These fluoroquinolone derivatives penetrate excellently to different tissues (excepting bone and ofloxacin O cerebrospinal fluid); they reach favourably high F COOH concentrations in the prostate, which otherwise is usually poorly accessible for other antibiotics. Ofloxacin , N N pefloxacin and ciprofloxacin belongs to this generation. N O H C CH These are active against all isolates susceptible to 3 3 norfloxacin, and are also active against Neisseria spp ., Moraxella (Branhamella ) catarrhalis , Haemophilus spp . They have better activity than norfloxacin against O Pseudomonas aeruginosa and members of the F COOH Enterobacteriaceae ; ciprofloxacin is the most effective. They are active against certain obligate intracellular N N N pathogens (Chlamydia , Rickettsia , Bartonella ), against N H C H C Mycoplasma spp . and Legionella spp . They have a 3 3 pefloxacin clinically useful activity against mycobacteria including M. tuberculosis and M. leprae . (Ofloxacin has the best O nd antimycobacterial activity of the 2 generation.) F COOH Activity of 2 nd generation fluoroquinolones is weak against streptococci and enterococci (against these N N N genera they have therapeutic value only in urinary tract HN infections, where the fluoroquinolone concentrations are very high). Obligate anaerobic bacteria exhibit intrinsic ciprofloxacin resistance. (This lack of activity has the advantage of preserving the anaerobic gut flora.)
3rd generation
Levofloxacin (the left-rotating enantiomer of the olfoxacin racemic mixture) belongs to the 3rd generation. Its activity is better than that of the former generation against Streptococcus pneumoniae and enterococci, but generally it has slightly weaker activity than ciprofloxacin against Gram negatives. This weaker activity is marked against Pseudomonas aeruginosa . In contrast, it may perform better than ciprofloxacin against Acinetobacter and Stenotrophomonas . It is still inactive against strict anaerobes.
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4th generation
O NH2 O O F COOH F COOH F COOH
H3C N N N N N N HN O HN F N O H3C H H3C CH CH sparfloxacin 3 gatifloxacin 3 moxifloxacin
This generation includes moxifloxacin , and several other drugs not marketed in Hungary (e.g. sparfloxacin , gatifloxacin , etc). Similarly to levofloxacin, these drugs have broader Gram positive spectrum, but poorer activity against Gram negatives than drugs of the 2nd generation. They also have activity against strict anaerobes, but its clinical utility is not yet established. They have good activity against Streptococcus pneumoniae and enterococci; in vitro they are also active against β-haemolytic streptococci, data are being collected presently to assess their clinical usefulness in infections caused by these streptococci. They are proven to be efficient against Chlamydia and Mycoplasma spp . (in airway infections). They have improved antimycobacterial activity compared to the 2 nd generation; moxifloxacin is presently the most active marketed fluoroquinolone against M. tuberculosis . Gram negative spectrum of this generation is narrower than that of the 2 nd generation; they are less active than ciprofloxacin against Pseudomonas spp . and enterobacteria. Their activity is variable against other non-fermenting Gram negatives ( Stenotrophomonas , Acinetobacter ). Against Gram negative cocci and coccobacilli ( Neisseria , Haemophilus , Moraxella ) their activity is comparable to that of the 2 nd generation.
„5th generation”
There are newer quinolone drugs in different stages of the drug development process, the ones closest to being marketed are prulifloxacin and garenoxacin . The spectrum of these drugs is generally similar to that of the 4 th generation; but they are more active against isolates carrying resistance mutation in the genes of the target molecules, and are less prone to provoke resistance mutations in susceptible strains.
O O COOH F COOH O N O N N N HN garenoxacin O S O N H C H C 3 3 CH3 prulifloxacin F F
Mechanisms of quinolone resistance
1. Mutational alteration of the target Point mutations in the gene of the primary quinolone target causes decreased quinolone binding to the enzyme. In such primary mutants the mutant primary target is less capable of quinolone binding and less prone to inhibition. This may result in a shift of the primary target to the non-mutated other (originally secondary target) enzyme, or simply in a
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primary target (same as the original primary target) enzyme which needs higher drug levels for similar inhibition. Both bring about a moderate increase in the MIC of the quinolone drugs; in most cases these primary mutants remain susceptible. The next step is the occurrence of a new mutation in the gene of the current primary target, i.e. in the gene of the enzyme inhibited more effectively in the primary mutant, either the original primary target now slightly more immune to quinolone action due to the primary mutation or the originally secondary target, which became the primary target through the affinity decrease, which was caused by the primary mutation in the original primary target. This causes the MIC to increase further and redefines the primary target enzyme. This stepwise acquisition of multiple mutations ultimately leads to high level quinolone resistance. The exact number of mutation necessary for high level resistance against a certain quinolone depends primarily on the bacterial species involved. Some species, e.g. Staphylococcus aureus or Pseudomonas aeruginosa may acquire relatively high level resistance through only one mutation. In contrast to other antibacterial classes, in case of quinolones the resistance development is hindered by using not narrower, but broader spectrum agents (later generation instead of the first generation or norfloxacin). The explanation for this is the stepwise manner of resistance development, i.e. the mutant strains with high level resistance (multiple mutations) evolve from strains with lower level resistance (less mutations), surviving quinolone therapy. Generally, the later the generation a quinolone belongs to, the more mutations are necessary to increase the MIC to the resistant category (to become resistant). More modern drugs kill primary mutants readily, preventing the development of further mutations leading to high level resistance. Using earlier generation quinolones carries the danger that primary mutants survive quinolone therapy, and allow for development of strains with multiple mutations and consequently with increasingly higher level resistance. Other resistance mechanisms causing low level quinolone resistance aids the accumulation of mutations and acquisition of high level resistance in a similar manner. (See in detail in the section ‘Pharmacodynamics of resistance development’. The findings described there are mostly drawn from experiments and observations on quinolone resistance.) This mechanism is currently the most frequently found in the background of quinolone resistance both in Gram positives and in Gram negatives; it is the sole known mechanism in case of Mycoplasma spp . 2. Protection of the target Plasmid-borne, easily transferable mechanism. The plasmid coding for this mechanism frequently carries genes causing resistance to other drug classes, e.g ESBL genes. The product of the gene (qnr protein) binds to DNA-gyrase and to topoisomerase IV, protecting them from quinolone binding and action. Though results in relatively low level resistance, its importance lies in that it increases the chance of the survival of primary mutants (see above) contributing to development of high level resistance. It was demonstrated mostly in members of the Enterobacteriaceae family. 3. Decreased quinolone uptake Intracellular quinolone concentration may be lowered by decreased uptake mediated by alteration of the density of porin proteins. This mechanism is coded by the chromosome and is observed mostly in Gram negatives. Provides low level resistance, alone it is insufficient to elevate the MIC above the susceptible breakpoint, but contributes to development of high level mutational resistance through increasing MPC. 4. Active efflux of quinolones This mechanism is chromosomal, may occur both in Gram positives and Gram negatives. Usually provides moderate or high level resistance. It is found most frequently in staphylococci and Pseudomonas aeruginosa . 5. Inactivation through enzymatic modification Recently, a variety of an aminoglycoside acetyltransferase (AAC(6’) mutant) has been described, which is capable of inactivating quinolones containing a piperazine ring substituent (norfloxacin, ciprofloxacin) through acetylation, but ineffective against quinolones not containing a piperazine ring (nalidixic acid or moxifloxacin) or containing methylated piperazine ring (ofloxacin, pefloxacin). This enzyme retains the
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activity against aminoglycosides. Provides very low level resistance, but may contribute to development of high level mutational resistance by increasing MPC and by widening the mutant selection window.
Rifamycins
The target of these compounds CH CH containing multiple rings is the bacterial 3 3 mRNA synthesis; they inhibit the HO O CH DNA-dependent RNA polymerase responsible 3 H3C C O OH O for transcription. Rifamycins inhibit the OH OH CH3 CH3 initiation of transcription, but not elongation. H3C O NH
They are bactericidal; additionally, they have CH3 rifampin N immunomodulatory, antiviral and antitumour O N activity. They penetrate well into tissues due to O OH N O CH good lipid solubility; they reach therapeutic 3 CH3 drug levels in the serous cavities, bile, bone; they penetrate excellently into abscesses and through the placenta and reach the cerebrospinal fluid. The natural rifampin and the semisynthetic rifabutin and rifapentin , as well as the experimental drugs rifalazil and the unabsorbed rifaximin designed for topical use belong here. They are active primarily against Gram positives, staphylococci are especially susceptible. Out of Gram negative aerobes and facultative anaerobes they are active against Neisseria , Haemophilus , Brucella and Legionella ; weakly active or inactive against other Gram negative aerobes and facultative anaerobes. They have excellent antituberculotic activity; they are active against all mycobacteria including atypical mycobacteria and M. leprae . Rifabutin and rifapentin are more active than rifampin against mycobacteria (see in detail in the section ‘Antituberculotic drugs’). They are also active against Chlamydia spp ., Bartonella spp . and rickettsias, but ineffective against Mycoplasma , Ureaplasma and Treponema . They have good activity against strict anaerobes, excepting some commensal clostridia. Though in vitro antifungal activity of rifamycins has been observed, it probably does not have clinical relevance.
Mechanisms of rifamycin resistance
The primary mechanism is the mutational alteration of the target RNA-polymerase. This mechanism is very fast to develop during rifamycin monotherapy; consequently rifamycins should be used as members of combinations. Rifamycins (rifampin) in monotherapy are indicated only for short-term prophylaxis against Neisseria meningitidis . Rifabutin and rifapentin may remain efficacious against certain Mycobacterium strains possessing low level resistance to rifampin.
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Drugs targeting f olic acid metabolism 1. Sulfonamides sulfamethoxazole, sulfaguanidine 2. Trimethoprim Drugs with miscellaneous mechanism of action 1. Metronidazole 2. Nitrofurantoin 3. Methenamine 4. Mupirocin 5. Nitazoxanide
Drugs targeting folic acid metabolism
Sulfonamides COOH O SN R Sulfonamides are synthetic analogues of the 2 H para-amino benzoic acid (PABA), and competitively inhibit the incorporation of PABA into the folic acid by inhibition of dihydropteroate synthase (DHPS). As folic NH 2 NH acid has a crucial role in bacterial metabolism as a carrier 2 of one carbon atom residues (e.g. in biosynthesis of p-aminobenzoic basic structure pyrimidine bases), lack of folic acid leads to inhibition of acid (PABA) of sulfonamides bacterial growth and multiplication. Sulfonamides are therefore bacteriostatic antibiotics. O The mechanism of action and antibacterial spectrum are the same O2SN N in case of all sulfonamides, they differ in pharmacokinetic parameters H (absorption, tissue penetration, distribution) only. Many compounds CH3 CH3 belong to this group, e.g. drugs not absorbed from the gut (bowel decontaminants, e.g. sulfaguanidine ), topical preparations (e.g. eye NH2 drops) and formulas combined with trimethoprim (e.g. sulfamethoxazole , Sumetrolim) are available. They exhibit synergy with sulfamethoxazole trimethoprim and other drugs targeting dihydrofolate reductase (DHFR). Their spectrum is broad; they are active against both Gram positives and Gram negatives, though the rate of secondary resistance is around 50% in case of most clinically relevant species. They are active against Nocardia spp ., actinomycetes, Chlamydia trachomatis , Bartonella spp ., but inactive against bacteria which do not synthesize folic acid and rely on environmental folic acid sources (e.g. Streptococcus pyogenes and enterococci). Sulfonamides are also active against certain protozoa (Toxoplasma , Plasmodium , Cyclospora , Isospora ) and fungi ( Pneumocystis , Paracoccidioides ); Sumetrolim is also used against certain ectoparasites (see below).
Mechanisms of sulfonamide resistance
1. Production of a new target A novel DHPS enzyme is used for sulphonamide synthesis, which binds sulfonamides with a very low affinity. This mechanism is mostly plasmid-borne, and widespread among Enterobacteriaceae . 2. Mutational alteration of the target Certain mutations result in a modified chromosomal DHPS with a low affinity to
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sulfonamides. This mechanism is common in Gram positives. 3. Overproduction of PABA The resulting high PABA concentration shifts the competitive antagonism in favour of PABA, leading to undisturbed folic acid synthesis. 4. Inactivation through modification Sulfonamides are inactivated by acetylation.
O CH Trimethoprim NH2 3 N H N C O CH Trimethoprim is a multisubstituted synthetic 2 3 H2 derivative of benzylpyrimidine. It is a bactericidal drug N O CH3 targeting, similarly to sulfonamides, folic acid trimethoprim metabolism. Its target is the bacterial dihydrofolate reductase (DHFR), which converts inactive dihydrofolate produced in biochemical reactions involving folic acid back into active tetrahydrofolic acid. If this pathway is inactive, the level of active folic acid (tetrahydrofolate) drops rapidly, leading to arrest of biochemical pathways involving folic acid. This is the explanation for the synergy with sulfonamides; both drugs decrease the level of active folic acid available for biosynthetic reactions. Trimethoprim penetrates into most tissues but not into the cerebrospinal fluid; its high levels in the prostate tissue is remarkable. As its teratogenicity is not unequivocal, it is not recommended during pregnancy, but may be used to treat children. Its spectrum is broad; it is active against most Gram positive aerobic and facultative anaerobic bacteria, against Gram negative enterobacteria, Haemophilus spp . and Legionella spp . Its activity is weak against Nocardia spp ., and atypical mycobacteria; it is inactive against Mycobacterium tuberculosis , Neisseria spp . and Moraxella (Branhamella) catarrhalis , Chlamydia spp ., Mycoplasma spp ., and Treponema pallidum . It is used in combination with sulfonamides (Sumetrolim) against the multiresistant nosocomial pathogen Stenotrophomonas maltophilia . The combination is the drug of first choice against certain fungi and protozoa (see below).
A new trimethoprim derivative iclaprim is in NH2 the clinical trial stage of the drug development process; N H N this new derivative can bind to DHFR resistant to 2 N trimethoprim. Iclaprim remains active against isolates O resistant to trimethoprim through mutational alteration iclaprim of the target enzyme. It also has a slightly broader O O spectrum; it is active against Neisseria spp . Moraxella CH3 CH3 (Branhamella) catarrhalis and Chlamydia spp . as well.
Mechanisms of trimethoprim resistance
1. Mutational alteration of the target enzyme The mutant chromosomal DHFR enzyme needs markedly higher trimethoprim concentration to be inhibited. 2. Overproduction of the target enzyme By the overproduction of the target enzyme, the isolate is capable of normal folic acid metabolism evening the presence of extremely high trimethoprim concentrations. 3. Production of a new enzyme The produced new DHFR is trimethoprim resistant and replaces the original in folic acid metabolism. This mechanism is plasmid- or transpozon-borne.
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4. Metabolic bypass This mechanism is based on losing the ability to synthesize timin; this pathway has the highest folic acid demand. Timin is taken up from environmental sources.
Drugs with miscellaneous mechanism of action
Metronidazole metronidazole OH Metronidazole is a nitroimidazole derivative; it exerts its N bactericidal activity after activation as an unknown toxic metabolite. O N CH This activation takes place only in an anaerobic environment; the toxic 2 3 metabolite is produced by a reductive pathway using the cellular electron N transport chain of the anaerobic respiration. Aerobic bacteria are resistant. Metronidazole penetrates well into tissues, including the bile, bone, abscesses and the cerebrospinal fluid; it can cross the placenta. Metronidazole is active only against strict anaerobic and microaerophilic organisms. Out of strict anaerobic bacteria Gram negatives are susceptible, but there are many species possessing intrinsic resistance among Gram positives (Propionibacterium acnes , Actinomyces spp .). Metronidazole is active against Gardnerella vaginalis , Helicobacter spp . and Campylobacter spp . Besides antibacterial activity, it is the drug of first choice against most strict anaerobic protozoa (see below). Resistance is mediated by inhibited uptake or decreased reductive activation. The decrease of reductive activation is based on decreased expression of the genes coding for the proteins involved in the electron transport system. In case of Bacteroides spp . another mechanism of resistance has recently been described. This novel mechanism is based on degradation of the drug without the production of toxic metabolites. Resistance has also been found in protozoa (see below). O Nitrofurantoin O O N C N NH 2 H N Nitrofurantoin is a nitrofurane derivative. It is O bactericidal; its mechanism of action and cellular target is nitrofurantoin unknown. Similarly to nitroimidazoles (metronidazole) it needs activation (but this activation is not dependent on anaerobic respiration), leading to production of active radicals, which in turn damage protein synthesis, respiration and the DNA. It may be antagonistic when combined with quinolones or fluoroquinolones. As it does not reach therapeutic concentration in the serum or in tissues, it is used only to treat urinary tract infections. Its spectrum is broad; it is active against both Gram negatives and Gram positives, but many species (members of the genera Micrococcus , Pseudomonas , Acinetobacter , Stenotrophomonas , Serratia , Proteus ) exhibit natural resistance. Both natural and acquired resistance is probably based on the lack or loss of the enzymatic step producing the toxic metabolite. methenamine Methenamine N
Methenamine is a compound containing four nitrogen atoms in a complicated three N N dimensional ring system. Alone it does not have antibacterial activity, but in acidic urine it N releases formaldehyde, which is a potent antimicrobial agent (see in more detail at the alkylating disinfectants). In neutral or basic urine formaldehyde is not released in sufficient concentration and methenamine remains inactive. As urease production of Proteus spp . increases the urinary pH, methenamine may remain ineffective in infections
136 Antimicrobial chemotherapy: antibacterial agents caused by Proteus spp . It can only be used for urine decontamination, it is inappropriate for systemic treatment. Its spectrum is wide due to the broad spectrum biocidal activity of the formaldehyde; it is active against all pathogens. Resistance has never been found.
Mupirocin
Mupirocin is a natural derivative of a short-chain fatty acid produced by Pseudomonas fluorescens . It inhibits the enzyme binding isoleucine to its specific tRNA (isoleucyl-tRNA synthase). It is bacteriostatic at lower, but bactericidal at higher concentrations. As it is inactivated very rapidly in the body, it is used only for topical treatment. Its spectrum is narrow; it is active against staphylococci, streptococci, Neisseria spp ., Moraxella (Branhamella ) catarrhalis , Bordetella spp . and Haemophilus spp . It is inactive against other Gram negative aerobes or facultative anaerobes, against enterococci, against most Gram positive rods and against strict anaerobic bacteria. It is frequently used to eradicate colonizing MRSA from the nasal cavity.
OH HO O CH3 COOH
H3C O CH3 O O mupirocin OH
Mechanisms of mupirocin resistance
1. Production of a new target This plasmid-borne mechanism is based on production of a isoleucyl-tRNA synthase resistant to mupirocin. It provides extremely high level resistance. 2. Mutational alteration of the target This mechanism is caused by a point mutation in the chromosomal isoleucyl-tRNA synthase gene. Mutant enzyme has lower affinity to mupirocin; this mechanism provides low level resistance.
Nitazoxanide
See in more detail in the section ‘Drugs against protozoa parasitizing body cavities’. It is active primarily against strict anaerobic and microaerophilic bacteria (its activity against clostridia is questionable, it cannot eradicate Helicobacter pylori in monotherapy); it was suggested that it is active also against Gram positive aerobes. Resistance to nitazoxanide has not yet been described.
Drugs inhibiting potential new targets
These drugs are in the preclinical phase of drug development. They include platensimycin, which is an inhibitor of the bacterial (type 2.) fatty acid synthesis; peptidyl-deformylase inhibitors and the drug reutericycline, which selectively dissolves the transmembrane proton gradient.
137 Antimicrobial chemotherapy: antibacterial agents
The most important natural resistances according to pathogens
GRAM POSITIVES polymyxines, 1st gen. quinolones, monobactams, temocillin, mecillinam, all Gram positives ceftazidime, cefsulodin + 3rd gen. cephalosporins staphylococci + oxacillin, norfloxacin, 2nd gen. quinolones, aminoglycosides streptococci oxacillin, all cephalosporins, macrolides, lincosamides, mupirocin, + enterococci sulfonamides, trimethoprim, aminoglycosides + oxacillin, all cephalosporins, mupirocin Listeria GRAM NEGATIVES oxacillin, glycopeptides, lipoglycopeptides, daptomycin, lincosamides, all Gram negatives oxazolidinones, mupirocin, fusidic acid + early penicillins, 1 st gen. cephalosporins Haemophilus, Moraxella + early penicillins, macrolides, lincosamides, streptogramins, rifamycins enterobacteria + 1st gen. quinolones, polymyxins, tetracyclines, nitrofurantoin P. mirabilis aminopenicillins, 1 st and 2 nd generation cephalosporins, + other Proteus spp . 1st gen. quinolones, polymyxins, tetracyclines, nitrofurantoin aminopenicillins, carboxipenicillins, ureidopenicillins, 1 st gen. + Klebsiella spp. cephalosporins + aminopenicillins, 1 st gen. cephalosporins Citrobacter spp. + aminopenicillins, 1 st ge. cephalosporins, polymyxines Serratia spp. + aminopenicillins, amox+clav., ampi+sulb., 1 st gen. cephalosporins Enterobacter spp. early and aminopenicillins, amox+clav., ampi+sulb., temocillin, 1 st , 2 nd and 3 rd gen. cephalosporins (except ceftazidime), ertapenem, + Pseudomonas aeruginosa chloramphenicol, macrolides, lincosamides, streptogramins, 1 st gen. quinolones, rifamycins all β-lactam drugs, macrolides, lincosamides, streptogramins, Stenotrophomonas + tetracyclines and glycylcyclines, 1st gen. quinolones, norfloxacin, maltophilia rifamycins, aminoglycosides penicillin derivatives (except pip+tazo.), cephalosporins (except ceftazidime), imipenem, ertapenem, macrolides, lincosamides, + Burkholderia cepacia streptogramins, all quinolones, polymyxins, aminoglycosides, rifamycins early and aminopenicillins, amox+clav., temocillin, 1 st and 2 nd gen. + cephalosporins , macrolides, lincosamides, streptogramins, 1 st gen. Acinetobacter spp. quinolones, norfloxacin, rifamycins + β-lactams Legionella spp. STRICT ANAEROBES aminoglycosides, 1 st and 2 nd gen. quinolones, norfloxacin all strict anaerobes + all cephalosporins (except cefamycins), sulfonamides, trimethoprim Bacteroides spp. + metronidazole Propionibacterium spp. + β-lactams, lincosamides Clostridium difficile MYCOPLASMA SPP. cell wall active drugs, aminoglycosides, rifamycins all Mycoplasma spp. + 14-membered macrolides, azalides M. hominis RICKETTSIA and EHRLICHIA SPP. cell wall active drugs, aminoglycosides, sulfonamides, trimethoprim all Rickettsia and Ehrlichia + macrolides spotted fever Rickettsia spp. + macrolides, chloramphenicol Ehrlichia spp. CHLAMYDIA SPP. polymyxines, aminoglycosides; (cell wall active drugs, chloramphenicol, all Chlamydia spp. lincosamides, sulfonamides has weak activity)
138 Antimicrobial chemotherapy: antituberculotic agents
Antituberculotic agents
Though the agents against mycobacteria (antituberculotic agents) may belong to drug classes already covered, hereby we discuss them separately. The reason for this is that the clinically most important mycobacteria are naturally resistant to most known antibacterial agents (e.g. to β-lactam drugs or to glycopeptides); moreover, many drugs penetrate poorly into the tubercolous granulomas and into the infected macrophages. These necessitate a chemotherapeutic approach different from that used in case of other bacterial infections. Natural resistance of mycobacteria to many antibiotics and biocides is the result of their highly hydrophobic cell wall, which serves as a barrier to many drugs and biocides, but other mechanisms, similar to those found in other bacteria (e.g. production of β-lactamases), also play a role in their resistance. Further difficulties derive from the fact that during an infection caused by mycobacteria, different subpopulations with different characteristics are present at the same time in the host. Members of these populations may easily be converted into members of other populations by change of phenotype. These populations have different physiological properties, have different growth and multiplication rates and these affect their susceptibility to antituberculotic drugs, leading to large differences in the drug susceptibility of these subpopulations. These populations are
1. an actively growing and replicating subpopulation, 2. a non-growing and non-replicating subpopulation with a slowed metabolism, and 3. a slowly growing intracellular subpopulation.
Cells belonging to the actively growing subpopulation are susceptible to most antituberculotic agents; in vitro susceptibility results reflect mainly the susceptibility of this population. Non-growing cells show phenotypic resistance to most antituberculotics (except pyrazinamide), these population may be eradicated only when the cells undergo a phenotypic change and start growing (enter another subpopulation). The slowly growing subpopulation exhibit decreased susceptibility to certain drugs, and their intracellular position offers additional protection as many drugs do not enter the intracellular space and thus cannot reach this subpopulation. A further difficulty arises from secondary resistance development during antituberculotic therapy due to the long duration of the treatment (from six months to even years), the risk of which is especially high when using monotherapy. For these reasons, antimycobacterial treatment is performed combining more than one drug. Resistance to antituberculotics is almost always chromosomally encoded, as members of the Mycobacterium tuberculosis complex ( M. tuberculosis , M. bovis , M. africanum, etc. ) do not carry plasmids. Therapeutic failure arises frequently from antibiotic tolerance as well. In case of mycobacteria the tolerance is attributed to the permanent presence of non-replicating mycobacteria in the host, against which the antituberculotic drugs are ineffective or active only weakly.
Antituberculotic susceptibility testing
As a result of the peculiar properties of mycobacteria, susceptibility testing is difficult, and differs markedly from traditional susceptibility testing in methodology. In case of Mycobacterium leprae , which is not cultivable on artificial media, traditional susceptibility testing is impossible. To test the susceptibility of fast-growing atypical mycobacteria
139 Antimicrobial chemotherapy: antituberculotic agents traditional techniques may be used, but in case of slow-growing species (including the M. tuberculosis complex ) the majority of these cannot be used to detect resistance. In case of slowly growing species techniques using serial dilutions (agar-, micro- and macrodilution) can be used, but there are special methods used only to test mycobacteria.
The proportion method
This method is used exclusively for susceptibility testing of slowly growing mycobacteria ( M. tuberculosis complex ). The principle of the method is the comparison of the colony numbers produced by the same inoculum on a medium containing the drug to be tested and on drug-free medium for multiple inocula containing different dilutions of the organism (different CFU numbers). This may be performed using a culture (indirect method) or a Ziehl-Neelsen positive specimen (direct method). The given strain is resistant if the colony number found on the medium supplemented with the drug reaches a certain percentage of the colony number found on the drug-free medium. Disadvantages of the method are its being labour-intensive and consuming many media. Besides, the results are not always reliable. Newer guidelines do not recommend the usage of the proportion method.
The radiometric method
This method is based on using the higher breakpoint (the breakpoint for resistance) in the test. Strains capable of growing in this critical concentration are considered resistant to the antituberculotic agent tested. Detection of growth is performed using radiometry, by 14 14 measuring the CO 2 liberated during the metabolism of fatty acids marked with C added to the test medium.
MGIT (Mycobacterium Growth Indicator Tube) method
The principle of the technique is similar to that of the radiometric method, but detection is based on demonstration of oxygen consumption during metabolism of mycobacteria. The test tube contains a fluorophore, which emits fluorescence upon decrease of oxygen concentration, and this fluorescence is detected. Similar methods exist, which are based on non-radiometric detection of CO 2 production rather then on measuring oxygen consumption.
140 Antimicrobial chemotherapy: antituberculotic agents
Antituber culoti c agents 1. first-line drugs 1.1. Isonicotinic acid hydrazide (isonicide, INH) 1.2. Rifamycins (RIF) rifampin, rifabutin, rifapentin 1.3. Pyrazinamide (PZA) 1.4. Ethambuthol (1.5. Streptomycin) it is presently considered a second-line drug 2. second-line drugs 2.1. Fluoroquinolones ofloxacin, moxifloxacin 2.2. Paraamino-salicylic acid (PAS) 2.3. Cycloserin 2.4. Ethionamide, prothionamide 2.5. Capreomycin, viomycin 2.6. Aminoglycosides amikacin, kanamycin, (streptomycin) 2.7. Thioacetazone 3. drugs against leprosy 3.1. Dapsone 3.2. Clofazimine 3.3. Rifamycins rifampin 3.4. Tetracyclines doxycycline, minocycline 3.5. Macrolides clarithromycin 3.6. Fluoroquinolones ofloxacin 4. drugs against atypical mycobacteria 4.1. Macrolides clarithromycin, azithromycin 4.2. Ethambuthol 4.3. Rifamycins (rifampin), rifabutin, rifapentin 4.4. Amikacin 4.5. Fluoroquinolones ofloxacin, moxifloxacin 4.6. Tetracyclines doxycycline, minocycline
First-line antituberculotics
Drugs with the best antituberculotic activity belong to this group; primarily these are used in combinations used for treatment of tuberculosis and for chemoprophylaxis. If the isolate is susceptible, the drugs used in the combination therapy should be chosen from this group. If the susceptibility is unknown, a combination of these drugs is used for empirical therapy. The most widely used combination against susceptible tuberculosis is INH+rifampin+pyrazinamide.
