Prof. Beata M. Sobieszczańska Department of Microbiology University of Medicine Antimicrobials
• Antibiotic is a low molecular substance produced by microorganisms (molds, bacteria) that at a low concentration inhibits or kills other microorganisms • Antimicrobial is any substance of natural, semisynthetic or synthetic origin that kills or inhibits the growth of microorganisms but causes little or no damage to the host All antibiotics are antimicrobials but not all antimicrobials are antibiotics Antimicrobials • Bacteriostatic = they inhibit bacterial growth but generally do not kill the bacteria e.g. protein synthesis inhibitors (macrolides, tetracyclines, aminoglycosides, streptogramins, lincosamides, oxazolidinones, chloramfenicol) • Bactericidal = agent that kills the target bacteria (β- lactams, glycopeptides, bacitracin, polymyxin, fluoroquinolones, metronidazole, rifampin) Drug’s spectrum of activity
Depending on the range of bacterial species susceptible to antimicrobials – they are classified as: narrow spectrum - have limited activity to some bacteria e.g. GP or GN (e.g. penicillin, glycopeptides) broad spectrum – are active against both GP and GN (e.g. amoxycillin, carbapenems, fluoroquinolones)
Sites of action of different antimicrobial agents Bactericidal - without cell wall, osmotic pressure causes bacteria to burst
Examples • β-lactams • Glycopeptides • Bacitracin
PBP=penicillin binding proteins (enzymes e.g. transpeptydases) :
Penicillins in blocking transpeptidase enzymes from assembling the peptide cross-links in peptidoglycan Cephalosporins
First generation: GP, little activity against GN cephalothin, cephalexin
Second generation: GN, less GP cefamandole, cefaclor, cefazolin, cefuroxime cefoxitin & cefotetan (cephamycins) – anaerobes
Third generation: GN, GP variable, resistant to β-lactamases cefotaxime, ceftriaxone, cefoperazone, ceftazidine
Fourth generation: GP & GN, enhanced stability against β- lactamases; cefepime & cefpirome
Fifth generation: GP, GN & MRSA; ceftaproline Monobactams (Aztreonam) Aerobic GN bacteria + Pseudomonas and other nonfermentative rods except Stenotrophomonas maltophilia Carbapenems Imipenem, meropenem, ertapenem, doripenem Active against GP, GN aerobic and anaerobic Reserved for severe, life-threatening infections caused by resistant bacteria Bacitracin Polypeptide antibiotic • Inhibits cell wall synthesis of GP bacteria – mostly Staphylococci and Streptococcus pyogenes (narrow spectrum) • Used as a topical preparations (toxic – causes kidney damage) Glycopeptides Vancomycin & teicoplanin are glycopeptides that are effective against GP bacteria Antimicrobial agents that alter/disrupt the cytoplasmic membrane these antimicrobials are cidal
Polymyxin B, polymyxin E (colistin) are used in severe infections caused by GN rods (also MDR) Several GN pathogens (Proteus, Burkholderia, Neisseria, Brucella) exhibit intrinsic resistance
Daptomycin (lipopeptide antibiotic) – disrupts membrane potential (depolarization) Spectrum: GP pathogens (VRE, MRSA etc.) Used to treat complicated skin and soft tissue infections Antimicrobial agents that inhibit protein synthesis
These agents prevent bacteria from synthesizing structural proteins and enzymes Alter bacterial ribosome, block translation & cause faulty protein synthesis These antibiotics are bacteriostatic or bactericidal depending on concentration Antimicrobial agents that inhibit protein synthesis Macrolides Glycylcyclines Tetracyclines Ketolides Aminoglycosides Fusidic acid Spectinomycin Mupirocin Streptogramins Oxazolidynones Lincozamides Chloramphenicol Tetracyclines • Broad-spectrum: GP & GN bacteria, Rickettsia, Chlamydia, Mycoplasma, spirochetes, some protozoa (malaria) tetracycline, oxytetracycline, doxycycline, minocycline • Resistance common • Used to treat: atypical pneumonia, syphilis, brucellosis, Lyme disease, acne, plague, leptospirosis, anthrax, cholera Glycylcyclines
