. TABLE OF CONTENTS

LIST OF FIGURES ...... …………...... ……………...... V LIST OF TABLES ...... ……………………………...... VI LIST OF APPENDICES ...... VII LIST OF ABBREVIATIONS & ACRONYMS ...... IX DEFINITIONS ...... XI AGRADECIMENTOS ...... XIV ABSTRACT ...... ………………………………...... XV RESUMO ...... ………………………………...... XVI STRUCTURE OF DISSERTATION ...... XVII INTRODUCTION ...………………………………...... 1 OBJECTIVES ...... 5 I. General Objective ...... ……………….……….…………………...... 6 II. Specific Objectives ....……...... ………….……….…………………...... 6 LITERATURE REVIEW ...... 7 Chapter I : The Big Picture on Antibacterial Resistance ...... 8 I. Antibacterial Resistance ....……………….……….…………………...... 9 1. Why is Antibacterial Resistance a Public Health Concern? …...... 9 2. Origins of Antibacterial Resistance ...... 10 3. Types of Antibacterial Resistance ...... 11 4. Antibacterial Modes of Action and Resistance Mechanisms …...... 12 4.1. Inhibition of Cell Wall Synthesis ...... 14 4.2. Inhibition of Protein Synthesis ...... 16 4.3.Inhibition of Metabolic Pathways & Interference with Nucleic Acid Metabolism 18 5. Genetics of Antibacterial Resistance Transfer ……...... 20 6. Pharmacodynamics and Pharmacokinetics of Antibacterials ...... 24 6.1. Time-dependent vs. Concentration-dependent Bacterial Decimation …...... 27 7. Laboratory Detection of Antibacterial Resistance ……...... 28 7.1. Antibacterial Susceptibility Breakpoints ...... 28 7.2. Definitions of Susceptibility Categories ...... 29 7.3. Organizations that Set Breakpoints ...... 30 7.4. The Nature of Minimum Inhibitory Concentrations …...... 31 7.5. Test Methods in Antibacterial Resistance Detection …...... 34 7.5.1. Broth Dilution Methods ...... 34 7.5.2. Disk Diffusion Method …...... 35 7.5.3. Antibacterial Gradient Diffusion Method ...... 36 7.5.4. Automated Antibacterial Susceptibility Testing Systems ...... 37 7.5.5. Current Test Methods and Future Directions ...... 38 II. The Use of Antibacterials ..……………….……….……...…...... 39 8. The Use of Antibacterial Agents in Human Populations ……...... 39 8.1. The Relationship Between Antibacterial Use and Resistance ……...... 39 8.2. Critically Important Antibacterials for Human Medicine ……...... 40 8.3. The Current Use of Antibacterials in Europe ……...... 41 8.4. The Current Use of Antibacterials in Portugal …...... 46 9. The Use of Antibacterials in Animal Husbandry …...... 48 9.1. Definitions of Antibacterial Use ...... 49 9.2. Pharmacodynamics and Pharmacokinetics of Antibacterial Use in Animal Husbandry …...... 50 9.3. Regulation and Authorization of Antibacterial Use in the EU ...... 51 9.4. The Current Use of Antibacterials in Europe ...... 52 9.5. The Current Use of Antibacterials in Portugal ...... 55 9.6. Applications of Antibacterials in Animal Husbandry Operations …...... 58 9.6.1. Antibacterial Use in Dairy Production Systems ...... 59 III. Dissemination and Transfer of Resistant Bacteria and Resistance Genes from Animals to Humans ...... 63 10. Sources and Routes for ABR Dissemination and Transfer ...... 64 11. Bacteria of Public Health Concern ...... 66 11.1. Foodborne Pathogens (Salmonella & Campylobacter) …...... 66 11.2. Indicator (Commensal) Organisms ...... …...... 69 11.2.1. Enterococci …...... …...... 70 11.2.2. Escherichia coli …...... …...... 73 11.3. Other Gram-negative Bacteria ...... 76 11.4. Staphylococcus aureus ...... …...... 76 11.5. Streptococcus pneumoniae …...... …...... 79 IV. Response to the Increasing Burden of ABR: Control Strategies and Interventions – The One Health Approach ...... 80 12. Surveillance Systems to Track Antibacterial Use and Resistance ...... 81 12.1 Surveillance of Antibacterial Resistance …...... 82 12.2 Surveillance of Antibacterial Usage …...... 83 12.3 Combined Surveillance …...... 83 13. Reducing Antibacterial Use in Humans ...... 84 13.1 Promoting Rational Antibacterial Use …...... 84 13.2 Infection Prevention and Control in Health-Care Facilities …...... 85 13.3 Fostering Innovation ...... 85 14. Reducing Antibacterial Use in Animal Husbandry ...... 87 14.1 Regulations to Restrict the Use of Antibacterials in Food-Producing Animals 87 14.2 Financial Incentives …...... 90 14.3 Prudent use Guidelines and Education ...... 90 14.4 Improving Animal Health ...... 90 14.5 Improving Hygiene in Food Production ...... 91 14.6 Applying Advances in Data Management Technology …...... 91

Chapter II: Antibacterial Resistance of Mastitis Pathogens ...... 92 I. Mastitis in Dairy Production Operations ...... ……………...... 93 1. Introduction …...... 93 2. Mastitis Pathogens …...... 95 3. Current Approaches for Mastitis Diagnosis ...... 97 II. Mastitis Antibacterial Therapy and the Use of Susceptibility Profiles for Treatment Decisions ...... …………….... 98 4. Assessing Efficacy …...... 98 5. Pharmacological Considerations …...... 101 6. Susceptibility Testing for Mastitis Pathogens …...... 102

6.1. Determination and Validation of Susceptibility Breakpoints for Mastitis Pathogens ...... 102 6.1.1. Limited Availability of MIC Values for Mastitis Pathogens ...... 103 6.1.2. Incomplete PK/PD Data for Lactating Dairy Cows ...... 103 6.1.3. Inadequate Number of Field Studies Validating Susceptibility Breakpoints ...... 103 6.2. Test Methods …...... 104 6.2.1. Milk Dilution Method ...... 104 6.3. Guidance for Antibacterial Selection Using Susceptibility Test Results and MIC Values ...... 106 6.3.1. Validity of Developing a ‘‘Herd Profile’’ for Susceptibility ...... 106 6.3.2. Validity of Selecting the Antibacterial with the Lowest In Vitro MIC Value ...... 106 6.3.3. Validity of Assumption that all Antibacterials Within a Class have Identical MIC Values ...... 107 6.3.4. Effect of Milk on MIC Values ...... 107 6.3.5. Deleterious Effects of Antibacterials on Normal Mammary Defense Mechanisms ...... 108 6.3.6. Distribution of Antibacterials in an Inflamed Mammary Gland ...... 108 7. Calculation of Antibacterial Dosage …...... 109 III. Resistance Patterns of Mastitis Pathogens ...... 112 8.Trends on Resistance Patterns Over Time in Response to Antibacterial Usage ...... 112 MATERIALS & METHODS ...... 115 I. Criteria for Selection of Cases ……...... …………………...... 116 II. Sample Collection and Microbiology ……...... …………………..... 116 III. In vitro Antibacterial Susceptibility Testing …....…………………..... 117 IV. Tested Antibacterials ...... ……….……….…………………...... 117 V. Selection of Pathogens ...... …..……….…………………...... 117 VI. Data Analysis ...... ………….……….…………………...... 118 RESULTS ...... 119 DISCUSSION ...... 128 I. Novelty Aspects of this Study ……...... …………………...... 129 II. Antibacterial Resistance Pattern and Trend Analysis ……...... 129 III. Data Analysis …....…………………...... 132 IV. Limitations of the Study ...... ……….……….…………...... 133 V. Improvement Suggestions for Future Similar Research ...... 134 VI. Further Research Ideas and Recommendations ...... 134 CONCLUSIONS ...... 136 REFERENCES ...... 139 APPENDICES …...... i - xl

LIST OF FIGURES

Figure 1: Broad depiction of major ABR mechanisms ...... 13

Figure 2: Protein synthesis. Aminoacyl-tRNA molecules are formed in the cytoplasm and bind to the cognate triplicate codon of mRNA at the ribosome. Peptide bond formation links the new amino acid to the growing polypeptide chain. The ribosome migrates to free the A site for the next aminoacyl-tRNA molecule and the cycle repeats until a stop codon is encountered and translation is terminated ...... 17

Figure 3: Activity of protein synthesis inhibitors. Schematic of the bacterial ribosome and the sites of action of select antibacterials that inhibit polypeptide biosynthesis ...... 18

Figure 4: Schematic of multiple antibacterial resistance accumulation on a plasmid ...... 22

Figure 5: Schematic representation of the complexity of interactions between patient, pathogen and antibacterial agent ...... 25

Figure 6: Concentration-versus-time curve with MIC superimposed and pharmacokinetic and pharmacodynamic markers ...... 26

Figure 7: MIC distributions for four microorganism-antibacterial pairs. In each case, the wild type appears as the log-normally distributed

population at the lower MICs. COWT – calculated wild-type cutoff value ...... 33

Figure 8: A broth microdilution susceptibility panel containing 98 reagent wells and a disposable tray inoculator ...... 35

Figure 9: Antibacterial susceptibility testing by disk diffusion. On this agar plate, a bacterial isolate is tested for resistance to each of four different antibacterials ...... 36

Figure 10: Antibacterial susceptibility testing by E-Test. On this agar plate, a bacterial isolate is tested for resistance to a specific antibacterial ...... 37

Figure 11: Vitek® 2 System – BioMérieux, France ...... 38

Figure 12: Total outpatient antibacterial use in 2009 in Europe ...... 42

Figure 13: Boxplotted distribution of outpatient antibacterial use between 1999 and 2009 among the participating European countries ...... 42

Figure 14: Trends of total outpatient antibacterial use in Europe from 1997 to 2009. Dark bars correspond to the year 2009 ...... 44

Figure 15: Outpatient antibacterial use in 2009 subdivided into the major antibacterial classes according to ATC classification ...... 44

Figure 16: Hospital use of antibacterials for systemic use in 2009 (N= 22 countries) ...... 46

Figure 17: Distribution of antibacterial classes in ambulatory (A) and hospital (B) care sectors in Portugal in 2009 ...... 47

Figure 18: Trends of antibacterial usage in ambulatory care sector in Portugal ...... 47

Figure 19: Annual antibacterial/antimicrobial use for human and veterinary practice in Denmark ...... 48

Figure 20: PCU (in 1.000 tons) of the major food-producing animal species in 2009, by country ...... 53

Figure 21: Proportion of US dairy operations in 2007 that treated cows with any antibacterial for the main diseases/disorders ...... 59

Figure 22: Proportion of US adult dairy cows treated with antibacterials for the main diseases/disorders in 2007 ...... 60

Figure 23: Proportion of preweaned and weaned heifers treated with antibacterials in 2007 for the main diseases/disorders ...... 60

Figure 24: Possible routes of transmission of antibacterial-susceptible or -resistant gastrointestinal pathogens or normal intestinal flora between animals and humans ...... 64

Figure 25: Reservoirs of ABR bacteria causing human infections. Schematic overview of some of the most important ABR pathogens and the overlap between the different reservoirs ...... 65

Figure 26: Trend and number of reported confirmed human campylobacteriosis cases by month, in the EU and EEA/EFTA countries, 2006–09 ...... 66

Figure 27: Trend and number of reported confirmed human salmonellosis cases by month, in the EU and EEA/EFTA countries, 2006–09 67

Figure 28: (A) E. faecalis: trends of high-level resistance to aminoglycosides by country, 2007–2010. (B) E. faecium: Trends of resistance to vancomycin by country 2007–2011 ...... 71

Figure 29: S. aureus: Trends of resistance to methicillin (MRSA) by country, 2007–2011 ...... 78

Figure 30: ECDC promotional One Health poster ...... 80

Figure 31: Discovery timeline of new antibacterial classes (1930s to 2000s) ...... 86

Figure 32: Macrolide use and resistance among enterococci in swine, Denmark ...... 88

Figure 33: Cephalosporin resistance in poultry industry in Quebec, Canada ...... 89

Figure 34: Reduction in antibacterial use after the introduction of vaccination in aquaculture in Norway ...... 91

Figure 35: Sliding scale for contagious and environmental origin of mastitis pathogens, based on insights from molecular epidemiology .... 95

Figure 36: Concentration-versus-time curve for drug concentration in milk and plasma ...... 109

______Balbino M. Rocha, 2013 V LIST OF TABLES

Table 1: Estimated annual burden due to selected antibacterial-resistant bacteria in EU-Member States, Iceland and Norway, 2007 ...... 9

Table 2: Evolution of resistance to major antibacterials ...... ….……....……………...... 10

Table 3: Mechanisms of action of main antibacterial agents ...... ….……....……………...... 12

Table 4: Mechanisms of ABR …...... ………...... ….……....……………...... 13

Table 5: World organizations with published breakpoints …...... ………...... ….……....…………...... 31

Table 6: Summary of the antibacterial classes included in the three categories of Critically Important Antimicrobials for Human Medicine 41

Table 7: Outpatient antibacterial use in 2009 subdivided into the major antibacterial classes according to ATC classification …...... 43

Table 8: Hospital use of antibacterials for systemic use in 2009 (N= 22 countries) …...... ………...... ….……...... 45

Table 9: Total sales of veterinary antibacterial agents (active ingredient) and PCU (1000 tons) in eight European countries (Switzerland not included) …...... ………...... ….……....……………...... 52

Table 10: Sales normalized by PCU (mg/PCU) for the years 2005-2009 …...... ……...... ….……....……………...... 53

Table 11: Difference between 2009 and 2005 sales, expressed as tons of active ingredient and as mg/PCU, for eight European countries (Switzerland not included) …...... ……...... ….……....……………...... 54

Table 12: Total 2010 sales of veterinary antibacterial agents (active ingredient, in tons and %) by antibacterial class in Portugal …...... 56

Table 13: Distribution of active ingredients (in tons) in each animal species, in Portugal in 2010 …...... ……...... 57

Table 14: Distribution of the antibacterial dosage forms in each animal species, in Portugal in 2010 …...... ……...... 58

Table 15: Examples of diseases on the different food-producing animal species including organ, pathogen and type of treatment …...... 62

Table 16: Salmonella serotypes most frequently reported from human salmonellosis cases in the EU and EEA/EFTA countries and percentage change, 2008-09 …...... 67