Isonicotinic acid-hydrazide (isoniazide, INH) N
Chemically INH is a pyridine derivative with a structure similar to nicotinamide. To exert its bactericidal activity it should be converted into an O unknown toxic metabolite; activation is due to the bacterial enzyme N NH2 catalase-peroxidase. The primary target of the active metabolite is the synthesis H of the mycolic acids responsible for the high hydrophobicity and the INH acid-fastness of the cell wall. INH is active against members of the M. tuberculosis complex , but inactive against most atypical mycobacteria and agains M. leprae . M. bovis is slightly less susceptible than M. tuberculosis . INH is active exclusively against the actively growing subpopulation.
141 Antimicrobial chemotherapy: antituberculotic agents
Mechanisms of INH resistance
1. Alternation of the activating enzyme This mechanism is based on loss or decrease of the activity of the activator catalase-peroxidase enzyme (or sometimes on mutational alteration of the enzyme activity). Though providing high level resistance, mutations leading to total loss of enzyme activity are extremely rare in clinical isolates, as this enzyme has an important role in virulence (in protection of the cell against the oxidative attack of host macrophages by detoxifying oxygen radicals). 2. Mutational alteration of the target enzyme This mechanism involves a mutation in one of the genes coding for a member of the enzyme complex responsible for mycolic acid synthesis, rendering the enzyme resistant to INH-mediated damage. Most frequently the enoyl-acylcarried reductase gene ( inhA ) and the ketoacid synthase genes are affected. These mutations cause low level resistance; against such mutants a high dose INH therapy may remain effective. The mechanism provides cross-resistance to ethionamide as well. 3. Overproduction of the target enzyme In case of overproduction of a key enzyme in the mycolic acid biosynthetic apparatus, the INH concentration available in vivo is not sufficient for effective inhibition of mycolic acid synthesis, consequently bacteria are not killed. This mechanism is based on mutations in the promoter region of genes coding for mycolic acid synthesis. The mechanism provides cross-resistance to ethionamide as well.
Rifamycins
Rifampin (RIF), rifabutin and CH3 CH3 rifapentin belongs to this group; they were HO discussed in the section ‘Antibacterial O CH3 H C C O OH O agents’ (structure of rifampin is shown 3 OH O CH3 CH there). A newer member of the group under 3 H C O NH clinical trials is the rifalazil. They exert their 3 CH rifabutin bactericidal activity by inhibition of the 3 O NH initiation of transcription. O N CH3 They are active against the M. O CH N C C CH 3 H 3 tuberculosis complex , against M. leprae and H2 against most atypical mycobacteria. Its combination with INH proved sufficient to CH3 CH3 eradicate M. tuberculosis from the tissues in HO O a murine model. Rifabutin and rifapentin CH3 H3C C O OH O may remain active against isolates resistant OH O CH3 CH3 to rifampin. This is exemplified by the H3C O NH natural resistance of M. avium-intracellulare CH3 rifapentin N complex , as these are naturally resistant to O N rifampin but susceptible to rifabutin and O OH N O rifapentin. They are active against the CH actively growing and the intracellular 3 subpopulations, but not against the non-growing subpopulation. In case of mycobacteria, the background of rifamycin resistance is mostly the
142 Antimicrobial chemotherapy: antituberculotic agents mutational alteration of the target RNA-polymerase; some mutations provide resistance solely to rifampin, while others provide cross-resistance to other rifamycins. This cross-resistance affects rifapentin more frequently than rifabutin. Rarely permeability changes may be observed as causes of resistance. Low level natural resistance of the apathogenic commensal M. smegmatis is mediated by enzymatic modification (ribosylation) and consequent inactivation of the drug.
Pyrazinamide
Pyrazinamide (PZA) is a pyrazine derivative, a structural analogue of nicotinamide. It is bactericidal, and it has excellent activity against the non-replicating subpopulation. To exert activity it should be activated by the enzyme pyrazinamidase, the resulting toxic metabolite probably targets the energy metabolism of the bacteria. It is more active in vivo than in vitro . Anaerobic conditions enhance its activity. Pyrazinamide is active exclusively against M. tuberculosis , it is inactive N against M. bovis , M. leprae , or atypical mycobacteria. Natural resistance of M. N bovis and certain atypical mycobacteria is due to possessing a pyrazinamidase O which is unable to convert pyrazinamide into the toxic metabolite. Other NH2 species with natural resistance do not take up pyrazinamide due to lack of the pyrazinamide necessary transport system. PZA Acquired pyrazinamide resistance maybe mediated by decreased pyrazinamidase activity due to mutational or gene expression alternations or by decreased pyrazinamide uptake.
Ethambuthol
Ethambutol is an aliphatic diamine. Its target is the OH arabinogalactane synthesis, which serves as an anchor for the H incorporation of mycolic acids into the cell wall. H C N 3 N CH Ethambuthol is bacteriostatic. H 3 It is active against the M. tuberculosis complex and ethambuthol OH against most atypical mycobacteria, but inactive against M. leprae and against fast-growing species. It is active against the actively growing subpopulation only. Resistance is attributed to overproduction or mutational alteration of the target enzyme.
Streptomycin
Streptomycin is an aminoglycoside antibiotic with bactericidal action (see also in the section ‘Aminoglycosides and aminocyclitoles’). According to new data, its efficacy is weaker than previously postulated; for this reason (and due to its marked toxicity) its usage is not recommended and was reclassified as a second-line drug (see below).
Second-line antituberculotics
This group include drugs with weaker antituberculotic activity and/or significant toxicity. These drugs are applied when an effective combination cannot be designed exclusively from first-line drugs due to resistance of the isolates involved. In these cases the
143 Antimicrobial chemotherapy: antituberculotic agents combination to be used for treatment will contain the effective first-line drug(s) complemented with one or more second-line agents. Fluoroquinolones
See in more detail in the section ‘Antibaterial agents’. Fluoroquinolones are bactericidal against mycobacteria; they target the DNA-gyrase (and topoisomerase IV as a secondary target). Fluoroquinolones of the 2 nd , 3 rd and the 4 th generation are the most effective antituberculotics; the most active agent is moxifloxacin . Many mycobacteria including the M. tuberculosis complex exhibit low level generic fluoroquinolone resistance due to poor penetration. Fluoroquinolones are totally inactive against certain fast-growing atypical mycobacteria ( M. chelonae , M. abscessus ). Ofloxacin (but not ciprofloxacin) is used against M. leprae . Resistance is mediated by the mutation of the target enzyme; this mechanism provides high level resistance. Active efflux of fluoroquinolones may also occur, causing low level resistance.
Paraamino-salicylic acid (PAS) NH 2 The putative target of PAS is folic acid synthesis; its action is probably similar to sulphonamide action. It is a bacteriostatic agent; its action is specific to mycobacteria. It is active only against the M. tuberculosis complex . OH It is inactive against the intracellular subpopulation. Due to its numerous side COOH effects it is rarely used. paraamino- salcylic acid (PAS) Cycloserine
Cycloserine is an analogue of D-alanine; it interferes with the O biosynthesis of the pentapeptide crucial for cell wall synthesis H N 2 NH (transpeptidation). It is active against most bacteria, but solely used as a H O second-line antituberculotic drug due to its marked toxicity. It is bacteriostatic against mycobacteria, active against all Mycobacterium cycloserine species excepting the fast-growing species and M. leprae. Resistance is due to mutational alteration of the enzyme responsible for biosynthesis of the D-alanine-D-alanine dipeptid (which comprises a part of the pentapeptide involved in transpeptidation.
Ethionamide, prothionamide
Ethionamide and prothionamide are thioether derivatives of the isonicotinic acid. Their putative target is the mycolic acid N R biosynthesis; their mechanism of action is probably similar to that of the active metabolite of INH (see above). They are weakly bactericidal. They are active primarily against the M. tuberculosis S complex , also show activity against M. leprae . INH resistance caused NH2 by mutational alteration or overproduction of inhA provides R=C 2H5: ethionamide cross-resistance to these drugs, but strains with INH resistance due to R=C 3H7: prothionamide altered or missing activation remain susceptible to ethionamide and prothionamide.
144 Antimicrobial chemotherapy: antituberculotic agents
Capreomycin, viomycin
Oligopeptide antituberculotics, they interfere with the normal function of both ribosomal subunits leading to bactericidal action. Capreomycin is probably active against the non-replicating subpopulation, viomycin has not yet been investigated in this regard. Viomycin is the more toxic drug. Both are active only against the M. tuberculosis complex . OH R NH2 O O H H NH2 N H2N N NH2 HN OH N N H H H N O O O H O NH O N HN O H H H NH2 O N N NH2 H2N N N N H O H H H H H O O O O NH HN viomycin capreomycin HO N NH H2N N H Resistance is mediated by mutations in the 16S rRNA gene or by loss of the natural methylation of the ribosomal binding site (mutational loss of activity of the enzyme responsible for methylation of the site). Certain mutations provide cross-resistance between the two drugs; some rRNA mutations (but not mutations in the methylase enzyme gene) result in cross-resistance to kanamycin and amikacin; streptomycin susceptibility is never affected.
Amikacin, kanamycin, streptomycin
Aminoglycoside antibiotics, they exert their activity by interfering with peptide synthesis (see above). Streptomycin was the first antituberculotic in clinical use, for this reason it had for long been used as a first-line drug. They do not cross the blood-brain barrier and are ineffective at acidic pH; consequently they are unsuitable to treat tuberculous meningitis and are inactive against bacteria located within the granulomas. They are effective only against the actively replicating subpopulation. They are active against the M. tuberculosis complex and against atypical mycobacteria, but not against M. leprae . Kanamycin has weaker activity against certain fast-growing atypical species ( M. abscessus , M. chelonae , M. fortuitum ). Resistance develops through mutational alteration of the 16S rRNA or one of the ribosomal proteins; cross-resistance is partial between aminoglycosides (amikacin may remain active against some kanamycin-resistant isolates.) Certain mutations in the 16S rDNA may provide cross-resistance to capreomycin and viomycin.
Thioacetazone H Thioacetazone is a thiosemicarbazone derivative acting N NH2 O N on an unknown target and with severe toxicity. It inhibits mycolic S acid biosynthesis, but its action is only bacteriostatic. It is active H C N against the M. tuberculosis complex . Due to its low price it is in 3 H use in developing countries, but in developed countries it is not thioacetazone marketed.
145 Antimicrobial chemotherapy: antituberculotic agents
Principles of antituberculotic therapy
As described in the introductory paragraphs, it is extremely important that the therapy applied should be effective against all three subpopulations. At the early period of therapy, the main aim is to rapidly decrease the number of actively growing cells, as these are responsible for the release of mycobacteria. Killing these cells will decrease and eventually stop the release and consequently cease disease transmission. This therapeutic period is the period of intensification therapy ; a threefold combination is commonly used (the first choice is INH+rifampin+pyrazinamide). After several weeks (or months) of therapy, the patients stop releasing bacteria; the further aim of the therapy is to eradicate the intracellular and the non-replicating subpopulations to avoid relapse or recurrence. In this period of maintenance therapy , the most toxic INH may be omitted and commonly the therapy is continued with rifampin+pyrazinamide for at least one or two months. Unfortunately, resistance to antituberculotics is a continuously growing concern, especially in developing countries. Resistance to some or most first-line drugs became frequent in some regions. We refer to multidrug resistant (MDR) mycobacteria , if they are resistant to at least two first-line drugs, commonly these are INH and rifampin. Against these isolates the most widely used threefold combination (INH+rifampin+pyrazinamide) is not efficacious, necessitating the use of second-line drugs. In case of MDR M. tuberculosis , therapy should only be conducted with at least five drugs proven to be active with in vitro susceptibility testing, because otherwise the risk of development of further resistance is very high and the consequent therapeutic failure cannot be avoided. Due to neglecting of these guidelines, new multiresistant strains appeared. These are called extensively drug resistant (XDR) M. tuberculosis , and are resistant not only to first-line drugs, but to a number of second-line drugs as well. Against XDR M. tuberculosis reliably effective therapy is presently unavailable, though anecdotal reports on successful therapy were published. However, most infections by these strains are finally fatal. With the further worsening of the resistance situation, extreme drug resistant (XXDR) M. tuberculosis strains appeared, against which only one or two drugs are active. Presently effective combination against these strains definitely cannot be composed.
Drugs against leprosy
Dapsone
Dapsone chemically is di-aminophenyl-sulfone. Its mechanism of action is thought to be similar to that of sulfonamides, its action is weakly bactericidal. This drug is the backbone of the therapy against leprosy, but cannot be used as a O monotherapy. It is combined with rifampin, or in case of H N 2 S NH2 multibacillary form with rifampin and clofazimine. O Resistance to dapsone is mediated by mutational alteration of dapsone the DHPS. Cl clofazimine Clofazimine H C CH 3 3 Clofazimine is a phenazine derivative. Data are Cl N N lacking on its mechanism of action; it is a weakly bactericidal agent. N N H
146 Antimicrobial chemotherapy: antituberculotic agents
Rifamycins (rifampin)
Their mechanism of action is discussed above. They are rapidly bactericidal against M. lepra , but resistance is fast to develop (see above).
Other drugs
Against M. leprae tetracyclines ( doxycycline , minocycline ), clarithromycin and certain fluoroquinolones (ofloxacin , pefloxacin ; ciprofloxacin is inactive), but these are not routinely used, mostly because of their cost. They may be indispensable as second-line drugs. Similarly, ethionamide and prothionamide are sometimes also used as second-line drugs. The efficacy of the combination of rifampin+minocycline+ofloxacin is presently under investigation; the combination seems to have good clinical efficacy, but the relapse rate to be expected after cessation of the therapy is yet unknown.
Drugs against atypical mycobacteria
Macrolides
Out of the macrolide family, clarithromycin and azithromycin are used against atypical mycobacteria. Their target and mechanism of action is the same as described above. They are active against most atypical mycobacteria (but not against M. tuberculosis complex or M. leprae ). Resistance develops through mutations in the 23S rRNA gene.
Miscellaneous drugs
Many first- and second-line antituberculotic agents ( ethambutol , rifamycins , amikacin , fluoroquinolones ) may also be used against atypical mycobacteria. Susceptibility is variable among species; against the clinically most important M. avium-intracellulare complex ethambutol , rifabutin (but not other rifamycins), amikacin and fluoroquinolones may be used, besides the first choice macrolides. Against the fast-growing M. chelonae and M. abscessus , besides macrolides, amikacin or possibly tigecycline proved to be effective. Against certain species ( M. fortuitum , M. marinum ) tetracyclines are also active. Linezolid is considered a possible alternative against all atypical species, but lack of clinical experience precludes drawing firm conclusions on efficacy of linezolid.
Other drugs
Usage of β-lactam antibiotics and sulfonamides had been suggested as well. As all mycobacteria are β-lactamase producers, only lactamase-stable drugs may have therapeutic value. As β-lactams penetrate poorly into the intracellular space, they are frequently ineffective even against strains appearing susceptible in vitro due to the intracellular localization of mycobacteria. Though in some infections caused by certain atypical species (M. fortuitum , M. abscessus ), cefoxitine and imipenem proved to be efficacious, presently available β-lactams are not considered suitable for therapy of infections caused by mycobacteria. Regarding sulfonamides even less information is available, but they might be efficacious against a few atypical species.
147 Antimicrobial chemotherapy: antifungal agents
Antifungal agents
Fungal infections have for long been thought to belong almost exclusively to the field of dermatology, systemic mycoses were extremely rare. However, with the constant increase of immunocompromised patients highly susceptible to opportunistic infections including systemic fungal infections, this situation underwent a profound change; life-threatening (and frequently fatal) fungal infections are becoming increasingly prevalent. These fungal infections are sometimes caused by extremely rare species, when resistance patterns are unknown due to the lack of available data. Against some species (e.g. Fonsecaea pedrosoi or Scedosporium prolificans ), none of the marketed antifungals used in systemic infections show sufficient activity. As many fungal species and diseases have recently emerged, it is worth overviewing the groups of human fungal infections and their causative agents.
• Infections of the skin and keratinized structures (never disseminate) – dermatophytoses (Trichophyton , Epidermophyton , Microsporum ) – other dermatomycoses (Candida , Aspergillus , Scopulariopsis , Alternaria ) – pityriasis versicolor (Malassezia )
• mucosal mycoses – candidiasis
• subcutaneous mycoses – sporotrichosis (Sporothrix schenkii ) – chromomycosis (fungi producing melanin: Cladophialophora , Fonsecaea , Wangiella , Exophiala , etc.) – eumycetoma (Madurella , Acremonium , Pseudallescheria , rarely Aspergillus , Fusarium , or causative agents of chromomycosis)
• invasive mycoses – candidiasis – cryptococcosis – other yeast infections (Rhodotorula , Saccharomyces , Trichosporon , Hansenula , Blastoschizomyces , Sporobolomyces ) – endemic systemic mycoses (Histoplasma , Coccidioides , Paracoccidioides , Blastomyces ) – opportunistic penicillosis (Penicillium marneffei ) – mucormycosis (zygomycosis) (Mucor , Rhizomucor , Rhizopus , Absidia , Cunninghamella ) – entomophthoramycosis (Basidiobolus , Conidiobolus ) – hyalohyphomycosis (Aspergillus , Fusarium , Paecilomyces , Pseudallescheria , Acremonium , etc.) – phaeohyphomycosis (Scedosporium , Cladophialophora , Bipolaris , Wangiella , Alternaria , Fonsecaea , Exophiala , Curvularia , Scopulariopsis , etc.)
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Out of the abovementioned pathogens, limited and frequently contradictory data are available on rare yeast infections or hyalohyphomycoses caused by rare fungal species as well as on phaeohyphomycoses. As activity and clinical efficacy of antifungals is not established in case of these fungal pathogens, they are not discussed here in detail. Many controversies exist in case of the therapy against aspergillosis and fusariosis as well. Due to their eukaryotic cellular structure, antibacterial agents are inactive against fungi, necessitating specific antifungal agents. However, the number of available clinically approved and marketed antifungals is very low, especially when compared to that of antibacterial agents. This has two causes; on one hand due to relatively low clinical importance of fungal infections in the past, intensive research on antifungal agents did not seem justified and was not performed. On the other hand, as the pathogen and the host shares many cellular biochemical properties both being eukaryotes, it is harder to find potential targets that can be inhibited selectively. (In other words, the number of biochemical processes unique to fungi is limited, and the risk that a drug will not be selective enough and will have unacceptable toxicity is higher due to biochemical similarity of many potential target processes.) In spite of the differences in targets and mechanisms of action, resistance mechanisms to antifungal and antibacterial agents are very similar. In case of fungi, resistance is always encoded on the chromosome; acquired resistance develops under selection pressure exerted by the antifungal, transmission of resistance between strains does not occur, in contrast to what seen in case of bacteria. Moreover, there is a profound difference between fungi and bacteria regarding interpretation of in vitro susceptibility data. In case of bacteria in vitro susceptibilities excellently predict the expectable therapeutic success (in case of in vitro susceptibility) or failure (in case of in vitro resistance), and based on the in vitro antibiogram, an expectably effective drug can almost always be suggested. In contrast, in fungal infections in vitro susceptibility testing has lower reliability to predict therapeutic outcome. The cause of this is twofold; most fungal infections are opportunistic infections, occurring in patients with seriously impaired immune function or with serious underlying diseases, when outcome is depends highly on the improvement of the underlying condition (recovery of the immune function). On the other hand, much fewer data are available on therapy and treatment outcome of fungal than of bacterial infections.
Antifung al agents 1. Polyenes 1.1. Amphotericin B 1.2. Nystatin 1.3. Natamycin 2. Azoles 2.1. Imidazoles clotrimazole, miconazole, ketoconazole 2.2. Triazoles 2.2.1. Fluconazole 2.2.2. Itraconazole 2.2.3. Voriconazole 2.2.4. Posaconazole 3. 5-fluorocytosine 4. Allylamines terbinafine, naftifine 5. Morpholines amorolfine 6. Griseofulvin 7. Echinocandins caspofungin, micafungin, anidulafungin
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The above list of the antifungal classes is not exhaustive. It includes all classes of agents used for systemic therapy, but some less important drugs used only topically (e.g. cyclopyrox olamine or tolnaftate ) has been omitted. These drugs are now used rarely, due to the widespread use of newer agents; and their mechanisms of action or mechanisms of resistance against them are virtually unknown.
Polyenes
Their chemical structure is characterized by a polydesaturated ring with a hydrophilic and a hydrophobic part. Fungicidal drugs, but at lower concentrations they are only fungistatic. Their mechanism of action is pore formation in the fungal membrane. Multiple polyene molecules bind to membrane sterols; their hydrophobic parts are incorporated into the lipid bilayer, while their hydrophilic parts form a hydrophilic pore in the membrane, leading to increased permeability and eventually cell death. Their selectivity arises from better binding to fungal sterols (mainly to ergosterol) than to mammalian sterols (cholesterol). Due to non-selective binding to mammalian sterols, their toxicity is marked. They are not absorbed from the gastrointestinal tract, parenteral use is necessary. As the clinical utility of different polyenes differs in many aspects, they are discussed separately.
Amphotericin B OH O OH OH OH OH O HOOC Amphotericin B is a OH he ptaenic polyene, slightly HO less toxic than other O O OH polyenes; therefore it may be used for systemic treatment. HO OH amphotericin B Nevertheless, it has NH significant toxicity (mainly 2 nephrotoxicity) arising from binding to mammalian cell membrane cholesterol and to its ability to form pores in the mammalian membranes as well. Besides antifungal activity, it modulates the immune response; causes alterations in the granulocyte function and leads to cytokine release. The role of these immunmodulatory activities in antifungal action is unknown. It is not absorbed when administered orally, but systemically given it crosses the placenta, but does not penetrate to the cerebrospinal fluid. It shows synergy when combined with 5-fluorocytosine. This synergy arises from the better penetration of 5-fluorocytosine through the pores formed by amphotericin B. Recently lipid associated amphotericin B formulations (liposomal, lipid complexed, etc.) have been marketed; these have the advantage of better tissue penetration and better tolerability profile. Spectrum of the traditional and different lipid formulations does not differ; these formulations are also inactive against amphotericin B resistant isolates. Spectrum of amphotericin B is broad; it is active against yeasts, moulds and dimorphic fungi. It has excellent activity against fungi causing endemic systemic mycoses, Cryptococcus neoformans , most Candida and other yeast species, Sporothrix schenkii, aspergilli (excepting A. terreus ), against Penicillium marneffei , pathogens of zygomycosis (except for Cuninghamella bertholletiae ) and against dermatophytic fungi. It is only weakly active against Fusarium spp . Some moulds ( A. terreus , C. bertholletiae , Scedosporium prolificans and Pseudallescheria boydii ) exhibit natural resistance, as well as Sporothrix , Geotrichum, Trichosporon , and some Candida lusitaniae , C. lipolytica and C. guilliermondii strains, together with the fungi causing chromomycosis (as well as causing phaeohyphomycosis: Cladophialophora , Fonsecaea , Exophiala , Wangiella , etc).
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It is active against certain protozoa, e.g. against Trichomonas , Leishmania , Naegleria and Acanthamoeba species. OH OH O Nystatin H3C OH
HO O OH OH OH OH O Nystatin is a hexaenic CH3 COOH polyene. It is not absorbed H C when administered orally; it is 3 O O CH used for topical treatment in 3 oral or gastrointestinal nystatin candidiasis mainly. Due to its HO OH toxicity, it is not used for NH2 systemic treatment. Its spectrum is broad; it is active against Candida spp ., Cryptococcus spp ., against fungi causing endemic systemic mycoses as well as against dermatophytic fungi and aspergilli. Regardless of its activity, itsmarketing was ceased.
Natamycin O OH O OH It is a pentaenic toxic antifungal. Due to H C O OH O its toxicity it is used only for topical treatment, 3 COOH mostly to treat vulvovaginal candidiasis; it is unsuitable for systemic treatment. Its spectrum O O CH is broad, it is active against yeasts and moulds; 3 it is suitable for treatment against Candida spp ., natamycin aspergilli and dermatophytic fungi. It has HO OH NH antprotozoal activity as well; it is used also 2 against Trichomonas vaginalis .
Mechanisms of resistance to polyenes
1. Alteration of the target Sterol composition of the membrane changes in polyene resistant isolates; ergosterol content is decreased, leading to decreased binding of polyenes to the membrane. The biochemical background is the loss of one of the enzyme activities involved in ergosterol synthesis. Cross-resistance to azole antifungal agents has been described in polyene resistant laboratory strains, but it is likely to be rare in polyene resistant clinical isolates. 2. Compensation of the damage done by the pore formation (protection of the cell) Increased catalase activity may contribute to polyene resistance through protecting the cells more efficiently from oxidative stress caused by pore formation.
Acquired polyene resistance is rare, and has been described mostly in Candida spp ., but has also been found in case of Cryptococcus neoformans and Aspergillus flavus . Biochemical and genetic background of resistance, especially in moulds, is not well understood.
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Azole antifungals
Two basic chemical structures are possible, older agents are imidazole, while newer ones are triazole derivatives. Azoles are fungistatic agents; their target is the inhibition of ergosterol synthesis through inhibition of 14-α-lanosterol-demethylase by binding to the enzyme cofactor cytochrome P 450 . As ergosterol play a key role in regulation of the membrane function, its loss leads to membrane disfunction and inhibition of growth. Besides demethylase, other enzymes involved in the ergosterol synthesis may be additional targets, but these are not inhibited by all azoles and are variable among species as well. (E.g. itraconazole also inhibits C 14 -sterol reductase, but only in case of Cryptococcus neoformans and Histoplasma capsulatum .) Azoles may be fungicidal against certain (mostly yeast) species, due to accumulation of toxic sterol by-products. Antifungal activity of azole-type antifungals is highly concentration dependent. For this reason, even less susceptible isolates may be eradicated with higher doses; therefore in case of azole antifungals, the category for isolates with decreased susceptibility, but without manifest resistance is called dose-dependent susceptible , instead of intermediate susceptible used in case of bacteria or in case of other antifungal classes. In other words, this category corresponds to the intermediate category, but expresses that though the isolate possesses decreased susceptibility (cannot be treated with normal therapeutic doses), the antifungal in question is suitable for eradication if used in higher than standard doses. (Naturally, against resistant isolates increasing drug dose will not lead to therapeutic success, as effective therapeutic concentration cannot be reached in vivo .)
Imidazoles Cl These include the topically Cl O used econazole, miconazole and N N clotrimazole. These are not used N R Cl for systemic treatment due to their N toxicity. For systemic treatment ketoconazole was used, despite its R=H: econazole clotrimazole Cl frequent side effects. Ketoconazole R=Cl: miconazole is orally bioavailable, but does not Cl penetrate to the cerebrospinal fluid. O Cl It should not be combined with C O N N CH H C O H 3 amphotericine B due to the possible 2 2 O antagonistic interaction. Its use is N ketoconazole outdated, and it has been replaced N by triazole drugs. Spectrum of imidazoles is broad; they are active against dermatophytones, Candida spp ., Malassezia spp . and against fungi causing endemic systemic mycoses, except Coccidioides immitis . Less susceptible fungi are aspergilli, Cryptococcus neoformans and Sporothrix schenkii ; ketoconazole is not suitable for treatment of infections caused by these species. Fungi causing mucormycosis possess natural resistance. Ketoconazole is also active against certain Leishmania species and against Acanthamoeba .