Tigecycline – derived from minocycline Broad-spectrum: GP+GN bacteria GP cocci: staphylococci, enterococci, streptococci - including resistant strains GN intestinal rods – except Proteus GN nonfermentative rods e.g. Acinetobacter spp. except Pseudomonas Obligatory anaerobic – Bacteroides fragilis Advantage over tetracyclines: active against bacteria resistant to tetracyclines, higher binding affinity, broader spectrum
Aminoglycosides Bactericidal – except for staphylococci Active against most GN aerobic bacilli but lack activity against anaerobic & most GP bacteria, except for staphylococci streptomycin, kanamycin, tobramycin, amikacin, gentamycin Spectinomycin • Bacteriostatic antibiotic chemically related to aminoglycosides • Its activity is restricted to gonococci • Spectinomycin is given for gonococcal urethritis, cervicitis, proctitis Macrolides (erythromycin, roxithromycin, clarithromycin, azithromycin) – mainly affects GP cocci (streptococci, staphylococci, but NOT enterococci) and intracellular pathogens (Mycoplasma, Chlamydia, Legionella); other: Campylobacter, Helicobacter, Borrelia, Treponema, Corynebacterium sp. and some anaerobes (Propionibacterium) Ketolides (derived from erythromycin) – Telithromycin Active against bacterial strains resistant to macrolides Lincosamides (lincomycin, clindamycin) active against GP bacteria (but NOT GN), most anaerobes Principal therapeutic indications are staphylococcal infections of bones and joints, and anaerobic infections They all static or cidal depending on concentration, bacterial inoculum and species Streptogramins A combination of quinupristin & dalfopristin (Synercid) – exhibit dose-dependent cidal activity work synergistically to inhibit protein synthesis Active against: GP cocci (also multi-resistant), modest activity: common respiratory pathogens (Moraxella, Str.pneumoniae, Mycoplasma, Legionella, Chlamydophila) & anaerobes Oxazolidynones Linezolid - narrow spectrum of activity: GP bacteria (staphylococci, streptococci, enterococci, pneumococci – resistant strains, Listeria, corynebacteria), and GN bacteria: Moraxella, Pasteurella, Bacteroides but other GN are resistant
Fusidic acid Fusidic acid Narrow spectrum of activity: staphylococci resistant strains in combination with other antibiotics (bone and joints infections) Activity against other GP cocci is poor Other: anaerobes (Bacteroides fragilis, Clostridium spp.) Moderate activity against mycobacteria: M. tuberculosis, bovis, leprae and certain protozoa (Giardia lamblia, Plasmodium falciparum) Mupirocin
Mupirocin - cidal or static activity depends on concentration Narrow spectrum of activity: staphylococci + resistant strains Used for topical treatment of superficial skin infections e.g. furuncles, impetigo and decolonization of MRSA (intranasaly) Chloramphenicol Spectrum of activity: GP bacteria: streptococci, staphylococci, enterococci, B. anthracis, Listeria GN bacteria: H. influenzae, Moraxella, Neisseria, E. coli, Proteus mirabilis, Salmonella, Shigella, Stenotrophomonas matlophilia Many anaerobic bacteria Penetrates to CSF – meningitis treatment Side effects: fatal aplastic anemia, dose-dependent leucopenia, bone marrow suppression etc. – limit its use
Antimicrobial that interfere with DNA synthesis bactericidal Rifampins Fluoroquinolones Metronidazole Fidaxomycin
Rifampin (Rifampicin) Prevent the synthesis of mRNA by inhibiting the enzyme RNA polymerase Effective against some GP & GN bacteria Mycobacterium tuberculosis, M. leprae, Legionella pneumophila Used primarily to treat tuberculosis Prophylaxis in meningococcal meningitis