Table 17: Number of invasive E. faecalis and E. faecium isolates and proportion of high-level aminoglycoside-resistant E. faecalis and vancomycin-resistant E. faecium (%R), including 95% CI, reported per country in 2011 ...... 70

Table 18: Number and proportion of invasive E. coli isolates resistant to aminopenicillins, 3rd-generation cephalosporins, fluoroquinolones, aminoglycosides and multi-drug resistant (%R), including. 95% CI, reported per country in 2011 ...... 74

Table 19: Number of invasive E. coli isolates resistant to 3rd-generation cephalosporins (CREC) and proportion of ESBL-positive (%ESBL) among these isolates, as ascertained by the participating laboratories in 2011 ...... 74

Table 20: Overall resistance and resistance combinations among invasive E. coli isolates tested against aminopenicillins, fluoroquinolones, 3rd-generation cephalosporins and aminoglycosides (n= 49847) in Europe, 2011 ...... 75

Table 21: Number and proportion of invasive S. aureus isolates resistant to methicillin (MRSA) and rifampin (RIF), including 95% CI, reported per country in 2011 …...... ……...... ….……....……...... 77

Table 22: ABR surveillance networks for common bacterial pathogens in the WHO Regions …...... 82

Table 23: Prevalence of mastitis pathogens in dairy herds from Northwestern Portugal, between 2005 and 2008 ...... 96

Table 24: Current SCC measuring methods and alternatives for detection of mastitis ...... 98

Table 25: Summary of three-compartment model to target mastitis pathogens ...... 101

Table 26: MIC data for several bacterial isolates from mastitic milk samples from the MAHDL, 1999-2001 ...... 111

Table 27: Conclusions from short- to long-term studies on the effect of antibacterials on resistance of mastitis pathogens worldwide ...... 113

Table 28: Antibacterial agents used for susceptibility testing of 47,413 bacterial pathogen isolates obtained from dairy cow milk samples and submitted for bacterial culture between January 2004 and September 2012 ...... 120

Table 29: Number of major bacterial pathogens isolated along the study period (2004-2012) ...... 120

Table 30: Results of resistance in antibacterial susceptibility testing, by antibacterial agent, of major mastitis bacterial pathogens ...... 121

Table 31: Results of logistic regression analysis to determine, for the isolated bacterial pathogens, whether the percentage of isolates resistant to the various antibacterial agents changed with year ...... 122

Table 32: S. aureus resistance proportions, among each tested antibacterial agent, along each tested year (n = 28,126 isolates) ...... 123

Table 33: S. agalactiae resistance proportions, among each tested antibacterial agent, along each tested year (n = 4,589 isolates) ...... 123

Table 34: S. uberis resistance proportions, among each tested antibacterial agent, along each tested year (n = 5,799 isolates) ...... 124

Table 35: S. dysgalactiae resistance proportions, among each tested antibacterial agent, along each tested year (n = 1,231 isolates) ...... 125

Table 36: Enterococcus spp. resistance proportions, among each tested antibacterial agent, along each tested year (n = 979 isolates) ..... 125

Table 37: E. coli resistance proportions, among each tested antibacterial agent, along each tested year (n = 5,916 isolates) ...... 126

Table 38: K. pneumoniae resistance proportions, among each tested antibacterial agent, along each tested year (n = 773 isolates) ...... 127

______Balbino M. Rocha, 2013 VI LIST OF APPENDICES

Appendix 1:

Table 39: Summary of some of the pertinent literature on the ABR of S. aureus isolated in milk from cows with mastitis worldwide ...... ii

Table 40: Summary of some of the pertinent literature on the ABR of CNS isolated in milk from cows with mastitis worldwide ...... iii

Table 41: Summary of some of the pertinent literature on the ABR of environmental Streptococcus sp. isolated in milk from cows with iv mastitis worldwide ......

Table 42: Summary of some of the pertinent literature on the ABR of E. coli isolated in milk from cows with mastitis worldwide ...... v Table 43: Summary of some of the pertinent literature on the ABR of other Gram-negative bacteria (besides E. coli) isolated in milk from vi cows with mastitis worldwide ......

Table 44: Summary of some of the pertinent literature on the ABR of S. uberis and S. dysgalactiae isolated in milk from cows with mastitis vii worldwide ......

Table 45: Summary of some of the pertinent literature on the ABR of S. agalactiae, esculin-positive Streptococcus sp. and Enterococcus viii spp. isolated in milk from cows with mastitis worldwide ......

Appendix 2:

Figure 36: Resistance proportions of S. aureus isolates, among each tested antibacterial agent, along each tested year (2004-2012) ...... ix

Figure 37: Resistance proportions of S. aureus isolates, among each tested antibacterial agent, along each tested year. Asterisks represent statistically significant changes (p<0.05) ...... x

Figure 38: Resistance proportions of S. agalactiae isolates, among each tested antibacterial agent, along each tested year (2004-2012) .. xi

Figure 39: Resistance proportions of S. agalactiae isolates, among each tested antibacterial agent, along each tested year. Asterisks represent statistically significant changes (p<0.05) ...... xii

Figure 40: Resistance proportions of S. uberis isolates, among each tested antibacterial agent, along each tested year (2004-2012) ...... xiii

Figure 41: Resistance proportions of S. uberis isolates, among each tested antibacterial agent, along each tested year. Asterisks represent statistically significant changes (p<0.05) ...... xiv

Figure 42: Resistance proportions of S. dysgalactiae isolates, among each tested antibacterial, along each tested year (2004-2010) ...... xv

Figure 43: Resistance proportions of S. dysgalactiae isolates, among each tested antibacterial agent, along each tested year. Asterisks represent statistically significant changes (p<0.05) ...... xvi

Figure 44: Resistance proportions of Enterococcus spp. isolates, among each tested antibacterial, along each tested year (2004-2010) ... xvii

Figure 45: Resistance proportions of Enterococcus spp. isolates, among each tested antibacterial agent, along each tested year. Asterisks represent statistically significant changes (p<0.05) ...... xviii

Figure 46: Resistance proportions of E. coli isolates, among each tested antibacterial agent, along each tested year (2004-2012) ...... xix

Figure 47: Resistance proportions of E. coli isolates, among each tested antibacterial agent, along each tested year. Asterisks represent statistically significant changes (p<0.05) ...... xx

Figure 48: Resistance proportions of K. pneumoniae isolates, among each tested antibacterial agent, along each tested year (2004-2012) xxi

Figure 49: Resistance proportions of K. pneumoniae isolates, among each tested antibacterial agent, along each tested year. Asterisks represent statistically significant changes (p<0.05) ...... xxii

Table 46: SPSS outputs for logistic regression analysis to determine, for the S. aureus isolates, whether the percentage of isolates resistant to the various antibacterial agents changed throughout the study period ...... xxiii

Table 47: SPSS outputs for logistic regression analysis to determine, for the S. agalactiae isolates, whether the percentage of isolates resistant to the various antibacterial agents changed throughout the study period ...... xxiv

Table 48: SPSS outputs for logistic regression analysis to determine, for the S. uberis isolates, whether the percentage of isolates resistant to the various antibacterial agents changed throughout the study period ...... xxv

Table 49: SPSS outputs for logistic regression analysis to determine, for the S. dysgalactiae isolates, whether the percentage of isolates resistant to the various antibacterial agents changed throughout the study period ...... xxvi

Table 50: SPSS outputs for logistic regression analysis to determine, for the Enterococcus spp. isolates, whether the percentage of isolates resistant to the various antibacterial agents changed throughout the study period ...... xxvii

Table 51: SPSS outputs for logistic regression analysis to determine, for the E. coli isolates, whether the percentage of isolates resistant to the various antibacterial agents changed throughout the study period ...... xxviii

Table 52: SPSS outputs for logistic regression analysis to determine, for the K. pneumoniae isolates, whether the percentage of isolates resistant to the various antibacterial agents changed throughout the study period ...... xxix

______Balbino M. Rocha, 2013 VII Appendix 3:

Article under submission for publication in international journal: Rocha, B.; Mendonça, D.; Niza-Ribeiro, J. (2013) "Trends in Antibacterial Resistance of Major Bovine Mastitis Pathogens in Portugal" ...... xxx

Appendix 4:

Slide presentation of short communication presented by the author: "Evolução de Padrões de Resistência a Antibióticos em Agentes Etiológicos da Mastite Bovina em Portugal". II Conferência Anual do Conselho Português de Saúde do Úbere (CPSU). Santarém, Portugal – February 23rd, 2013 ...... xxxviii

Appendix 5:

Poster presentation: Rocha, B., Mendonça, D., Niza-Ribeiro, J. (2013). “Evolução de Padrões de Resistência a Antibióticos em Agentes Etiológicos da Mastite Bovina em Portugal”. XV Jornadas da Associação Portuguesa de Buiatria. Ílhavo, Portugal – May 24th to 26th, 2013 ...... xl

______Balbino M. Rocha, 2013 VIII LIST OF ABBREVIATIONS & ACRONYMS

3GCREC – E. coli isolates resistant to 3rd-generation cephalosporins ABR – Antibacterial Resistance AI - Active Ingredient AMP – Ampicillin ATC – Anatomical Therapeutic Chemical (Classification System) ATCC – American Type Culture Collection AUC – Area under the (concentration–time) curve AUG – Amoxicillin/Clavulanic acid AHVLA – Animal Health and Veterinary Laboratories Agency bp – Base pair BSAC – British Society for Antimicrobial Chemotherapy BTSCC – Bulk tank somatic cell count CATs – Chloramphenicol acetyltransferases CDS-AST – CDS disk diffusion method - Australia CFP – Cefoperazone CFU – Colony Forming Unit CI – Confidence Interval CLSI – The Clinical and Laboratory Standards Institute CM – Clinical Mastitis

Cmax – Peak concentration CN – Gentamicin

COWT – Calculated wild-type cutoff value CSF – Cerebrospinal fluid CSN - Coagulase-negative staphylococci CVMP – Committee for Medicinal Products for Veterinary Use DDD – Defined Daily Dose DGAV – Direção Geral de Alimentação e Veterinária DID – Defined Daily Doses per 1000 inhabitants per day DIN – Deutsches Institut für Normung DNA – Deoxyribonucleic Acid EARS-Net – The European Antimicrobial Resistance Surveillance Network EEA – European Economic Area EFTA – European Free Trade Association EMA – European Medicines Agency ESAC-Net – European Surveillance of Antimicrobial Consumption Network ESBL – Extended Spectrum β-Lactamase ESVAC – European Surveillance of Veterinary Antimicrobial Consumption Project EU – European Union EU-MS – European Union Member State EUCAST – European Committee of Antimicrobial Susceptibility Testing FAO – Food and Agriculture Organization of the United Nations HACCP – Hazard Analysis and Critical Control Point HHPM – Herd Health and Production Management HGT – Horizontal gene transfer IMI – Intramammary Infection IMM – Intramammary IS – Insertion sequence ISCR1 – Insertion sequence common regions ______Balbino M. Rocha, 2013 IX IV – Intravenous KZ – Cefazolin LA-MRSA – Livestock-associated MRSA LPS – Lipopolysaccharides LR – Logistic Regression mg/PCU – Milligram of antimicrobial per Population Correction Unit MIC – Minimum Inhibitory Concentration MIS – Management Information Systems mL – Milliliter MLS (group) – Macrolides, Lincosamides and Streptogramins Group MRSA – Methicillin-resistant Staphylococcus aureus mRNA – Messenger RNA NAG – N-acetylglucosamine NAHMS – National Animal Health Monitoring System of the USDA NAM – N-acetylmuramic acid NMC – National Mastitis Council OIE – Office International des Epizooties (World Organization for Animal Health) OMPs – Outer Membrane Proteins P – Penicillin G PABA – p-aminobenzoic acid PAE – Post-antibiotic effect PBPs – Penicillin-binding proteins PCU – Population Correction Unit PD – Pharmacodynamic PK – Pharmacokinetic PNCUM – Plano Nacional de Controlo e Utilização de Medicamentos PRK – People's Republic of Korea PRSP – Penicillin G-resistant S. pneumoniae QACs – Quaternary ammonium compounds QRDR – Quinolone resistance-determining region rRNA – Ribossomal RNA RNA – Ribonucleic Acid S – Streptomycin SCAN – European Union’s Scientific Committee on Animal Nutrition SCC – Somatic Cell Count SCM – Subclinical Mastitis SFM – Société Française de Microbiologie SGI – Salmonella Genomic Island SHV – Sulphydryl variable SRGA – Swedish Reference Group for antibiotics ST398 – Sequence type 398 t – ton TEM – From Temoniera, the name of the patient from whom the original β-lactam resistant isolate was acquired TMP-SMX – Trimethoprim/Sulfamethoxazole tRNA – Transfer RNA URI – Upper Respiratory Infections USDA – United States Department of Agriculture VMP – Veterinary Medicinal Products VRE – Vancomycin-resistant enterococci VRTI – Viral Respiratory Tract Illness WFE – Whole Fish Equivalent WHO – World Health Organization ______Balbino M. Rocha, 2013 X Definitions

DEFINITIONS

Anatomical Therapeutic Chemical (ATC) Classification System: This system is used for the classification of drugs. It is controlled by the WHO Collaborating Centre for Drug Statistics Methodology (WHOCC), and was first published in 1976. This pharmaceutical coding system divides drugs into different groups according to the organ or system on which they act and/or their therapeutic and chemical characteristics. 2

Antibacterial (agents): Synthetic (chemotherapeutics) or natural (antibiotics) substances that destroy bacteria or suppress bacterial growth or reproduction. 3

Antimicrobial (agents): This broad term normally covers antibacterial, antiviral, coccidiostatic and antimycotic agents. 4

Bactericidal (activity): Refers to a substance or a condition capable of killing bacteria. 5

Bacteriologic cure: Failure to isolate the same pathogen from affected quarter within a specific time interval. 6

Bacteriostatic (activity): Refers to a substance or condition that inhibit or prevent further growth of bacteria. 5

Breakpoints: Breakpoints are specific MIC values used to assign bacteria to one of three categories – susceptible, intermediate and resistant – using recommendations for testing veterinary pathogens from organizations created specifically to set those breakpoints (e.g., EUCAST or CLSI). 7

Clinical cure: Return to visibly normal secretion in a specific time interval. 6

Control: Administration of an antimicrobial to animals, usually as a herd or flock, in which morbidity and/or mortality has exceeded baseline norms. 8