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Triazoles OH N N Triazoles have the great advantage of an acceptable N CH CH N N 2 2 N toxicity profile compared to imidazoles and polyenes. They F are orally bioavailable. This group contains fluconazole , itraconazole , voriconazole and posaconazole ; some other fluconazole drugs are under development. F The spectrum of fluconazole is markedly different from those of other triazoles; it is active mainly against yeasts, including most Candida species, with weaker activity against C. glabrata . C. krusei and C. inconspicua possess natural fluconazole resistance (but in vitro this does not always exceed the MICs corresponding to the dose-dependent category. It is also active against Cryptococcus neoformans , Sporothrix schenkii , fungi causing endemic systemic mycoses and dermatophytic fungi; but inactive against most rare yeasts (Rhodotorula , Trichosporon ). Excepting dermatophytic fungi, it is inactive against moulds, including aspergilli, Zygomycetes and most rare opportunistic moulds. The other three triazoles are generally active against all yeasts including Candida spp . with natural fluconazole resistance as well as fluconazole resistant rare yeasts ( Rhodotorula , Trichosporon ), against dermatophytic fungi and fungi causing endemic systemic mycoses. In contrast to fluconazole they show good to excellent activity against aspergilli and other agents of hyalohyphomycosis (but not against Fusarium spp .), against Penicillium marneffei and against many agents causing phaeohyphomycosis (though considerably less clinical data are available on efficacy against the latter group due to infrequency of phaeohyphomycosis). They are active in chromomycosis (the causative agents are mostly those causing phaeohyphomycoses, see above). They are unequivocally inactive in vitro against Fusarium spp ., but anecdotal reports described therapeutic success in some fusariosis cases. (It is unknown whether these successes are due to pharmacological reasons or to improvement of the underlying condition.) N N N itraconazole O CH3 O H3C O N H N N N O Cl Cl N
The spectrum of itraconazole is identical to that described above; it is used mainly in the therapy of dermatophytoses and aspergillosis. Against Candida spp . and in cryptococcosis it did not prove to be more active clinically than fluconazole. Itraconazole has excellent activity against Penicillum marneffei , and is active in phaeohyphomycosis as well. It is inactive against most agents causing zygomycosis ( Mucor spp ., Rhizopus spp ., etc.), but Absidia spp . are susceptible. It is also inactive against fusaria and against rare moulds (Paecilomyces , Pseudallescheria ). With the introduction of voriconazole, its importance in the therapy of invasive infections has decreased considerably; presently it is rarely used, but still is the drug of first choice in sporotrichosis, chromomycosis, and combined with amphotericin B against Penicillium marneffei . Voriconazole is active against all Candida and Cryptococcus species as well as against rare yeasts (e.g. Trichosporon spp .). It is also active against fungi causing endemic systemic mycoses. Out of moulds it as good activity against aspergilli, Pseudallescheria and
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Paecilomyces , but has a weaker activity against agents of phaeohyphomycosis. Voriconazole is inactive in zygomycosis. It N H C OH has extremely weak activity against fusaria, however, it is used 3 N N C C N H H due to the lack of other therapeutic options. It is fungicidal 2 N F against aspergilli, and the possibility of fungicidal activity has F also been suggested in case of some yeasts ( C. krusei , C. lusitaniae , Trichosporon asahii ) as well. Spectrum of posaconazole is broader than that of F voriconazole, being the broadest spectrum azole presently voriconazole available. It is active against all Candida and Cryptococcus species (excepting C. glabrata ). Posaconazole is active against agents of the endemic systemic mycoses and against aspergilli (excepting A. niger ). It has a promising in vitro and in vivo activity against certain fungi causing hyalohyphomycosis (e.g. Pseudallescheria boydii ) as well as against agents of phaeohyphomycosis, Trichosporon and Rhodotorula species. It has a weak, but clinically relevant activity against agents of mucormycosis. It has weak activity against C. glabrata . Though it is weakly active against fusaria, it is used due to the lack of other therapeutic options. It may possess fungicidal activity against certain species ( C. inconspicua , C. lusitaniae , C. kefyr , Trichosporon asahii ). N N posaconazole N H3C O O H3C N N N N O OH N H F F
Cross-resistance between imidazoles and triazoles is practically nonexistent. Cross-resistance between different triazoles is only partial; isolates resistant to fluconazole and/or itraconazole remain susceptible to newer triazoles in most cases.
Mechanisms of resistance to azole antifungals
Mainly mechanisms of fluconazole resistance in yeasts has been studied, these mechanisms are primarily mechanisms of fluconazole resistance and mostly refer to Candida species. 1. Active efflux This mechanism is based on overproduction of efflux pumps. Two types of efflux are known; there are pumps providing resistance to all azole-type agents, and there are pumps specific to fluconazole. Efflux is a frequent cause of resistance in Candida species, plays a role in the natural fluconazole resistance of C. krusei as well. 2. Mutational alteration of the target This mechanism is mediated by point mutations in the target lanosterol-demethylase gene. It was described mostly in Candida species. It may provide cross-resistance to all azole-type antifungals. Besides the lanosterol-demethylase gene, other enzymes involved in ergosterol biosynthesis may also undergo mutational alteration and provide a certain level of azole resistance, but the importance of these mutations is smaller. 3. Metabolic bypass This mechanism is caused by mutational loss of an enzyme activity catalyzing a step in ergosterol synthesis earlier than lanosterol demethylation (this activity is frequently the 5,6 -desaturase activity). These mutant cells still cannot produce ergosterol in the
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presence of azoles, but avoids the production of sterols toxic to the cell (due to loss of desaturase). This mechanism provides cross-resistance to all azoles and amphotericin B (as ergosterol is not produced, amphotericin B cannot bind to the membrane. Besides desaturase loss, a number of different mutations involved or possibly involved in azole resistance have been described, but these were found in only a few strains. Some of these have been shown to provide cross-resistance to all azoles and/or amphotericin B. 4. Overproduction of the target It may be based on gene amplification or increased expression of the demethylase gene. A rare mechanism of minor importance. 5. Decreased uptake The possibility was raised that alteration of cell membrane (in case of C. albicans ) or of cell wall (in case of Aspergillus flavus ) may lead to decreased azole uptake, but the existence of such a mechanism has not yet been proven.
Acquired resistance to fluconazole and voriconazole is frequently found in C. glabrata .
5-fluorocytosine (flucytosine)
NH Flucytosine is a nucleoside analogue antifungal; its action may be 2 F fungicidal or fungistatic depending on the species. Its uptake is mediated by a N specific permease (cytosin-permease). Within the cell it is deaminated by O N cytosin-deaminase to yield 5-fluorouracyl, which in turn yields a metabolite H (5-fluorodeoxy-uridylic acid) inhibiting the timin biosynthesis by inhibition of 5-fluorocytosine the enzyme timidylate synthase. Another metabolite (5-fluorodeoxyuridine-monophosphate) inhibits RNA-synthesis. Specificity is provided by the deamination step, which is inefficient in mammalian cells. Flucytosine is orally bioavailable and penetrates to the cerebrospinal fluid. When given as a monotherapy, resistance develops rapidly, therefore it is mostly used in combination with amphotericin B; the combination is synergistic. Due to toxicity its use is presently negligible. Its spectrum is narrow, it is active primarily against yeasts. It has excellent activity against Candida species and Cryptococcus neoformans . It seems to have activity against rare yeasts, except Trichosporon spp . and Yarrowia lipolytica . It has activity against certain Aspergillus spp . and most fungi causing phaeohyphomycosis. It is inactive against agents of the endemic systemic mycoses and of the mucormycosis as well as against dermatophytic fungi. It is also inactive against agents of hyalohyphomycosis excepting the abovementioned aspergilli.
Mechanisms of flucytosine resistance
1. Decreased uptake This is a rare mechanism based on loss of the cytosin-permease. 2. Alternative biosynthetic pathway in nucleoside metabolism This mechanism involves the loss of cytosine-deaminase activity or the activity of one of the enzymes responsible for synthesis of 5-fluorodeoxyuridine-monophosphate (one of the UMP-pyrophosphorylase enzymes). As a consequence, flucytosine is not converted into toxic metabolites. Most flucytosine resistant Candida and Cryptococcus isolates possess this mechanism. 3. Overproduction of pyrimidine nucleosides The higher amounts of the normal nucleosides competitively antagonize the drug effect.
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Allylamines
Allylamines are tertiary amines containing an CH3 CH CH aromatic ring. Their fungicidal effect is based on 3 3 CH inhibition of squalene-epoxydase, which catalyzes one N 3 of the initiating steps of fungal sterol biosynthesis. The systemically used terbinafine and the topical naftifine terbinafine belong to this antifungal class. They are active primarily against moulds, and have a weaker activity against yeasts. Dermatophytic fungi, Malassezia furfur , aspegilli and some agents of hyalohyphomycosis ( Pseudallescheria , Paecilomyces ) are susceptible to allylamines, similarly to agents of endemic systemic mycoses. They have excellent activity in chromomycosis, it is active even against Fonsecaea pedrosoi , which is resistant to all other antifungals, but their efficacy in phaeohyphomycosis (caused by the same agents as chromomycosis) is not established. (In chromomycosis, terbinafine showed a synergistic activity when combined CH3 with itraconazole.) They are inactive in mucormycosis N or in fusariosis. The activity of allylamines against Candida and Cryptococcus spp . is only fungistatic. The naftifine activity of terbinafine in disseminated mycoses is not proven. Acquired allylamine resistance is presently rare, but with the spread of their use, this will expectably increase. A mechanism based on active efflux has been suggested, and this may provide cross-resistance to azole antifungals as well.
CH3 H C Morpholines (amorolfine) H 3
Amorolfine inhibits two enzymes ( 14 reductase and O N 7,8 isomerase) involved in ergosterol biosynthesis. This generally results in a fungistatic effect, but may be fungicidal against certain H CH species. Fungicidal activity may be mediated by accumulation of toxic 3 C H sterols similarly to that described for azoles. Amorolfine is used only for amorolfine 2 5 H3C CH topical treatment. 3 Its spectrum is narrow; it is active primarily against dermatophytic fungi. It has a weak activity against Candida spp ., but is active against certain naturally multiresistant species (e.g. Alternaria spp. Scopulariopsis spp.). This is its main therapeutic use. It is inactive against aspergilli. Resistance has not yet been described.
Griseofulvin
Griseofulvin is a benzofurane derivative. Its fungistatic effect O CH O CH3 is based on interfering with the development of the mitotic spindle. 3 Though it is given orally, therapeutic concentration is reached only in O keratinocyte precursor cells. For this reason, its activity is confined to O newly synthesized keratinized epithelium and its keratinized accessories O CH3 O (nails, hair). Its spectrum is narrow, it is used only against dermatophytic H3C Cl fungi, and it is inactive against Candida spp . and Malassezia furfur . It is unsuitable for treatment of systemic mycoses. Resistance to griseofulvin griseofulvin is largely unknown; its use is outdated now and has been replaced by modern azole agents.
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Echinocandins H2N H N OH These are lipopeptide antifungals O OH O inhibiting the fungal cell wall synthesis. N H Their biochemical target is the NH 1,3-β-glucane synthase, responsible for H N N O 2 H C the synthesis of the main cell wall O HN 3 OH CH CH component 1,3-β-glucane. Their action 3 3 HO NH O CH is fungicidal against yeasts, but 3 O N fungistatic against moulds. H HO Caspofungin , micafungin and N OH anidulafungin belongs to this antifungal OH O class. Echinocandins are active against Candida spp ., Coccidioides immitis , HO caspofungin aspergilli, Penicillium spp ., Paecilomyces spp . and Pneumocystis jiroveczii cysts (but inactive against the HO OH O trophozoite form). Their activity is OH O H C weaker against agents of 3 NH phaeohyphomycosis. They are inactive N O H2N against species containing low amounts O HN OH N of β-glucane as Cryptococcus spp ., O O HO NH Trichosporon spp . and Zygomycetes O CH3 O N (agents of mucormycosis and H entomophthoramycosis), as well as HO N against Rhodotorula spp ., Fusarium spp ., SO H OH 3 OH O Pseudallescheria boydii and the O pathogenic yeast forms of agents of the O H C endemic systemic mycoses (they are 3 HO micafungin active against the mould forms founding the soil, but this, of course, does not have clinical value). They are inactive against fungi growing in HO OH O biofilms. OH O The mechanism of H C N 3 H echinocandin resistance is the NH mutational alteration of the target N O H C O HN OH glucane-synthase. Clinical 3 isolates resistant to echinocandins HO NH O CH were found in case of C. krusei 3 O N O and C. glabrata . Different H HO N H C echinocandins show at least 3 OH partial cross-resistance. OH O
anidulafungin HO
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Drugs active against Pneumocystis jiroveczii ( carinii )
The drug of first choice is trimethoprim+sulfamethoxazole (Sumetrolim). (It also has activity against Paracoccidioides brasiliensis , but not against any other clinically relevant fungi.) The doses must exceed significantly (five-ten times) those active against bacteria. Some Pneumocystis jiroveczii strains may become resistant by means of a mutational alteration of the target dihydropteroate synthase (DHPS). Pentamidine (see in the section ‘Antiprotozoal drugs’) may be used as an alternative (for inhalational therapy and in cases when Sumetrolim is not tolerated by the patient), or primaquine+clindamycin, atovaquone, trimethoprim+dapsone may be used.
Drugs used in microsporidiosis
Microsporidia is a group of unicellular CH pathogens, found to be related to Zygomycetes by means 3 O CH3 of molecular taxonomy. They may cause enteritis H CH3 (Enterocytozoon bieneusi , Encephalitozoon intestinalis ) O both in otherwise healthy and in immunocompromised O CH individuals, while they cause ocular ( Encephalitozoon 3 O COOH hellem , E. cuniculi , Vittaforma corneae ) and disseminated (E. hellem , E. cuniculi , E. intestinalis , as O fumagillin well as some other, rare species) disease in immunocompromised patients. Against Enterocytozoon bieneusi and Vittaforma corneae oral or topical fumagillin is used; against other species albendazole (see also in the section on anthelminthic drugs) seems to be effective. Consequently, in intestinal infection fumagillin, in disseminated disease albendazole is used. In case of an ocular infection, the two drugs should be combined to cover all species. Besides these two drugs, successful therapy with furazolidone, clindamycin, itraconazole, metronidazole and nitazoxanide has been reported; however, the efficacy of these drugs against Microsporidia is not yet proven.
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Antiprotozoal drugs
Some protozoa are well-known human pathogens (plasmodia, Trypanosoma spp.), but many protozoa known for long as apathogenic or pathogenic only to animals were found to cause human diseases (Blastocystis spp ., Cryptosporidium spp .). The role of the latter protozoa is especially important in the immunocompromised population; they can be life- threatening and fatal in these patients. There are well-established drugs to treat long-known protozoal infections, but development and spread of resistance is a serious threat. On the other hand, on chemotherapy of emerging protozoal infections the available data is scant. There are protozoal pathogens, against which therapy is seriously limited due to resistance. For these reasons, knowledge on antiprotozoal therapy and on resistance to antiprotozoal drugs gains more and more importance. Antiprotozoal therapy differs from antibacterial therapy in many aspects. An important difference is that we usually do not distinguish cidal and static action; most drugs kill protozoa. (The only exception is the antitrypanosomal eflornithine, which causes growth arrest but not cell death.) Targets of the antiprotozoal drugs are very diverse, due to the differences in protozoal physiology and consequently in the available targets. Besides antiprotozoal activity, these drugs may be active against bacteria, fungi, and sometimes even against helminths, but due to unfavourable toxicity of most antiprotozoal drugs, these are not used against other pathogens. Notable exceptions to this statement are the antibiotics targeting the bacterial ribosome and the fluoroquinolones, which exert their antiprotozoal activity on the prokaryote-type ribosomes or genome of the plastids of the protozoa, with a mechanism identical to their antibacterial action. An antiprotozoal drug may be active against many different protozoal groups, but are used only against a few. A drug is frequently active during only certain parts of the life-cycle of the parasites (against certain developmental forms), but inactive against all other developmental forms. There are drugs with a very narrow spectrum, with activity against only one group of parasites due to acting on a highly specific target; these are inactive against all other protozoa. For these reasons, antiprotozoal drugs are classified according to their spectrum of activity, and this section will also follow this classification.
Drugs against protozoa parasitizing body cavities
Drugs agains protozoa parasitizing body cavities 1. Nitroimidazoles metronidazole, tinidazole, ornidazole 2. Quinacrine 3. Furazolidone 4. Paromomycin 5. Emetine 6. Iodoquinol 7. Diloxanide furoate 8. Tetracyclines 9. Nitazoxanide 10. Albendazole (benzimidazoles) 11. Sumetrolim 12. Ciprofloxacin (fluoroquinolones) 13. Chloroquine
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Nitroimidazoles
These drugs are the derivatives of nitroimidazole. Their action is based on DNA-degrading capacity of an unknown derivative yielded after intracellular activation performed by the cellular electron transport system. This activation takes places only in pathogens with anaerobic respiration, and depends also on the presence of the nitro group, but the activating enzyme may differ between different pathogens. Metronidazole, tinidazole, as well as some newer and rarely used drugs (ornidazole, misonidazole, nimorazole, secnidazole) belongs to this group. Metronidazole is the preferred drug.
CH3 CH3 CH3 N N N H N O 2 N N N CH3 N C R S N O OH OH NO NO NO NO 2 2 O 2 2 metronidazole tinidazole R=H: secnidazole nimorazole R=Cl: ornidazole
Their spectrum regarding strictly anaerobic pathogens is wide; but they are inactive against aerobic or facultative anaerobic species. Against strictly anaerobic bacteria metronidazole is used, see in detail in section ‘Antibacterial agents’. Out of protozoal pathogens they are active against Giardia , Entamoeba , Dientamoeba , Trichomonas , Balantidium and Blastocystis . They are inactive against blood- and tissue protozoa as well as against intestinal Apicomplexa ( Cryptosporidium , Isospora , Cyclospora ). They are (especially metronidazole) the drug of first choice in giardiasis, in infections caused by Trichomonas vaginalis and Entamoeba histolytica ; they are active both in intestinal and in invasive amebiasis. They have negligible activity against the more resistant cyst forms. Mechanisms of resistance are similar in case of bacteria and protozoa; activation of the drugs is impaired through decreased activity of the activating electron transport system. This is based on the down-regulation of the expression of the genes coding for the enzymes involved. In different protozoal genera the gene and protein involved may differ, more than one protein may suffer changes in their activity simultaneously. Besides decreased activation, role of other mechanisms has also been suggested; in case of Giardia active efflux plays a probable additional role in resistance. Cross-resistance between metronidazole and tinidazole is only partial, similar data on other nitroimidazole drugs are not available.
Quinacrine
Quinacrine is an acridine dye, also known as an antimalarial agent (see below). Its mechanism of action against non-malarial protozoa is unknown; the target of its antimalarial activity is not present in protozoa parasitizing body cavities. It inhibits NADH-oxidase (which is an activator of furazolidone), and increases the fragility of the cellular membrane, but none of these effects were proven to play a role in antiprotozoal activity. It intercalates DNA, but has not been demonstrated in the nucleus of the protozoa. Its selectivity is probably provided by the slow uptake of quinacrine into mammalian cells. It is used rarely. Its activity against body cavity protozoa in confined to Giardia , but also it possesses antimalarial activity. It is inactive against other protozoa. It is also active against adult tapeworms, but this activity is not utilized; it is not used to treat tapeworm infections. Resistance may be mediated by active efflux.
160 Antimicrobial chemotherapy: antiprotozoal drugs
Furazolidone furazolidone O O2N N O Furazolidone is a nitrofurane derivative, structurally N related to oxazolidinone antibiotics. Its mechanism of action is O similar to that of nitroimidazoles, but activation is provided by different cellular enzymes. It is active against Giardia , Trichomonas vaginalis and Blastocystis , has some (clinically not utilized) antibacterial activity, and exhibits a weak activity against Isospora belli . It is inactive against Entamoeba histolytica , Cryptosporidium , Cyclospora , as well as against blood- and tissue protozoa. Resistance to furazolidone may be based on decreased drug uptake or on increase in the activity of mechanisms protecting against radicals, the latter arises from overproduction of enzymes involved in the thiol cycle.
Paromomycin
Paromomycin is an aminoglycoside; it exerts its activity on the ribosomes of the protozoal mitochondria. Its mechanism of action and its chemical structure is presented in the section ‘Antibacterial agenst’. It is used in non-invasive amoebiasis, and in infections caused by Dientamoeba fragilis , Trichomonas , Giardia and Blastocystis . It is also active against Leishmania . Paromomycin may be partially effective in cryptosporidiosis, but relapse is inevitable when the drug is withdrawn. Data are not available on resistance to paromomycin.
H C O Emetine 3 N H C O Emetine is a plant alkaloide. Its mechanism 3 H of action is unknown. Its synthetic derivative, CH CH H 2 3 dehydroemetine is less toxic, but also less active. H emetine O CH Emetine is used primarily in invasive amoebiasis; HN 3 its drawback is that it is active only against O CH trophozoites, it does not kill cysts. Due to its 3 toxicity, emetine is used only in severe extraintestial amoebiasis. It may show activity against Blastocystis , or against certain helminths (e.g. Fasciola hepatica ). Information on resistance is scant, but the role of active efflux in resistance has been proven.
Iodoquinol OH It is a iodinated quinoline derivative. Its mechanism of action is I N unknown. It is applicable in the treatment of non-invasive amoebiasis (confined to the intestinal lumen); it has activity against cysts. It is also active against Blastocystis , Dientamoeba and Balantidium coli . Data on I iodoquinol occurrence and mechanisms of iodoquinol resistance are lacking.
Diloxanide furoate CH Cl 3 N Diloxanide furoate is a derivative of acetyl-aniline. O Cl Its mechanism of action is unknown. It is used exclusively O O O in invasive amoebiasis; its advantage is that it kills cysts. diloxanide furoate Similarly to the case of iodoquinol, no data are available on diloxanide resistance.
161 Antimicrobial chemotherapy: antiprotozoal drugs
Tetracyclines
For their chemical structure and mechanism of action we refer to the section ‘Antibacterial agents’, their antiparasitic activity is due to inhibition of prokaryote-type ribosomes of protozoal mitochondria or plastids. They are rarely used as antiprotozoals against body cavity protozoa. They are applied against Balantidium coli , in amoebiasis confined to the gut lumen or affecting the gut wall, as well as an antimalarial agent (see below). Due to their rare use, data on resistance developing in protozoa are scant.
Nitazoxanide
Nitazoxanide is a thiazole derivative, its active H S form is tizoxanid produced in the host by O2N N deacetytilization. Its target and mechanism of action is N O O CH unknown. It is active against all intestinal protozoa 3 including Cryptosporidium , Blastocystis , and nitazoxanide O Cyclospora ). Besides antiprotozoal activity, it has activity against strict anaerobic and microaerophilic bacteria (but its activity against clostridia is ambiguous and it cannot not eradicate Helicobacter pylori when given as monotherapy), as well as against most human helminth infections, including Echinococcus multilocularis infection untreatable with any other known drug. An activity against hepatitis C virus has also been suggested. It is the drug of first choice against Cryptosporidium and Blastocystis . Acquired resistance to nitazoxanide has not yet been described.
Albendazole (benzimidazoles)
Their chemical structure is shown in the section ‘Anthelminthic drugs’. Similarly to their anthelmithic activity, their antiprotozoal action is mediated most probably by inhibition of tubuline polymerisation. In clinical practice only albendazole is used. They are active against Giardia , have weak and clinically unimportant activity against cryptosporidia. Resistance has never been described.
Other drugs
Besides the abovementioned, Sumetrolim (see in section ‘Antibacterial agents’) is the drug of first choice in Cyclospora and Isospora infections; it also has activity against Blastocystis . Ciprofloxacin is effective in Cyclospora and Isospora infections; macrolides may be used in amoebiasis confined to the gut lumen and the gut wall, (their activity in cryptosporidiosis has been suggested, but they did not become an established therapy); chloroquine is active in invasive amoebiasis.
Antimalarial agents
The aims of antimalarial chemotherapy are twofold; while targeting and killing plasmodia in the host to cure human malaria is of utmost importance, killing out sexual forms maintaining the human-mosquito cycle and developmental forms dwelling in the salivary glands of mosquitoes, should also be kept in mind in order to inhibit the spread of malaria. Naturally, the number of drugs suitable for curing of infected individuals is higher, and chemotherapeutics inhibiting the spread are fewer. Different developmental forms differ in drug susceptibility; even drugs with similar mechanism of action may show profound differences in activity. Most drugs are active against erythrocytic developmental forms, but
162 Antimicrobial chemotherapy: antiprotozoal drugs inactive against gametocytes. Mechanism of action of and resistance to antimalarial drugs are known primarily in case of the most frequent Plasmodium falciparum causing the most severe disease; and to a lesser extent in case of the not infrequent P. vivax . Data on the other two Plasmodium species are fewer. Antimalarial drugs have two main traditional targets; the folic acid metabolism (folic acid is indispensable for the parasite as it is heavily involved in pathways responsible for biosynthesis and salvage of purine and pyrimidine nucleotides) and the biochemical processes in the food vacuole, the site of the nutrient uptake for the parasite. Additionally, some newer drugs target the mitochondria and the plastids of plasmodia. In case of many drugs, the mechanism of action is unknown, sometimes even classification into the abovementioned two large groups is uncertain. In spite of these uncertainties and due to its being accepted in the literature, we discuss the antimalarial drugs according to this abovementioned classification.
Antimal arial agents 1. Folic acid antagonists 1.1. type I folic acid antagonists (inhibiting DHPS) 1.1.1. Sulfonamides sulfadoxine 1.1.2. Sulfones dapsone 1.2. type II. folic acid antagonists (inhibiting 1.2.1. Diaminopyrimidines pyrimethamine 1.2.2. Biguanides proguanil, chlorproguanil 2. Drugs targeting mitochondria 2.1. Naphtoquinones atovaquone 2.2. 8-aminoquinolines primaquine, pamaquine 2.3. Tafenoquine 3. Blood-schizontocidal drugs 3.1. Quinoline type drugs 3.1.1. type I blood schizontocidal drugs 3.1.1.1. 4-aminoquinolines chloroquine, amodiaquine, cycloquine 3.1.1.2. Aminoacridines quinacrine, pyronaridine 3.1.2. type II blood schizontocidal drugs 3.1.2.1. Cinchona-alkaloides quinine, quinidine, cinchonine, cinchonidine 3.1.2.2. Quinoline-methanoles mefloquine 3.1.2.3. Halofantrinee 3.1.2.4. Lumefantrine (benflumetol) 3.2. Artemisinin derivatives artemether, arteether, artesunate 4. Drugs targeting the plastid 4.1. Tetracyclines doxycycline 4.2. Clindamycin 4.3. Macrolides erythromycin, azithromycin, spiramycin
163 Antimicrobial chemotherapy: antiprotozoal drugs
Drugs targeting folic acid metabolism sulfadoxine dapsone
N O CH NH Sulfonamides and sulfones (type 1 folic acid antagonists) 3 2 N O CH Both groups have a chemical structure resembling 3 para-amino-benzoic acid (PABA), a component of folic acid; NH SO SO their activity is exerted by competitive inhibition of the enzyme 2 2 dihydropteroate synthase (DHPS), which is involved in folic acid biosynthesis (see also in the section ‘Antibacterial agents’). They show synergy when combined with diaminopyrimidines and biguanides (inhibitors of the dihydrofolate reductase), NH2 NH2 similarly to the antibacterial combination sulfonamide+trimethoprim. Mainly sulfadoxine (a sulphonamide) and dapsone (a sulfone) are used as antimalarial drugs. As resistance develops fast when they are used in monotherapy, they are always used in combination with other antimalarials, most frequently with inhibitors of the dihydrofolate reductase. They are active against late erythrocytic forms of Plasmodium falciparum , but their activity is poor against early erythrocytic stages. They are less active against the other three human pathogenic Plasmodium species (P. vivax , P. ovale and P. malariae ). They are inactive against gametocytes and sporozoites of all four species as well as against hypnozoites of P. vivax and P. ovale . Besides their antimalarial activity, they are effective against Toxoplasma gondii and against other protozoa, and they also have antibacterial activity. Resistance is based on different mutations in the gene of the DHPS enzyme; mutant enzymes do not bind sulfonamides and are not inhibited. As only one mutation may be enough for high level resistance, and the first mutation is followed fast by further mutations causing further increase of resistance, the resistance is very fast to develop during therapy. Cross-resistance is total within the groups of sulfonamides as well as among sulfones, but only partial between the two groups. When used in combination, the clinical response is determined by the resistance to the drug inhibiting dihydrofolate reductase; the resistance to the DHPS inhibitors becomes important only in case of high level of resistance to the dihydrofolate reductase inhibitor. As inside the red blood cells there is practically no PABA or folate, susceptibility to sulfonamides or sulfones can only be measured correctly under PABA and folate free circumstances.