Fluoroquinolones Synthetic chemicals Inhibit topoisomerases (DNA gyrases) involved in bacterial nucleic acid synthesis Generations: I – nalidixic acid (GN bacteria) – UTI II – ciprofloxacin, norfloxacin, ofloxacin (GN+ P. aeruginosa, S. aureus, some atypical) – UTI, STD, skin, soft tissue infections, GITI III – levofloxacin, gatifloxacin, moxifloxacin (GP, GN, atypical) – RTI, GITI IV – trovafloxacin (GP, GN, atypical, anaerobes) Nitroimidazoles: Metronidazole An antibiotic active against anaerobic bacteria & certain parasites (Entamoeba histolytica, Trichomonas, Giardia) Metronidazole puts nicks in the microbial DNA strands
Macrocyclic antibiotics Fidaxomycin - binds to bacterial RNA polymerase active against GP bacteria, especially C. difficile (treatment of CDAD=Clostridium difficile associated diarrhea) Minimally absorbed into the bloodstream
Competitive antagonistic antibiotics
Inhibitors of metabolic pathways via competitive antagonism include: • Sulphonamides • Trimethoprim • Isoniazid These all inhibit folic acid biosynthesis
Competitive antagonism Competitive antagonism - a drug chemically resembles a substrate in a metabolic pathway Because of their similarity, either the drug or the substrate can bind to the enzyme While the enzyme is bound to the drug, it is unable to bind to its natural substrate and blocks that step in the metabolic pathway Competitive antagonism Sulfonamides & trimethoprim
Synthetic chemicals Co-trimoxazole is a combination of trimethoprim + sulfamethoxazole (TMP-SMX) Both of these drugs block enzymes in the bacterial pathway required for the synthesis of tetrahydrofolic acid - a cofactor needed for bacteria to produce nucleic acids
Antimicrobial agents active against anaerobes β–lactams (penicillins; carboxypenicillins; β-lactams with β-lactamases inhibitors; cephamycins, carbapenems) Chloramphenicol Nitroimidazoles (Metronidazole) Clindamycin Macrolides & tetracyclines Fidaxomycin & Vancomycin (AAD caused by C. difficile – per rectum and intravenously in severe cases)
Antimicrobial agents active against staphylococci β–lactams (if susceptible) Chloramphenicol TMP-SMX Lincosamides Fusidic acid Macrolides & tetracyclines, tigecyclin Fluoroquinolones Daptomycin Glycopeptides Streptogramins Rifampin Mupirocin Aminoglycosides Oxazolidynones
Antimicrobial agents active against meningococci
Penicillin (if susceptible - MIC) Ampicillin Cephalosporins (III gen. Cefotaxime, ceftriaxone) Chloramphenicol Fluoroquinolones Rifampin
Antimicrobial agents active against Haemophilus spp.
Ampicillin Cephalosporins (III gen. cefuroxime, ceftriaxone) Monobactams (aztreonam) Carbapenems Macrolides, tetracyclines Chloramphenicol Fluoroquinolones Rifampin
Antimicrobial agents active against pneumococci Penicillins and ampicillin, amoxycillin (if susceptible) Cephalosporins (III) Macrolides, tetracyclines, tigecycline, ketolides (Telithromycin) Glycopeptides TMP-SMX Chloramphenicol Lincosamides Fluoroquinolones Rifampin+vancomycin Oxazolidynones Daptomycin
Antimicrobial agents active against enterococci
Penicillin, ampicillin Glycopeptides Aminoglycosides (gentamycin, streptomycin) – combined with β-lactams or glycopeptides Chloramphenicol Fluoroquinolones Rifampin Tigecycline Streptogramins (Synercid) Oxazolidynones (Linezolid)
Antimicrobial agents active against GN fermentative rods Aminopenicillins and anti-pseudomonal penicillins Cephalosporins Monobactams (aztreonam) Carbapenems Tigecycline Aminoglycosides Chloramphenicol Fluoroquinolones TMP-SMX
Antimicrobial agents active against GN non-fermentative rods Anti-pseudomonal penicillins, ampicillin (+sulbactam – Acinetobacter spp.) Cephalosporins (III, IV, V gen.) Monobactams (aztreonam) Carbapenems Tigecycline Tetracyclines (minocycline) Aminoglycosides Polymyxins (colistin) Chloramphenicol Fluoroquinolones TMP-SMX
Microbial resistance to antimicrobial agents What is a drug resistance?