Defined Daily Dose (DDD): Unit of measurement that corresponds to the assumed average maintenance dose per day for a drug used for its main indication in adults. Drug consumption data presented in DDDs only give a rough estimate of consumption and not an exact picture of actual use. The DDD provide a fixed unit of measurement independent of price and dosage form (e.g., tablet strength) enabling the researcher to assess trends in drug

______Balbino M. Rocha, 2013 XI Definitions

consumption and to perform comparisons between population groups. Doses for individual patients and patient groups will often differ from the DDD and will necessarily have to be based on individual characteristics (e.g., age and weight) and pharmacokinetic considerations. 2

Dry cow: A cow that is not lactating or secreting milk after it has completed a lactation period following calving. 9

Fresh cow: A cow that has recently given birth to a calf. 9

Growth promotion: Administration of an antimicrobial, usually as a feed additive, over a period of time, to growing animals that results in improved physiological performance. 8

High-level ABR: Increased antibacterial potency within the clinically susceptible range. 10

Level of antibacterial resistance: Percentage of resistant isolates from the tested isolates.11

L-forms: Antibacterial-induced variants of S. aureus that lack a cell wall. 12

Low-level ABR: Diminished antibacterial potency within the clinically susceptible range. 10

Minimum Inhibitory Concentration (MIC): Corresponds to the lowest antibacterial concentration (expressed in g/mL or mg/L) that under defined in vitro conditions prevents the growth of bacteria within a defined period of time. 7 It is generally accepted that MIC

values are very repeatable. Statistics such as MIC50 (the median MIC for all isolates) and

MIC90 (the MIC value that exceeds or equals the MIC for 90% of the isolates) are frequently used to summarize population data. 13

Minimum Bactericidal Concentration (MBC): Corresponds to the lowest concentration of an antibacterial that under defined laboratory conditions reduces by 99.9% (three-log reduction) the number of organisms in a medium containing a defined bacterial inoculum within a defined period of time. 7

Multidrug-resistant bacteria: Bacteria with resistance to three or more antibacterial drug classes. 14

______Balbino M. Rocha, 2013 XII Definitions

Population Correction Unit (PCU): The amounts of veterinary antimicrobial agents sold in the different countries are, among others, linked to the animal demographics in each country, which may vary over time. In this dissertation's section 9.4., the annual sales figures in each country were divided by the estimated weight at treatment of livestock and of slaughtered animals in the corresponding year. The PCU is therefore used as the term for the estimated weight and is purely a technical unit of measurement used only to estimate temporal trends in individual countries and across countries. 1 PCU = 1 kg of different categories of livestock and slaughtered animals. 15

Prevention/prophylaxis: Administration of an antimicrobial to exposed healthy animals considered to be at risk, but before expected onset of disease and for which no etiological agent has yet been cultured. (Metaphylaxis is a term sometimes used when there is clinical disease in some animals, but all are treated). 8

Somatic Cell Counts (SCC): Somatic cells are present in a certain normal physiological amount in milk. These consist of various cell types and their relative proportions depend on the health status of the udder. In a healthy lactating mammary gland, the major proportion of somatic cells is constituted by leukocytes. 16 These are primarily macrophages and lymphocytes, but a small fraction consists of neutrophils and epithelial cells. 17, 18 Microbial infection results in rapid accumulation of large numbers of somatic cells in the udder, and these are predominantly neutrophils. 16-18 The increase in SCC constitutes an important part of the cow’s immune response, and SCC is, therefore, along with bacterial culture examination, a widely used indicator of mastitis. According to the International Dairy Federation, a threshold of 200×103 cells/mL of milk is suggested for subclinical mastitis. 19

Therapy: Administration of an antimicrobial to an animal or group of animals, which exhibit frank clinical disease. 8

Tripartite Concept Note: FAO, OIE and WHO issued, in 2010, this document that sets a strategic common direction and proposes a long-term basis for international collaboration aimed at coordinating global activities to address health risks at the human-animal-ecosystems interfaces. New synergies between the three partners include normative work, public communication, pathogen detection, risk assessment and management, technical capacity building and research development. 20

Withdrawal period: Length of time needed to allow an antimicrobial to be removed from edible tissue. 10

______Balbino M. Rocha, 2013 XIII Agradecimentos

AGRADECIMENTOS

À minha família, por todo o apoio e incentivo, sem os quais não teria podido levar a bom termo esta dissertação.

Ao Professor Doutor João Niza Ribeiro pelo interesse que me incutiu neste tema, bem como pela orientação e apoio prestados.

À Professora Doutora Denisa Mendonça, pela preciosa ajuda prestada na análise estatística e interpretação dos dados.

À Segalab - Laboratório de Sanidade Animal e Segurança Alimentar, S.A., pela disponibilização dos dados necessários.

Ao Departamento de Serviços Técnicos da Segalab, nomeadamente Dr.ª Helena Madeira, Dr.ª Abigail Barbosa, Eng.ª Paula Santos e Eng.ª Marta Barbosa, pela disponibilização dos dados e pela ajuda em questões de natureza técnica.

Ao Doutor Jorge Ferreira pelo apoio prestado na correção da dissertação.

______Balbino M. Rocha, 2013 XIV Resum o

RESUMO

O incremento de resistências a compostos antibacterianos tem-se revelado, ao longo dos anos, alvo de crescente preocupação a nível Mundial, quer do ponto de vista da Saúde Pública, quer na perspectiva da Segurança Alimentar. A mastite bovina é a causa mais frequente de utilização destes fármacos em efectivos leiteiros, sendo o seu uso apontado como factor selectivo na ecologia bacteriana do úbere bovino. Os padrões de resistências antibacterianas em agentes etiológicos de mastite têm, deste modo, vindo a suscitar um crescente interesse por parte da comunidade científica veterinária. A monitorização e análise destes padrões tem fornecido informações de grande utilidade na escolha de antibacterianos no tratamento de animais afectados, especialmente no que diz respeito a agentes etiológicos isolados em diferentes regiões geográficas.

Esta dissertação pretende dar a conhecer os padrões de resistência a sete fármacos antibacterianos disponíveis no mercado Português e a sua evolução, em 47.413 testes de susceptibilidade antibacteriana. Analisaram-se os principais agentes etiológicos da mastite – Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Streptococcus dysgalactiae, Enterococcus spp. (E. faecium e E. faecalis), Escherichia coli e Klebsiella pneumoniae. Os isolados foram obtidos a partir de amostras de leite provenientes de explorações leiteiras das regiões Litoral Norte, Centro e Sul de Portugal, entre 2004 e 2012. Os testes de susceptibilidade foram executados através do método de difusão em disco. Os fármacos utilizados foram penicilina, amoxicilina/ácido clavulânico, cloxacilina, cefazolina, cefquinoma, gentamicina e trimetoprim/sulfametoxazol.

Os resultados obtidos dos níveis de resistência não diferem significativamente dos de estudos semelhantes. Porém, a avaliação da evolução desses padrões, ao longo dos nove anos do estudo, revelou tendência para um aumento estatisticamente significativo dos níveis de resistência entre certos agentes testados. Estudos adicionais devem ser elaborados no esforço de se encontrar a origem destes aumentos, de modo a serem tomadas medidas concretas na inversão destas tendências.

______Balbino M. Rocha, 2013 XVI Abstract

ABSTRACT

Worldwide, the development of resistance to antibacterial agents has proved to be a growing concern over the years, from both public health and food safety perspective. Bovine mastitis reports as the most common pathology in dairy herds, and therefore, to which these drugs are mostly used. This use accounts as a selective factor in the bacterial ecology of the bovine udder. Antibacterial susceptibility patterns in mastitis pathogens have, for this reason, accordingly raised a growing interest in the veterinary scientific community. The long-term monitoring and analysis of these patterns has provided useful information in guiding the selection of antibacterial therapy of affected animals, especially with regard to etiologic agents isolated from different geographic regions.

This dissertation aims to provide patterns of resistance to seven antibacterial drugs available in the Portuguese market and their evolution over time, in 47,413 antibacterial susceptibility tests. The major etiological agents of mastitis isolated were: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Streptococcus dysgalactiae, Enterococcus spp. (E. faecium and E. faecalis), Escherichia coli and Klebsiella pneumoniae. Isolates were obtained from milk samples from dairy farms in the northwestern, central and southern regions of Portugal, between 2004 and 2012. Susceptibility testing was performed by the disk diffusion method. Tested antibacterials were penicillin, amoxicillin/clavulanic acid, cloxacillin, cefazolin, cefquinome, gentamicin and trimethoprim/sulfamethoxazole.

The overall antibacterial resistance levels found for these mastitis isolates were, in most cases, comparable to what other relevant similar studies have reported worldwide. When assessing the evolution of these patterns over the nine years of data, significant increases were determined among certain agents tested. Further research should be addressed in an effort to find the source of these increases and take actual steps in reversing these trends in the region.

______Balbino M. Rocha, 2013 XV STRUCTURE OF DISSERTATION Structure of the Dissertation

The current dissertation was organized into two main parts. The first part consists of an introduction, the objectives of the dissertation and a literature review. The second part describes the respective research project.

In the introduction, the author sets up the research topic, as well as its scope and significance; followed by the definition of the general and specific objectives. In order to contextualize the dissertation, a literature review was prepared, consisting of two chapters with relevant information concerning the subject in question: Antibacterial resistances by bacteria and the impact on animal and human health, with emphasis on the dairy environment, especially to mastitis pathogens.

The first chapter of the review corresponds to an introductory chapter, with the aim of covering the subject in its generality, and is divided into four subchapters. The first subchapter focuses on the origins and types of antibacterial resistance, as well as modes of action and resistance mechanisms by bacteria; pharmacodynamics and pharmacokinetics of the different antibacterial agents; and a section on laboratory detection breakpoints and diverse test methods used in identifying antibacterial resistances. The second subchapter describes the usage statistics of antibacterials among both humans and animals, mainly in Europe and Portugal. The third subchapter centers on the potential sources and routes of dissemination and transfer of resistant bacteria and resistance genes from animals to humans, describing the diverse bacteria of public health concern. The fourth subchapter focuses on the strategies and interventions available nowadays to monitor and control antibacterial resistance, with the One Health approach taking centre-stage.

Chapter II covers a more specific and detailed approach on antibacterial resistance, as it pertains to the dairy production systems. This chapter exposes the reader to an introduction on mastitis, mastitis pathogens and antibacterial therapy, as well as the use of susceptibility profiles for treatment decisions. Still in this chapter, a review on relevant available literature regarding resistance patterns of mastitis pathogens is made.

The second main part of this dissertation concerns the research project per se, describing the methodologies, results and conclusions obtained in its completion.

______Balbino M. Rocha, 2013 XVIII INTRODUCTION Introduction

Since the launch of large-scale production in the 1940s, antibacterial agents are conceivably the foremost medical advance in history, having become imperative tools in decreasing morbidity and mortality associated with a multitude of infectious diseases in both humans and animals. In addition to the considerable contribution to human wellbeing and quality of life, their use in food-producing animal agriculture has resulted in healthier and more productive animals, yielding high-quality and low-cost food products for human consumption. 10, 21-23

Even though most antibacterial agents currently remain effective in both humans and animals, their intensive and often inappropriate use over the years has triggered an increase in the emergence and dissemination of antibacterial resistance, consequently reducing their efficiency. In fact, resistance mechanisms have been reported in every major bacterial pathogen for all known antibacterial agents currently available for clinical use in human and veterinary medicine. 24-27

The evolution of bacteriology over the past seven decades has made us much better equipped to recognize that we were clearly unaware of the implications associated with the indiscriminate use of these compounds and underestimated the genetic flexibility of the targeted microorganisms. Over the last decades, serious public health concerns about antibacterial resistance from hospital-acquired, community-acquired and foodborne pathogens have been raised at international levels. The World Health Organization (WHO), the Food and Agriculture Organization of the United Nations (FAO) and the World Organization for Animal Health (OIE) consider antibacterial resistance in zoonotic pathogens as a growing global public health threat and recognize that documented emerging resistance phenotypes may be the outcome of the administration of therapeutic and subtherapeutic levels of antibacterial agents to food-producing animals. 20 Although this issue has taken center stage over the years, there is still, however, no complete agreement among researchers on the significance of these deliberations. This is mainly due to major gaps in information and evidence regarding the development and dissemination of bacterial resistance to antibacterial agents in various animal production settings. The understanding of the complete effects of these compounds in the animal production environment is particularly difficult because the involved ecosystems are extremely complex, containing hundreds of bacterial genera and species in constant interaction and adaptation to countless variables, in addition to the antibacterial selection pressure itself. 10,21,23,28

Regardless of the enduring debate, the facts are clear: bacterial pathogens of animal and human origin are becoming increasingly resistant to most vanguard antibacterials, including last-generation cephalosporins, aminoglycosides and even fluoroquinolones. If current resistance trends persist, we may certainly encounter, in the near future, bacterial pathogens that are impervious to the effects of all current therapeutic antibacterials to a point ______Balbino M. Rocha, 2013 2 Introduction

where therapeutic options could become very limited. This reality has pushed for important changes in awareness and priorities of government agencies worldwide with regard to antibacterial management. Hence the One Health concept. This concept embraces a worldwide strategy for expanding interdisciplinary collaborations (e.g., FAO-OIE-WHO collaboration, through the Tripartite Concept Note) and communications in all aspects of healthcare for humans, animals and the environment. Only with such a holistic approach can we better understand why the problem of antibacterial resistance is currently so pervasive and how we should best intervene to improve the situation. 20,27

Monitoring of resistance in bacteria circulating in animals should focus on bacteria of public health importance from food animals, considering that the food chain is a major route for transfer of bacteria from animals to humans. Bacterial species included in the surveys are foodborne pathogens and foodborne commensal organisms, preferably isolated from healthy animals at slaughter. To represent foodborne commensal organisms, sentinel or indicator organisms are used that may be a source for transmission of resistance determinants to pathogens. E. coli and enterococci are included as indicator for Gram-negative and -positive bacteria, respectively. The inclusion of animal pathogens is of indirect importance for public health, but still very relevant. These pathogens usually represent a worst-case scenario because they are isolated from diseased animals that were treated with antibacterials. Therefore the surveillance of these strains in animal husbandry can be used for early warning purposes regarding the detection of new emerging resistances. 1, 28-30

Although contamination of carcasses at slaughter and meat products at retail are considered the major routes of transmission, other important routes may exist. Routes such as contamination of the environment, direct contact and contaminated products like milk or eggs may contribute to the dissemination of bacteria and their genes. 1,10,29,30