Diaminopyrimidines (type II folic acid antagonists)
These drugs inhibit the enzyme dihydrofolate reductase NH2 (DHFR), which plays a key role in the folic acid metabolism; N Cl they also interfere with uptake or utilization of exogeneous NH2 folic acid through a yet uncharacterized mechanism distinct N pyrimethamine from inhibition of DHFR. They most widely used member is C2H5 the pyrimethamine. As resistance develops fast during pyrimethamine monotherapy, it is used in combination only. The most frequently used drugs in the combination are sulfonamides or sulfones, due to the synergy between the two drug classes. Out of these combinations Fansidar® ( pyrimethamine+sulfadoxine ), and the combination dapsone+pyrimethamine is used. They are active against the late erythrocytic forms; they are sporontocidal and inhibit sexual stages in case of P. falciparum esetében, but early erythrocytic forms and gametocytes
164 Antimicrobial chemotherapy: antiprotozoal drugs are resistant. They have a weaker activity against the other three species; hypnozoites are resistant definitely. Pyrimethamine is also used to treat toxoplasmosis. The mechanism of resistance is the mutational alteration of the target enzyme (DHFR). The drug does not bind to the mutant enzyme and therefore cannot inhibit it. Only one point mutation is enough to provide resistance, but multiple mutants show higher level of resistance. Resistance development is fast upon drug exposure. Some mutations provide only diaminopyrimidine resistance; others also cause cross-resistance to biguanides and/or trimethoprim. Different Plasmodium strains may have significant differences in their liability to mutations.
Biguanides (type II folic acid antagonists)
These drugs inhibit the catalytic activity of the enzyme dihydrofolate reductase (DHFR) similarly to diaminopyrimidines; they are more efficient inhibitors than the latter. The most important members of this class are proguanil and chlorproguanil ; they exert their activity after being converted to active cycloguanil and chlorcycloguanil, respectively. They are used in combinations, frequently combined with type I folic acid antagonists.
NH NH R R 2 NH N
Cl NH NH Cl N NH2
H C H C 3 CH3 3 CH3 R=H: proguanil R=H: cycloguanil R=Cl: chlorproguanil R=Cl: chlorcycloguanil
Proguanil is also used in combination with atovaquone, but in this combination it is not solely used for its DHFR inhibiting activity, but it binds to the membrane of the parasite mitochondrion in a non-activated proguanil form, and in this manner synergistically enhances the activity of atovaquone. They are active against the late erythrocytic forms, are sporontocidal and inhibit sexual stages in case of P. falciparum , but gametocytes are resistant. They have weak activity against early erythrocytic forms of P. falciparum (during the first 24 hours of the cycle), and against all forms of the other three species. They are definitely inactive against hypnozoites. The mechanism of resistance is the mutational alteration of the DHFR target enzyme. Similarly to the situation with diaminopyrimidines, the mutant enzyme is resistant to biguanide binding and inhibition. Resistance development is fats upon drug exposure, but slower than in case of diaminopyrimidines. Only one mutation is known, which confers an isolated biguanide resistance, most mutations confer cross-resistance to diaminopyrimidines as well.
Drugs targeting mitochondria
Naphtoquinones
This group presently includes only atovaquone . Its target is the electron transport chain in the parasite mitochondrion; it is structurally similar to ubiquinone, the substrate of the cytochrome bc 1 complex (consequently it inhibits the function of this complex). This inhibition leads to disfunction of the mitochondrial membrane, leading to loss of the membrane-associated enzyme activities. The killing of the parasite is due to the loss of
165 Antimicrobial chemotherapy: antiprotozoal drugs activity of a key enzyme in the pyrimidine synthesis, Cl dihydroorotate-dehydrogenase, and to the consequent arrest atovaquone of the synthesis of pyrimidine nucleotides and nucleic O acids. Activity of atovaquone is synergistically enhanced by proguanil. Atovaquone is active against erythrocytic forms; it OH is gametocytocidal and kills sporozoites and tissue O schizonts in the liver developing during primary infection. ubiquinone It is inactive, however, against hypnozoites. O Besides its antimalarial activity it is effective H3C O H against Toxoplasma gondii and Pneumocystis jiroveczii n (carinii ); combined with azithromycin it is the drug of first H C O CH choice against Babesia spp . It has a weak activity against 3 3 O Cryptosporidium . Resistance is fast to develop; to prevent resistance development it is always administered in combination with proguanil (Malarone®). The mechanism of resistance is the mutational alteration of the ubiquinone binding region of the target cytochrome bc 1 complex. (The mutation occurs in the gene of cytochrome b). This alteration leads to altered binding site, which is unable to bind atovaquone. Resistance was mainly found in P. falciparum .
8-aminoquinolines H C O 3 Though there are structurally related to blood schizontocidal drugs sharing a quinoline structure, the N target and mechanism of action of these drugs is HN NR different. They need activation in the liver for activity; R=H: primaquine 2 R=C 2H5: pamaquine the active metabolite is a quinone derivative. The target CH3 for the active metabolite is the mitochondrion of the parasite, for this reason a mechanism of action similar to that of atovaquone is postulated. Primaquine and pamaquine are representatives of this drug class. A serious side effect is that they cause severe haemolysis in individuals with glucose-6-phosphate dehydrogenase deficiency. They kill hypnozoites and sporozoites of P. vivax and P. ovale (they have a tissue schizontocidal effect), as well as the gametocytes of all four human pathogenic plasmodia. As they penetrate poorly to red blood cells, they are only active against blood forms when given in extremely high doses. For this reason they are used only to eradication of P. vivax and P. ovale infections, to prevent relapse and, recently, for prophylaxis. P. vivax strains resistant to primaquine were reported, but the mechanism of resistance is not yet known.
Tafenoquine tafenoquine O CF Tafenoquine is the structural analogue of 3 H C O primaquine. Besides a tissue schizontocidal and 3 gametocytocidal activity similar to that of primaquine, it N O CH also exerts blood schizontocidal action. Its blood 3 HN schizontocidal activity is a result of its better penetration NH2 to red blood cells and its markedly longer half-life. CH3
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Blood schizontocidal drugs
The chemical structure of blood schizontocidal drugs, with the exception of artemisinins, is similar. Based on the chemical structure they can be classified into two different structural groups, both substituted at the carbon atom number 4 of the heterocyclic (quinoline) ring structure. In type 1 drugs, the substituents are bound through a secondary amino group, while in case of type 2 drugs through a hydroxymethyl group. Targets and mechanism of actions are most probably shared among drugs of the same group, consequently between members of the same group there is partial cross-resistance. Artemisinins represent a drug class different from both groups, with distinct mechanism of action and exhibiting no cross-resistance.
Quinolines and their structural analogues
4-aminoquinolines (type 1 blood schizontocidal drugs)
In 4-aminoquinolines the substituents are linked through an amino group at the 4 th position (type 1). Their target is an unknown process of the food vacuole, which is clearly distinct from the target of the type 2 blood schizontocidal drugs. 4-aminoquinolines are accumulated in the food vacuole, this accumulation is thought to be mediated by a specific transport system, but possibly a trap mechanism is also involved. The latter is thought to be caused by the weakly basic nature of the drugs, which are lipophilic and can penetrate the vacuole membrane in unprotonated froms, while at the vacuolar low pH they become protonated, gain a positive charge, lose their lipophilic nature and remain trapped in the vacuole, where they are thus accumulated. They form a complex with the hemine derived from haemoglobin degradation, which inhibits the polymerization of the hematine toxic to parasites to the insoluble non-toxic haemozoin, leading to inhibition of the haemoglobin degradation. Antimalarial effect is derived from accumulation of hematine and hematine-chloroquine complexes, which are highly toxic for plasmodia. These toxic complexes cause lipid peroxidation, membrane disfunctions, calcium release, glutathion depletion and inhibition of proteolytic (haemoglobinolytic) enzymes. Out of these effects the most important are most probably the membrane disfunctions and calcium release, which lead to disturbances in haemoglobine digestion, accumulation of vacuoles with undigested haemoglobine in the parasite and to consequent parasite death. Representatives are the most frequently used antimalarial drug chloroquine as well as the HN N(C H ) amodiaquine . Rarely used derivatives are cycloquine 2 5 2 and amopyroquine. The drugs are active against 4 erythrocytic forms of all species, but are inactive chloroquine against sporozoites, tissue forms and hypnozoites. Cl N Gametocytocidal activity is species dependent, mature OH gametocytes of P. falciparum are resistant, but the drugs are active against immature P. falciparum HN N(C H ) gametocytes and the gametocytes of P. vivax . 2 5 2 According to some investigations, chloroquine 4 increases the oocyst production of plasmodia in the amodiaquine infected mosquito, contributing to spread of malaria. Cl N This effect is mediated by an unknown metabolite with
167 Antimicrobial chemotherapy: antiprotozoal drugs extremely long half-life. This phenomenon represents an especially serious problem in chloroquine resistant P. falciparum , where the gametocytocidal activity against immature gametocytes does not decrease the gametocyte numbers. Cross-resistance is common among 4-aminoquinolines as well as between 4-aminoquinolines and aminoacridines (see later). Many chloroquine resistant strains are hypersusceptible to quinoline-methanoles, cinchona alkaloids and to halofantrine (type 2 drugs). Presently known mechanisms of resistance in case of P. falciparum are linked to mutations of the PfCRT ( Plasmodium falciparum chloroquine resistance transporter) gene. This gene codes for a transporter protein located in the membrane of the food vacuole. Two possible explanations exist regarding the biochemical background of chloroquine resistance in P. falciparum . The product of the gene PfCRT may be involved in vacuolar proton transport. This hypothesis is supported by the finding that chloroquine resistance is associated with lower vacuolar pH. As chloroquine forms complexes with hemine only at a narrow pH range, further acidification of the vacuole may lead to inefficient complex formation leading to loss of efficacy of chloroquine. Alternatively, PfCRT may directly mediate chloroquine efflux from the food vacuole. Development of a resistant phenotype requires multiple mutations of the PfCRT gene, which decrease the viability of the parasite, explaining the slow development of chloroquine resistance. Consequently, spread of chloroquine resistance is mostly due to spread of resistant strains, not to development of resistance multiple times. PfCRT mutants show hypersusceptibility to mefloquine and artemisinins. Besides PfCRT mutations, other mechanisms, e.g. mutations of the PfMDR1 pump involved in mefloquine-quinine-halofantrine resistance may also play a role. More data are needed to fully resolve the problem of chloroquine resistance of P. falciparum . Chloroquine resistance of P. vivax is mediated by a mechanism distinct from that described above; its mechanism is yet unknown. Chloroquine resistance in P. ovale and P. malariae has not yet been proven. quinacrine
Aminoacridines (type 1 blood schizontocidal drugs) HN N(C2H5)2 4 O CH3 The basic structure of these compounds is slightly different from the quinoline structure, an additional aromatic ring is condensed to the quinoline structure, yielding a three-ringed structure. To this ring system Cl N the substituents are linked through an amino group corresponding to the amino group of 4-aminoquinolines. Due to the structural similarities, the mechanism of action of aminoacridines is presumably the same as that of N the 4-aminoquinolines. pyronaridine This group of drugs includes rarely used, less important OH N antimalarial agents, e.g. quinacrine (mepacrine) and pyronaridine . Their spectrum is identical to the spectrum of 4-aminoquinolines and there is cross-resistance between aminoacridines and 4-aminoquinolines. This HN cross-resistance confirms that the mechanisms of aminoacridine resistance 4 O CH are similar to the mechanisms of 4-aminoquinoline resistance. 3
Bis-quinolines (type 1 blood schizontocidal drugs) Cl
These newer antimalarials are composed of two 4-aminoquinoline rings and the linker structure. N N Only piperaquine is used presently, but several other N N derivatives are under development. Their mechanism of action is presumably the same as that of the piperaquine 4-aminoquinolines, their spectra are identical, but Cl N N Cl bisquinolines proved to be effective against some chloroquine resistant strains in vitro .
168 Antimicrobial chemotherapy: antiprotozoal drugs
Cinchona alcaloids (type 2 blood schizontocidal drugs)
In cinchona alkaloids a complicated ring structure is attached to the quinoline base structure through a hydroxylated carbon atom (type 2 drugs). Quinine, in the form of an extract of R=OCH 3: quinine wood bark of Cinchona spp , a tree native to tropical South R=H: cinchonine N America, was the first antimalarial used. Their target is a yet HO unknown process of the malarial food vacuole; their mechanism 4 H of action is similar to that of mefloquine (see there). They are the R drugs of first choice in life-threatening high level parasitemia, when a rapid decrease of the parasite burden is crucial for N survival. They have many side effects and are also used to treat cardiac arrhythmias. Representative drugs are quinine and its stereoisomer, quinidine as well as the close structural relative cinchonine and its stereoisomer cinchonidine . Quinine is used almost exclusively as an antimalarial agent. They are active against late erythrocytic forms of all four human pathogenic plasmodia, but inactive against sporozoites, tissue forms, hypnozoites, gametocytes and early erythrocytic forms (during the first 24 hours of the developmental cycle). Due to less frequent use, resistance is uncommon. Strains resistant to cinchona alcaloides show partial cross-resistance to quinoline-methanoles, halofantrine and lumefantrine. Mutations of the gene PfMDR1 , coding for a transporter protein of the food vacuole, plays a role in resistance. Single or double mutants are resistant only to cinchona alkaloids (and may even show increased mefloquine susceptibility), but triple mutations or gene duplications lead to total cross-resistance (see also in subsection ‘Quinoline- methanoles’).
Quinoline-methanoles (type 2 blood schizontocidal drugs)
In quinoline-methanoles substituents are linked to the quinoline ring through a hydroxylated carbon atom located at position corresponding to the amino group of 4-aminoquinolines (type 2). As they contain only one basic nitrogen, they are weaker bases than the 4-aminoquinolines, therefore the ion trap mechanism described in case of 4-aminoquinolines does not play a role in uptake; quinoline-methanoles are probably taken up by a specific transport system. Their mechanism of action is distinct from that of 4-aminoquinolines; they do not inhibit hemine polymerization in vivo , moreover, they inhibit the morphological changes caused by chloroquine. Their mechanism of action is unknown, the most probable explanation is that they bind to the membrane and interfere with metabolism of membrane phospholipids, leading to the net effect of inhibition of haemoglobin uptake. Their parasiticidal effect is slower than the action of 4-aminoquinolines. mefloquine H N The most important representative, mefloquine , is active against the late erythrocytic forms of P. falciparum and P. vivax ; data on the HO activity against P. ovale and P. malariae are scant, but it is presumably 4 H active against late eryhrocytic forms in case of the latter species as well.
It is inactive against gametocytes, tissue forms (sporozoites and N CF hypnozoites) and early erythrocytic forms (during the first 24 hours of 3 CF the developmental cycle). 3 In the background of mefloquine resistance multiple mutations or duplication of the
169 Antimicrobial chemotherapy: antiprotozoal drugs gene PfMDR1 have been implicated. (Some mutations of the gene were found surprisingly to increase mefloquine susceptibility, see above.) A similar gene duplication mechanism has also been described in case of P. vivax . This mechanism causes cross-resistance between quinoline-methanoles, cinchona alkaloids, halofantrine and lumefantrine, and also decreases artemisinin susceptibility. Conversely, susceptibility to 4-aminoquinolines is enhanced.
Halofantrine (type 2 blood schizontocidal drug)
Halofantrine has a phenanthrene base structure, to which HO N substituents are linked through a hydroxylated carbon atom, 4 H similarly to the case of quinoline-methanoles and cinchona alkaloids. For this reason a mechanism of action similar to that of quinoline-methanoles is assumed. This is supported by the partial Cl F3C cross-resistance between halofantrine and cinchona alkaloids and quinoline-methanoles. Halofantrine has proven efficacy against late halofantrine erythrocytic developmental stages of P. falciparum and P. vivax , but Cl has no activity against tissue and early erythrocytic forms. The activity pattern is most probably the same in case of P. ovale and P. malariae as well. Mutations of the PfMDR1 gene found in the background of mefloquine resistance may provide resistance also to halofantrine. Presently it has fallen out of use due to its cardiotoxicity.
Lumefantrine (benflumetol) (type 2 blood schizontocidal drug)
CH Structure of lumefantrine resembles the halofantrine 3 structure, but does not contain a phenanthrene ring system. CH Lumefantrine also contains the hydroxylated carbon atom N 3 structure characteristic to quinoline-methanoles; its HO H mechanism of action is probably similar. Lumefantrine is 4 Cl active only against erythrocytic forms of P. falciparum and P. vivax , shows no activity against tissue forms and gametocytes. Its activity pattern in P. ovale and P. malariae is not yet Cl Cl established, but it is putatively similar to that seen in the lumefantrine former two species. It is used in combination with artemether. Data on lumefantrine resistance are scant.
Artemisinin-derivatives
Artemisinin (Qinghaosu) is a sesquiterpene-lactone alcaloid of the plant Artemisia annua ; the group also includes its semisynthetic derivatives (artesunate, artemether , arteether ).
artemisinin artemether arteether artesunate CH CH 3 CH CH H 3 H H 3 H 3 O O O O H C H3C 3 H3C H3C O O O O O O O O
H H H H O O O O CH CH3 3 CH3 CH3 H H H H O O O O CH 3 C H 2 5 CHOH
CH2 COOH
170 Antimicrobial chemotherapy: antiprotozoal drugs
They are activated by the hemin derived from hemoglobine degradation leading to decomposition of the peroxide bond and to formation of free radicals, which alkylate the macromolecules of the parasite, causing its death. Out of the presently known antimalarial agents artemisinins kill erythrocytic forms with the greatest efficacy; they are active against early developmental forms as well (in the first 24 hours of the cycle). They are also active against immature gametocytes; they inhibit the differentiation into gametocytes, efficiently decreasing the number of gametocytes in the circulation. They are inactive against tissue forms. Artemisinins are equally active against all four human pathogenic (as well as against the investigated animal pathogenic) species. Artemisinins in monotherapy rarely lead to parasite clearance, and recurrences of the disease after cessation of the therapy do occur. For this reason, artemisinins are mostly used in combinations (see below). Resistance develops relatively rapidly; the most probable mechanism is a mutational alteration or overproduction of an efflux pump system. Involvement of the pump PfMDR1 seems to be proven, and the role of another protein of the ABC transporter family is also suspected. Another possible mechanism may be a point mutation in the gene coding for the calcium ATPase of the endoplasmic reticulum, but the issue is far from resolved.
Rarely used antimalarials
Tetracyclines
Their chemical structure is described in the section ‘Antibacterial agents’. They are presumed to act on the plastids of the plasmodia; they inhibit the prokaryote type ribosomes of the plastids with a mechanism similar to their antibacterial activity. As plastids code for and express a number of protein indispensable for the parasite, inhibition of the protein synthesis of the plastids lead to parasite death. Doxycycline as an antimalarial is used primarily in combinations, for chemoprophylaxis and against multiresistant strains of plasmodia. Our knowledge on the tetracycline resistance in plasmodia is little.
Clindamycin
For its chemical structure see the section ‘Antibacterial agents’. Its putative mechanism of action is similar to that of tetracyclines; it acts on the plastid ribosomes in a manner similar to its antibacterial activity. It is used primarily as a combination of quinine+clindamycin against multiresistant P. falciparum. Besides plasmodia it is also active against Toxoplasma and Babesia . Data on the frequency and mechanisms of clindamycin resistance in plasmodia are unavailable.
Macrolides
Their chemical structure is described in the section ‘Antibacterial agents’. Their mechanism of action is presumed to be their inhibition of the prokaryote type ribosomes of the plastids as in case of tetracyclines and clindamycin. Mainly erythromycin , azithromycin and spiramycin are used to treat multiresistant P. falciparum infections. They also show efficacy against Toxoplasma (see below). Azithromycin combined with atovaquone is the drug of choice in human babesiosis. Occurrence and mechanisms of macrolide resistance in plasmodia is unknown.
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Antimalarial drug combinations
The reasons of the use of combinations in malaria therapy are twofold. On one hand, against certain antimalarials (e.g. sulfonamides or atovaquone) the resistance rapidly develops, and using combinations, the risk of resistance development may be decreased significantly. This is the main reason for the usage of the combinations Fansidar® (pyrimethamine+sulfadoxine ), dapsone+pyrimethamine, proguanil+atovaquone (Malarone®), and chlorproguanil+dapsone . In these combinations drug synergy is exploited. Another advantage of the combination therapy is that due to different mechanisms of action of the drugs, the combination may result in faster decrease of the parasite burden and better clinical response. Besides, the different drugs in the combination may be effective against different developmental stages, complementing the action of each other. Thus the combination may be capable of elimination of different parasite forms simultaneously. These were the reasons for using the combinations chloroquin+pyrimethamine, chloroquin+primaquine, pyrimethamine+primaquine, as well as the combination pyrimethamine+sulfadoxine+mefloquine (Fansimef®). These, however, did not prove to be beneficial, as resistance develops rapidly to pyrimethamine, and for this reason the clinical efficacy of the combination was not better than the efficacy of chloroquine or mefloquine monotherapy. Presently the combinations containing artemisinin derivatives (artesunate+mefloquine , artesunate+chlorproguanil+dapsone , artemether+lumefantrine ) are used increasingly. The advantage of these is that the rapid schizontocidal action of artemisinins is followed by the more prolonged activity of the other component. The danger of their usage is that if the infection takes place soon after prophylaxis or a reinfection occurs during therapy, it is possible that the artemisinin component with faster elimination has degraded, but the other component (e.g. mefloquine) is still present in subtherapeutic levels, leading to an increased risk of resistance development to the component with longer half-life, which eventually leads to decrease of the utility of the combination. Further combinations include clindamycin+quinine (also used in babesiosis), doxycycline+chloroquine , and Sumetrolim ( sulfamethoxazole+trimethoprim ). The antimalarial activity of the latter is less marked than the activity of the former drugs, but it is used in some countries for chemoprophylaxis and treatment of children.
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Treatment and chemoprophylaxis of malaria
USA (2007) Drug of first choice Alternatives chloroquine (in case of P. treatment: Plasmodium spp ., excepting P. vivax and P. ovale
per os falciparum resistant to chloroquine combined with primaquine) quinine+tetracycline, P. falciparum resistant to mefloquine, quinine+clindamycin, chloroquine atovaquone+proguanil quinine+Fansidar parenteral quinidine+tetracycline, (in a life- in case of all Plasmodium spp . quinidine+clindamycin, threatening quinidine+Fansidar attack in regions free form chloroquine chloroquine resistance in regions where P. falciparum chloroquine+Fansidar, prophylaxis mefloquine or doxycycline resistant to chloroquine is endemic chloroquine+proguanil relapse prevention in case of primaquine P. vivax and P. ovale United Kingdom (2007) quinine+tetracycline, treatment: quinine+clindamycin, Plasmodium falciparum per os atovaquone+proguanil, artemether+lumefantrine quinine+tetracycline, quinine+clindamycin, P. malariae , P. vivax , P. ovale chloroquine atovaquone+proguanil, artemether+lumefantrine parenteral (in a life- in case of all Plasmodium spp . quinine artesunate+doxycycline threatening attack) in regions free form chloroquine chloroquine+proguanil resistance in regions where P. falciparum mefloquine, doxycycline, chloroquine+Fansidar, prophylaxis resistant to chloroquine is endemic atovaquone+proguanil chloroquine+proguanil relapse prevention in c ase of primaquine P. vivax and P. ovale
Drugs against Toxoplasma
Against Toxoplasma gondii the drug of first choice is the combination of pyrimethamine+sulfadiazine . For their chemical structure and mechanism of action see the section Antimalarial agents (sulfadiazine is a sulphonamide with a mechanism of action analogous to that of sulfadoxine). This combination kills tachyzoites but is inactive against bradyzoites. To prevent transplacental spread of Toxoplasma the macrolide spiramycin is used (pyrimethamine is toxic to the fetus). Spiramycin (for structure refer to the section Antibacterial agents) acts targeting the plastid of Toxoplasma , with a mechanism similar to that described of macrolides action against plasmodia (see above). While spiramycin efficiently prevents transplacental transmission, it is not suitable to treat symptomatic toxoplasmosis. For chemoprophylaxis of toxoplasmosis in the immunocompromised the combination of sulfamethoxazole+trimethoprim is also used, but this combination is not
173 Antimicrobial chemotherapy: antiprotozoal drugs suitable for treatment either. Mechanism of resistance to the pyrimethamine+sulfadiazine combination is the same as described for plasmodia (see above). The chemoprophylaxis and therapy described above may be replaced; possible alternative agents are clindamycin and azithromycin , acting by inhibiting protein synthesis of the plastid ribosomes (see in sections Antibacterial agents and Antimalarial agents). An advantageous characteristic of azithromycin is its activity against bradyzoites. Atovaquone also proved to be effective; its mechanism of action is probably similar to that described in plasmodia, i.e. inhibition the mitochondrial function of Toxoplasma . Atovaquone is active against both tachyzoites and bradyzoites. Ketolides (telithromycin), tetracyclines, streptogramins, and fluoroquinolones also proved to be active in vitro ; the target is the plastid in all cases, and the mechanism of action is the inhibition of the prokaryote type translation or the DNA metabolism of the plastid.
Drugs against Trypanosoma spp.
Drugs against Trypanosoma spp. 1. Pentamidine 2. Suramin 3. Arsenicals melarsoprol 4. Eflornithine 5. Nitrofurans nifurtimox 6. Nitroimidazoles benznidazole, megazole
Diamidines (pentamidine)
Diamidines are aromatic diamine derivatives. Besides the antiparasitic activity some derivatives are used as disinfectants (see above). They penetrate the Trypanosoma by means of specific transport systems. Their mechanism of action is largely unknown. They bind to kinetoplast DNA, but this does not seem to play a role in the antiparasitic effect. It is possible that the net effect of inhibition of a number of different targets is responsible for the trypanocidal action. Out of the diamidine drugs pentamidine , stilbamidine and propamidine , pentamidine is used almost exclusively against trypanosomiasis; a new derivative furamidine is before marketing.
pentamidine NH2 H2N O O NH HN
Pentamidine is active against T. brucei ssp. gambiense in the haemolymphatic phase of the disease. In the late (central nervous system) phase it is ineffective as it cannot penetrate the blood-brain barrier. It is inactive against T. brucei ssp.rhodesiense in both phases of the disease. Furamidine was primarily designed against T. cruzi . Besides trypanosomiasis, diamidines may be useful against antimony-resistant Leishmania donovani and against Pneumocystis jiroveczii (see above). Pentamidine resistance is thought to be caused by mutational loss of transporters responsible for drug uptake; this mechanism provides cross-resistance to arsenicals as well.
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Suramin
Suramin is a polysulfonated colourless naphthalene dye derivative. Drug uptake is mediated by the LDL (low density lipoprotein) receptor, probably by receptor-mediated endocytosis. The target and exact mechanism of action is unknown. It is capable of binding to different enzymes due to its polyanionic nature; this effect is presumed to play a role in trypanocidal activity.
SO3- SO3-
-O3S SO3- suramin
SO3- NH NH SO3-
CH3 H3C O O NH NH N N H H O O O
It is active against both human pathogenic subspecies of T. brucei in the haemolymphatic phase of the disease, though for several reasons it is rarely used against T. brucei ssp. gambiense . It is inactive in the central nervous system phase, as it cannot penetrate into the cerebrospinal fluid. It is also inactive against T. cruzi . Suramin kills the mature macrofilarias of Onchocerca volvulus and Wuchereria bancrofti , but it has no effect on microfilarias. The problem of suramin resistance is largely uninvestigated; decreased LDL uptake or specific efflux may be involved.