Antimicrobial resistance is the ability of a microorganism to survive and multiply in the presence of an antimicrobial agent that would normally inhibit or kill this species of microorganism
Intrinsic resistance
• Bacteria may be resistant because either – they have no mechanism to transport the drug into the cell – they do not contain or rely on the antibiotic’s target process or protein – an outer membrane establishes permeability barrier against antibiotic • Specific examples of bacterial strains with known natural resistance: – tetracycline-resistant Proteus mirabilis – ampicillin-resistant Klebsiella pneumoniae
NATURAL RESISTANCE ORGANISMS MECHANISM AGAINST: Lack of oxidative metabolism to drive Anaerobic bacteria Aminoglycosides uptake of aminoglycosides Inability to anaerobically reduce drug to its Aerobic bacteria Metronidazole active form Lack of penicillin binding proteins (PBPs) GP Aztreonam (β-lactam) that bind and are inhibited by this β-lactam antibiotic Lack of uptake resulting from inability of GN Vancomycin vancomycin to penetrate outer membrane
Production of enzymes (β-lactamases) that Klebsiella spp. Ampicillin (β-lactam) destroy ampicillin before the drug can reach the PBP targets
Production of enzymes (β-lactamases) that Stenotrophomonas Imipenem (β-lactam) destroy imipenem before the drug can reach the PBP targets.
Sulfonamides, trimethoprim, Lack of uptake resulting from inability of P. aeruginosa tetracycline, or antibiotics to achieve effective intracellular chloramphenicol concentrations Lack of sufficient oxidative metabolism to Aminoglycosides drive uptake of aminoglycosides Enterococci Lack of PBPs that effectively bind and are All cephalosporins inhibited by these β-lactam antibiotics Acquired resistance
Horizontal gene transfer = genes pass from a resistant strain to a non-resistant strain conferring resistance on the latter
The introduction of an antibiotic into the bacterial environment acts as a selective pressure Acquired resistance - mutation Horizontal gene transfer Bacterial resistance to antimicrobials Conjugation • Transmission of resistance genes via plasmid exchange • Bacteria have circles of DNA called plasmids that they can pass to other bacteria during conjugation • This type of acquisition allows resistance to spread among a population of bacterial cells much faster than simple mutation R (resistance)-Plasmid Conjugation
Transduction A virus serves as the agent of transfer between bacterial strains Transformation DNA released from a bacterium is picked up by a new cell
Most bacteria become resistant to antimicrobial agents by one or more of the following mechanisms: 1. Producing enzymes which detoxify or inactivate the antibiotic 2. Altering the target site to reduce or block binding of the antibiotic 3. Preventing transport of the antimicrobial agent 4. Developing an alternate metabolic pathway to by-pass the metabolic step being blocked by the antimicrobial agent
Inactivation of antimicrobials via modification or degradation= enzyme-based resistance β-lactamase enzymes
• β-lactamase enzymes can destroy the β-lactam ring of penicillins through hydrolysis • without a β-lactam ring penicillins are ineffective against the bacteria • Produced by many GP and GN bacteria
β-lactamases inhibitors
Clavulanate, tazobactam, sulbactam • Resemble beta-lactam antibiotics • Have no antimicrobial action of their own • Bind to the bacterial β-lactamases & neutralize them • They protect β-lactams from degradation e.g. Amoxycillin+ clavulanic acid
Extended-Spectrum β-Lactamases (ESBL) β-lactamases conferring resistance to the penicillins, 1st , 2nd, 3rd generation cephalosporins & monobactams, but not to carbapenems & cephamycins which are inhibited by β-lactamase inhibitors e.g. clavulanic acid Produced by GN bacilli Extended-Spectrum β-Lactamases (ESBL) Produced by GN bacilli Extended-spectrum β-lactamases (ESBL) Genes encoding for ESBL are frequently located on plasmids (horizontal transfer) that also resistance carry resistance genes for aminoglycosides, tetracycline, TMP-SMX, fluoroquinolones Clinical implications: treatment failure increased morbidity & mortality outbreaks
Metallo-β-Lactamases (Carbapenemases/MBL & KPC) • Hydrolyze virtually all β-lactams • Mediate broad spectrum β-lactam resistance • No clinical inhibitor available • Present on plasmids • Genes are continuously spreading • Associated (80%) with aminoglycoside resistance • Produced by GN bacilli – mostly nonfermentative rods Metallo-β-Lactamases (Carbapenemases/MBL & KPC)