The dairy industry, over the last century, and in response to broad economic forces ultimately driven by the price elasticity of consumer demand, has turned out to be a major segment in the food production sector. The top goal of modern dairy operations is to produce maximum quantities of high-quality milk with a longer shelf-life. However, the intensification of this industry has brought vast advantages, but also great challenges when it comes to disease control and management, with the dairy producer relying on increasingly advanced veterinary diagnostic methods, treatment protocols and management strategies in an attempt to control and prevent herd pathology, consequently increasing animal performance. As a result, antibacterial agents are, nowadays, used extensively in the dairy industry for the prophylaxis/metaphylaxis and treatment of a variety of bacterial diseases affecting calves/heifers and adult females in the different rearing and milk production stages, respectively. In general, most of these antibacterials belong to the same general classes as

______Balbino M. Rocha, 2013 3 Introduction

those used in human medicine, and in many cases even if they are not exactly the same compounds, their mode of action is identical. 31

Bovine mastitis accounts as the most common pathology in dairy operations worldwide and also the one on which antibacterials are mostly employed on. In addition to mastitis therapy in lactating animals, one of the more consistent uses of antibacterials in dairy herds takes place at drying-off. Dry cow therapy, which is in fact, a mastitis prophylactic measure, consists in the application of high levels of intramammary antibacterials in a slow release base, following the last milking of lactation in adult cattle. 32

Resistance to antibacterial agents in mastitis pathogens discloses three relevant aspects: 1) Reduction in cure rates after treatment of mastitis cases; 2) Potential risk of transmission of resistant bacteria to humans via the food chain; and 3) Potential source of resistance genes between mastitis pathogens and other environmental pathogens, which may consequently, through other routes, affect humans. 33-35

At present, most authors consider that, although antibacterial resistance does occur, the advantages of using antibacterials for mastitis treatment far outweigh the disadvantages. Consequently, the ultimate challenge will be to find a balance between antibacterial use and its risk outcomes (e.g., antibacterial residues, antibacterial resistances, etc.) always having in mind that the implications of this challenge are far reaching and include aspects like public health, animal welfare, and the impact on food quantity, quality, costs, among others. 21,31

______Balbino M. Rocha, 2013 4 OBJECTIVES Objecti ves

I. General Objective

The aim of the present dissertation was to determine whether antibacterial resistance patterns of major mastitis pathogens isolated from milk samples from dairy cattle of northwestern, central and southern Portugal have changed over time.

II. Specific Objectives

In order to accomplish the referred general objective, the specific objectives were, firstly, to establish antibacterial resistance patterns of major bovine mastitis pathogens isolated from submitted milk samples from dairy herds in northwestern, central and southern Portugal in a nine-year period and, in consequence, determine if those patterns have changed over the referred period, presumably in response to antibacterial use among the herds in those regions.

______Balbino M. Rocha, 2013 6 LITERATURE REVIEW CHAPTER I:

The Big Picture on Antibacterial Resistance Cha pte r I: The Big Picture on Antibacterial Resistance

I. Antibacterial Resistance

1. Why is Antibacterial Resistance a Public Health Concern?

There are several reasons why bacterial resistance should be of great concern for the medical community. First, resistant bacteria, particularly staphylococci, enterococci, Klebsiella pneumoniae and Pseudomonas spp. are becoming increasing threats in health- care institutions. 36-42 In addition, many of those microorganisms have been identified with the potential to employ simultaneous multiple resistance mechanisms towards different antibacterials, rendering combination treatments ineffective. 22

Bacterial resistance makes it difficult and more expensive to treat a variety of common infections, causing delays in effective treatment, or in worst cases, failure to provide appropriate therapy, which can have serious consequences, especially in critically ill patients. Estimates from Europe indicate that the excess mortality due to multidrug-resistant bacterial hospital infections exceeds 25 000 each year (Table 1) and the death rate is presumed to be higher in other parts of the world. Apart from additional patient morbidity/mortality, the attributable health-care costs and productivity losses are estimated to be at least €1.5 billion annually. This reality places an added burden on health-care management, especially with the cost-containment pressures of today’s health-care environment. 43

Table 1: Estimated annual burden due to selected antibacterial-resistant bacteria in EU-Member States, Iceland and Norway, 2007 (Adapted from ECDC/EMA, 2009). 43

No. Cases of No. Extra No. Extra Antibacterial-Resistant Bacteria

Infection* Deaths Hospital Days

Methicillin-resistant S. aureus (MRSA) 171.200 (12%) 5.400 (37%) 1.050.000 (16%)

Vancomycin-resistant E. faecium (VRE) 18.100 (9%) 1.500 (28%) 111.000 (22%) Gram+ rd

3 generation cephalosporin-resistant E. coli 32.500 (27%) 5.100 (52%) 358.000 (27%) –

3rd generation cephalosporin-resistant K. pneumoniae 18.900 (27%) 2.900 (52%) 208.000 (27%) Gram Carbapenem-resistant P. aeruginosa 141.900 (3%) 10.200 (7%) 809.000 (3%)

* Bloodstream infections, lower respiratory tract infections, skin and soft tissue infections, and urinary tract infections. Numbers in parentheses indicate percentage of bloodstream infections.

Resistant bacteria may also spread and become broader infection-control problems, not only within health-care institutions, but in communities too. Clinically important bacteria, such as Methicillin-resistant S. aureus (MRSA) 39,44 and extended-spectrum β-lactamase (ESBL)–producing E. coli 45,46 are increasingly isolated in the community. Infected individuals, including children, often lack identifiable risk factors for MRSA, and appear to have acquired their infections in a variety of community settings. 47,48 Laboratory reports show increasing resistance among pneumonia causing bacteria, which kills about 1.8 million children annually. 49 Community-associated MRSA strains are typically less resistant to antibacterial agents than health-care-associated MRSA, but are more likely to produce toxins, such as

______Balbino M. Rocha, 2013 9 Cha pte r I: The Big Picture on Antibacterial Resistance

Panton-Valentine leukocidin. 47 The spread of resistant bacteria within the community poses obvious additional problems for infection control, not just in long-term care facilities but also among sport teams, military recruits, and even children attending day care centers – a task that is complicated by the increased population mobility. 14

Finally, a recent development and cause for concern is an apparent shift in the burden of ABR which may be occurring between the main classes of pathogenic bacteria. This shift, from Gram-positive to Gram-negative pathogens, could further stretch the already limited resources of health-care services as the infections due to resistant Gram-negative organisms will likely outweigh recent achievements in the control of Gram-positive pathogens. 50 The evolution of ABR (Table 2), coupled with a shortage of new antibacterials in the pipeline, raises the possibility that untreatable multidrug-resistant infections will become more and more common. It is particularly worrisome that once it develops, ABR is either irreversible or very slow to reverse, despite the introduction of ABR containment and stewardship programs. 4

Table 2: Evolution of resistance to major antibacterials (Adapted from Palumbi, 2001). 51

Antibacterial Year Year of Observed Agent Deployed Resistance

Sulfonamides 1930s 1940s Penicillin 1943 1946 Streptomycin 1943 1959 Chloramphenicol 1947 1959 Tetracycline 1948 1953 Erythromycin 1952 1988 Vancomycin 1956 1988 Methicillin 1960 1961 Ampicillin 1961 1973 Cephalosporins 1960s Late 1960s

2. Origins of Antibacterial Resistance

Antibacterial resistance, via specific genes and mechanisms, represents an outcome of bacterial evolution that characterizes to perfection Darwin’s biological principle of ‘survival of the fittest’. This phenomenon has been, thus, occurring in nature for thousands of years, long before the introduction of commercial antibacterials. 52 In fact, Dancer et al. (1999) have isolated resistant bacteria estimated to be over 2 000 years old from deep within glaciers in Canada’s high Arctic regions. 53 Another study detected TEM-type β-lactamases from a metagenomic library in cold-seep sediments of Edison Seamount (south of Lihir Island, Papua New Guinea), estimated to be approximately 10 000 years old. 54 Furthermore, resistance to sulfadiazine, spectinomycin and tetracycline has been identified among E. coli isolates collected before the introduction of large-scale commercial antibacterials. 52 Barlow & ______Balbino M. Rocha, 2013 10 Cha pte r I: The Big Picture on Antibacterial Resistance

Hall (2002) also showed that ampC ß-lactamase genes recovered from Citrobacter freundii strains collected prior to the clinical use of antibacterials, in the 1920s, were shown to be as effective at providing ß-lactam-resistance in E. coli as were the plasmid-borne alleles from ß- lactam-resistant clinical isolates. 55

ABR is likely to have had its origin when originally susceptible organisms managed to acquire protection from the competitive attack of antibiotic-producing organisms. In addition, these organisms had to obtain protection from their own toxic products as well. 10,52,53,56-58 This presumption has been substantiated by the finding of aminoglycoside-modifying enzymes in aminoglycoside-producing organisms that display marked homology to modifying enzymes found in aminoglycoside-resistant bacteria. 59 Also, the essential genetic determinants associated with resistance to vancomycin – vanA, vanH, and vanX – appear to be very similar to the self protection mechanism employed in the vancomycin producing strains of Actinomyces spp. 60 Moreover, researchers have shown that several antibacterial preparations employed for human and animal use were found to be contaminated with chromosomal DNA of antibiotic-producing organisms, including identifiable ABR gene sequences. 61,62 It was further postulated that this presence of DNA encoding ABR in antibacterial preparations has been a factor in the rapid development of multiple resistance 22,26 by providing the resistance sequences that can then be taken up by the causative pathogen. With this said, it is even so imperative to recognize that the bacterial evolutionary response has not been limited to the acquisition of resistance genes. Bacteria have also developed means for stabilizing the resistance phenotype, thus dashing initial hopes of reversing resistance by merely reducing antibacterial usage. 23

Resistance to natural antibacterial agents (antibiotics) and synthetic derivatives has been observed in a collection of soil-dwelling actinomycetes, with some displaying resistance mechanisms not usually observed in clinical bacterial pathogens. Unexpectedly, researchers found that every isolate tested displayed resistance to at least six different antibacterial agents and in other cases, as many as twenty. The use of novel resistance mechanisms by these organisms, coupled with the fact that these soil microorganisms are not as intensively exposed to antibacterial selective pressures as are the clinical pathogens, emphasizes the fact that resistance is not a new phenomenon. Additionally, the identification of this new reservoir of resistance genes highlights the possibility of future horizontal transfer of novel ABR determinants to bacteria of human and veterinary significance. 58

3. Types of Antibacterial Resistance

Bacteria can display one of three fundamental ABR phenotypes: susceptibility, intrinsic resistance, or acquired resistance. 25 Intrinsic resistance can be described as a natural ______Balbino M. Rocha, 2013 11 Cha pte r I: The Big Picture on Antibacterial Resistance

phenomenon that is displayed by all strains of a species and is a function of the physiological or biochemical makeup of that species. Of greater concern are cases of acquired resistance, where initially susceptible populations of bacteria become resistant to an antibacterial and proliferate in exposed host-animal populations under the selective pressure of usage of that agent. 10 This type of resistance may be consequent to the mutation of regulatory or structural genes (vertical evolution), to the acquisition of foreign resistance genes (horizontal evolution), or to a combination of these two mechanisms and is present not in the entire 14, 26, 59, 63 species but only within a certain lineage of bacteria derived from a susceptible parent.

ABR defined in this way is a microbiological phenomenon, which may have clinical implications, depending on pharmacokinetic and pharmacodynamic parameters as they apply to specific antibacterial agents. Nevertheless, even low-level resistance is worth mentioning since it may be a first step towards clinical resistance. 10 These considerations have always been important in definitions of rational antibacterial therapy, 64 and have been reemphasized by recent calls for prudent therapy in human and veterinary medicine. More on this matter will be described in detail in upcoming sections.

4. Antibacterial Modes of Action and Resistance Mechanisms

Antibacterial agents function as selective toxins that inhibit enzymes that are either unique to the prokaryotic cell or sufficiently different such that toxicity to the mammalian host is minimal. 25 Most antibacterials may be classified according to their main mechanisms of action (Table 3): 1) inhibition of cell wall synthesis, 2) inhibition of protein synthesis, 3) interference with nucleic acid synthesis, and 4) inhibition of a metabolic pathway. Although less well characterized, some authors consider the disruption of bacterial phospholipid membrane structure to be a fifth major mechanism of action. 14, 65-67

Table 3: Mechanisms of action of main antibacterial agents (Adapted from Tenover, 2006). 68

Mechanisms of Action of Main Antibacterial Agents

I. Inhibition of cell wall synthesis  β-lactams: penicillins, cephalosporins, carbapenems, monobactams  Glycopeptides: vancomycin, teicoplanin II. Inhibition of protein synthesis  Binding to 50S ribosomal subunit: macrolides, chloramphenicol, clindamycin, linezolid, lincomycin, quinupristin-dalfopristin  Binding to 30S ribosomal subunit: aminoglycosides, tetracyclines  Binding to bacterial isoleucyl-tRNA synthetase: mupirocin III. Interference with nucleic acid metabolism  Inhibition of DNA synthesis: quinolones (fluoroquinolones)  Inhibition of RNA synthesis: rifamycins (rifampicin) IV. Inhibition of metabolic pathways: sulfonamides (trimethoprim/ormetoprim), folic acid analogues V. Disruption of bacterial phospholipid membrane structure: polymyxins, daptomycin

______Balbino M. Rocha, 2013 12 Cha pte r I: The Big Picture on Antibacterial Resistance

In spite of these mechanisms of action and even with a great variety of currently available antibacterial agents for human and veterinary clinical use, ABR mechanisms are known to exist for them all. 25,26 The most common mechanisms by which resistance occurs are listed and illustrated in Table 4 and Figure 1, respectively. Resistance generally develops through one or more of these mechanisms. 22 In the first example of resistance (Table 4), the antibacterial is either degraded or modified by enzymatic activity before it can reach the target site and damage the cell. A very large number of drug-modifying enzymes have been identified for numerous antibacterial agents. For example, there are now more than ninety TEM-type β-lactamases and more than 25 SHV-type enzymes identified that are capable of hydrolyzing penicillins and cephalosporins. 69 In the second resistance mechanism, changes in the target molecule occur as a result of spontaneous mutation, resulting in decreased affinity of the target for the antibacterial. Both decreased uptake and increased efflux mechanisms, due to their non-specific nature, are normally responsible for the creation of multiple ABR phenotypes. 70 Lastly, organisms may acquire antibacterial-insensitive enzymes that circumvent a specific metabolic pathway that antibacterial sensitive enzymes drive, allowing the microorganism to survive and continue growth in the presence of the antibacterial. 25

Figure 1: Broad depiction of major ABR mechanisms (Adapted from www.scq.ubc.ca).