Organic arsenicals
These are organic molecules with diverse chemical salvarsan NH structures containing tri- or pentavalent arsenic. Drugs H2N 2 containing pentavalent arsenic are not used due to their toxicity. One of the first chemotherapeutic ever used, HO As As OH salvarsan (no longer used), is an arsenical used against trypanosomiasis. Currently melaminophenyl arsenicals containing trivalent arsenic are used. H2N Their most important representative is melarsoprol N S N N As (MelB), but cymelarsene , trimelarsene H N S CH OH (melarsonyl), and melarsene oxide also belong to 2 H N this drug group. All arsenicals are trypanocidal. 2 melarsoprol For drug uptake a specific purine transport system involved also in pentamidine uptake is necessary (see above). Regarding their mechanism of action two hypotheses exist. They may act by inhibiting parasite enzymes containing sulfhydryl- (-SH) groups (e.g. enzymes of the glycolysis), other data suggest inactivation of trypanothion (a molecule analogous to mammalian glutathione) as a mechanism. Arsenicals have serious, potentially fatal side effects (agranulocytosis, encephalopathia). Arsenicals are active against both subspecies of T. brucei in both phases of the disease, but they are never used in the haemolymphatic phase to avoid toxicity. They are not used
175 Antimicrobial chemotherapy: antiprotozoal drugs against T. cruzi . Data on arsenical resistance are available only regarding melarsoprol. The mechanism of resistance elucidated is the loss of function of the purine uptake system involved in drug penetration. This mechanism provides partial cross-resistance to other melaminophenyl arsenicals and to pentamidine. The role of active efflux has also been suggested.
Eflornithine
Eflornithine is difluoromethyl-ornithine. Its penetration takes place by passive diffusion, its mechanism of action is the irreversible inhibition of ornithine-decarboxylase. This enzyme plays a key role in polyamine (putrescin, spermidin) biosynthesis of the parasite; these polyamines participate in stabilization of the DNA of the CHF parasite, are indispensable for normal cell division and cell 2 H N COOH differentiation, and they are components of the trypanothione. 2 NH Eflornithine inhibits equally the mammalian and the parasite eflornithine 2 enzymes; its specificity is due to the decreased synthesis of trypanosomal ornithine-decarboxylase. Eflornithine does not kill trypanosomes; its action is trypanostatic only. Eflornithine is active only against T. brucei ssp. gambiense . T. brucei ssp. rhodesiense possesses generic resistance, due to the shorter half-life and consequent faster synthesis of ornithine-decarboxylase in T. brucei ssp. rhodesiense . In spite of this it proved to be effective against some T. brucei ssp. rhodesiense strains. Against T. brucei ssp. gambiense it is active in both phases of the disease. It is not used against T. cruzi . Resistance to eflornithine seems to be mediated by changes in drug uptake, but the exact biochemical background is unresolved.
Nitrofurans (Nifurtimox)
O N O C N N SO The active metabolite of nifurtimox is the nitro anion 2 H 2 formed by reduction of the molecule, which induces the H C generation of reactive oxygen radicals. These radicals nifurtimox 3 damage the macromolecules of the parasite leading to trypanocidal action (see also in the sections ‘Nitrofurantoin’ in section ‘Antibacterial agents’ and in ‘Drugs against protozoa parasitizing body cavities’). The main target for the radicals seems to be the trypanothion. Specificity of nitrofurans is due to the lack of mechanism for detoxification of oxygen radicals in trypanosomes. Toxicity of nifurtimox is significant. It is used primarily against T. cruzi . Its efficacy is variable; it eradicates the parasite in approximately 80% of the cases. It has only marginal activity in chronic infections. Nifurtimox is presently the drug of first choice in acute T. cruzi infection. It is also active against T. brucei ssp. gambiense and T. brucei ssp. rhodesiense in both phases of the disease, its efficacy is under investigation against arsenical resistant strains. Little is known about the resistance to nifurtimox.
Nitroimidazoles
benznidazole The active metabolite of N NO nitroimidazoles is also the nitro 2 S NH H O N 2 N 2 N anion generated by reduction of NN NN CH the drug molecule, but they do 3 O not provoke oxidative stress, megazole
176 Antimicrobial chemotherapy: antiprotozoal drugs but exert their activity by covalently modifying the macromolecules of the parasite. Benznidazole and megazole are the nitroimidazoles used against trypanosomes. Toxicity of benznidazole is somewhat more marked than nifurtimox toxicity; megazole was withdrawn from the market due to mutagenicity. Benznidazole is used mainly against T. cruzi , it can eradicate the acute infection in only approximately 80% of the patients. Benznidazole also possesses weak activity in chronic infections. It is slightly more active than nifurtimox as is capable of slowing the progression of chronic infections. Its utility is being tested in arsenical resistant African trypanosomiasis as well.
Other drugs against Trypanosoma spp.
Out of currently available antiprotozoal drugs, besides those described above, primaquine (see also in section ‘Antimalarial drugs’), allopurinol (see also in section ‘Drugs against Leishmania spp .’), amphotericin B (see also in sections ‘Drugs against Leishmania spp .’ and ‘Antifungal agents’), different azole derivatives (see also in sections ‘Drugs against Leishmania spp .’ and ‘Antifungal agents’) and alkyl-phosphocholines e.g. miltefonsine (see also in section ‘Drugs against Leishmania spp .’) also possess activity against T. cruzi . Experiments to develop novel less toxic drugs and drugs efficacious against strains resistant to available drugs are being performed. Against T. cruzi the most promising are the pyrophosphate analogue bisphosphonates (used in the therapy of osteoporosis), and the drugs inhibiting the main protease of the parasite cruzapain. None of these drugs has been entered the drug development process. The efficacy of certain combinations is also being investigated; pentamidine+nifurtimox has shown a degree of therapeutic success against arsenical resistant T. brucei ssp. gambiense .
Drugs against Leishmania spp .
Drugs against Leishmania spp . 1. Antimony derivatives sodium-stibogluconate, meglumine antimoniate 2. Amphotericin B 3. Paromomycin (aminosidine) 4. Phosphocholines miltefosine 5. Azoles ketoconazole 6. Diamidines pentamidine 7. Allopurinol
Different Leishmania species (including Viannia spp .) may differ markedly regarding drug susceptibility, therefore knowledge on the species distribution within a region or isolation and species-level identification of the parasite is important for choosing the appropriate therapy. It is also important that the two developmental stages found in humans also differ in drug susceptibility; promastigotes for instance are significantly more resistant to antimonials than the tissue amastigotes. Acquired resistance is relatively slow to develop, with the important exception of L. donovani , because other species are not or rarely are transmitted from human to human, therefore the spread of the strain acquiring secondary resistance is slow in the human population. Chemotherapy of animal leishmaniasis, however, will ultimately lead to human infections by drug resistant parasites.
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Organic antimony derivatives (antimonials) OH OH meglumine antimoniate H O N + O Sb H Chemical structure of these drugs is H3C OH variable; the only structural relationship is the OH OH O antimony component. Antimony may be trivalent III V (Sb ) as in antimony tartarate or pentavalent (Sb ) as in OH HO sodium stibogluconate or in meglumine antimoniate . Uptake of drugs containing trivalent and pentavalent antimony is OH HO mediated by different mechanisms. Uptake efficacy may differ O O significantly in different species; L. donovani takes up one to O Sb O Sb O two orders of magnitude more antimony than Viannia ( L. ) O OH O O O + O panamensis . + Na + Sb V is reduced to trivalent antimony in the parasite; the O Na Na O sodium stibogluconate process may be enzymatic or non-enzymatic involving trypanothione. Host macrophages may also contribute to reducing the drug. The active form is Sb III . Its antiparasitic activity is mediated most probably by decreasing the trypanothion levels in the parasite. Decreasing of the trypanothion level occurs through two mechanisms. i) The parasite is capable of extruding Sb III conjugated with trypanothion with active efflux, and ii) Sb III compounds inhibit the trypanothion reductase enzyme reducing inactive (-S-S-) trypanothione to the active (-SH HS-) form. Decrease of the intracellular trypanothion level leads to parasite death through drastic decrease in the tolerance to oxidative stress. Many authors claim that antimony-induced parasite death is similar to apoptosis of mammalian cells. Host cytokine response modulation may also contribute to leishmanicidal activity of antimonials. Amastigotes are much more susceptible than promastigotes to Sb V, this was not observed in case of Sb III drugs. (A study conducted with L. donovani found inhibition of Sb III activity by Sb V in promastigotes, while the effect was additive in amastigotes.) This is caused by lack of intracellular activation (Sb V⇒Sb III conversion) in promastigotes. Antimonials accumulate in macrophages; therefore they kill intracellular amastigotes more readily than extracellular promastigotes. Antimonials are toxic, Sb III drugs show more serious toxicity; therefore presently only Sb V drugs are used exclusively. Antimonials inhibit other parasites and helminths as well, but due to their toxicity they are used only for treatment of leishmaniasis. They are leishmanicidal against all Leishmania (and Viannia ) species, but the degree of their efficacy is species dependent. Total eradication is possible only in patients with good Th 1 type immune response. Efficacy of antimonial therapy is determined by the species, the developmental form and the acquired resistance of the parasite. Reliable data on resistance to antimony derivatives is scant, and the existing data are mostly derived from studying of laboratory-selected mutants. Species differences in susceptibility are explained by differences in drug uptake and differences in activation, consequently mutational alteration of these mechanisms may play a role in acquired resistance as well. It was proven in an antimony resistant L. donovani strain that the resistance of amastigotes to Sb V was mediated by decrease in activation (conversion to Sb III ). Resistance to Sb III derivatives is most probably caused by overproduction of the trypanothione. This overproduction may be mediated by overexpression of a number of enzymes involved in polyamine or trypanothione biosynthesis. This mechanism directly counteracts with the activity of antimonials and, additionally, leads to accumulation of trypanothione-Sb III drug complexes, which in turn leads to increased efflux of the complexes. This increased efflux may also be aided by duplication of the efflux pump gene. (A similar mechanism was proven in case of arsenical resistance of the reptile pathogenic species L. tarentolae .) Mechanism of Sb III resistance, naturally, will decrease the efficacy of not only directly administered Sb III , but also of Sb III derived from activation of Sb V drugs. Alterations in trypanothione metabolism provide resistance both to Sb III and to Sb V, while mechanisms leading to decreased activation (reduction) of Sb V drugs affect only the latter group. Cross-resistance within a group is usually complete; however, a strain resistant to meglumine antimoniate but not to sodium stibogluconate has been reported.
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Amphotericin-B
For its chemical structure we refer to the section ‘Antifungal agents’. Its activity against Leishmania is identical to its antifungal activity. (The main sterol component of Leishmania promastigotes is ergosterol and its derivatives, while amastigotes contain sterols different from both ergosterol and mammalian cholesterol.) As amphotericin-B also inhibits amastigotes in vitro , it is postulated that it may also bind to the membrane sterols of amastigotes, but this remains to be proved. Penetration to macrophages is an important step in leishmanicidal activity. This is mediated by the LDL- (low density lipoprotein-) receptor. Due to better intracellular penetration, lipid formulations (liposomal, lipid complex, etc.) show better activity against Leishmania spp . Amphotericin-B is active against all Leishmania species, but its activity is inferior to that of antimonials. Newer lipid formulations are equal to antimonials in efficacy with significantly more favourable toxicity. However, due to high price of these formulations, antimonials remain the most widely used drugs in leishmaniasis. Mechanisms of resistance to amphotericin-B in Leishmania species are largely unknown. In laboratory selected resistant promastigotes alterations were found in sterol biosynthesis pathways, similarly to that described in fungi. In these mutants the end-product of sterol biosynthesis and thus the main membrane sterol is not ergosterol, consequently amphotericin-B cannot bind to the parasite membrane. Resistance in amastigotes has not been addressed.
Paromomycin (Aminosidine)
Paromomycin is an aminoglycoside antibiotic (see also in sections ‘Antibacterial agents’ and ‘Drugs against protozoa parasitizing body cavities’). Its mechanism of action most probably involves inhibition of mitochondrial translation with a mechanism identical to the aminoglycoside antibacterial action, but action on other targets (inhibition of mitochondrial respiratory chain and phospholipids synthesis) is also possible. It has for long been in use to treat cutaneous leishmaniasis as a topical drug, a suitable parenteral formulation is being developed. Its combination with antimony derivatives also seems to be promising. Its activity is best against L. major and L. tropica (in Old World cutaneous leishmaniasis), its activity is less marked against L. donovani (in visceral leishmaniasis) and is weak against L. mexicana and Viannia ( L.) braziliensis (in New World cutaneous and mucocutaneous leishmaniasis). Mechanisms of resistance are largely unknown, decreased drug uptake was described in some paromomycin resistant L. donovani strains.
Phosphocholines
These drugs are alkyl- and alkylglicerol derivatives of phosphocholine. They damage the membranes of the parasite (cause the vacuolization of the mitochondrial and the periflagellar membrane), interfere with the phospholipid synthesis and with the signal transduction process mediated by phosphatidyl inositol, and inhibit an important enzyme of the sterol biosynthetic pathway (C-22-desaturase). However, it is unknown which mechanism(s) are responsible for clinical leishmanicidal activity. This drug group includes the alkylphosphocholine miltefosine , and the alkylglycerol-phosphocholine ilmofosine and edelfosine. At present, only miltefosine is marketed for the treatment of visceral leishmaniasis caused by L. donovani . The advantage of the phosphocholines compared to other antileishmanial drugs is their oral bioavailability (all other drugs are parenteral).
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O O O + CH P N 3 H C CH O 3 3 miltefosine
All phosphocholines are more active against the promastigote developmental form, the extent of activity proved to be species dependent. The most susceptible species is L. donovani , the least susceptible are L. major , Viannia ( L. ) panamensis and L. mexicana . The susceptibility pattern is similar, but not identical, in case of the amastigote forms. Phosphocholines are active also against T. cruzi amastigotes and epimastigotes, but has significantly weaker activity against the trypomastigote form. Their activity is similarly weak against T. brucei trypomastigotes. They are suitable for treatment of canine leishmaniasis, allowing for the decrease of the prevalence in the most important animal reservoir. Though relapses were reported after miltefosine therapy, data on miltefosine resistance are yet unknown. In laboratory mutants, mutations of the transporter responsible for drug uptake led to high level resistance to all phosphocholine derivatives. Multidrug resistance due to efflux pumps decreases phosphocholine susceptibility as well.
Azoles
Ketoconazole and itraconazole are used against leishmaniasis; for their chemical structure and mechanism of action we refer to the section ‘Antifungal agents’ and to the description of the antileishmanial activity of amphotericin B. Their activity against Leishmania spp . is weaker than that of antimonials; they are drugs used rarely, mainly against antimony-refractory disease. They are also suitable for topical treatment in cutaneous leishmaniasis. Data on resistance is lacking.
Diamidines
Out of these aromatic diamidine derivatives pentamidine is used as an antileishmanial drug. The mechanism of its leishmanicidal action is unknown. Its most probable target is the parasite’s mitochondrion, but possible targets also include polyamine biosynthesis. Due to its weak activity and its toxicity, it is used increasingly rarely, but may find its role as a member of antileishmanial combinations enhancing the activity of other drugs. It is used in antimony-refractory leishmaniasis. It also shows activity against T. brucei ssp. gambiense as well as against certain fungi (e.g. Pneumocystis jiroveczii ; also see above). Pentamidin resistance may probably be mediated by alteration of the uptake transport system and/or by active efflux; these data are derived from laboratory mutant Leishmania strains. The active efflux may provide cross-resistance to antimonials as well. Mechanisms of resistance developing spontaneously during pentamidine therapy are unknown.
Allopurinol allopurinol This pyrazolopyrimidine derivative behaves as a purine base analogue, is metabolized similarly to purine bases, therefore, it interferes with the purine uptake of the OH H parasite and upon being combined to (deoxy)ribose it inhibits nucleic acid synthesis in N N triphosphate form. It may interfere with protein synthesis if incorporated into mRNAs. Its N selectivity derives from the marked differences in leishmanial and mammal purine metabolism; Leishmania spp . are unable to synthesize purine bases de novo , these are N obtained from the host. Allopurinol is usually used as member of antileishmanial combinations; it is marginally active in monotherapy. It is suitable, however, to treat canine leishmaniasis, and may be used to reduce transmission risk by decreasing the prevalence in the canine reservoir. It is also active against T. cruzi . Data on allopurinol resistance is lacking.
Other drugs
Several alternative drugs are under development. Out of these the primaquine analogue sitamaquine is in
180 Antimicrobial chemotherapy: antiprotozoal drugs the clinical evaluation phase. An alternative approach is the utilization of immunomodulants; drugs stimulating the Th 1 immune response crucial to parasite elimination (tucaresole) and drugs stimulating the macrophage killing activity inhibited by the parasite (imiquimod) are given as an attempt to control the infection.
Drugs against free-living amoebae
Drug with proven activity is available only against Naegleria fowleri ; naeglerial primary amoebic meningoencephalitis can be treated with amphotericin B . The traditional formulation is more active than the newer lipid-associated (e.g. liposomal) formulations. Besides amphotericin B, different azole antifungal agents and azithromycin are effective. However, mortality remains high in spite of the effective drug therapy. Against Acanthamoeba and Balamuthia there are no drugs with sufficient activity; therapeutic attempts may involve combinations of different drugs, including amphotericin B , flucytosin, azole antifungals , pentamidine and other diamidines, azithromycin and the sulfonamide sulfadiazine. Therapeutic success is expectable only in Acanthamoeba keratitis, granulomatous encephalitides are likely to result in death even when treated. Locally applied disinfectants (cationic detergents) also play an important role in treatment of keratitis.
Drugs of first choice in protozoal infections
Parasite drug of first choice alternatives
Acanthamoeba spp. not available 1 pentamidine, itraconazole, ketoconazole 2 Babesia spp. atovaquone+azithromycin clindamycin+quinine Balantidium coli tetracyclines metronidazole, iodoquinol Blastocystis hominis nitazoxanide metronidazole, iodoquinol, TMP+SMX 3 Cryptosporidium spp. nitazoxanide Cyclospora spp. TMP+SMX 3 Dientamoeba fragilis metronidazole, iodoquinol, paromomycin, tetracyclines Entamoeba histolytica 4 metronidazole emetine, tinidazole Giardia lamblia metronidazole, nitazoxanide quinacrine, paromomycin, furazolidon Isospora belli TMP+SMX 3 Leishmania spp. antimony compounds 5 amphotericin B, pentamidine Naegleria spp. amphotericin B Plasmodium spp. see above Toxoplasma gondii pyrimethamine+sulfadoxine spiramycin, atovaquone Trichomonas vaginalis metronidazole tinidazole Trypanosoma cruzi nifurtimox, benznidazole Trypanosoma brucei (early stage) suramin, eflornithine pentamidine Trypanosoma brucei (late stage) melarsoprol, eflornithine 1 There is no therapy proven to be effective. Drugs listed as alternatives were effective in certain cases. 2 Successful treatment of Acanthamoeba encephalitis is anecdotal, these drugs proved to be effective mostly in keratitis. 3 Trimethoprim+sulfamethoxazole (Sumetrolim) 4 The listed drugs are active both in intestinal and extraintestinal (invasive) amoebiasis. In treatment of intestinal amoebiasis other drugs are also used (see text). 5 sodium stibogluconate, meglumine antimoniate
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In vitro testing of the susceptibility of protozoa
The importance and utility of in vitro susceptibility testing is increasing in case of certain protozoa due to the spread of resistance. Most protozoal parasites are cultivable in artificial media or in cell cultures providing a means for susceptibility testing. Similarly to antibacterial susceptibility testing, the most frequently utilized approach is to observe the effect of the drug on the viability of the protozoon to be tested. The viability may be assessed in several different manners. 1. monitoring certain viability signs 1.1. through microscopic evaluation 1.1.1. observing motility ( Entamoeba ) 1.1.2. observing phagocytic activity ( Entamoeba ) 1.1.3. observing adhesiveness ( Giardia ) 1.2. through uptake of radiolabelled nutrients ( Leishmania , Entamoeba , Plasmodium ) 2. monitoring the differentiation of the parasite ( Plasmodium ) Methodologies based on nutrient uptake can be automatized. Molecular biology methods (demonstration of genetic alterations associated with resistance) are gaining ground; their main drawback is that they detect only previously known mechanisms of resistance.
182 Antimicrobial chemotherapy: anthelminthic drugs
Anthelminthic drugs
Besides being a major cause of morbidity and mortality in developing countries, the importance of human helminthic infections is increasing in the developed countries as well. New human pathogenic helminths emerge (e.g. Baylisascaris procyonis , a racoon-associated nematode, which may cause severe, frequently fatal encephalitis), and well-known pathogenic helminths are shown to cause unexpectedly serious infections in vulnerable patients, i.e. mainly in the immunocompromised (e.g. Strongyloides stercoralis ). Similarly to other groups of human pathogenic organisms, resistance to chemotherapy became a problem in helminthic infections; presently only in animal helminthioses, but the appearance of resistance in human helminths also seems to be expectable. Human helminthioses, similarly to protozoal diseases, are mainly diseases of the tropical, subtropical areas, and those found in temperate regions usually produce less severe symptoms, therefore the clinical importance of helminthioses is considered relatively low. For this reason, the research activities on human helminthioses are limited, and the knowledge on the chemotherapy of human helminthic infections (data on the mechanisms of action of and resistance to anthelminthic drugs) are mostly deduced from data collected on related animal helminths. Resistance to anthelminthic drugs spreads in certain helminths of veterinary importance; in human infections resistance has not yet been unequivocally proven. For this reason the mechanisms of resistance described reflect experience with resistance in animal pathogenic helminths closely related to human pathogenic species. Resistance develops slowly in helminths, but once resistance develops, reversion to susceptibility never occurs. A repeated exposure to the anthelminthic drug favours the propagation of the resistant subpopulation, thus in case of regular drug exposure even extremely low proportions of resistant individuals is sufficient for preservation of resistance genes; these genes are preserved in the population for a long time even in the absence of drug. On the other hand, regular drug exposure leads to superseding of the susceptible subpopulation by the resistant subpopulation over time. These susceptible genes may eventually be lost, which leads to uniform resistance of the whole population or species to the particular drug group. This loss of susceptibility is irreversible in such a case; susceptibility, due to lack of the gene conferring it, never returns. As the number of available anthelminthic drugs is limited, such a resistance development may make therapy of that particular infection impossible. Fortunately, resistance in human helminthioses was only described in case of Schistosoma mansoni (and it is not yet proven even in that case), but the possibility has been suggested in other human helminthic infections (e.g. hookworm infections) as well. For the abovementioned reasons, however, the prevention of resistance development is of key importance in human helminths. This, in turn, requires a deeper knowledge on the anthelminthic drugs, their mechanism of action and the possibilities of resistance.
183 Antimicrobial chemotherapy: anthelminthic drugs
Anthelminthic drugs 1. Drugs affecting the microtubular system of worms 1.1. Benzimidazoles 1.1.1. Benzimidazole-carbamates mebendazole, flubendazole, albendazole 1.1.2. Benzimidazole-thiazoles thiabendazole 1.1.3. Benzimidazol-thioethers triclabendazole 2. Drugs affecting the nervous system of the worms 2.1. Drugs acting on excitatory (cholinerg) synapses 2.1.1. Imidazolothiazoles levamisole 2.1.2. Tetrahydropyrimidines pyrantel, oxantel 2.1.3. Bephenium 2.1.4. Metrifonate 2.2. Drugs acting on inhibitory synapses 2.2.1. Avermectins (makrocyclic lactones) ivermectin 2.2.2. Piperazine 3. Drugs affecting energy metabolism of the worms 3.1. Niclosamide 3.2. Bithionol 3.3. Albendazole (in form of albendazole-sulfone) 3.4. Cyanine dyes pyrvinium 4. Drugs with other or unknown mechanism of action 4.1. Diethylcarbamazine 4.2. Praziquantel 4.3. Oxamniquine 4.4. Artemisinin-derivatives 4.5. Nitazoxanide 4.6. Tribendimidine
Drugs affecting the microtubular system of worms
Benzimidazoles N S thiabendazole Their structure is based on condensed ring N N system formed by a benzene and an imidazole ring. O Their target is the microtubular system of the N O worms; they inhibit the polymerisation of the N O CH H 3 N tubuline monomers by binding to them. This leads mebendazole to inhibition of the microtubular functions including e.g. vesicular transport, which is crucial for nutrient N O N O CH uptake. Selectivity of benzimidazoles is attributed H C H 3 3 S N to the fact that the helminthic tubuline monomers albendazole bind them with markedly higher affinity than F N O mammalian tubuline. This drug group includes N O CH H 3 thiabendazole containing a thiazole ring, as well as N mebendazole , albendazole and flubendazole , O flubendazole which are carbamate-methylesters. Their spectrum is broad; they are active against most human- and animal pathogenic nematodes. They kill both larvae and mature worms. Generally, albendazole and mebendazole is more or less active against almost all human nematodes, weaker activity is shown against Strongyloides stercoralis ; they are not used in filariasis (excepting Mansonella perstans ).
184 Antimicrobial chemotherapy: anthelminthic drugs
Thiabendazole is no longer used in developed countries; it was used against Strongyloides stercoralis and Toxocara infection as well as in cutan and visceral larva migrans. Albendazole and/or mebendazole is considered the drug of first choice against Trichuris trichiura , Ascaris lumbricoides , hookworms ( Ancylostoma duodenale and Necator americanus ). They are active against Enterobius vermicularis ; their activity against Strongyloides stercoralis is weaker. Mebendazole or albendazole is recommended in toxocariasis and in Trichinella infections, against Gnathostoma , Capillaria and Mansonella perstans . Albendazole is the drug of first choice in cysticercosis, but in this case the mechanism of action is not the inhibition of the microtubular system (see in the section ‘Drugs affecting energy metabolism of the worms’). Besides its anthelminthic activity, albendazole is also active against Giardia . Cl Triclabendazole (a benzimidazole-thioether) is N S CH the drug of first choice against Fasciola hepatica . It 3 Cl O N does not exhibit activity against nematodes. Cl They are not recommended (excepting triclabendazole triclabendazole) in fluke infections (against Schistosoma spp ., Paragonimus westermani , etc.), in cestode infection of the intestines (against Taenia spp ., Diphyllobothrium latum , Hymenolepis spp ., etc.), and against most agents of the filariasis (against Onchocerca volvulus , Brugia spp ., Loa loa , Wuchereria bancrofti , etc.; an exception is the therapy of Mansonella perstans infection). The mechanism benzimidazole resistance is the mutational alternation of one of the tubuline monomers leading to decreased drug binding by the target. In laboratory mutant worms subsequent deletion of the other tubuline gene led to higher level resistance. Resistance has not yet been described in human worm infections.
Drugs affecting the nervous system of the worms
Drugs acting on excitatory (acetylcholinerg) synapses
The common target of these drugs is the acetylcholinerg neurotransmission of the nerve-muscle synapsis of the worms. The drugs included are acetylcholine-agonists (imidazolothiazoles, tetrahydropyrimidines, bephenium) and the acetylcholine-esterase inhibitor metrifonate. These drugs cause spastic paralysis of the musculature of the worms.
Imidazolothiazoles
These drugs contain a condensed ring system of an imidazole and a thiazole ring bound to a benzene ring. Their target is the cholinerg synapsis of the helminthic muscle, where they behave as acetylcholine agonists causing a prolonged N S contraction of the muscles (spastic paralysis). The immobile worms are removed quickly from the host gut by the peristaltic movement. N Levamisole is the member of the group used against human levamisole pathogenic worms. Their spectrum is narrow, they are active only against enteric nematodes, out of the human helminths they are useful against only Ascaris , Necator and Ancylostoma (hookworms). Levamisole also has an immunostimulant activity. Resistance is hypothesized to be caused by the decrease of the number and drug affinity of the cholinerg receptors in the synapsis, but these presumptions need confirmation. Levamisole resistance has only been described in animal helminths. Increase of dose may lead to desensitization of the target receptors leading to decrease or loss of drug activity.
185 Antimicrobial chemotherapy: anthelminthic drugs
Tetrahydropyrimidines OH
The drugs pyrantel and oxantel pyrantel N N belonging to this group exert their anthelminthic S activity with a mechanism identical to that N N oxantel CH CH described for levamisole. Similarly to 3 3 levamisole, these drugs are active only against enteric nematodes, pyrantel is active against Ancylostoma , Necator , Enterobius and Trichinella (they are not active against tissue larvae of Trichinella ), while oxantel is active against Trichuris . Pyrantel is considered the drug of first choice against Enterobius in some guidelines. Mechanism of resistance to tetrahydropyrimidines is probably similar to that of levamisole resistance, but has never been described in case of human pathogenic species.