• KPC – Klebsiella pneumoniae carbapenemase – isolates resistant to many antimicrobials also aminoglycosides and quinolones • Genes encoding KPC are on mobile genetic elements e.g. plasmids • KPC strains - multiresistant
Metallo-β-Lactamases (Carbapenemases/MBL & KPC) Mechanisms of Aminoglycoside Resistance There are three mechanisms of aminoglycoside resistance: • reduced uptake or decreased cell permeability • alterations at the ribosomal binding sites • production of aminoglycoside modifying enzymes – most common There are three types of aminoglycoside modifying enzymes: 1. N-Acetyltransferases (AAC) – catalyzes acetyl CoA-dependent acetylation of an amino group 2. O-Adenyltransferases (ANT) – catalyzes ATP-dependent adenylation of hydroxyl group 3. O-Phosphotransferases (APH) – catalyzes ATP-dependent phosphorylation of a hydroxyl group Enterococci Enterococci are inherently resistant to cephalosporins, lincosamides, polymyxin B, low concentrations of aminoglycosides and TMP-SMX Synergistic combination therapy with a cell wall active agent + high concentration of aminoglycoside often provides effective therapy (treatment of endocarditis caused by enterococci)
Enterococci
HLAR (High Level of Aminoglycoside Resistance) – enterococci that acquired genes encoding aminoglycoside inactivating enzymes The synergism of aminoglycosides with cell wall active agent is lost
Most bacteria become resistant to antimicrobial agents by one or more of the following mechanisms: 1. Producing enzymes which detoxify or inactivate the antibiotic 2. Altering the target site to reduce or block binding of the antibiotic 3. Preventing transport of the antimicrobial agent 4. Developing an alternate metabolic pathway to by-pass the metabolic step being blocked by the antimicrobial agent
Producing an altered target site to which the antibiotic no longer binds Resistance to β-lactams
PBP (Penicillin Binding Proteins = transpeptidase enzyme) Examples: Streptococcus pneumoniae resistant to penicillin PRP (Penicillin Resistant Pneumococci) Staphylococci
• MRSA (Methicillin Resistant Staphylococcus Aureus) • GISA (Glycopeptide Intermediate Resistant Staphylococcus Aureus) • VISA (Vancomycin Intermediate Resistant Staphylococcus Aureus) • VRSA (Vancomycin Resistant Staphylococcus Aureus)
Resistance to glycopeptides & ampicillin
Enterococci GRE (Glycopeptide Resistant Enterococci) VRE (vancomycin resistant enterococci) Ampicillin resistant enterococci (altered PBP)
Resistance to macrolides
• Resistance to Macrolide, Lincosamide & Streptogramin B antibiotics (MLSB phenotype) – staphylococci, streptococci Most bacteria become resistant to antimicrobial agents by one or more of the following mechanisms: 1. Producing enzymes which detoxify or inactivate the antibiotic 2. Altering the target site to reduce or block binding of the antibiotic 3. Preventing transport of the antimicrobial agent 4. Developing an alternate metabolic pathway to by-pass the metabolic step being blocked by the antimicrobial agent
Producing an altered transport (carrier) protein in the cytoplasmic membrane Producing an altered porin in the outer membrane of a Gram-negative cell wall Producing transporter molecules to pump the drug out (efflux) of the bacterium Resistance to tetracyclines
• Common problem among both GP & GN bacteria • Cross-resistance with other tetracyclines Mechanisms: • decreased penetration of the drug into organisms (lack the necessary transport system) • drug efflux – most common • alteration of the target ribosome site Most bacteria become resistant to antimicrobial agents by one or more of the following mechanisms: 1. Producing enzymes which detoxify or inactivate the antibiotic 2. Altering the target site to reduce or block binding of the antibiotic 3. Preventing transport of the antimicrobial agent 4. Developing an alternate metabolic pathway to by-pass the metabolic step being blocked by the antimicrobial agent
4. Developing an alternate metabolic pathway to by-pass the metabolic step being blocked by the antimicrobial agent (e.g. overcoming drugs that resemble substrates and tie-up bacterial enzymes) Increased production of a certain bacterial enzyme
Production of greater amounts of the limited enzyme that is being tied up or inactivated by the antibiotic