Table 4: Mechanisms of ABR (Adapted from McDermott, 2003). 25

Mechanisms of Antibacterial Resistance

I. Modification of the antibacterial agent  Aminoglycosides, chloramphenicol, β-lactams, streptogramins II. Mutation at target site  Aminoglycosides, β-lactams, macrolides, quinolones, rifampicin, trimethoprim, tetracyclines, mupirocin III. Decreased antibacterial accumulation  Decreased uptake: Numerous antibacterials  Increased efflux: Tetracyclines, macrolides, chloramphenicol, quinolones IV. Bypass of antibacterial-sensitive step through acquisition of drug-insensitive enzymes: sulfonamides, trimethoprim ______Balbino M. Rocha, 2013 13 Cha pte r I: The Big Picture on Antibacterial Resistance

The following subsections will provide an overview of the major mechanisms of action of the most commonly used antibacterial agents, emphasizing key examples of resistance mechanisms to these compounds.

4.1. Inhibition of Cell Wall Synthesis

Many antibacterial agents function by targeting bacterial cell wall synthesis (Table 3). Cell walls are not found in mammalian cells, and differ between various bacterial species. As a result, cell wall synthesis provides a number of potential therapeutic targets in anti-infective drug development. 25, 71

Peptidoglycan or murein is an exclusive mucopolysaccharide component of the bacterial (except in Archaea) cell wall. The quantity of this polymer and its position within the cell envelope is different between Gram-positive and Gram-negative bacteria. The polymer consists of repeating disaccharide subunits of N-acetylglucosamine (NAG) and N- acetylmuramic acid (NAM). The NAM subunit has a short peptide chain attached, which mediates cross-linking of parallel glycan molecules in mature peptidoglycan. This peptide consists of L- and D-amino acids, which typically end in D-alanyl-D-alanine (D-Ala-D-Ala). Cross-links between adjacent peptide side chains convey mechanical strength to the molecule, and also offer opportunities for biochemical diversity in the types of cross-links within and between different bacterial species. 25, 71

Peptidoglycan biosynthesis has four major stages: 1) synthesis of precursors in the cytoplasm; 2) transport of lipid-bound precursors across the cytoplasmic membrane; 3) insertion of glycan units into the cell wall; and 4) transpeptidation linking and maturation. 71 D-Cycloserine and bacitracin inhibit the first two steps, respectively. The most commonly used inhibitors of cell wall biosynthesis, β-lactams (penicillins, cephalosporins, carbapenems, and monobactams) and glycopeptides, act at stages 3 and 4. These compounds act by forming covalent complexes with enzymes that generate the mature peptidoglycan molecule. Because the functions of these enzymes were studied in the context of penicillin binding and resistance, they are acknowledged collectively as penicillin-binding proteins or PBPs. In Gram-negative bacteria, β-lactams must pass through cell wall protein channels called porins, to reach the target PBPs. The effects of drug binding on cell growth differ, depending on the agent and the PBP involved. Some inhibit cell division, leading to long filamentous forms, whereas others lead to the formation of cell wall deficient types that readily lyse under osmotic pressure. 63

Resistance to β-lactams arises through one or more of the following mechanisms: 1) mutations in the target PBP or acquisition of new PBPs with decreased affinity for the drug; 2) production of one or more β-lactamases that inactivate the drug; 3) changes in cell wall ______Balbino M. Rocha, 2013 14 Cha pte r I: The Big Picture on Antibacterial Resistance

porins that limit movement of drug to the target site; and 4) active efflux of the drug out of the cell through energy-dependent pumps. 69 The latter two mechanisms frequently lead to multidrug ABR because different classes of agents may pass through the same porin or be extruded from the cell by the same low-specificity efflux pump. In other cases, drug-specific porins or pumps can mediate single resistance phenotypes. The most effective and frequent mechanism of resistance among Gram-negative bacteria is via production of β-lactamases, which inactivate the antibacterial by hydrolysis of the β-lactam ring. 72 Over 300 β-lactamase isoenzymes associated with a diversity of β-lactam resistance phenotypes have been identified. 73,74 Over the last decades many new β-lactams have been developed, that were specifically designed to be impervious to the hydrolytic action of β-lactamases. However, with each new class introduced into clinical use, new β-lactamases have emerged resulting in resistance to both newer and older β-lactams. 69

Glycopeptides comprise the second major group of antibacterial agents that inhibit cell wall synthesis, with the foremost examples being vancomycin and teicoplanin (Table 3). These are large complex heterocyclic molecules consisting of a heptapeptide backbone to which are attached various substituted sugars. Because these molecules are generally too large to pass across the Gram-negative outer membrane, their activity is generally limited to Gram-positive organisms, with enterococci and staphylococci conversely corresponding to the biggest resistance setbacks to these compounds. Glycopeptides function by binding to D-Ala-D-Ala dipeptide terminus of the peptidoglycan pentapeptide side chains. This binding blocks the transglycosylation and transpeptidation reactions necessary to add new subunits to the growing peptidoglycan chain. This contrasts to the β-lactam mode of action, which bind to anabolic enzymes rather than structures in the cell wall. 63

Resistance to glycopeptides arises in cells that synthesize a dipeptide terminus consisting of D-Ala-D-Lac in place of D-Ala-D-Ala. In enterococci with high-level glycopeptide resistance, two new enzymes are acquired, a ligase and a dehydrogenase. The resulting modified subunit weakly binds to vancomycin only, allowing the cell to continue growth in the presence of the antibacterial. Six vancomycin resistance types in enterococci have been described: VanA, VanB, VanC, VanD, VanE and VanG. 75-82 VanA type resistance, characterized by high-level inducible vancomycin and teicoplanin resistance, arises due to the acquisition of the VanA transposon Tn1546 or Tn1546-like elements, and has been the most commonly described in both human and animal enterococcal isolates. 78 The scientific community has expressed, over the years, some concern towards the dissemination of the van gene cluster from Enterococcus spp. to Staphylococcus spp., especially when vancomycin is a last resort drug in the therapy of multiresistant infections. 83,84 In fact, the first reported case of a glycopeptide-resistant Staphylococcus strain isolate containing the vanA

______Balbino M. Rocha, 2013 15 Cha pte r I: The Big Picture on Antibacterial Resistance

gene took place in 2002. 85 The long term Public Health threat posed by this development, although troubling, remains to be seen though.

4.2. Inhibition of Protein Synthesis

Protein synthesis occurs in three general steps (Figure 2). First is the synthesis of aminoacyl-tRNAs, where specific aminoacyl-tRNA synthetase enzymes attach tRNA molecules to the corresponding amino acids, which are then transported to the ribosome. At the ribosome, RNA polymerase transcribes a mRNA from the coding region of a gene. During initiation of protein synthesis, the 30S ribosomal subunit binds to the start codon of the mRNA. Formylmethionyl (f-Met) tRNA binds to the 30S-mRNA complex at the peptidyl site (P site, location of growing peptide chain), and the 50S ribosomal subunit binds to complete the 70S initiation complex. A tRNA corresponding to the next triplicate codon then binds to the aminoacyl site (A site), and a transpeptidation reaction links the amino acids into the growing peptide chain. The growing chain moves to the P site, and the next tRNA enters the A site. The nascent protein builds in this way until a translational stop codon enters the A site, translation is terminated, and the peptide is released. The prokaryotic ribosome is sufficiently different from that of eukaryotic cells that a large number of antibacterials function by interfering with various stages of bacterial protein synthesis (Table 3; Figure 3). 63,86

Although aminoglycoside activity is incompletely understood, antibacterial activity appears to take place from multiple changes at the ribosome. One mechanism interferes with proofreading, resulting in the misreading of certain mRNAs. Additionally, some aminoglycosides appear to block formation of a functional initiation complex, while others inhibit the translocation step in polypeptide synthesis. Bacterial resistance to aminoglycosides is most commonly associated with the expression of modifying enzymes that can phosphorylate, adenylate or acetylate these agents. 87

Chloramphenicol binds to the 70S ribosome and inhibits the peptidyl transferase reaction, which forms the peptide bond between amino acids. Chloramphenicol resistance among bacteria is frequently mediated by chloramphenicol acetyltransferases (CATs), and numerous cat genes have been identified among many different bacterial genera. 88,89 Resistance is usually due to inactivation of the drug by acetylation of the two hydroxyl groups. Additionally, chloramphenicol resistance efflux mechanisms have also been described among a variety of bacteria. 90,91

Macrolides, like erythromycin for instance, are antibacterial agents that prevent protein synthesis by binding to the tRNA binding site on the 50S subunit and causing the tRNA molecules to dissociate from the ribosomes. Resistance in Gram-positive organisms is often due to mutation or modification, through methylation, of the 23S ribosomal RNA subunit ______Balbino M. Rocha, 2013 16 Cha pte r I: The Big Picture on Antibacterial Resistance

along with removal by efflux pumps. 102 Gram-negative bacteria are intrinsically resistant to the activity of macrolides because these agents are slow to traverse the cell wall and are also removed by constitutively expressed efflux pumps. 101

Figure 2: Protein synthesis. Aminoacyl-tRNA molecules are formed in the cytoplasm and bind to the cognate triplicate codon of mRNA at the ribosome. Peptide bond formation links the new amino acid to the growing polypeptide chain. The ribosome migrates to free the A site for the next aminoacyl-tRNA molecule and the cycle repeats until a stop codon is encountered and translation is terminated (Adapted from McDermott, 2003). 25

Tetracyclines, such as doxycycline and minocycline, consist of broad-spectrum, bacteriostatic agents, that function at the 30S subunit by blocking aminoacyl tRNA binding at the A site. Their activity is against a wide range of microorganisms including Gram-positive and Gram-negative bacteria, chlamydiae, mycoplasmas, rickettsiae and protozoan parasites. These compounds are used extensively in the prophylaxis and treatment of human and animal infections and also at subtherapeutic levels in animal feed as growth promoters. They are toxic to both bacterial and mammalian ribosomes, although can attain much higher concentrations within bacterial cells. Resistance results from active efflux and from modification of the ribosome that protects it from tetracycline binding. Unfortunately, there has been widespread emergence of efflux- and ribosome-based resistance to both older and newer tetracyclines. 92 This pressed towards the development of the antibacterial class of glycylcyclines which have been shown to be active against tetracycline-resistant strains possessing known efflux and ribosomal protection resistance determinants. 93

______Balbino M. Rocha, 2013 17 Cha pte r I: The Big Picture on Antibacterial Resistance

Other inhibitors of bacterial protein synthesis include the streptogramins (i.e., quinupristin-dalphopristin, virginiamycin and pristinamycin), the oxazolidinones (e.g., linezolid) and mupirocin. High-level streptogramin resistance is due to inactivation by acetylation, although ribosomal mutations can confer low-level resistance. 94 Oxazolidinones represent a relatively new class of antibacterial agents that are chemically unrelated to currently available compounds, and are used for treating Gram-positive infections. As of 2009, linezolid is the only oxazolidinone in this class approved for clinical use in humans, although others are in development. Linezolid selectively binds to the bacterial 50S ribosomal subunit and inhibits initiation of protein synthesis. 95 Resistance is poorly understood, but is due in part to mutations in the 23S rRNA subunit. 96

Figure 3: Activity of protein synthesis inhibitors. Schematic of the bacterial ribosome and the sites of action of select antibacterials that inhibit polypeptide biosynthesis (Adapted from McDermott, 2003). 25

4.3. Inhibition of Metabolic Pathways and Interference with Nucleic Acid Metabolism

In general, antibacterials that disrupt DNA and RNA synthesis do so by interfering with either nucleotide (e.g., sulfonamides) or nucleic acid (e.g., quinolones and rifamycins) biosynthetic processes in the cell (Table 3). 25

Sulfonamides are structural analogues of p-aminobenzoic acid (PABA). PABA is a substrate in the synthesis of tetrahydrofolic acid, a donor of one-carbon units in the synthesis of purine and pyrimidine nucleotides. Sulfonamides competitively compete for the dihydropteroate synthetase enzyme active site, blocking the formation of nucleotide precursors. Resistance in both Gram-negative and Gram-positive bacteria is usually due to the acquisition of a new enzyme that is unaffected by sulfonamides. Potentiated

______Balbino M. Rocha, 2013 18 Cha pte r I: The Big Picture on Antibacterial Resistance

sulfonamides consist of a combination with either trimethoprim or ormetoprim. These compounds block a subsequent sequential enzymatic step in folic acid metabolism (dihydrofolate reductase), and so these agents are often given in combination with sulfonamides (Table 3). 97,98 As with sulfonamides, resistant strains produce a new trimethoprim insensitive enzyme, allowing the organisms to circumvent the susceptible enzyme and grow in the presence of the drug. Genes encoding resistance to sulfonamides and trimethoprim are widespread among both commensal and pathogenic Gram-negative bacteria and are often located on extrachromosomal DNA mobile elements. 99,100

Compounds that inhibit enzyme function in nucleic acid synthesis typically act on RNA polymerase (rpoB) or DNA topoisomerases (gyrA, gyrB, parC, parE). The rifamycins, which include the antituberculosis drug rifampin, bind to the bacterial RNA polymerase, selectively inhibiting the initiation of bacterial transcription. Resistance in Mycobacterium tuberculosis arises from mutations in the structural gene (rpoB) for RNA polymerase. 101 Enzymatic modification of rifampin (e.g., ribosylation) and efflux have been described in other organisms. 102

DNA topoisomerases are essential enzymes that catalyze supercoiling of DNA, a vital process in cellular metabolism. 103,104 Members of the quinolone class bind to the active site of these enzymes and inhibit their activity (Table 3). Fluoroquinolones are potent antibacterial agents that target two related enzymes: DNA topoisomerase II (DNA gyrase) and DNA topoisomerase IV. Resistance to fluoroquinolones is mediated by three mechanisms: 1) target mutations in the topoisomerase genes; 2) decreased permeability of the bacterial cell wall; and 3) energy-dependent efflux pumps. High-level fluoroquinolone resistance is attributed primarily to mutations in the gyrA and parC genes, which reduce quinolone binding to the gyrase-DNA complex. This absence of binding allows DNA replication to continue in the presence of fluoroquinolone concentrations that are inhibitory to wild-type bacterial cell growth. 103,104 Other quinolone-resistance mutations have been identified, to be exact in parC and parE, the genes encoding topoisomerase IV. In Gram-negative organisms, the primary target of fluoroquinolones is the active site of DNA topoisomerase II, with secondary mutations in topoisomerase IV contributing to higher levels of resistance. Mutations causing resistance have been localized to a subdomain, termed the quinolone resistance-determining region (QRDR), within the gyrA and parC genes. In the Escherichia coli QRDR, most mutations associated with quinolone resistance occur at serine 83 and aspartate 87 of gyrA, and at serine 79 and aspartate 83 of parC. DNA sequence analysis of fluoroquinolone resistance genes from Staphylococcus aureus and Streptococcus spp. implies that the situation is reversed in Gram-positive bacteria, where topoisomerase IV is the primary fluoroquinolone target. 104