Bephenium CH3 O C C N C Bephenium is a quaternary amine; its mechanism of action is H H H 2 2 CH 2 similar to that described for levamisole, though recent research has 3 bephenium shown that they act on different receptors. Bephenium is also a drug against enteric nematodes; it is active primarily against Ascaris , Necator and Ancylostoma . Sometimes it is used combined with pyrantel. Data on bephenium resistance is lacking.
Metrifonate O O
H C O P C CCl H C O P C CCl Metrifonate is an organic phosphorus derivative, 3 3 3 H 2 H2 its active metabolite, dichlorvos, inhibits acetylcholine O O metrifonate dichlorvos esterase. Without acetylcholine esterase activity, a CH3 CH3 permanent acetylcholine excess develops in the synapses, leading to spastic paralysis of the musculature. Its selectivity is due to the higher sensitivity of the helminthic esterase compared to the mammalian enzyme. It is active exclusively against Schistosoma haematobium . Metrifonate resistance has not been reported.
Drugs acting on inhibitory synapses
These drugs target the chloride ion O CH channels of the worm muscles, which play a 3 HO key role in inhibition of muscle contraction. O CH3 The channel targeted may be γ-amino-butiric O H C O acid- (GABA-) or glutamate-regulated. These 3 H O anthelminthics cause a flaccid paralysis of the H C O 3 H CH musculature. H C 3 3 Macrocyclic lactones (avermectins) ivermectin H
O O Chemical structure of these drugs is O OH characterized by a complicated, H O polyglycosilated lactone ring. Members of this HO H CH3 drug class are the natural avermectin and its H H H3C semisynthetic derivatives; their most CH3 important representative is ivermectin . (A CH3 structurally related compound group,
186 Antimicrobial chemotherapy: anthelminthic drugs milbemycins, exhibiting a similar mechanism of action is used only in veterinary medicine.) Their activity is exerted on a glutamate-mediated inhibitory synapsis, on the postsynaptic glutamate-dependent chloride ion channel. Macrocyclic lactones augment the effect of glutamate, and at higher concentration they keep the channel open even in the absence of glutamate (i.e. of the excitatory stimulus). (They also act on GABA-dependent channels, but this latter effect seems to be of minor importance in the anthelminthic effect.) The net effect of avermectins is the predominance of inhibitory signals leading to flaccid paralysis of the musculature. The primary cause of the nematocidal effect is the paralysis of the pharyngeal muscles and the consequent starvation, but immobilization also plays a role. Selectivity is due to the fact that in mammals glutaminerg neurotransmission is confined to the central nervous system, and as avermectins cannot cross the blood-brain barrier they do not take effect on the mammalian nervous system. They have nematocidal activity, both against helminths parasitizing the gastrointestinal tract and against tissue nematodes. Ivermectin is the drug of first choice against Onchocerca volvulus , as they have potent microfilaricidal activity. Ivermectin causes less severe local inflammatory reaction accompanying the death of the microfilariae than the other widely used microfilaricidal drug diethycarbamazine (see below). This fact has important implications when the microfilariae are located in the eyes, where the immune response and inflammatory reaction to the decomposing worms may lead to increased eye damage. Besides Onchocerca , avermectins are also active against other agents of the filariasis ( Loa , Brugia , Wuchereria ). They are active against enteric nematodes, but presently are used only against Strongyloides stercoralis against which ivermectin is the drug of choice. Besides nematodes, they are active against blood-sucking arthropode ectoparasites as well as against Sarcoptes scabiei . They are inactive against flukes ( Trematoda ) and tapeworms (Cestoda ). Avermectin resistance is an issue in animal helminths; resistance in human pathogenic helminths has not yet been reported. The mechanism of avermectin resistance is the mutational alteration of the target ion channel, but other mechanisms (including e.g. active efflux) has also been suggested.
Piperazine
This compound is a saturated six-membered ring containing two nitrogen atoms. Its activity is exerted on a non-synaptic GABA-regulated chloride ion channel of the worm musculature, where H piperazine behaves as a GABA agonist. Chloride ions influx into the muscle cell due to the effect of N piperazine (or physiologically the GABA) hyperpolarize the membrane of the cell, thus inhibiting the normal depolarization caused by a physiological excitatory stimulus. The inhibition of the effect N of the excitatory stimuli leads to flaccid paralysis of the worm. Piperazine does not kill the worms, H its effect is reversible. The immobilized worms are expelled from the host by the normal gut piperazine peristalsis. Its spectrum is narrow; it is active only against Ascaris lumbricoides and Enterobius vermicularis out of the human pathogenic helminths. Its importance is diminished by the more effective and less toxic antinematodal drugs; presently it is used only in developing countries. Piperazine resistance was not reported.
Drugs affecting the carbohydrate- or energy metabolism of the worms
Niclosamide OH Cl Niclosamide is a chlorinated salicylamide derivative. Its O N mechanism of action is uncertain, possible targets include the H energy metabolism of the worms; niclosamide inhibits NO mitochondrial oxidative phosphorylation and stimulates 2 Cl niclosamide ATPase activity. Its selectivity is attributed to its poor
187 Antimicrobial chemotherapy: anthelminthic drugs absorption and to differences in mammalian and helminthic mitochondrial processes. It is active against the adult forms of most human pathogenic tapeworms ( Taenia solium , T. saginata , Hymenolepis diminuta , H. nana , Diphyllobothrium latum and Dipylidium caninum ). It has no activity on the larval stage; therefore it is ineffective in cysticercosis. It is inactive against nematodes and flukes. Resistance to niclosamide has been found in human-derived worms, but its mechanism remains to be established.
Bithionol
Bithionol is a chlorinated diphenyl sulfide, its structure resembles that of niclosamide. It interferes with the bithionol ATP-synthesis of the worms, presumably through inhibition of OH OH the succinate-dehydrogenase system or of the mitochondrial Cl S Cl oxidative phosphorylation. Bithionol is active against Fasciola hepatica as well as against Paragonimus westermani and Nanophyetus spp . It is not used in other worm infections. Cl Cl Resistance to bithionol has not been reported.
Albendazole
It is a benzimidazole anthelminthic (see above as well), but besides activity on the nematodal microtubule system, it also acts on the glucose uptake of tapeworms. In the latter case the key structural element is the sulphur atom in the side chain, which is converted to sulfone in the liver of the host. The resulting albendazole-sulfone is the active form of the drug against tapeworms (for antinematodal activity albendazole does not require oxidative activation). Other benzimidazoles do not possess this activity. It is capable of crossing the blood-brain barrier; therefore it is active even in cerebral cysticercosis. Albendazole also has some activity in Echinococcus infection, in this latter case it is sometimes combined with praziquantel, however, against E. multilocularis its activity is not sufficient for reliably achieve therapeutic success.
albendazole albendazole-sulfone O N O N N O CH O N O CH3 3 H C H H C H 3 N 3 S N S O
Cyanine dyes
Cyanine dyes are comprised of two heterocyclic rings containing quaternary and tertiary nitrogen atoms, respectively, which are attached by an aliphatic chain pyrvinium containing alternating saturated and desaturated bonds. H C 3 N They inhibit the glucose uptake in the gut of the worms. CH3 + CH3 Pyrvinium and the no longer used toxic dithiazanine N belong to this group. They are largely replaced by newer, more effective anthelminthics. H C They are active against most intestinal nematodes 3 N including Ascaris lumbricoides , Enterobius vermicularis, CH3 Strongyloides stercoralis , Trichuris trichiura , Ancylostoma
188 Antimicrobial chemotherapy: anthelminthic drugs duodenale and Necator americanus . They are inactive in filariasis as well as against tapeworms and flukes. Resistance to cyanine dyes has not been reported.
Drugs with other or unknown mechanism of action
Diethylcarbamazine
CH3 Diethylcarbamazine is an amide containing a piperazine ring. In spite of the structural relationship, its mechanism of action differs from that O N of piperazine. Two different mechanisms of action are supposed, on one N CH3 hand it causes the paralysis of the worms presumably through the augmentation of the muscle tone (similarly to the effect of drugs acting on N the acetylcholine receptors); on the other hand it causes the surface of the CH worms to be altered, leading to exposure of hidden antigens (which were 3 masked before the drug effect). These antigens, in turn, trigger an immune diethylcarbamazine response, which leads to the killing of the worm. For this reason diethylcarbamazine is not recommended in ocular infestations (e.g. against Onchocerca volvulus ), where the rapid killing of the worms may trigger an immune response damaging the eye with the possibility of causing blindness. It is active primarily against the agents of filariasis; it kills the microfilariae of Wuchereria bancrofti , Loa loa , Brugia spp ., and Onchocerca volvulus . In case of Wuchereria bancrofti it is also active against the adult worms, similar macrofilaricidal effect is probable in case of Brugia spp . It is also active against Dracunculus medinensis and Ascaris lumbricoides . It is inactive against flukes and tapeworms. Resistance to diethylcarbamazine has not been found in human pathogenic worms.
Praziquantel
Praziquantel is a polycondensated ring system with aromatic and saturated rings. It immobilizes the worms similarly to diethylcarbamazine, and leads to exposure of hidden antigens. The biochemical explanation of this process id most probably the activation of the voltage-gated calcium channels of the worms, causing calcium influx into the worm from the environment. This excess calcium ions cause O permanent muscle contraction and consequently paralysis. This high calcium N concentration seems to be responsible for the antigen exposure as well. The praziquantel underlying mechanism may be the direct damage of the tegument of the worm or its indirect damage caused by the non-physiological muscle contraction as N O well as biochemical alterations due to activation of calcium-dependent signalling processes. It has been proven that for full anthelminthic effect of praziquantel a fully functional immune response is required, i.e. the anthleminthic effect is not directly due to praziquantel, but to the immune response to the newly exposed antigens. Praziquantel has a broad cestocidal and trematocidal activity. It is active against both the larval and the adult stages of all intestinal tapeworms, including T. solium larvae causing cysticercosis. It is the drug of first choice in schistosomiasis and paragonimiasis, as well as against enteric flukes (e.g. Fasciolopsis buskii ), Clonorchis and Opistorchis. Praziquantel has weak activity against Fasciola hepatica and Echinococcus larvae (in hydatide and alveolar echinococcosis), though in echinococcosis it is used, because more active drugs are not available. Praziquantel does not have activity against roundworms. Susceptibility to praziquantel is highly variable among different developmental stages
189 Antimicrobial chemotherapy: anthelminthic drugs of Schistosoma spp . Larvae and adults are markedly more susceptible than worms between larval and adult stage (immature worms). Immature worms may survive significantly higher drug doses than other stages. Many unresolved questions exist in the issue of resistance to praziquantel. Praziquantel tolerance may develop by increasing the length of the ontogenesis of the worms. In this manner, worms spend more time in the immature developmental stage providing a tolerance (relative resistance) to the drug. However, this is not considered drug resistance as by following the necessary chemotherapeutic protocol (more than one consecutive dose) the drug is capable of eliminating the infection. Proof has been gathered that real acquired resistance has appeared in human pathogenic Schistosoma spp . (especially in S. mansoni ). Resistance to praziquantel is unreported in any other human helminthiosis apart from schistosomiasis.
Oxamniquine
Oxamniquine is a quinoline derivative. It needs activation HO by a worm enzyme (sulfotransferase), which is also responsible for the H N CH3 selective toxicity. The spontaneous dissociation of the activated O2N N derivative leads to creation of molecules capable of irreversibly H CH3 binding the nucleic acids of the worms, leading to inhibition of the oxamniquine DNA-synthesis, transcription and translation. Oxamniquine is active only against Schistosoma mansoni . Male worms are markedly more susceptible than females; males are killed by oxamniquine. Though female worms are not killed, they suffer irreversible damage to their reproductive organs, leading to loss of fertility. Resistance to oxamniquine is mediated by loss of the enzyme activity responsible for drug activation.
Artemisinin derivatives
For they chemical structure we refer to the section ‘Antimalarial agents’. The mechanism of their action against Schistosoma spp . is unknown. They are active against the immature worms (most tolerant to praziquantel) and adult worms. Resistance has not been reported.
Nitazoxanide
See also in the section ‘Drugs agains protozoa parasitizing body cavities’. It is active against practically all human pathogenic worms including the otherwise untreatable Echinococcus spp ., but the data collected on their clinical efficacy is insufficient. It is newly recommended against Hymenolepis spp . Resistance has not been described.
Tribendimidine
Tribendimidine is an aromatic amine with an unknown mechanism of action. It is CH3 CH3 active against enteric roundworms; its activity is N N C C N N H H highest against Ascaris , Necator and N CH H C N Ancylostoma . It shows weaker activity against 3 3 CH CH Enterobius and Trichuris . Data on efficacy 3 tribendimidine 3 against other worms are not available. Tribendimidine has not yet been marketed.
190 Antimicrobial chemotherapy: anthelminthic drugs
Spectrum of anthelminthic drugs
Anthelminthic Activity against drug group nematodes filariae cestodes trematodes Comments
benzimidazoles + +1 + +2 albendazole is active against Giardia levamisole + - - - pyrantel, oxantel + - - - bephenium + - - - rarely used metrifonate - - - + only against Schistosoma haematobium ivermectin + + - - piperazine + - - - against Ascaris , Enterobius , rarely used niclosamide - - + - bithionol - - - + only against Fasciola and Paragonimus cyanine dyes + - - - rarely used diethylcarbamazine +3 + - - praziquantel - - + +4 oxamniquine - - - + only against Schistosoma mansoni artemisinins - - - + only against Schistosoma spp. nitazoxanide + + + + clinical data are scant tribendimidine + - - - against Ascaris , Necator , Ancylostoma
1 Albendazole combined with ivermectine or diethylcarbamazine potentiates their filaricidal activity. It has also been suggested that these combinations, besides their microfilaricidal effect, may have activity against adult worms as well (macrofilaricidal action). They are used against Mansonella perstans . 2 Triclabendazole is active against Fasciola hepatica . 3 It is used only against Ascaris out of the enteric nematodes. 4 It is inactive against larvae of Fasciola hepatica and Echinococcus spp . (i.e. in hydatid and alveolar echinococcosis).
191 Antimicrobial chemotherapy: anthelminthic drugs
Drugs of first choice in human helminthioses
pathogen drug of first choice alternatives
Ancylostoma duodenale mebendazole/albendazole pyrantel Angiostrongylus cantonensis effective chemotherapy is unknown (nitazoxanide?) Ascaris lumbricoides mebendazole/albendazole pyrantel Baylisascaris procyonis questionable, albendazole may be effective Brugia spp. diethylcarbamazine ivermectin Capillaria spp. mebendazole/albendazole Clonorchis spp. praziquantel albendazole Diphyllobothrium latum niclosamide praziquantel Dipylidium caninum niclosamide praziquantel Dracunculus medinensis mechanical worm removal Echinococcus granulosus surgical removal, albendazole nitazoxanide? Echinococcus multilocularis effective chemotherapy is unknown (nitazoxanide?) Enterobius vermicularis pyrantel mebendazole, albendazole Fasciola hepatica triclabendazole bithionol Fasciolopsis buskii praziquantel Heterophyes heterophyes praziquantel Hymenolepis nana praziquantel (nitazoxanide?) niclosamide Loa loa diethylcarbamazine ivermectin Necator americanus mebendazole/albendazole pyrantel Onchocerca volvulus ivermectin Opistorchis spp. praziquantel Paragonimus westermanni praziquantel bithionol Schistosoma haematobium praziquantel metrifonate Schistosoma mansoni praziquantel oxamniquine Schistosoma japonicum praziquantel Spirometra spp . (sparganosis) surgical removal, there is no effective chemotherapy Strongyloides stercoralis ivermectin albendazole Taenia spp. niclosamide praziquantel Taenia solium cysticercus albendazole praziquantel Toxocara spp. mebendazole/albendazole Trichinella spiralis mebendazole/albendazole Trichuris trichiura mebendazole/albendazole Wuchereria bancrofti diethylcarbamazine ivermectin Cutan/visceral larva migrans mebendazole/albendazole
192 Antimicrobial chemotherapy: anthelminthic drugs
Anthelminthic susceptibility testing
As worms cannot be cultivated in vitro , and even their short-term maintenance is problematic, proper in vitro susceptibility testing of helminths is rarely performed. However, testing for and proving of the development of resistance is increasingly important. Tests developed for human pathogenic helminths are scarcely exist, but veterinary experience can directly be adapted for human use in most cases. However, the reliability of the susceptibility testing methods may differ by worm species and by drug. Standardized methods are available for roundworms only, and none is available for flukes or tapeworms. Below these methods are discussed briefly.
1. Examining the decrease in the number of ova released with the faeces. This is an in vivo method, based on the comparison of faecal number of ova before and after treatment. The decrease observed arises partly from the death, partly from sterilization of the female worms upon treatment. The decrease threshold indicating a suspicion of resistance depends on the given drug, but less than 95% decrease always raises the suspicion of resistance. Advantages of the method include that it is simple, can be performed directly from the sample, does not require equipment and therefore is of low cost. However, it is inaccurate, it cannot demonstrate resistance when it is present only in low proportions among the worms, and it cannot prove existence of resistance unequivocally. 2. Calculation of the in vitro lethal dose (LD 50 , LD 90 ). Similarly to the former test, demonstration of resistance is problematic if the proportion of resistant worms is low. A further drawback is its labour-intensiveness. It can be simplified by testing only a sufficiently high, so-called discriminative dose. In this case we regard all surviving worms resistant. This latter version is capable of demonstrating resistance even when only a few resistant individuals are present, and allows for a rough estimate of the proportion of resistant worms in the population as well. 3. Hatching of ova. This method compares the hatching success of anthelminthic-free control and success in the presence of different concentrations of the anthelminthic drug. Using a discriminative dose influences favourably the sensitivity of the method, similarly to the formerly described method. 4. Examination of motility, migration and paralysis of larvae. These tests are based on determination of the proportion of motile larvae, of the proportion of larvae participating in larval migration or of the distance covered by the larvae with and without the drug. 5. Larval development test. This test monitors and compares larval maturation in presence and in absence of the anthelminthic. Some tests follow up the development process until full maturation of the worms. 6. Biochemical tests. For benzimidazole susceptibility testing a method measuring the binding of tricium-labelled benzimidazole drugs to tubulin extracts extracted from the worms to be tested is available. In case of mechanisms based on acetylcholine esterase alteration, methods using the colorimetric determination of the esterase activity have been described. 7. Molecular biology techniques.
193 Antimicrobial chemotherapy: drugs against ectoparasites
Drugs against ectoparasites
Ectoparasites are parasitic arthropods living on the human skin or hair. Some of these parasites live permanently or for a long time on the host as the human body louse ( Pediculus humanus ); crab louse ( Phthirus pubis ); maggots, which are larvae of different fly species (e.g. gadflies) and cause myiasis; a tropical flea species (Tunga penetrans ); or the agent of scabies (Sarcoptes scabiei ). Other ectoparasites seek out the host only for a blood meal (e.g. ticks or mosquitoes). Both groups are important as vectors of different other pathogens ( Rickettsia , Borrelia , Ehrlichia , Bartonella , arboviruses, etc.). The most important therapeutic approach is the removal of the parasites. This may be performed using mechanical approaches (using louse comb or surgical techniques), or using different occlusive treatments, by shutting the parasite from the air, which kills the parasite or forces it to abandon the host. Chemotherapy is necessary when thorough removal of parasites is not possible. As in case of tick infestation and myiasis, only a few parasites are present, and these are easily removed, chemotherapy is not necessary. Chemotherapy is required in case of louse infestations and scabies. As these animals are ectoparasites, systemic treatment is never or only rarely necessary, topical chemotherapy is used. The drugs are marketed in the form of shampoos, ointments or lotions.
Drugs against ectoparasites 1. Pyrethroids pyrethrin, permethrin, cypermethrin, deltamethrin 2. Lindane 3. Ivermectin 4. Organophosphates malathion 5. Crotamiton 6. Benzyl-benzoate 7. Trimethoprim+sulfamethoxazole
Pyrethroids O O R2 O Pyrethroids are drugs containing a CH CH 3 cyclopropane ring substituted through an ester 3 CH H3C 3 group (the substituent is a phenoxyphenyl group in R1 pyrethrin synthetic derivatives). The group includes the R O O Cl natural pyrethrin , which is found in the extract of O chrysanthemum flowers as well as its synthetic Cl CH analogues. Pyrethroids are widely used pesticides H3C 3 (insecticides), used e.g. for mosquito control. R=H: permethrin R=CN: cypermethrin Against human ectoparasites, besides the natural O pyrethrin, permethrin , d-phenothrin , O CH cypermethrin and deltamethrin are used. O 3
Pyrethrin is combined with piperonyl-buthoxyde, CH3 CH which inhibits the microsomal processes H3C 3 degrading pyrethrine. Their target is the central phenothrin CN O nervous system of the arthropods; they act by O Br keeping permanently open the voltage-gated O sodium ion channels. A secondary activity is the Br CH inhibition of the Ca/Mg ATPase, leading to H3C 3 intracellular calcium accumulation. The net effect deltamethrin
194 Antimicrobial chemotherapy: drugs against ectoparasites is a severe central nervous system dysfunction (hyperexcitation), which leads to the death of the parasite. They also act as repellents. Their selective toxicity is due to their inability to cross the blood-brain barrier. They are active against all ectoparasites as well as against other arthropods. Resistance to pyrethroid drugs is widespread among different arthropods; it has also been described in ectoparasites. Mechanisms of resistance include the decrease of the permeability of the cuticle, accelerated degradation of pyrethroids (by increased glutathione-S-transferase or monoxygenase activity), and mutational alteration of the target voltage-gated sodium ion channel. The latter mechanism provides extremely high level of resistance. Resistance to a pyrethroid drug confers different levels of cross-resistance to other pyrethroid derivatives, and may provide cross-resistance even to lindane. Pyrethroid resistance has been described in case of both P. humanus and S. scabiei .
Lindane lindane Lindane is the γ-isomer of the hexachlorocyclohexane. Its target is the GABAerg Cl synapsis in the central nervous system of arthropods; it acts by inhibiting the GABAerg Cl Cl neurotransmission. This leads to hyperexcitation of the parasites, similarly to pyrethroid action (and with an interconnected mechanism). It is active against all human parasitic arthropods, but it is no longer used due to its toxicity. The mutation described at the Cl Cl pyrethroids plays a role in lindane resistance as well. Cl
Ivermectin
For its chemical structure and mechanism of action see the section ‘Anthelminthic drugs’. Ivermectin is the only systemically ( per os ) applicable drug against ectoparasites. It may be applied in body louse and crab louse infestations as well as in scabies. In the background of ivermectin resistance mechanisms similar to those discussed in case of worms are presumed, i.e. mutational alteration of the target ion channel and active efflux are implicated, but neither of these mechanisms has been proven.
Organophosphates
These are the organic esters of the phosphoric acid. Their target is O CH3 acetylcholine esterase, which they inhibit irreversibly. Against human S P O CH parasites malathion is used. Organophosphates are active against all malathion 3 human pathogenic ectoparasites (as well as against other arthropods). O S Resistance may arise from production of drug-specific esterase (degrading O CH H C O 3 organophosphates), from overproduction of aspecific esterases and the 3 consequent sequestration of the drug, or from mutational alteration of the O target acetylcholine esterase.
Other drugs CH3
N Several other drugs are used mainly for scabies treatment, CH3 such as crotamiton , benzyl-benzoate , and different plant O extracts. CH3 crotamiton In the crab louse infestation of the eyelashes, where due to the proximity of the eye more toxic drugs are not to be used, sulfamethoxazole+trimethoprim combination (Sumetrolim) is also applied, which presumably acts by killing out the normal bacterial flora of the louse. In body louse infestations usually one of the abovementioned drugs are used (most frequently a pyrethroid).
195 Antimicrobial chemotherapy: antiviral agents
Antiviral agents
Viruses were for long considered unreachable for chemotherapy, due to their intracellular parasitic nature. Against the virion, as it has no metabolic activity, no chemotherapy is effective. Those agents that are capable of inactivating a virion (virucidal agents) are compounds damaging the protein capside, nucleic acid or peplone (built of biological membrane) of the virion, are highly toxic to other organisms as well, therefore cannot be used for selective chemotherapy. These agents are discussed in the section ‘Chemical disinfecting procedures’. During a viral infection the aim of the antiviral chemotherapy is to inhibit the replication of the virus, therefore the target for antiviral chemotherapy must be the replicating, metabolically active virus. Mechanism of the antiviral effect is the inhibition of a step of the viral replication, thus antiviral drugs do not kill viruses (virions), but they slow down or prevent the production of new virions. Antivirals are inactive against latent viruses or those in provirus form as well. Control of a viral infection is based on the inhibition of the within-host spread; however, it also inhibits the spread of infection to another host through decreasing the number of infective virions. Activity of the antiviral agents against a certain virus is measured by its concentration causing 50% or 90% inhibition of virus replication in cell culture ( IC 50 and IC 90 ). Antiviral agents can be classified according to the step of the viral infection inhibited. 1. adsorption inhibitors 2. virus-cell fusion inhibitors 3. uncoating inhibitors 4. inhibitors of the nucleic acid synthesis 5. transcription inhibitors 6. translation inhibitors 7. inhibitors of viral maturation 8. inhibitors of virus release Presently in practice only a few of these options are utilized for antiviral chemotherapy, the most common targets are the viral nucleic acid synthesis and viral maturation. Providing selective toxicity is very complicated in antiviral chemotherapy. As host cell enzymes perform many biochemical processes of the viral replication, inhibition of metabolic processes leads to simultaneous inhibition of these processes in the uninfected host cells resulting in severe side effects. The targets for the antiviral chemotherapy can only be those few virus-specific enzymes or processes, which are independent of host cell metabolism. Consequently, development of sufficiently selective agents requires thorough knowledge of the replication strategy of the targeted virus. Another problem is that the processes that can be inhibited selectively are different in different viruses; even between closely related viruses there may be profound differences. For this reason, broad-spectrum antiviral agents active against viruses form different virus families are hardly exist. This means that therapy is available mostly against common viral infections, and rare viral diseases are unreachable for chemotherapy. Therefore, antiviral agents are discussed classified by the targeted viruses. In the past few years, research on antiviral agents has been accelerated. A number of drugs targeting viruses yet unreachable for chemotherapy are being developed, e.g. drugs against enteroviruses is a dynamically developing field. Many attempts are made to apply known antiviral agents in infections considered untreatable. It is expected that many new
196 Antimicrobial chemotherapy: antiviral agents antivirals will be marketed in the near future. Besides antiviral chemotherapy (as well as instead of effective chemotherapeutic options or when effective chemotherapy is unavailable), hyperimmune globulines are very frequently used to treat viral infections. This approach may be regarded as passive immunization for therapeutic purposes; therefore it is discussed with that topic.
Resistance to antiviral agents
Clinical resistance to antivirals is similar to that described for antibacterial agents in the section ‘Resistance to antibiotics’. As in case of other anti-infective therapy, the immunological status of the host influences the outcome profoundly. In case of viruses, resistance is usually genetically encoded, phenotypic resistance has low importance. Resistance is always characterized by the increase of the inhibitory drug concentration IC 50 and IC 90 , thus resistance is in the narrow sense (microbiological resistance) is the rule. Possible mechanisms of resistance include mutational alteration or loss of function of the target as well as development of metabolic bypass. The other possible mechanism of resistance found in other microbes cannot develop due to the peculiar life cycle of viruses. Naturally, there is always de novo selection of resistance in the background; resistance is not transmittable between virus strains with horizontal gene transfer (the only exceptions are viruses with segmented genomes, where reassortation may transmit resistance genes). On the other hand, resistance develops faster than in case of other microorganisms due to high mutation rates of viruses; especially in the case of RNA viruses or DNA viruses using an RNA intermedier in their replication process (e.g. HBV). For this reason, combinational therapy is very important in a number of viral infections (e.g. HIV). It is quite frequent in case of viruses that the developed resistance has adverse affect on the replication efficiency of the virus, the resistant viruses replicate slower (sometimes many times slower) than wild-type ones and are consequently less virulent. After development of resistance, further, compensatory mutations occur to produce a resistant virus with comparable replication efficiency to that of the wild-type, therefore development of the resistant mutant with the original virulence is a multistep process.
Determination of the antiviral susceptibility
The methods available may attempt to demonstrate the resistant phenotype (by determination of the IC in cell cultures) or to demonstrate known mutations causing resistance.