______Balbino M. Rocha, 2013 19 Cha pte r I: The Big Picture on Antibacterial Resistance

Outer membrane proteins (OMPs) provide channels of entry for molecules, including antibacterials, to the cell membrane and internally into Gram-negative bacteria based on charge, shape and size. Although some fluoroquinolones may diffuse directly across the lipid bilayer, most fluoroquinolones cross the Gram-negative outer membrane through these channels. 103 Common OMPs in E. coli are OmpF, OmpC and OmpE, and the loss of function of one of these porins due to mutation can result in decreased susceptibility to a wide variety of structurally unrelated antibacterials. 74,105,106 For example, in E. coli, the over-expression of the transcriptional activator marA results in the decreased expression of the OmpF porin protein, in addition to increased expression of the AcrAB multidrug efflux pump. 107,108 This specific mutation showed about a two-fold increase in this microorganism’s resistance to quinolones. 109 Resistance due to decreased drug influx is generally low-level resistance. Efflux is a mechanism of fluoroquinolone resistance in Gram-negative and Gram-positive bacteria. This is an energy-dependent mechanism that can confer resistance against a particular antibacterial agent, class, or a number of antibacterials resulting in multidrug resistance. 106,110,111 It is generally thought that full clinical resistance requires mutations in gyrA even in the presence of efflux mechanisms. Recent reports on E. coli, however, demonstrated that deletion of the gene encoding the AcrAB efflux pump reduced ciprofloxacin MICs to near wild-type levels in cells carrying topoisomerase mutations. 112

5. Genetics of Antibacterial Resistance and Transfer

Despite the abundance of resistance phenotypes observed among bacteria, there are only a limited number of mechanisms by which these resistance traits are acquired. The genes encoding ABR determinants may be located on the chromosome, where they are inherited by daughter cells, or they might be horizontally transmitted on mobile DNA elements such as plasmids and transposons. 113

Plasmids are extrachromosomal segments of DNA that replicate independently of the chromosome and can be exchanged among various bacteria. 114 In general, plasmids are not essential for survival, but typically carry genes that impart some selective advantage to the host bacterium, such as virulence, adhesion and ABR determinants. 26 Plasmids are very diverse in size, copy number (the number contained within a bacterial cell), and the number of accessory genes that they contribute to the host cell. 25 Plasmids that carry resistance genes are called R-plasmids or R-factors. Since their discovery in the 1950s, 115 R-plasmids have been increasingly associated with both Gram-positive and Gram-negative bacterial pathogens and commensal organisms. Plasmid-associated resistance genes have been characterized for the majority of clinically available antibacterials, 116-118 and it is not unusual for a single plasmid to simultaneously mediate resistance to multiple antibacterials and be

______Balbino M. Rocha, 2013 20 Cha pte r I: The Big Picture on Antibacterial Resistance

shared among different bacterial genera. 119 For example, interserovar exchange of plasmids between Salmonella serovar Muenchen and S. typhimurium has been shown in animals where both serovar strains shared similar plasmid profiles and carried similar ABR genes on their plasmids. 120 Also, the spread of multiple ESBLs has been partly attributed to their presence on multidrug resistance plasmids. 74,121

Until recently, quinolone resistance was believed to arise solely from chromosomal mutations in genes encoding target enzymes or via active efflux. However, in 1998, a novel mechanism of plasmid-mediated quinolone resistance, termed qnr, was discovered in a Klebsiella pneumoniae isolate. 122 Qnr was found in an integron-like structure near Orf513 on the multidrug resistance plasmid, pMG252. 123 Qnr, the gene product, is a member of the pentapeptide repeat family of proteins and has been shown to block the action of ciprofloxacin on purified DNA topoisomerases II and IV. 118 Subsequently, qnr plasmids have been reported from clinical isolates of E. coli, Citrobacter freundii, Enterobacter spp., K. pneumoniae, Providencia stuartii, and Salmonella spp. from across the globe. 117 The different qnr genes reported to date include qnrA, qnrB, and qnrS. 118,124 The plasmid- encoded qnr proteins derived from E. coli, K. oxytoca and K. pneumoniae isolates recovered from different geographic sources (China, Europe and USA) show almost identical residues, indicating these proteins most likely have similar origins. 116

The potential arrangement of accessory resistance genes on a plasmid is nearly unlimited and may arrive as constituents of other mobile elements such as transposon and integrons. This phenomenon has been compared to the arrangement of Russian matrioshka dolls, where each element is contained within a larger and more complex unit (Figure 4). Transposons and integrons have been shown to possess a number of genetic determinants encoding resistance to certain biocides such as quaternary ammonium compounds (QACs) and heavy metals, in addition to antibacterials. 125 Transposons are gene sequences that can move from one location to another within the bacterial cell’s DNA (chromosomal and plasmid). The most basic form of a transposon is an insertion sequence (IS) containing only those genes required for transposition. An advancement on the IS model is seen in composite transposons. Composite transposons consist of a central region containing genes (passenger sequences) other than those required for transposition (e.g., ABR) flanked on both sides by IS that are identical or very similar in sequence, usually in inverted orientation.38 A large number of resistance determinants in many different bacterial species are transmitted via composite transposons. 25

As to integrons, these are DNA elements with a specific structure consisting of two conserved segments flanking a central region in which ABR “gene cassettes” can be inserted. Gene cassettes exist as free circular DNA structures with 500 to 1000 base pairs (bp) that are not expressed on their own due to the lack of the promoter region. A 59 bp ______Balbino M. Rocha, 2013 21 Cha pte r I: The Big Picture on Antibacterial Resistance

element is located downstream of the promoter-less resistance gene and serves as the recombination site. Insertion of the cassette into the integron structure via the recombination process at the attI recombination site downstream from a promoter helps in the expression of the gene encoded by the cassette. Multiple gene cassettes can be arranged in tandem, and more than 60 distinct cassettes have been identified. 126 Cassette-associated genes have been shown to confer resistance to β-lactams, aminoglycosides, trimethoprim, chloramphenicol, streptothricin and QACs. 99, 126-128

Figure 4: Schematic of multiple antibacterial resistance accumulation on a plasmid. (A) Class 1 integron with conserve termini containing the integrase gene (int), gene cassette attachment site (attI), a truncated gene for resistance to quaternary ammonium compounds (∆qacE) and a resistance gene for sulfonamides (suII). Block arrows represent resistance gene cassettes. (B) Basic structure of a composite transposon, with terminal insertion sequences flanking accessory genes. (C) Composite transposon bearing a multiple ABR integron, which may locate to a plasmid backbone (D) carrying other resistance determinants and mobile DNA elements (Adapted from McDermott, 2003). 25

There are four major integron classes with class I being the most prevalent among clinical enteric isolates. Class I integrons and integrases have been identified in a number of different bacterial genera and appear to be widespread in nature. 129 As the microorganisms with integrons pass from one environment to another, so too does the possibility of transmitting its resident resistance genes to new bacterial hosts. Integrons demonstrate an

______Balbino M. Rocha, 2013 22 Cha pte r I: The Big Picture on Antibacterial Resistance

especially interesting situation, since the resistance determinants are often under the control of a single promoter and are therefore co-expressed. As a result, selective pressure exerted by an antibacterial will maintain the co-resistance phenotypes mediated by the adjacent genes. This raises the possibility that exposing integron-carrying bacteria to sub- inhibitory or residual concentrations of disinfectants, such as QACs, may enhance ABR strains, even in the absence of chemotherapeutic antibacterials. 125-128

Integrons are usually identified in conjunction with streptomycin and TMP-SMX- resistant bacteria isolated from food-producing animals and human infections. 130,131 The inclination to exchange genes increases concern about the possible dissemination of ABR determinants from commensal or nonclinical organisms in animals to human pathogens. 3 An example of rapid dissemination of integron-associated ABR genes is Salmonella Typhimurium phage type DT104, commonly known as R-type ACSSuT. This phage type has had a significant Public Health impact and is a global concern. It was first documented in cattle from UK in 1984 and was soon reported in animals and humans over the world. 132-135 The genetic determinants for this R-type are contained in a 43 kb island (Salmonella

Genomic Island [SGI]), comprised of integrons containing respectively the ASu (blaCARB-2 and sul1) and SSp (aadA2) genes with intervening plasmid-derived genes coding for resistance to chloramphenicol/florfenicol (flo) and tetracyclines (tetG). 136 All isolates of multidrug- resistant DT104 with the ACSSuT phenotype have contained the same gene cassettes regardless of the source or country of origin. Of note in recent years has been the identification of SGI1 in several different Salmonella serovars, including serovars Agona, Albany and Paratyphi B variant Java. 137 Furthermore, it has been shown experimentally that the multidrug-resistant DT104 cluster can be efficiently transduced by P22-like phages. 138 Upstream of the first integron in the multidrug resistance locus is a gene encoding a putative resolvase enzyme, which demonstrates greater than 50% identity with the Tn3 resolvase family. These findings support the potential for horizontal spread of the multidrug-resistant gene cluster among Salmonella and other bacteria. 139

More recently, a new element called orf513 has been increasingly identified in association with multiple ABR genes and with class I integrons, spawning the idea of ‘complex class I integrons’. This complex integron, termed insertion sequence common regions (ISCR1), can mediate resistance to chloramphenicol, trimethoprim, aminoglycosides, tetracyclines and an assortment of β-lactams. 140 The orf513 region is thought to be similar to a common region element, which is a group of potentially mobile DNA elements found in the Salmonella pathogenicity islands and on the TMP-SMX conjugative element in Vibrio cholerae. 141 Some studies suggest that common region elements replicate by rolling circle replication and may be a subset of a family of unusual IS elements, IS91. Replication using the rolling circle mechanism allows for genetic rearrangements that may not be possible by ______Balbino M. Rocha, 2013 23 Cha pte r I: The Big Picture on Antibacterial Resistance

traditional rearrangement mechanisms, and therefore may present a new evolutionary advancement in the class I integron, along with new clinical concerns. 140

Bacteria are very skilled in acquiring the necessary genetic information to survive in environments where antibacterials are present. The movement of mobile DNA elements between bacteria is a consequence of natural horizontal genetic exchange, also known as horizontal gene transfer (HGT). Three general mechanisms of HGT are currently recognized. Plasmids are transferred to new strains in the process of conjugation, which is mediated by surface pili and requires intimate contact between donor and recipient cells. 59,125,142 Some specialized transposable elements, termed conjugative transposons, can also be spread in this way. The second mechanism of exchange is transduction, which occurs by means of transducing bacteriophages. As part of the replicative cycle, their DNA is inserted into the host genome. When exiting, they may pick up pieces of host DNA and transfer them to subsequent hosts. By chance, some of these fragments of host DNA will contain resistance determinants. Lastly, DNA can be acquired by a cell that takes up free DNA from the immediate environment in a process termed transformation. 114

In resistant clinical isolates, the great predominance of resistances appears to be plasmid mediated. The importance of transduction in generating multidrug-resistant strains is thought to be a relatively rare event. 14 It is difficult to assess the contribution of transformation in ABR evolution, and it may be negligible, but its demonstration in the laboratory indicates a theoretical role. 143 Regardless, these mechanisms taken together provide an adequate thoroughfare for mobile DNA elements to spread readily among distantly related bacterial genera and produce the rapid emergence and dissemination of multiple ABR witnessed in many bacterial pathogens. 25

6. Pharmacodynamics and Pharmacokinetics of Antibacterials

Since the introduction of antibacterials, human and veterinary clinicians have been challenged with the issue of how to achieve optimal outcomes in patients with bacterial infections. While the discovery of these life-saving agents revolutionized modern medical treatment, balancing their use with the loss of activity resulting from bacterial resistance has become a challenge. For as long as these agents have been used clinically, there has been ongoing research into the best way to use them. As early as the 1940s, the effect of dose and dosing interval on bacteriostatic and bactericidal activities has been investigated, 144 and strategies to optimize antibacterial selection and dosing remain at the forefront of present clinical research. The appropriate use of antibacterial agents requires an understanding of the characteristics of the drug, the host factors and the pathogen, all of which, through multiple complex interactions (Figure 5), will influence the selection of the antibacterial agent ______Balbino M. Rocha, 2013 24 Cha pte r I: The Big Picture on Antibacterial Resistance

and dose. Characteristics of the patient that must be considered include those that affect the interaction between the patient and the infection, such as comorbidity factors and underlying immune status, as well as patient-specific factors such as organ function and weight, which will impact the pharmacokinetics of the antibacterial agent. Characteristics of the bacteria include its role as a pathogen in causing infection at the site, the pattern of susceptibility to antibacterials, and possible consequences of resistant bacterial subpopulations. Lastly, considerations for selection of the antibacterial include antibacterial activity, clinical efficacy and safety, and potential for drug interactions. Pharmacologic properties such as tissue penetration, protein binding and metabolism/elimination characteristics will affect the resulting pharmacokinetic profile and must be also evaluated. 145

Antibacterial pharmacodynamics integrates the complex correlation between organism susceptibility and patient pharmacokinetics (Figure 5). Pharmacodynamics is simply the indexing of the total drug exposure in a body site to a measure of microbiological activity of the agent against the organism, 146,147 whereas pharmacokinetics describes the fundamental processes of absorption, distribution, metabolism and elimination, as well as the resulting concentration-versus-time profile of an agent administered in vivo. Pharmacokinetic studies

describe parameters such as peak concentration (Cmax), the serum half-life (t1/2), and the cumulative exposure to an agent (‘Area under the concentration–time curve’ or AUC) for a 24 hour period (Figure 6). 145

Figure 5: Schematic representation of the complexity of interactions between patient, pathogen and antibacterial agent (Adapted from McKinnon & Davis, 2004). 145