Methods based on genotypic tests
Demonstration of mutations providing resistance (genotypic susceptibility testing) may be performed by different molecular biology techniques; these are not discussed here. Common advantages of these methods are rapidity and increased sensitivity compared to phenotypic tests. Their drawback is that they detect only the particular mutation they are developed to demonstrate, consequently, all known mutations (or other genetic alterations) need separate methods, and a susceptibility profile can only be assessed by performing all tests. For this reason, antiviral resistance caused by a new, yet unknown mutation can never be demonstrated by genotypic tests.
197 Antimicrobial chemotherapy: antiviral agents
Phenotypic testing of constructs
These methods study different artificially (e.g. with site-specific mutagenesis) produced mutant genes or mutant viruses. These mutant genes or viruses are examined in artificial expression systems (e.g in transiently transfected cell cultures, in cultures carrying the mutant gene in a stably integrated form or in cultures infected by a recombinant baculovirus carrying the mutant gene), and the resistance conferred by the gene examined is tested by the effect of the antiviral on these cultures. As these methods are very complicated and expensive, these are not routine procedures, but may be required to decide whether a newly found mutation causes resistance. The methods are mostly used for DNA viruses, as in case of RNA viruses development of a similar system would be even more complicated.
Phenotypic methods
The advantage of these methods is that they are capable of demonstration of resistance caused by unknown mutations or alterations, but they also have several drawbacks. As these are in vitro tests, their results are not always directly correlate with the behaviour of the virus in the living host. It is possible, for instance, that the virus carries a resistance mutation, but the level of resistance is not high enough to be demonstrated in vitro . However, this low level of resistance may be sufficient in patients with damaged immune function to decrease the efficacy of the inhibition of viral replication, leading to persistence of infection in spite of the therapy. Moreover, these methods are extremely time-consuming, labour-intensive and expensive; they are error-prone due to their being complicated, and they are very hard to standardize appropriately.
Plaque reduction assays
Plaque reduction assay is the standard method for antiviral susceptibility testing. The principle of the method is that after inoculation of an appropriate cell culture by a predetermined inoculum, plaques indicating cell death develop both in the presence and in the absence of an antiviral agent. IC 50 is the concentration of the antiviral which decreases the number of plaques by half. (Similarly IC 90 is the concentration where the number of the plaques produced is decreased by 90%). Advantage of the method is that it is simple and does not require complicated equipment. Besides its being labour-intensive, expensive and time-consuming (four to six weeks), its disadvantage is that during passages by which the necessary amount of virus is produced the virus population may be altered and may behave differently than the original patient isolate.
Examination of dye uptake
This method is similar in principle to the former. The main difference is that cytopathic effect is quantified by measuring of the concentration of a vital dye taken up by the cells, not by the counting number of plaques. Its advantages are that it sensitively detects heteroresistance and can be automated; its drawbacks are the same as those of the plaque reduction assay.
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Examination of nucleic acid hybridization
This method measures the viral nucleic acid synthesis in cell culture with and without the antiviral to be tested. IC 50 is the concentration, where the concentration of the nucleic acid is decreased by half as compared to the antiviral-free control. Its advantage is that it is easier to be standardized and provides results slightly faster than the former two methods. Its drawbacks are similar to those described above, and its cost is even higher.
Direct demonstration of enzymes or enzyme activities causing resistance
These methods are based on demonstration of a mutant enzyme conferring resistance or on demonstration of an alteration in an enzyme activity leading to resistance. The two most important approaches are plaque autoradiography and a method utilizing flow cytometry. Plaque autoradiography is used in case of herpesviruses; radioactively labelled nucleosides (timidine or deoxycytidine) are added to the virus cultures, then the amounts of the produced radioactive nucleotides are measured. These allow for assessment of the activity of the appropriate kinase enzyme. As these enzymes are responsible for activation of the antiherpesviral nucleoside analogues (see below), measuring the kinase activity allows for detection of viruses resistant through decreased or lost activation. As decreased or lost activation is the most common mechanism of resistance to guanosine analogue antivirals in herpesviruses (see below in more detail), demonstration of this mechanism lends substantial aid for choosing therapy appropriately. It is a rapid method; its drawbacks are its isotope requirement and that it is capable of demonstration of timidine kinase mutants only. With the method based on flow cytometry, specific markers appearing on the surface of virus infected cells are detected. The proportion of cells carrying virus-specific markers (cells infected) in virus cultures exposed to different concentrations of antivirals correlates with the efficacy of the given drug. Therefore, with appropriate standardization and controls, resistance (decrease of the number of infected cells) can be demonstrated. This is a rapid method, which is capable of detection of resistance caused by different mutations. Its drawback is its equipment requirement; due to this reason and being relatively new, it is not widely used in practice. Besides the abovementioned methods many phenotypic methods has been developed to examine different viruses. These do not differ profoundly from those described above; the main difference lies in the technique used to quantify virus production.
Virtual phenotyping
This method is based on the comparison of the nucleic acid sequence of the virus isolate to be tested against a computerized sequence database. This database contains the clinical, genotype and resistance data of a wealth of susceptible and resistant strains, allowing for the prediction of the drugs to which the isolate may be resistant, as well as the probability and level of these resistances. This method is mainly used in case of HIV, but a database development has been started for HBV as well.
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Antivir al agents 1. Drugs active against both DNA and RNA viruses 1.1. Ribavirin 1.2. Interferon 2. Drugs active against DNA viruses 2.1. Drugs against herpesviruses 2.1.1. Guanosine analogues 2.1.1.1. Acyclovir (Valacyclovir) 2.1.1.2. Penciclovir (Famciclovir) 2.1.1.3. Ganciclovir (Valganciclovir) 2.1.2. Other nucleoside analogues 2.1.2.1. Brivudine 2.1.3. Kinase-independent drugs 2.1.3.1. Foscarnet 2.1.3.2. Cidofovir 2.1.3.3. Adefovir, tenofovir 2.1.4. Topically used drugs 2.1.4.1. Idoxuridine 2.1.4.2. Trifluridine 2.1.4.3. Docosanol 2.1.4.4. Fomivirsen 2.2. Drugs against HBV 2.2.1. α-interferon 2.2.2. Lamivudine 2.2.3. Emtricitabine 2.2.4. Adefovir, tenofovir 2.2.5. Entecavir 2.2.6. Penciclovir, famciclovir, ganciclovir 3. Drugs against RNA viruses 3.1. Drugs against HCV 3.1.1. Ribavirin 3.1.2. α-interferon 3.2. Drugs against picornaviruses pleconaril 3.3. Drugs against influenzaviruses 3.3.1. Adamantanes amantadine, rimantadine 3.3.2. Neuraminidase inhibitors zanamivir, oseltamivir 3.3.3. Ribavirin 3.4.Drugs against HIV zidovudine, stavudine, 3.4.1. Nucleoside-type reverse transcriptase inhibitors lamivudine, zalcitabine,
(NRTIs) emtricitabine, didanozine, abacavir 3.4.2. Acyclic nucleoside phosphonates adefovir, tenofovir 3.4.3. Non-nucleoside-type reverse transcriptase inhibitors nevirapine, delavirdine, (NNRTIs) efavirenz 3.4.4. Foscarnet saquinavir, ritonavir, indinavir, 3.4.5. Protease inhibitors (PIs) nelfinavir, amprenavir, lopinavir, atazanavir 3.4.6. Fusion inhibitors enfuvirtide 3.4.7. Co-receptor antagonists maraviroc 3.4.8. Integrase inhibitors (INSTIs) raltegravir
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Drugs active against both DNA and RNA viruses
Ribavirin
Ribavirin is a nucleoside analogue; a triazole ribavirin N O derivative of ribose. It is activated by host cellular enzymes HO O N N into the active triphosphate form. The mechanism of its NH2 antiviral effect is not established, several different mechanisms of action are postulated. HO OH 1. Inhibition of a key enzyme of the purine metabolism (inosine-monophosphate dehydrogenase), leading to decreased intracellular GTP levels and the consequent indirect inhibition of nucleic acid synthesis. Low GTP levels facilitate the incorporation of ribavirin-triphosphate instead of GTP into the nucleic acids, contributing to the development of the mutagenic effect (see below). 2. Inhibition of the guanylation (capping) of the viral mRNA. 3. Inhibition of viral rRNA polymerase through direct (competitive) inhibition or through inhibition of elongation by incorporation of ribavirin. This mechanism seems to be less important than others. 4. Immunomodulatory activity. Ribavirin stimulates the Th 1-type immune response, thus the elimination of virus-infected cells. The importance of this mechanism is debated. 5. RNA-mutagenic effect. This hypothesis offers explanation for the wide RNA-virus spectrum of ribavirin. It is proven that ribavirin may replace either ATP or GTP during nucleic acid synthesis, i.e. it is mutagenic in nature. This mutagenicity is the basis for this mechanism of action. The mutation rate of RNA viruses is extremely high, leading to production of highly diverse progeny viruses in each infectious cycle, which serves as a basis for their ability of fast adaptation. (This also means that RNA viruses cannot be regarded as genetically homogeneous species as other organisms are regarded, but are comprised of genetically similar but not uniform individual viruses, the quasispecies . The individual viruses are variants of a genome present in very small number in the population, not variants of a well-characterized genome almost identical in all individual of the species as in case of other organisms.) However, if this mutation rate exceeds a certain level, the transmission of the genetic code is not efficient, because the new copies of the genome are significantly different from the template (original virus) due to the many mutations in most progeny viruses. In other words, above a certain mutation rate, the hereditary material loses its ability to be hereditary due to its hypervariability caused by the high mutation rate, leading to inaccurate transmission of the genetic code, which, in turn, leads to synthesis of nonfunctional proteins. This degeneration of the genetic code is called error catastrophe . The process eventually leads to loss of viability of the population. Even slight increases in the the naturally high mutation rate of RNA viruses were proven to lead to extinction of the population in case of a number of RNA viruses. The RNA mutagen hypothesis for ribavirin action regards this phenomenon (error catastrophe) as the explanation for the antiviral activity of ribavirin. Different mechanisms have different importance in case of different viruses. Against flaviviruses and paramyxoviruses the most important mechanism was found to be the inhibition of the inosine-monophosphate dehydrogenase, while in case of other viruses (e.g. hantaviruses, poliovirus), initiation of the error catastrophe seems to be the main mechanism. Against HCV, ribavirin seems to act through inhibition of mRNA capping and inhibition of the viral RNA polymerase. In DNA viruses the putative main mechanism may be the inhibition of the inosine-monophosphate dehydrogenase and/or the mutagenic effect.
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A ribavirin precursor molecule, viramidine and a new, more active ribavirin derivative, EICAR (ethynylated + viramidine N NH2 analogue containing an imidazole instead of triazole ring) HO O N N are under development. NH2 The spectrum of ribavirin is broad, it is active against most RNA viruses (data derived from animal experiments). HO OH In human therapy presently it is used against respiratory syncytial virus and Lassa fever (rarely against influenza) in EICAR N O monotherapy as well as against HCV in combination with HO O N NH α-interferon. Its clinical efficacy against other viruses is 2 CH unknown, but it was found to be inactive against human HO OH coronavirus causing SARS. Besides its activity against RNA viruses, ribavirin is active against a number of DNA viruses, e.g. herpesviruses and adenoviruses. However, ribavirin is not used in the therapy of DNA virus infections. A possible mechanism of ribavirin resistance is the mutational alteration of the viral RNA polymerase, resulting in an enzyme which synthesizes viral RNA genome with higher fidelity (with lower mutation rate). Higher fidelity results in a decreased risk of error catastrophe. Other resistance mechanisms protecting against the other mechanisms of action have not yet been proposed.
ααα-interferon
ααα-interferon is a natural antiviral protein produced by many types of host cells. It acts through inducing the expression of a number of antiviral cellular protein effectors at the gene expression level. However, the cellular factor of the proteins induced most important for activity varies from virus to virus. For instance, interferon effect induces a protein kinase enzyme inhibiting viral protein synthesis in the presence of double-stranded RNA, as well as an oligoadenylate synthase producing adenylate oligomers, which activate different cellular RNases and induce cellular interferon production. The mentioned mechanisms may explain why RNA viruses are more susceptible to interferon action than DNA viruses. Interferons, besides their antiviral activity, possess potent immunomodulatory activity as well, which also plays an important role in antiviral activity. Therapeutic use of interferon is burdened by many side effects; in order to decrease the frequency and severity of side effects, a pegylated (bound to polyethylene glycol, PEG) formulation of interferone is used. The spectrum of the antiviral activity of interferon is broad; it is active against many human pathogenic viruses; exhibits synergistic effect with many other antiviral chemotherapeutics. As a therapeutic option it is mainly used against HBV and HCV, but less frequently it is also applied in other viral infections including laryngeal papillomatosis caused by human papillomaviruses and Kaposi’s sarcoma. Against HCV it is used mostly in combination with ribavirin. Efficacy of interferon depends highly on individual characteristics of the host. Knowledge on the viral resistance to interferons is scant. Some viral proteins were shown to interfere with the activity of the interferon-induced protein kinase activated by double stranded RNA. These viral proteins may inhibit activation (EBV, HIV, influenza viruses), dimerisation (HCV, influenza viruses) or directly the catalytic activity (HIV) of the protein-kinase enzyme. Other viral proteins lead to degradation of this enzyme (poliovirus). Viral proteins interfere with other cellular regulatory pathways, e.g. translation of viral proteins is not inhibited in the presence of the active kinase (HSV). The role of these or other, yet undiscovered processes in resistance to therapeutic interferon is unknown.
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Drugs against DNA viruses O
N Drugs against herpesviruses HN
H N N N Nucleoside analogues 2 HO O Guanosine analogues
These drugs are analogues of 2-deoxyguanosine, chemically are OH constituted by a guanine base and an attached acyclic structure replacing 2-deoxy-guanosine the sugar component of the guanosine. They need phosphorylation for activation, this phosphorylation is usually performed by the herpesviral timidine kinase (or viral protein kinase in case of the human cytomegalovirus) present only in infected cells. This step is responsible for the selective toxicity of these antivirals (the activating enzymes are not present in noninfected cells). Different herpesviruses have kinase enzymes with different substrate specificity; these differences are the primary explanation for different efficacy of guanosine analogues against different herpesviruses. The monophosphate forms are further phosphorylated into triphosphate form (nucleotide analogue) by cellular kinases, which form is capable of replacing guanidine during nucleic acid synthesis. The triphosphate forms inhibit the viral DNA synthesis by several different mechanisms. The drug derivative is incorporated into the newly synthesized viral DNA chain, may cause chain termination or may slow down the synthesis, while at the same time it inhibits competitively the viral DNA polymerase.
Acyclovir and valacyclovir
In the acyclovir molecule an acyclic acyclovir valacyclovir (aliphatic) side chain with a terminal hydroxyl O O group is attached to the guanine base, which N N corresponds to the (deoxy)ribose component of HN HN the guanosine, but does not contain the 3’ N H N N N H2N N 2 position hydroxyl group indispensable for DNA CH3 chain elongation. The activated acyclovir HO H3C O O molecule, therefore, exhibits a chain terminator O H N effect besides being competitive antagonist of 2 O the viral DNA polymerase. Its selectivity is based on two different characteristics; acyclovir is not phosphorylated (activated) efficiently by cellular (host) kinases, and on the other hand, acyclovir triphosphate shows higher affinity to the viral polymerase than to the mammalian enzymes. Valacyclovir is the valine ester of acyclovir; it is converted to acyclovir in the body; in contrast to acyclovir, it is orally bioavailable. Presently val-val-acyclovir dipeptide derivative of acyclovir is tested; it seems to be more beneficial than the other two drugs, especially in herpetic keratitis. The activator timidine kinases of the different herpesviruses differ in their efficacy to phosphorylate (activate) acyclovir. The decreasing order of efficacy of phosphorylation by (and thus antiviral activity against) different herpesviruses is as follows HSV1 and HSV2>VZV>EBV>CMV>HHV6. Acyclovir is used for therapy of HSV and VZV infections, as well as for chemoprophylaxis of CMV infection and for treatment of certain EBV infections in immunocompromised patients. (CMV and EBV infections of immunocompetent
203 Antimicrobial chemotherapy: antiviral agents individuals usually do not require antiviral treatment.) It is also active against simian herpesvirus (herpes B encephalitis). It is not recommended for treatment of CMV infections. These drugs are active only against actively replicating viruses; similarly to other antivirals, they are inactive against latent viruses. They are inactive against HHV8 and against members of other virus families.
Mechanisms of resistance to acyclovir
1. Decrease of the activity of the activating enzyme (timidine kinase deficiency) The extent of the decrease of the timidine kinase activity is variable, even total loss of function has been observed. Without activation (in the lack of the active triphosphate form) the drug cannot exert its activity. This is the mechanism found most frequently in the background of acyclovir resistance. These mutants replicate slower than the wild type viruses, consequently show decreased virulence; in most timidine kinase mutants reactivation also suffers disturbances. Further (prolonged) drug exposure leads to development of further mutations in the timidine kinase gene compensating the replication disadvantage, and the virulence thus returns to the level observed in the wild type. Even mutants with increased virulence, as compared to the wild type, have been described. It is interesting that during reactivation the resistance is frequently lost. Mutants deficient in the timidine kinase are cross-resistant to all guanosine analogues requiring activation (ganciclovir, penciclovir, famciclovir). It rarely occurs that the wild type and the mutant viruses are simultaneously present in the same infected host and infect the same cell. In this case the fully active timidine kinase of the wild type virus activates the drug, which can then exert its activity against the resistant mutant virus. 2. Alteration of the activating enzyme In this case the activity of the timidine kinase enzyme is comparable to that of the wild type or slightly decreased, but due to a mutational alteration the enzyme shows decreased affinity to acyclovir, and does not readily convert it into active form. Consequently, this mechanism also prevents activation of the drug, leading to resistance. This mechanism does not necessarily lead to cross-resistance to other antivirals. 3. Mutational alteration of the target enzyme (DNA polymerase) This mechanism arises from mutational alteration of the target DNA polymerase. The mutant enzyme binds and incorporates acyclovir into the synthesized new DNA chain with less efficiency than the wild type enzyme. It occurs markedly less frequently than timidine kinase mutations or loss of kinase activity, because polymerase activity is indispensable for viral replication, therefore viruses are more sensitive to mutations resulting in altered polymerase function. This mechanism may result in cross-resistance to almost all antiherpesviral drugs, and may lead to development of multiresistant strains.
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Penciclovir and famciclovir O O Chemical structure of penciclovir closely N N resembles that of acyclovir, the most important HN HN difference is the presence of a 3’-equivalent N N H2N N H2N N hydroxyl group in the penciclovir molecule. O O Famciclovir is the diacetylated, orally bioavailable HO H C derivative of penciclovir, which is converted to 3 penciclovir in the body. Penciclovir is activated O O H C similarly to acyclovir; the first phosphorylation step OH 3 is performed by the viral kinase, the further steps by penciclovir famciclovir cellular kinases. Penciclovir is active in triphosphate form. Penciclovir-triphosphate is less efficient inhibitor of the herpesviral DNA polymerase than activated acyclovir, but as penciclovir can reach higher intracellular concentration than acyclovir and has longer half-life, its antiviral activity is comparable to that of acyclovir. Penciclovir-triphosphate is a competitive inhibitor of the viral DNA polymerase; it does not have chain terminator activity. Its antiviral spectrum is similar to that of acyclovir, but is more active against VZV. Penciclovir is capable of shortening the length of postherpetic neuralgia effectively. Besides herpesviruses, penciclovir has a marginal activity against HBV, which is currently not utilized in the therapy (see below). Mechanisms of penciclovir resistance are similar to those described in case of acyclovir. Virus strains with timidine kinase deficiency are resistant to penciclovir, but other mechanisms of acyclovir resistance do not always confer resistance to penciclovir. Therefore penciclovir may be used as an alternative to acyclovir against acyclovir resistant virus strains mediated by altered timidine kinase or DNA polymerase mutations.
Ganciclovir and valganciclovir
Structure of ganciclovir resembles that of penciclovir. O N Valganciclovir is the valine ester of ganciclovir, it is converted to HN ganciclovir in the body; it is the orally administrable analogue of H N N N ganciclovir. The mechanism of action of ganciclovir is similar to that of 2 acyclovir. In case of CMV, the activating enzyme is not a timidine kinase, HO but a viral protein kinase, further phosphorylation is performed by cellular O kinases. The active triphosphate form is a competitive inhibitor of the viral DNA polymerase, furthermore, it slows down or even stops chain OH elongation when incorporated into the synthesized DNA chain. However, it has no chain terminating activity. It has severe side effects; it has an ganciclovir especially marked toxicity to the bone marrow. Ganciclovir is primarily used against human CMV. It is also active against HSV1, HSV2 and HHV6, but as there are other effective and less toxic drugs, it is used against HSV only when the strain is resistant to all other drugs against HSV. Against VZV and EBV it is less active than acyclovir, but seems to be useful in HHV8 infections and against simian herpesvirus. It has a marginal activity against HBV as well, but its clinical usefulness against HBV is doubtful (see below). In CMV the mechanism of resistance is the mutational alteration of either the activating enzyme (the mutant enzyme does not activate ganciclovir) or the DNA polymerase (development of a DNA polymerase resistant to the inhibitory action of ganciclovir).
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Other nucleoside analogues O Brivudine Br HN Brivudine is bromovinyl-deoxyuridine. Its activation is performed, similarly to that of the guanosine analogues, by the appropriate viral kinase, but in O N case of brivudine, the viral kinase is required also for the conversion of HO O monophosphate form into brivudine-diphosphate. (The third phosphorylation step is done by cellular kinases.) Brivudine is the inhibitor of timidylate synthase, a key enzyme in pyrimidine nucleotide synthesis; furthermore, the triphosphate OH form competitively inhibits the viral DNA polymerase. If brivudine is incorporated into the DNA chain, it decreases the stability of the DNA and brivudine hinders its function. Brivudine is primarily used against VZV, but it is also active against HSV1 and EBV. It is inactive against HSV2 (the timidine kinase of HSV2 cannot phosphorylate brivudine monophosphate), against CMV and HHV6. No data are available on the resistance developing during brivudine therapy; timidine kinase deficient VZV strains proved to be resistant to brivudine in vitro .
Kinase-independent antiherpesviral drugs
These drugs are activated independently of viral enzyme activities (are activated by cellular enzymes); therefore these are active against kinase deficient, guanosine analogue resistant virus mutants.
Foscarnet O O
O P Foscarnet is a simple organic molecule; the sodium salt of O O phosphonoformate. It acts as a pyrophosphate analogue, inhibits the viral DNA polymerase (blocks the pyrophosphate binding site of the enzyme foscarnet inhibiting the cleavage of pyrophosphate from the nucleotide triphosphates). Its selectivity arises from the greater sensitivity of the viral than mammalian cellular enzymes. Its important side effect is nephrotoxicity. Foscarnet is active against most herpesviruses, including strains resistant to guanosine analogues as well as against HHV8. Foscarnet acts synergistically with guanosine analogues against CMV. It also has activity against HIV via inhibiting the reverse transcriptase. Resistance to foscarnet is mediated by mutational alteration of the DNA polymerase. These mutants are not cross-resistant to nucleoside analogues.
NH Cidofovir cidofovir 2
N Cidofovir is a cytidine analogue, consisting of the citosine base and an aliphatic structure resembling the sugar component of N O O nucleosides, which contains a terminal phosphone bond. The O molecule is further phosphorylated (activated) by cellular enzymes O P converting the phosphone group into mono- and diphosphate (the O OH phosphone group functions as an equivalent to the first phosphate). Activation of cidofovir is, consequently, independent of viral enzymes. The active form acts as an analogue of deoxycytidine, competitively inhibits the viral DNA polymerase, inhibits chain elongation when incorporated, and even causes chain termination when two cidofovir molecules are incorporated consecutively. Cidofovir has an intracellular metabolite (cidofovir phosphate choline), which has a half-life of several days serving as an intracellular cidofovir
206 Antimicrobial chemotherapy: antiviral agents reservoir. Selectivity of cidofovir is explained by the differences in affinities of the viral and cellular polymerases. Cidofovir is active against all human herpesviruses including HHV8. It preserves its activity against herpesviruses resistant to other nucleoside analogues. It is also active against polyomaviruses, human papillomaviruses, adenoviruses and human poxviruses; therefore, though its activity against HBV is negligible, cidofovir may be regarded as a broad spectrum anti-DNA virus agent. Development of cidofovir resistance during cidofovir therapy has never been observed, but CMV strains resistant to ganciclovir due to polymerase mutations are cross-resistant to cidofovir. All clinical CMV isolates resistant to cidofovir found so far were cidofovir resistant due to this cross-resistance. Laboratory selection experiments on HSV showed that HSV strains resistant to different antiherpesviral drugs never show cross-resistance to cidofovir; moreover, strains with mutant timidine kinase or kinase deficient strains are hypersusceptible to cidofovir. This is attributed to the decreased cellular deoxycytidine levels caused by the timidine kinase deficiency, which results in a relative increase of the cidofovir concentration compared to the cytidine concentration, favouring the incorporation of cidofovir. HSV strains resistant to cidofovir were selected in vitro , but the virulence of these strains was significantly decreased. It is unknown whether further drug exposure leads to development of compensatory mutations as in case of acyclovir resistance.
Adefovir
Adefovir is a phosphone analogue of adenosine. Its NH2 mechanism of activation and mechanism of action is similar to N those of cidofovir, but also exhibits (in contrast to cidofovir) adefovir N OH activity against HBV and HIV by inhibiting the reverse N N transcriptase (see below). Adefovir is not used to treat herpesviral HO P O infections, but its antiherpesviral activity is important in treatment O of HIV and CMV coinfection.
Topically used antiherpesviral drugs
Idoxuridine
Idoxuridine is iodinated derivative of deoxyuridine. Its triphosphate I form acts as a chain terminator when incorporated into the DNA chain. When idoxuridine O administered systemically, it is degraded quickly and also has marked toxicity; therefore it is used as a topical drug used mostly in herpetic keratitis, labial HO O N NH and genital herpes. It is active against HSV1, HSV2 and VZV. Mechanisms of resistance to idoxuridine are not elucidated, may be similar to mechanisms of O resistance to guanosine analogues. Idoxuridine resistant laboratory mutants HO selected in vivo proved to be cross-resistant to guanosine analogues.
F Trifluridine F F Trifluridine is a fluorinated derivative of timidine. Its activity is exerted trifluridine O following an activation most probably similar to that observed in case of guanosine analogues. In monophosphate form it inhibits irreversibly a key HO O N NH enzyme in pyrimidine metabolism (timidylate synthase); in triphosphate form it is a competitive inhibitor of DNA polymerase. Its specificity is low, it is also O incorporated into the host DNA, precluding its use in systemic therapy; it is used HO exclusively topically. It is active against HSV1, HSV2, VZV and CMV; it has
207 Antimicrobial chemotherapy: antiviral agents some activity agsint adenovirus and vacciniavirus (the mechanism of these two activities is unknown). Data are lacking on trifluridine resistance.
Docosanol
Docosanol is a saturated, long-chain aliphatic alcohol. It inhibits the fusion of the viral envelope and the target cell membrane. It is active in vitro against a number of enveloped viruses (HSV, VZV, HIV, RSV, influenza A virus, etc.), but can only be used topically. Its clinical application is topical treatment against HSV and VZV. It seems to be applicable in local therapy of the Kaposi’s sarcoma of the skin as well. Data on resistance are lacking.
docosanol
H C OH 3
Fomivirsen
Fomivirsen is an antisense oligonucleotide complementer to the mRNA of the CMV immediate-early IE2 protein. Its activity is exerted by hybridizing to the target mRNA and by consequent inhibition of translation of this mRNA. It is active exclusively against CMV and is used only in CMV retinitis in the form of local ocular injections. It is unsuitable for systemic treatment. Resistance to fomivirsen has been reported, but the mechanism of resistance remains unknown. Resistant viruses showed no cross-resistance to any other drugs against CMV.