The major objective in the use of antibacterial agents for the treatment of infections is the decimation of the pathogen as quickly as possible with minimal adverse effects on the recipient. In order to accomplish this goal and ensure a successful outcome, three basic conditions must exist. First, the antibacterial must bind to a specific target-binding site or ‘active site’ on the microorganism. Although the active sites are different for different classes

______Balbino M. Rocha, 2013 25 Cha pte r I: The Big Picture on Antibacterial Resistance

of antibacterials, the principle is the same  namely to disrupt a point of biochemical reaction that the bacterium must undergo as part of its life cycle. If the biochemical reaction is critical to the survival of the bacteria, then the antibacterial will have a deleterious effect on the life of the microorganism. The second condition is that the antibacterial is in adequate concentrations such as to occupy a critical number of these specific active sites on the microorganism. Finally, it is important that the agent occupies a sufficient number of active sites for an adequate period of time. 145 The relationship between the antibacterial concentration and the time that this concentration remains at these active sites above the target MIC during any dosing interval is known as the area under the concentration–time curve (Cp × time = AUC). 147 In essence, the AUC indirectly measures these two major factors for bacterial eradication and quantifies the amount of exposure of the organism to the antibacterial during any dosing interval. 148

Antibacterial concentrations still remain immeasurable directly at the different active sites of attachment to the bacterium. However the antibacterial levels can be measured in serum and other tissues as a function of time. These surrogate levels predict the concentrations of the antibacterial that are necessary to inhibit (Minimum Inhibitory Concentration, [MIC]) or to be bactericidal (Minimum Bactericidal Concentration, [MBC]) to microorganisms. Blood (plasma/serum) AUC has been the most frequently used surrogate concentration, demonstrating an optimal correlation to in vivo bacterial eradication. Although this is a good surrogate in the majority of situations, certain infections may require different body sites as more accurate surrogates. 147

Figure 6: Concentration-versus-time curve with MIC superimposed and pharmacokinetic and pharmacodynamic markers (Adapted from McKinnon & Davis, 2004). 145

______Balbino M. Rocha, 2013 26 Cha pte r I: The Big Picture on Antibacterial Resistance

When evaluating the concentration-versus-time curve for an antibacterial agent (Figure 6), the introduction of the corresponding MIC onto the graph provides an illustration of the relationship between the concentration and the MIC. An increase in dose will provide a

significant increase in antibacterial peak concentration (Cmax), while a shorter dosing interval will predominantly increase the time that concentrations remain above the MIC of the

infecting pathogen. It should be noted that an increase in dose will increase both Cmax and AUC, frequently resulting in covariance of these PD targets. Different dosing strategies may be used, therefore, to optimize dosing for the various classes of antibacterials (Figure 6).

6.1. Time-dependent vs. Concentration-dependent Bacterial Decimation

Certain antibacterials like β-lactams, clindamycin, macrolides and oxazolidinones can be effective because of the extensive amount of time bound to the microorganism and the inhibitory effect can be effective because their concentration exceeds the MIC for the microorganism. Hence, these agents are referred to as time-dependent antibacterials. Other classes of antibacterials, such as aminoglycosides and quinolones, have high concentrations at the binding site which eradicates the microorganism. These drugs are considered to have a different kind of bacterial decimation, termed concentration-dependent killing. 149

Antibacterials with time-dependent decimation exhibit optimal responses when the time that the drug remains above the MIC is equal or greater than 50% of the dosing interval (t>MIC) (Figure 6). 149

For agents with concentration-dependent decimation, the best responses occur when the concentrations are ≥ 10 times above the MIC for their target organism at the site of 148 infection. The PD parameter for these agents can be simplified as a Cmax/MIC ratio, predicted by measuring the AUC over the dosing interval and dividing that value by the antibacterial's MIC against the target microorganism. In essence, the AUC/MIC ratio

becomes a “default” PD concept for the Cmax/MIC ratio for antibacterials with concentration- dependent decimation. 146,147 This parameter represents the degree to which the serum concentration and time exposure of the antibacterial exceed the minimum needed to interfere with the bacterial life cycle. The higher the AUC/MIC ratio, the greater the probability of maximum decimation of the organism (Figure 6). 147 Resistance can occur as a result of using low doses, selecting organisms in a population that have higher MIC values. 150 On the other hand, the use of higher AUC/MIC ratios not only maximizes decimation but can also minimize the risk of selection of resistant organisms. Excessive AUC/MIC ratios may however produce unwanted adverse reactions in the patient by disrupting the normal gastrointestinal flora and producing organ dysfunction. 149

______Balbino M. Rocha, 2013 27 Cha pte r I: The Big Picture on Antibacterial Resistance

7. Laboratory Detection of Antibacterial Resistance

Historically, physicians and veterinary practitioners have prescribed antibacterials based on expected mode of action, spectrum of activity and clinical experience. With the emergence and spread of ABR, treatment of bacterial infections has become increasingly difficult and is no longer as straightforward as it was many years prior. Practitioners now need to consider that a particular pathogen they wish to treat may be resistant to some or all

of the available antibacterials, making antibacterial susceptibility testing a standard procedure.

Antibacterial susceptibility testing methods are in vitro procedures used to detect ABR in individual bacterial isolates. Because these laboratory detection methods can determine resistance or susceptibility of an isolate against an array of possible therapeutic candidates, the respective results can be a useful clinical guideline in selecting for the best antibacterial treatment option for each particular patient. These same methods can also be used for monitoring the emergence and spread of resistant microorganisms in a population.

7.1. Antibacterial Susceptibility Breakpoints

Breakpoints are currently an integral part of modern microbiology laboratory practice and are used to define susceptibility and resistance to antibacterial agents. Depending on the testing method, they are expressed as either a concentration (in mg/L or g/mL) or a zone diameter (in mm). In general, all susceptibility testing methods require breakpoints, also known as interpretive criteria, so that the results of the tests can be interpreted as susceptible, intermediate, or resistant and reported as such to a broad range of clinicians. The interpretative criteria for these are based on extensive studies that correlate laboratory resistance data with serum achievable levels for each antibacterial agent and a history of successful and unsuccessful therapeutic outcomes. It is acknowledged that sophisticated prescribers may not require (or desire) breakpoints but rather use MIC and PD information to optimize antibacterial selection and dosing. However, given the volume of specimens that a typical clinical microbiology laboratory receives and the diversity of clinicians that a laboratory serves, categorical interpretation of antibacterial susceptibility testing results is a practical necessity, preferred by most clinicians. 151

The term ‘breakpoint’ has been used in a variety of ways in research literature. 125 The first and most obvious one refers to the MIC for any given antibacterial that distinguishes wild-type populations of bacteria from those with acquired or selected resistance mechanisms, named wild-type breakpoints or microbiological breakpoints. Data for obtaining this type of breakpoint are generated from moderate to large numbers of in vitro MIC tests, sufficient to describe the wild-type population. In this context, the wild-type strain is defined

______Balbino M. Rocha, 2013 28 Cha pte r I: The Big Picture on Antibacterial Resistance

as a strain of a bacterium which does not harbor any acquired or selected resistance to the particular antibacterial being tested or to antibacterials with the same mechanisms of action. The second are so-called clinical breakpoints, which refer to those MICs that separate strains where there is a high likelihood of treatment success from those bacteria where treatment is more likely to fail. In their simplest form, these breakpoints are derived from prospective human clinical studies comparing outcomes with the MICs of the infecting pathogen. The third use of the term breakpoint refers to antibacterial concentrations calculated from knowledge of a pharmacodynamic parameter and the dimension of that parameter that predicts efficacy in vivo. These are the pharmacokinetic/pharmacodynamic (PK/PD) breakpoints, where data that have been generated in an animal model are extrapolated to humans and other animals by using mathematical or statistical techniques. In an attempt to reduce confusion about the meaning of the term ‘breakpoint’, the European Committee on Antibacterial Susceptibility Testing (EUCAST) proposed the use of the term ‘epidemiological (or wild-type) cutoff value’ to replace the term microbiological breakpoint. 153 In this dissertation, the author will use the term ‘cutoff’ to describe the three types of breakpoints (wild-type/epidemiological cutoff, PK/PD cutoff and clinical cutoff) and the term ‘breakpoint’ be reserved for the final selected value to be applied in the clinical laboratory.

7.2. Definitions of Susceptibility Categories

Breakpoints are used to define susceptibility and resistance. While these terms should be universally understood, they are frequently used ambiguously because they can refer to the direct interaction between the antibacterial agent and the organism or to the likelihood that the patient will respond to treatment. The first can be measured simply in vitro, while the second involves in vivo complexities, already described in section 6 of this dissertation.

Two sets of category definitions are given below to accommodate the two types of classifications. In vitro definitions are as follows: 1) Susceptible: growth of the bacterial strain is inhibited by an antibacterial agent concentration in the range found for wild-type strains; 2) Resistant: growth of the bacterial strain is inhibited by an antibacterial agent concentration higher than the range seen for wild-type strains; and 3) Wild type: strains that harbor no acquired resistance mechanism to the tested antibacterial, specifically no resistance attributable to (i) mutation, (ii) acquisition of foreign DNA, (iii) up-regulation of an efflux pump, (iv) up-regulation of target production, or (v) any combination of these. Pharmacodynamic and clinical definitions, currently listed in the international reference method ISO 20776-1, are as follows: 1) Susceptible: the bacterial strain is inhibited by a concentration of an antibacterial agent that is associated with a high likelihood of therapeutic success; 2) Intermediate (susceptibility): the bacterial strain is inhibited by a concentration of an

______Balbino M. Rocha, 2013 29 Cha pte r I: The Big Picture on Antibacterial Resistance

antibacterial agent that is associated with an uncertain therapeutic effect; and 3) Resistant: the bacterial strain is inhibited by a concentration of an antibacterial agent that is associated with a high likelihood of therapeutic failure. 154

While intuitively appealing, these definitions do not capture all of the concepts embedded in susceptibility categories. A more encompassing set of definitions is provided by the Clinical and Laboratory Standards Institute (CLSI): 1) Susceptible: isolates are inhibited by the usually achievable concentrations of the antibacterial agent when the recommended dosage (dosage regimen) is used for that site of infection; 2) Intermediate: isolates with antibacterial agent MICs that approach usually attainable blood and tissue levels and for which response rates may be lower than those for susceptible categories; and 3) Resistant: isolates are not inhibited by the usually achievable concentrations of the agent with normal dosage schedules and/or demonstrate MICs/zone diameters that fall in the range where specific microbial resistance mechanisms (e.g., β-lactamases) are likely and that clinical efficacy against the isolate has not been shown reliably in treatment studies. The intermediate susceptibility category has multiple purposes, including: 1) to provide a buffer zone between the resistant and susceptible categories, in order to prevent small, uncontrolled technical factors from causing major discrepancies in interpretations, especially for drugs with narrow pharmacotoxicity margins; and 2) to imply clinical efficacy if the antibacterial is concentrated at the site of infection (e.g., quinolones and β-lactams in urine); or suggest that higher doses of antibacterial should be used where it is safe to do so to achieve efficacy. 151,155

7.3. Organizations that Set Breakpoints

Various organizations worldwide have developed and published breakpoints (Table 5). Unfortunately the differences in the methodologies used and resultant breakpoints between these organizations have originated confusion for clinical microbiologists, antibacterial susceptibility testing device manufacturers and clinicians. 151 Only two international standard- setting groups  CLSI and EUCAST  have published guidelines on which data are required for (and how these data are applied to) breakpoint determination. 155,156 Harmonization of breakpoints among these organizations should clearly be the aim, taking into account possible differences in doses and dosing schedules used in different parts of the world. 151

Although veterinary laboratories originally based interpretations on standards established using human pathogens, it became apparent by the early 1980s that such an approach did not reliably predict clinical outcomes when applied to veterinary practice. Consequently, groups within some of the mentioned organizations were created for the purpose of developing veterinary-specific standards. 157 ______Balbino M. Rocha, 2013 30 Cha pte r I: The Big Picture on Antibacterial Resistance

Table 5: World organizations with published breakpoints (Adapted from Turnidge, 2007). 151

Organization or test Breakpoint-setting Method(s) and main media used [method reference(s)] parameters [reference(s)]

Arbeidsgruppen for antibiotikaspørsmål Resistance markers, MIC distributions, Disk diffusion — Mueller-Hinton or Iso- (Norwegian Working Group on Antibiotics PK/PD, clinical and bacteriological Sensitest [APA]) 158 outcomes159, 160 Agar dilution, broth dilution, broth British Society for Antibacterial PK and protein binding (formula), MIC microdilution, disk diffusion — Iso-Sensitest Chemotherapy (BSAC) 161,162 distributions 163 agar and broth Calibrated dichotomous sensitivity test CDS; promulgated by a single laboratory in Disk diffusion — Sensitest agar Mainly zone diameter distributions 164 Sydney, Australia) 164 For aerobic and facultative bacteria, broth Clinical and Laboratory Standards Institute dilution, broth microdilution, disk diffusion — MIC distributions, PK/PD, (CLSI) 155 and the U.S. Food and Drug Mueller-Hinton agar and broth; for anaerobic clinical/bacteriological outcome Administration bacteria, agar dilution, broth microdilution — correlations167 supplemented Brucella agar and broth Commissie Richtlijnen MIC distributions, PK/PD, clinical and Disk diffusion — Iso-Sensitest Gevoeligheidsbepalingen (CRG) 165,166 bacteriological outcome correlates 167 Agar dilution, broth microdilution, disk Comité de l’Antibiogramme de la Société diffusion — Mueller-Hinton MIC distributions, PK, correlation with clinical Française de Microbiologie (CA-SFM) 168 and bacteriological outcomes 163

169 Deutches Institut für Normung (DIN) Agar dilution, broth microdilution, disk MIC distributions, PK, correlation with clinical 169 diffusion — Mueller-Hinton and bacteriological outcomes In vitro drug characteristics, MIC Agar dilution, broth dilution, broth EUCAST 7,170 distributions, PK/PD, clinical outcome microdilution — Mueller-Hinton correlations 13 Japanese Society for Chemotherapy Broth microdilution — Mueller-Hinton MIC - clinical outcome correlations 175 (JSC)173,174 Zone diameters are calibrated against a Disk (pressed tablet) diffusion — Mueller- range of different national and international Rosco Diagnostica (a commercial company Hinton, Iso-Sensitest, PDM, and Danish MIC breakpoints as well as unique based in Denmark) 179 blood agar breakpoints for tests performed on Danish blood agar 176 Mesa Española de Normalización de la Disk diffusion, broth dilution, agar dilution — MIC distributions, PK/PD, clinical and Sensibilidad y Resistencia a los Mueller-Hinton bacteriological outcomes 178 Antimicrobianos (MENSURA [Spain]) 177 Swedish Reference Group for Antibiotics Agar dilution, disk diffusion, gradient “Pharmacological breakpoints” with species (SRGA) 179 diffusion — Iso-Sensitest related adjustments