Novel investigational antiherpesviral agents
Non-nucleoside DNA polymerase inhibitors (hidroxyquinoline-carboxamide derivatives) are under development, which target a conserative domain of the herpesviral DNA polymerase and consequently are active against all human herpesviruses excepting HHV6 and HHV7. Several different compounds are considered as alternatives in the therapy of CMV infections. Thiourea derivatives target the glycoprotein responsible for fusion of the viral envelope and the cellular membrane; benzimidazole ribosides inhibit the terminase protein involved in viral maturation; many compounds are known that inhibit the CMV protease (thieno-oxazinone-, spirocyclopropyl-oxazolone-, and benzimidazole-sulfoxide derivatives). Besides these drugs some reports consider agents with yet unknown mechanism of action (naftiridines, cyclotriaza-disulfonamides, isoquinoline-carboxamides). The most promising candidate is maribavir (a structural relative CH3 Cl of benzimidazole-ribosides), which inhibits the protein kinase of CMV H3C N (therefore antagonizes the action of gancyclovir activated by that protein N H kinase), and shows activity against EBV as well. Maribavir is presently N Cl undergoing the clinical trial stage of drug development. HO O 4-oxodihydroquinolines inhibit the DNA-polymerase of HSV1, HSV2, VZV, CMV, EBV and HHV8. Thiazolylamides inhibit the viral helicase-primase enzyme HO OH maribavir complex, which is responsible for decoiling of the viral genome and consequently is indispensable for viral replication. They effectively inhibit HSV1, HSV2 and CMV, their activity against other herpesviruses has not yet been tested. The cobalt chelate doxovir with unknown mechanism of action is active against all human herpesviruses excepting HHV8; it also shows activity against other viruses, e.g. adenoviruses and vesicular stomatitis virus. All of these drugs, excepting maribavir, are in the preclinical phase of the drug development process.
Drugs against HBV
Most of the drugs to be listed in this section are nucleoside analogues and target the DNA polymerase (reverse transcriptase) of HBV. Many of them are also active against HIV. Their general drawback is that they cannot prevent the formation of the covalently closed circular HBV DNA, which remains to serve as a source of reactivation. For this reason, upon withdrawal of these drugs, the virus replication restarts in many cases. The mechanism of resistance to nucleoside analogues in HBV is most frequently the mutational alteration of the
208 Antimicrobial chemotherapy: antiviral agents
DNA polymerase (reverse transcriptase). Many such mutations have been reported; these confer different level of resistance and different cross-resistance patterns. Experiments are going on to enhance the efficacy of anti-HBV therapy using drug combinations. Combinations of α-interferon-lamivudine and lamivudin-famciclovir proved to be more active than monotherapy. Some combination regimens utilizing newer nucleoside analogues show some promise in the eradication of the circular HBV DNA (reactivation reserve) as well. It is important to note that appropriate therapy for treatment of acute and hyperacute HBV infection is presently unavailable. The drugs listed below are used to treat chronic HBV infection. Their efficacy in acute infection is being tested.
ααα-interferon
α-interferon is discussed in the section ‘Drugs active against both DNA and RNA viruses’.
Lamivudine lamivudine NH2 Lamivudine is a cytidine analogue; instead of the (deoxy)ribose, a five-membered heterocyclic ring containing both sulphur and oxygen as N hetetoatoms is attached to the cytosine base. Its activation is independent of O N viral functions; it is activated by cellular enzymes. As it contains a hydroxyl group only in the position corresponding to the 5’ hydroxyl group, it has a HO S chain terminating activity; its target is the reverse transcriptase. Besides its anti-HBV activity, it is also active against both HIV types (HIV-1 and HIV- O 2). Relapses after cessation of the lamivudine therapy are common. Resistance to lamivudine arises from mutational alteration of the DNA polymerase; the replicative ability (and consequently the virulence) of these mutants is markedly decreased. This disadvantage is compensated by further mutations, leading to different levels of compensation of the replication disadvantage and to further increase in lamivudine resistance. Some mutations provide cross-resistance to other drugs targeting the DNA polymerase. Such mutations may develop without lamivudine exposure, e.g. due to exposure to penciclovir, ganciclovir or foscarnet.
Emtricitabine
Emtricitabine is a derivative of lamivudine fluorinated F NH on the base. Its activation, target, mechanism of action and emtricitabine 2 spectrum is identical to those of lamivudine. It is more active HO O N N than lamivudine, and induces resistance less frequently at least in case of HIV. However, HBV or HIV strains resistant to S O lamivudine are cross-resistant to emtricitabine as well.
Adefovir, tenofovir
Adefovir and tenofovir are phosphonated adenosine analogues; they are marketed as orally bioavailable dipivoxyl precursor drugs. Another adefovir precursor with oral administration, remofovir is under development. Adefovir is used primarily against HBV; tenofovir is a drug rather against HIV (see below). They do not contain hydroxyl group
209 Antimicrobial chemotherapy: antiviral agents corresponding to the 3’ position, therefore act as chain NH terminator drugs in active (diphosphate) form. Activation does 2 N not depend on viral functions. Their target is the DNA adefovir N polymerase (reverse transcriptase). Besides this effect, adefovir OH N N was found to promote the activity of NK cells and to induce HO P O endogeneous interferon production. Their drawback is that O relapses are not uncommon after cessation of the therapy. They are active against HBV and both HIV types, their efficacy is comparable to that of lamivudine. They are active against lamivudine resistant strains. Resistance to adefovir and tenofovir is due to mutational alteration of the DNA polymerase, but develops slower than in case of lamivudine. There is no cross-resistance between adefovir and lamivudine.
Entecavir
It is a guanosine analogue. Entecavir is activated by entecavir O cellular enzymes; it is active in triphosphate form. Its target is the N viral DNA-polymerase, it is a competitive inhibitor of DNA NH synthesis, and moreover, it is capable of inhibiting the synthesis of N N NH the primer necessary for DNA synthesis. Its efficacy is due to the 2 H C higher affinity of the DNA polymerase to entecavir than to its 2 natural substrate (GTP). It is active only against HBV and HIV of the human pathogenic viruses. It is more active than lamivudine; it HO OH inhibits lamivudine resistant strains as well. Resistance to entecavir can develop only on the basis of prior lamivudine resistance, with a further DNA polymerase mutation in addition to those conferring lamivudine resistance. Entecavir resistance develops very slowly.
Penciclovir, famciclovir and ganciclovir
For their chemical structures and mechanisms of action we refer to the section ‘Drugs against herpesviruses’. An important difference is that while in case of herpesviruses these drugs are activated by virus-specific mechanisms, in case of HBV the first phosphorylation (activation) step is also performed by cellular enzymes. As chronic HBV infection requires long-term therapy, mostly the orally bioavailable famciclovir was used. Their target in HBV is the DNA polymerase, which they inhibit competitively; they also cause early termination of DNA synthesis. Penciclovir ( famciclovir ) also inhibits the function of the protein responsible for the synthesis of the primer required for DNA replication. They are significantly less active than lamivudine. Resistance to penciclovir (famciclovir) and ganciclovir arises from the mutational alteration of the DNA polymerase; these lead frequently to cross-resistance to lamivudine, but the susceptibility to adefovir is generally preserved. Due to their ability to induce mutations providing cross-resistance to lamivudine and their poor activity, they were used rarely and mostly in combinations, but their use against HBV is now contraindicated.
Drugs under development
A number of nucleoside analogue drugs are in the preclinical or clinical phase of drug development against HBV. These include telbivudine ( L-enantiomer of deoxytimidine); elvucitabine and valtorcitabine (both cytidine analogues) planned to be marketed in combination; as well as two new guanosine analogues. Besides these, there are phosphonate type nucleotide analogues under development. All abovementioned drug candidates are inhibitors of the viral DNA polymerase (reverse transcriptase), therefore they share the drawback of being incapable of eradicating the integrated viral DNA; consequently, they rarely lead to cure. To reach this aim, nucleoside analogues are tried out in combinations with interferon or drugs acting on targets other than DNA polymerase. Such alternative target may be the inhibition of the capsid assembly (heteroaryl-pyrimidines), but these drugs have not yet reached the clinical trial phase.
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Drugs against other DNA viruses
Drugs against adenoviruses
Against adenoviruses acyclic nucleoside phosphonates (cidofovir , adefovir , tenofovir ), ganciclovir and ribavirin proved to be active. For their chemical structure and mechanism of action we refer to the respective sections. (On their mechanism of action against adenoviruses hardly any data are available, but it is assumed that they act similarly as against other DNA viruses.) Some anti-HIV nucleoside-type reverse transcriptase inhibitors (e.g. zalcitabine ) are also active against adenoviruses targeting the DNA polymerase of the adenoviruses. Other active, but not yet marketed drugs include the polysulfonated sialyl-lipid NMSO3, which interferes most probably with virus adsorption; the locally applicable n-chlorotaurine to be used in adenovirus conjunctivitis, which is also produced by polymorphonuclear granulocytes, with a probable mechanism of action of oxydizing thiol- and amino groups in proteins; doxovir, a cobalt chelate molecule with unknown mechanism of action; and stampidine, which is a monophosphate derivative of stavudine (see in the section Drugs against HIV) substitutionized on the phosphate group and is active against both types of HIV besides its activity against adenoviruses. The clinical efficacy of none of these drugs has been confirmed in systematic clinical trials. Similarly data on resistance do not exist.
Drugs against papillomaviruses
imiquimod NH Against papillomaviruses the acyclic nucleoside phosphonates (cidofovir, 2 N adefovir and tenofovir) discussed in the sections ‘Drugs against herpesviruses’ and N ‘Drugs against HBV’ (see above) as well as imiquimod are active. Out of the acyclic nucleoside phosphonates cidofovir is used against N papillomaviruses. Its mechanism of action is unknown; as papillomaviruses do not H3C contain viral DNA polymerase, the mechanism described for herpesviruses is not applicable. It was proven that cidofovir induces apoptosis in cells infected with CH3 papillomaviruses. Data on resistance are unavailable. Imiquimod contains a substitutionized quinoline ring condensed with an imidazole ring. Its antiviral effect is not specific; it acts through immunomodulatory activity. Besides HPV, it is also active against molluscum contagiosum virus. It is used exclusively for local treatment. As its activity is not virus-specific, development of resistance does not occur.
H Drugs against poxviruses N NH2 N Historically a thiosemicarbazone derivative of methisazone S (methisazone-thiosemicarbazone ) was used against smallpox. Its target is O the translation of the viral mRNA, but due to its weak activity it was efficient N only as a prophylactic drug and had little or no therapeutic value. Upon CH eradication of smallpox, the development of drugs against smallpox ceased, 3 but the fear of bioterrorism has given it new actuality. methisazone-thiosemicarbazone Out of the presently marketed antiviral drugs the ones active against poxviruses include acyclic nucleoside phosphonates (primarily cidofovir ), ribavirin , and, for local therapy of molluscum contagiosum, the immunomodulatory imiquimod (see also in the section Drugs against papillomaviruses). EICAR, a structural analogue of ribavirin (see above) exerts its activity against poxviruses by inhibition of inosin-monophosphate dehydrogenase. Drugs acting on miscellaneous targets (S-adenosyl-methionine hydrolase; different enzymes of the pyrimidine biosynthesis; DNA polymerase) have been described and some are under development.
211 Antimicrobial chemotherapy: antiviral agents
Drugs against RNA viruses
Drugs against HCV
Ribavirin
See in detail in the section ‘Drugs active against DNA and RNA viruses’. It is used mostly in combination with interferon.
ααα-interferon
See in detail in the section ‘Drugs active against DNA and RNA viruses’. It is used mostly in combination with ribavirin.
New agents
Experiments are going on with different nucleoside analogues, which act as competitive inhibitors of the viral RNA polymerase as well as with non-nucleoside RNA polymerase inhibitors. A novel agent merimepodib, an inhibitor of the inosine-monophosphate dehydrogenase, seems to enhance the activity of ribavirin. Promising targets are the viral protease involved in the viral maturation by cleavage of the viral polyprotein (telaprevir, boceprevir) and the RNA helicase responsible for decoiling of the virus genome.
N Drugs against picornaviruses CH3 O CH O 3 Though none of these drugs have yet reached the market, intensive drug development is going on with some N CH promising results. O 3 Pleconaril (disoxaril) is a drug consisting of two N heterocycles connected by an aliphatic chain and a benzene ring. The basis for the mechanism of action is a complete F F pleconaril complementarity with and fitting into the hydrophobic pocket F close to the receptor recognition site of the viral capsid. Binding of pleconaril leads to inhibition of decapsidation and, in case of some but not all viruses, adsorption. It is orally bioavailable. It is active against enteroviruses and rhinoviruses, it is probably active against echoviruses and Coxsackie A and B viruses, but its activity against poliovirus and HAV is questionable. Resistance is based on mutational alternation of one of the capsid proteins. It is presently under clinical trials against rhinoviruses. Enviroxime is an oxime derivative of benzimidazole. Its target is a virus protein with a key role in the formation of the replication intermedier during virus replication. It is active against all human pathogenic picornaviruses excepting HAV; data are not available on its activity against HAV. Resistance is mediated by the mutational alteration of the target protein. Enviroxime has failed as a drug candidate, for this reason its derivatives are being tested currently. There are attempts to develop drugs inhibiting the protease of the viruses (e.g. ruprintrivir).
H C NH NH 3 2 Drugs against influenzaviruses 2
Adamantanes H H H H These drugs are derivatives of adamantane (a symmetric H H three-ring system) containing an amino group. Adamantanes amantadine rimantadine inhibit the decapsidation of the virus through inhibition of the M2 ion channel, which is responsible for the proton influx providing the acidic pH necessary for alteration of the hemagglutinine conformation. As this conformation change is crucial for decapsidation, without the proper function of the ion channel the decapsidation of the virus fails to occur. In
212 Antimicrobial chemotherapy: antiviral agents case of viruses with H7 hemagglutinine adamantanes also inhibit viral maturation. Adamantanes include amantadine and rimantadine . Due to their highly specific target, they are active exclusively against influenza A virus, as other influenzaviruses (influenza B and C) and members of other virus families do not contain M2 protein. Resistance develops rapidly during treatment. The mechanism of resistance includes the mutational alteration of the M2 protein; the mutant M2 is not sensitive to the inhibitory effect of adamantanes. Another described mechanism is the mutational alteration of the hemagglutinine; the conformation of the mutant hemagglutinine alters at higher pH, therefore decapsidation becomes less dependent on M2 protein activity. The two adamantanes show total cross-resistance, but resistant viruses remain susceptible to neuraminidase inhibitors and to ribavirin.
Neuraminidase inhibitors COOH
These drugs are structural relatives of neuraminic acid. NH O OH OH The drugs zanamivir and oseltamivir belonging here inhibit H N N 2 H H the neuraminidase of the influenza viruses. The function of NH OH the neuraminidase is to cleave the carbohydrate structure O CH zanamivir serving as the receptor of the hemagglutinin, to allow for 3 detachment of the mature virions from the cell as well as for O O CH3 preventing aggregation of virions. Without the neuraminidase CH activity mature virions remain cell-bound or bound to each 3 other. Consequently, neuraminidase inhibitors inhibit virus CH3 release and therefore virus spread within the host. H2N O Their spectrum is narrow; they are active only against NH O influenza A and B viruses, as neither influenza C virus nor CH oseltamivir other human pathogenic viruses contain neuraminidase. 3 Two mechanisms of resistance to neuraminidase inhibitors have been described; mutational alteration of neuraminidase and of hemagglutinine. Hemagglutinine of hemagglutinine-mutants binds its receptor with weaker affinity, thus virus release is less dependent on neuraminidase activity. This mechanism provides resistance to both zanamivir and oseltamivir, but decreases the virulence of the virus. Neuraminidase-mutants possess a neuraminidase insensitive to the inhibitory action of the inhibitors. The virulence of most neuraminidase-mutants is also decreased; the extent of this decrease depends on the site of the mutation within the neuraminidase gene. The position of the mutation also determines whether the mutants are resistant to zanamivir only, to oseltamivir only, or to both drugs. Double mutant strains with mutation in both the hemagglutinine and neuraminidase genes have been isolated; these exhibit high level resistance and their virulence is comparable to that of the wild-type.
Ribavirin
For its chemical structure and spectrum we refer to the section ‘Drugs active against both DNA and RNA viruses’. Its mechanism of action against influenza viruses is not characterized. It is proven that it decreases the cellular GTP levels (through inhibition of the inosine-monophosphate dehydrogenase), leading to indirect inhibition of virus replication and transcription of virus genes. Ribavirin-triphosphate inhibits the RNA-polymerase of influenza viruses, capping of viral mRNA and initiation of virus replication. The role of the mechanism based on error catastrophe is also concievable. It is used rarely against influenzaviruses. Ribavirin resistance has never been reported in influenza viruses.
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Drugs against HIV
Nucleoside-type reverse transcriptase inhibitors (NRTIs)
This group includes nucleoside analogues, with a common characteristic of the lack of 3’ hydroxyl group, and consequently all of these drugs act as chain terminating agents during viral nucleic acid synthesis. For activation they need to be phosphorylated by cellular enzymes, they are active in triphosphate form. Their target is the viral reverse transcriptase (DNA polymerase), which they inhibit competitively besides their chain terminating activity. Both of these mechanisms play important role in their antiretroviral action. They are active against both HIV-1 and HIV-2; emtricitabine and lamivudine are also active against HBV. Unfortunately they have many side effects. The group includes zidovudine (azidotimidine, AZT), didanozine (dideoxy -inosine, ddI, adenosine analogue), zalcitabine (dideoxy-cytidine, ddC), stavudine (timidine analogue, d4T), lamivudine (cytidine analogue, 3TC, see also at drugs against HBV), abacavir (guanosine analogue, ABC), entecavir (guanosine analogue, see also at drugs against HBV) and emtricitabine (cytidine analogue, see also at drugs against HBV). Racivir (sulphur-containing timidine analogue) is in the developmental phase; it is also active against HBV. Resistance to NRTIs develops rapidly; therefore they are always used in combinations with each other (excepting the combination zidovudine+stavudine, as these antagonize each other) or with other antiretroviral agents (see below). Lamivudine is a frequent member of these combinations as many mutations causing resistance to other NRTIs do not affect lamivudine. Resistance develops through mutational alterations of the target enzyme; mutations occur mostly in the nucleotide binding pocket of the enzyme. For high level resistance usually at least two different mutations are necessary. Many mutations provide cross-resistance to some or all other NRTIs, but not to any other antiretroviral drug group. Besides mutational resistance, insertions were also shown to cause resistance. Some mutations causing resistance to NRTIs lead to hypersusceptibility to NNRTIs (see below).
CH3
azidotimidine O CH3 (zidovudine) O HO O N NH stavudine HO O N NH O N 3 O F NH NH NH lamivudine 2 emtricitabine 2 zalcitabine 2 HO O N N HO O N N HO O N N
S O S O O O HN
didanosine N abacavir N NH N
HO O N HO N N N NH2
214 Antimicrobial chemotherapy: antiviral agents
Acyclic nucleoside phosphonates (ANPs)
NH These are nucleotide analogues containing a phosphate 2 N group analogue at the 5’ position bound through a adefovir N carbon-phosphorus bond. Consequently, they need only two OH N N phosphorylation steps for activation. Though their chemical HO P O structure slightly differs, their target and mechanism of action are O equivalent to those of NRTIs; many authors do not separate ANPs from NRTIs. The group includes adefovir (as adefovir NH dipivoxyl precursor drug; see also at drugs against HBV) and 2 N tenofovir (as tenofovir disoproxyl precursor drug), which are tenofovir N adenosine analogues. Their spectrum is slightly broader than that OH N N of the former group; they are active against HBV as well. HO P O Resistance, similarly as in case of NRTIs, is mediated by O mutational or insertional alteration of the reverse transcriptase. CH3 Some mutations cause cross-resistance between NRTIs and ANPs.
Non-nucleoside-type reverse transcriptase inhibitors (NNRTIs)
Drugs belonging to this group have O unrelated chemical structures; many chemically delavirdine HN S CH3 N unrelated NNRTIs are known. However, their O mechanism of action is similar; all of these drugs N are non-competitive inhibitors of the reverse H C NH N 3 N transcriptase, they inhibit the binding of H nucleotides and the primers to the enzyme. Their CH3 O spectrum is narrower compared to that of NRTIs; they are active only against HIV-1. efavirenz Nevirapine, delavirdine and efavirenz belong to this F F group, recently etravirin has been marketed. Several other drug F Cl candidates (e.g. rilpivirin) are under development. O Resistance to NNRTIs is fast to develop (with the N O exception of efavirenz); therefore they are never used in H monotherapy. Resistance is mediated by mutational alteration of nevirapine the reverse transcriptase; the mutant enzyme is not inhibited by NNRTIs. Such mutations frequently cause cross-resistance to other NNRTIs, but usually not to NRTIs. However, certain mutations N N N induced by efavirenz were shown to lead to hypersusceptibility to delavirdine, other mutations induced by delavirdine caused N CH nevirapine hypersusceptibility. Because of these issues, H 3 susceptibility pattern to all NNRTIs can only be determined by O sequencing the reverse transcriptase gene.
Foscarnet
For its chemical structure and spectrum see the section ‘Drugs against herpesviruses’. Foscarnet is an uncompetitive inhibitor of the reverse transcriptase; its target is the pyrophosphate binding site of the enzyme. The mechanism of resistance in retroviruses
215 Antimicrobial chemotherapy: antiviral agents
(similarly to herpesviruses) is the mutational alteration of the reverse transcriptase; mutant enzyme binds foscarnet less efficiently. Some mutations lead NRTI hypersusceptibility. It is rarely used due to its toxicity.
Protease inhibitors (PIs)
Their chemical structure is variable, all are large molecules containing multiple rings; a peptide bond is always present (peptidomimetics). They interfere with viral maturation; their target is the protease of the HIV, which cleaves the viral polyprotein to functional viral enzymes. Without functional protease the produced virions are defective and non-infectious; therefore the virus is unable to infect new lymphocytes and to spread effectively within the host. Unlikely to NRTIs and NNRTIs, PIs inhibit virus production in chronically infected cells as well. They are active against both HIV-1 and HIV-2, but inactive against viruses belonging to other virus families. Further drawback of PIs is their poor penetration to the central nervous system. At present ten PIs are available on the market, saquinavir , ritonavir , indinavir , nelfinavir , amprenavir , lopinavir , atazanavir and fosmaprenavir; darunavir and tipranavir are licensed for patients with long therapy history. Resistance develops through mutational alteration of the protease. More point mutation sites are known, these confer different levels of resistance. Cross-resistance is not uncommon among PIs, but it is rare that a virus would be resistant to all PIs. (This requires at least four independent point mutations.) The pattern of the PIs the virus is resistant to is determined by the number and site of the mutations. PI-resistant mutants replicate slower (have lower virulence) than the wild-type virus, but this drawback can be compensated by mutations in the cleavage sites of the polyprotein, leading to development of resistant mutants with normal growth rate.
CH CH H 3 3 OH H O N CH3 O N CH O O 3 H CH CH N H 3 3 N N N N N H H O O OH OH N N H saquinavir indinavir NH2
H C 3 CH3 O O H H C N N S 3 N N N O H H CH OH H3C S 3 O N
ritonavir
216 Antimicrobial chemotherapy: antiviral agents
S H3C CH3 CH3 O O O CH HO OH H 3 N N O H H HN N N H N O OH H H C H3C N 3 nelfinavir H C H lopinavir 3 CH 3 O
Fusion inhibitors
This group includes enfuvirtide, which is an oligopeptide identical to a 36 amino acid fragment of the receptor binding gp41 glycoprotein of the HIV. Enfuvirtide binds to the receptor binding protein inhibiting the fusion of the virus and the host cell membrane. It is active only against HIV-1. It is used to supplement the combinational therapy (see below) further enhancing the efficacy. Resistance is mediated by the mutational alteration of the target protein.
Chemokine receptor antagonists H F N These agents have a very unique and peculiar mechanism of action; they inhibit a specific receptor-ligand F O interaction crucial for virus adsorption via binding to the host N H receptor, causing such changes in the conformation of the host molecule, which render it unaccessible for the viral maraviroc ligand. The receptor targeted is the human chemokine H3C receptor CCR5, which in its natural form binds the gp120 of N N the CCR5-tropic HIV subpopulation after gp120 is bound to N CH the main receptor CD4. Chemokine receptor antagonists alter 3 CH the conformation of CCR5, making it unable to bind the viral 3 gp120 and leading to inhibition of virus adsorption. These are the first antimicrobial drugs which interact with a host molecule to prevent infection. The only marketed chemokine receptor antagonist is maraviroc ; several other molecules with a similar mechanism of action (e.g. vicriviroc) are currently tested in clinical trials. These drugs act only against the HIV viruses. Most trials concentrated on HIV-1, but anecdotal reports suggest that they are active against HIV-2 as well. They are inactive against all other viruses. It is important, that these drugs inhibit only the HIV virus subpopulations using the CCR5 coreceptor; subpopulations with CXCR4- or dual tropism are not affected. For this reason, these drugs are to be used when the preferred co-receptor is the CCR5. An extremely small minority of HIV strains possesses natural resistance to these agents, mediated by nucleotide polymorphisms in the gene coding for the gp120 glycoprotein. Acquired resistance to maraviroc may be mediated by mutational alteration of the envelope glycoprotein responsible for CD4 and CCR5 binding; the mutant ligand may bind to the co-receptor altered by maraviroc as well. Another, possibly more important mechanisms are the selection of the pre-existing subpopulations using CXCR4 co-receptor instead of CCR5, or a shift in subpopulations in a mainly CCR5-tropic population.
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Integrase inhibitors O raltegravir H C O F These compounds inhibit the integrase N N 3 N H H of the HIV, which is responsible for H3C N N integration of proviral DNA into the host cell O N O H C CH O genome. Inhibition of the integration of 3 3 proviral DNA leads to suppression of virus replication. Two types exist, 3’-processing inhibitors, which inhibit the free integrase enzyme and strand-replacement inhibitors targeting the enzyme-DNA complex (integrase strand transfer inhibitors, INSTIs). Only the latter group is represented in the therapy at present. The only marketed representative is raltegravir , but elvitegravir is also close to being marketed. Raltegravir is provenly effectively only against HIV-1; activity against HIV-2 is probable, but unconfirmed in clinical trials. It is inactive against other viruses. Raltegravir resistance is mediated by the mutational alterations of the integrase, i.e. by mutations in the integrase-coding region of the HIV polyprotein gene.
Other drugs against HIV
Besides the drugs discussed above, experiments are going on with a number of compounds with other targets, including adsorption inhibitors acting locally (dextrane-sulphate, carragenane) and systemically (coreceptor-antagonists, recombinant CD4-fragments), decapsidation-inhibitors (bicyclams), drugs inhibiting the dimerization of the reverse transcriptase, antisense oligonucleotides inhibiting the reverse transcriptase or the integrase, ribozymes, compounds interfering with virus release (cyclosporine analogues) and interferon. Out of these, development of two drug groups with new targets is in an advanced stage, i.e. coreceptor-antagonists acting on CXCR4 coreceptors and the maturation inhibitor bevirimat. Experiments are going on with oligopeptides and a bicyclam derivative acting as CXCR4 coreceptor anagonists. Bevirimat inhibits the maturation of the capsid precursor protein.
Combination therapy against HIV
At present, the therapeutic options against HIV can provide long-term inhibition of the viral replication and allows for efficient postexposure prophylaxis. The aim of the therapy is to stop virus replication in the host (which in turn helps in preventing resistance development). The combinational therapy aimed at such a goal is called Highly Active AntiRetroviral Therapy ( HAART ). This combinational therapy includes at least two reverse transcriptase inhibitors (two NRTIs or an NRTI plus a NNRTI) and a PI, but four-drug combinations (two NRTIs plus an NNRTI plus a PI) are also common. To newer regimens enfuvirtide is also added. The place of the new drug classes in the therapy is currently evaluated. HAART is capable of keeping the virus numbers below the detection limit for extended periods, and by carefully providing the necessary serum drug levels, resistance development may be avoided for a long time. Unfortunately, the available treatment options will not result in total eradication of the virus. Additionally, the spread of multiresistant strains resistant to more than one antiretroviral drug group represents a growing problem.
Other drugs against RNA viruses
Though significant effort has been exerted to find efficient drugs against flaviviruses (dengue), rabiesviruses and filoviruses, promising results remain elusive.
218 department of medical microbiology medical and health science center university of Debrecen
Dr. Krisztina Szarka – Dr. Gábor Kardos
Pharmaceutical microbiology I. Antimicrobial Procedures and Chemotherapy
Debrecen University Press