7.4. The Nature of Minimum Inhibitory Concentrations

MICs, as currently measured, are presently the most straightforward estimates of the antibacterial effect in vitro. Despite semiquantitative, they have significant utility and there is currently no better measure of the antibacterial effect. 151

As mentioned before, all breakpoints are either MICs or zone diameter values correlated with MICs. As a consequence, an understanding of the nature of the MIC is essential for breakpoint setting. The central concept of a MIC is that it is a measurement of the activity of an antibacterial agent against an individual strain of an organism. It has become the reference measuring tool for susceptibility testing. The value of MIC measurement is frequently criticized because of the “unnatural” conditions under which it is performed, but that criticism misses the point. It is unnecessary for it to reflect exactly the conditions at the site of infection, and of course in most circumstances it cannot. Hence, the ______Balbino M. Rocha, 2013 31 Cha pte r I: The Big Picture on Antibacterial Resistance

common practice of comparing MICs with levels measured in various body compartments is qualitative at best. The true value of a MIC is as a measuring tool that generates values to which other parameters, such as PD endpoints and clinical outcomes, can be reliably compared. This requires that MICs have a reasonable level of reproducibility, a subject that has not received a great deal of attention over the years. Indeed, it is frequently quoted that the “error” associated with measuring a MIC is “plus or minus one two-fold dilution.” While this can work as a rule of thumb, results from so-called “tier 2 studies” described by the CLSI 155 for establishing quality control ranges show that precision of MIC measurements can be less than or greater than this, depending on the microorganism-antibacterial combination. 180

The origins of the MIC can be traced back to the original Fleming article (1929) on penicillin. Introduced in this paper were the ideas of (i) serial two-fold dilution of an antibacterial agent in broth to measure its activity against different species and (ii) reading the endpoint by “noting the opacity of the broth”. 181 The development and adoption of the serial two-fold dilution series for MIC measurement, while originally done for convenience in the macrobroth method (see subsection 7.5.1), has serendipitously turned out to be valuable from at least one point of view. When the MICs of a particular antibacterial for a large number of strains of a single species are plotted on a histogram, it appears that the wild-type population follows a log-normal distribution (Figure 7). 180 This means that wild-type MICs appear to be normally distributed on a logarithmic scale. Furthermore, strains with the same type of acquired resistance also have a log-normal distribution of MICs. It is therefore usual to see a bimodal distribution for species in which a single resistance mechanism to an antibacterial predominates. 151

Another important feature of the MIC as we currently measure it is that it actually represents a range of MICs. By way of example, Figure 7 shows that 51.082 of 71.360 strains of S. aureus have a vancomycin MIC of 1 mg/L. In reality, this represents the individual MICs for those strains, each of which is >0.5 mg/L and ≤1 mg/L. In other words, there are 51.082 strains whose MICs lie in the range of 0.5 to 1 mg/L. Indeed, it is quite possible to determine MICs between the two conventional two-fold dilution series values by setting up such concentrations or by using gradient diffusion products (e.g., E-test [AB Biodisk, Solna, Sweden]). From the clinical and PD perspectives, such a discriminatory ability may be quite useful. It would actually be preferable, In some settings, to have a more finely divided range of MICs than conventional two-fold dilutions. For example, “actual” MICs for amikacin for a P. aeruginosa strain of >4 to ≤8 µg/mL will be recorded, using serial two- fold dilution series, as 8 µg/mL. Yet if the PD parameter that best predicts amikacin success for P. aeruginosa is a ratio of peak concentration achieved to MIC, 182 a measured amikacin peak concentration of 40 µg/mL and a recorded MIC of 8 µg/mL will result in a ratio of 5. The true ratio may actually be closer to 10 if the “actual” MIC was just over 4 µg/mL. No ______Balbino M. Rocha, 2013 32 Cha pte r I: The Big Picture on Antibacterial Resistance

commercially available antibacterial susceptibility testing products give suitable divided ranges of MICs, and space limitations prevent long ranges of dilutions. However, their development for research purposes is hindered by a misunderstanding of the precision of current MIC tests, often stated to be “plus or minus one two-fold dilution,” as discussed above. 151

Figure 7: MIC distributions for four microorganism-antibacterial pairs. In each case, the wild-type appears as the log-normally distributed population at the lower MICs (Adapted from Turnidge, 2007). 151

The values generated by MIC tests will of necessity be influenced by the method employed. 183 The results may differ by the selection of the technique (broth macro and microdilution, agar dilution or gradient diffusion); the medium (Mueller-Hinton, Iso-Sensitest, or Sensitest medium, lot-to-lot variation, divalent cation concentrations, and the effects of additives, such as blood); the inoculum size and concentration; the incubation conditions (temperature and duration of incubation); and the precision in the preparation of different concentrations of the antibacterial agent being used. Thus, a MIC is only meaningful when the methods and conditions of the test are known. 151

______Balbino M. Rocha, 2013 33 Cha pte r I: The Big Picture on Antibacterial Resistance

7.5. Test Methods in Antibacterial Resistance Detection

Several antibacterial susceptibility testing methods are currently available and all have one same goal, which is to provide a reliable prediction of whether an infection caused by a bacterial isolate will respond therapeutically to a particular antibacterial treatment. This data may be utilized as guidelines for chemotherapy, or at the population level as indicators of emergence and spread of resistance based on passive or active surveillance. Selection of the appropriate method will depend on the intended degree of accuracy, convenience, urgency, availability of resources, availability of technical expertise and cost. Among these available tests, the most widely used methods in human and veterinary laboratories include broth microdilution or rapid automated instrument methods that use commercially marketed materials and devices. Manual methods that provide flexibility and possible cost savings include the disk-diffusion and gradient-diffusion methods (e.g., E-test). Each method has strengths and weaknesses, including organisms that may be accurately tested by the method. Some methods provide quantitative results (e.g., MIC), and all provide qualitative assessments using susceptibility categories. In general, current testing methods provide accurate detection of common ABR mechanisms. 184,185

7.5.1. Broth Dilution Methods

Broth dilution methods involve subjecting the bacterial isolate to a series of concentrations of antibacterial agents in a broth environment. For decades, the conventional method of determining MICs was in normal test tubes containing 1 to 2 mL of broth, termed “macrobroth method”. 155 Since the 1960s, the miniaturization and mechanization of the test by use of small, disposable, plastic “microdilution” trays (Figure 8) has made broth dilution testing practical and the preferred method for performing MIC tests in broth. 186 In fact, this quantitative method is internationally accepted as the reference standard. 187 Standard trays have 96 wells, holding a maximum volume of 0.1 mL each. This allows approximately 12 antibacterials to be tested in a range of 8 two-fold dilutions in a single tray. Microdilution panels are typically prepared using dispensing instruments that aliquot precise volumes of preweighed and diluted antibacterials into the individual wells. Hundreds of identical trays can be prepared from a single master set of dilutions in a relatively brief period. Few clinical microbiology laboratories prepare their own panels; instead frozen or dried microdilution panels are purchased from one of several commercial suppliers. Inoculation of panels with the standard 5 x 105 CFU/mL is accomplished using a disposable device that transfers 0.01 to 0.05 mL of standardized bacterial suspension into each well of the microdilution tray or by use of a mechanized dispenser. Following incubation, MICs are determined using a manual or automated viewing device for inspection of each of the panel wells for growth. Growth is

______Balbino M. Rocha, 2013 34 Cha pte r I: The Big Picture on Antibacterial Resistance

recorded by monitoring the turbidity of each well, and the first dilution with non-visible growth is considered to be the MIC for that isolate. 188

Figure 8: A broth microdilution susceptibility panel containing 96 reagent wells and a disposable tray inoculator (Adapted from Jorgensen et al., 2009). 184

The advantages of the microdilution procedure include the generation of MICs, the reproducibility and convenience of having pre-prepared panels, and the economy of reagents and space that occurs due to the miniaturization of the test. There is also assistance in generating computerized reports if an automated panel reader is used. The main disadvantage of the microdilution method is some inflexibility of drug selections available in standard commercial panels. 184

7.5.2. Disk Diffusion Method

This method, also called the Kirby-Bauer method, is a simple, practical and well standardized test. This test is performed by applying a bacterial inoculum of approximately 1 to 2 x 108 CFU/mL to the surface of a Mueller-Hinton agar plate. Up to 12 commercially prepared disks, each pre-impregnated with a standard concentration of a particular antibacterial agent, are then evenly distributed and lightly pressed onto the inoculated agar surface (Figure 9). The tested antibacterial immediately begins to diffuse outward from the disks, creating a concentration gradient in the agar such that the highest concentration is found close to the disk with decreasing concentrations further away from it. Plates are incubated for 16–24 h at 35 ºC prior to determination of results. If the test isolate is susceptible to a particular antibacterial agent, a clear no growth area will be observed around that particular disk. Those zones of growth inhibition are measured to the nearest millimeter and the obtained diameter of each drug is interpreted using the charted criteria published, usually, by either one of the international standard-setting groups  CLSI or EUCAST. The results of the disk diffusion tests are qualitative, classifying isolates by susceptibility category – as susceptible, intermediate or resistant – rather than ascribing a MIC measurement. 188-190 ______Balbino M. Rocha, 2013 35 Cha pte r I: The Big Picture on Antibacterial Resistance

However, some commercially available zone reader systems claim to calculate an approximate MIC with some organisms and antibacterials by comparing zone sizes with standard curves of that species-drug interaction stored in an algorithm. 191,192

Figure 9: Antibacterial susceptibility testing by disk diffusion. On this agar plate, a bacterial isolate is tested for resistance to each of four different antibacterials. The clear zones around each disc are the zones of inhibition that indicate the extent of the test organism’s inability to survive in the presence of the test antibacterial. The ATB1 disk shows a large zone of inhibition, whereas ATB2 shows no zone of inhibition, indicating resistance of the isolate to the test antibacterial (Adapted from http://ABRls.cvm.msu.edu, 2012). 185

The advantages of this method are the test simplicity that does not require any special equipment (low costs), the provision of categorical results easily interpreted by all clinicians, and flexibility in selection of disks for testing. The disadvantages are the lack of mechanization or automation of the test. Although not all fastidious or slow growing bacteria can be accurately tested by this method, the disk test has been standardized for testing streptococci, Haemophilus influenzae and N. meningitidis through use of specialized media, incubation conditions and specific zone size interpretive criteria. 190

7.5.3. Antibacterial Gradient Diffusion Method

The antibacterial gradient diffusion method uses the principle of establishment of an antibacterial concentration gradient in an agar medium as a means of determining susceptibility. The E-test (AB Biodisk, Solna, Sweden) is a commercially available version, among several others, that employs a plastic test strip impregnated on the underside with a gradually decreasing concentration of a particular antibacterial. The strip also displays, on the upper surface, a numerical scale that corresponds to the antibacterial concentration contained therein (Figure 10). As many as 6 strips may be placed in a radial fashion on the

______Balbino M. Rocha, 2013 36 Cha pte r I: The Big Picture on Antibacterial Resistance

surface of an appropriate agar plate that has been inoculated with a standardized organism suspension like that used for a disk diffusion test. After overnight incubation, the tests are read by viewing the strips from the top of the plate. The MIC is determined by the intersection of the lower part of the ellipse shaped growth inhibition area with the test strip. 185

Figure 10: Antibacterial susceptibility testing by the E-Test. On this agar plate, a bacterial isolate is tested for resistance to a specific antibacterial (Adapted from LeCorn et al., 2007). 193

The gradient diffusion method has intrinsic flexibility by being able to test the drugs the laboratory chooses. However, a separate strip is needed for each antibacterial, and therefore this method can turn out to be costly. This method is best suited to situations in which a MIC for only 1 or 2 drugs is needed or when a fastidious organism requiring enriched medium or special incubation atmosphere is to be tested. 194-196 Generally, E-test results have correlated well with MICs generated by broth or agar dilution methods. 194-198 However, there are some systematic biases toward higher or lower MICs determined by the E-test when testing certain organism-antibacterial agent combinations. 196,199 This can represent a potential shortcoming when standard MIC interpretive criteria derived from broth dilution testing are applied to E-test MICs that may not be identical. 199

7.5.4. Automated Antibacterial Susceptibility Testing Systems

Several commercial computer-assisted antibacterial susceptibility testing systems have been developed, providing convenient automated inoculation, reading and interpretation of samples. These methods are intended to reduce technical errors and lengthy preparation times. However, the one major limitation for most laboratories is the cost entailed in the initial purchase, operation and maintenance of the equipment. Some examples of these include: Vitek® 2 System (BioMérieux, France – Figure 11), 200 MicroScan® WalkAway®-96 Plus System (Siemens AG, Germany), 201 Sensititre ARIS (Trek Diagnostic Systems, East ______Balbino M. Rocha, 2013 37 Cha pte r I: The Big Picture on Antibacterial Resistance

Grinstead – UK), 202 Avantage Test System (Abbott Laboratories, Irving, Texas – USA), 203 Micronaut (Merlin, Bornheim-Hesel, Germany), 204 PhoenixTM (Becton, Dickinson and Company Diagnostic Systems, Maryland – USA), 205 and many more. 185,206

Figure 11: Vitek® 2 System – BioMérieux, France (Image from www.biomerieux-diagnostics.com).

7.5.5. Current Test Methods and Future Directions

The antibacterial susceptibility testing methods described above provide reliable results when used according to the procedures defined by the CLSI/EUCAST or by the manufacturers of the commercial products. However, there is considerable opportunity for improvement in the area of rapid and accurate recognition of ABR. There is, in fact, a need for development of new automated instruments that could provide faster results and also save money by virtue of lower reagent costs and reduced labor requirements.

The direct detection of resistance genes by PCR or similar techniques has limited utility, because only a few resistance genes are firmly associated with phenotypic resistance (e.g., mecA, vanA and vanB). 207 There are hundreds of β-lactamases and numerous mutations, acquisitions and expression mechanisms that result in fluoroquinolone, aminoglycoside and macrolide resistance; too many to be easily detected by current molecular techniques. 208 However, other promising molecular techniques have been developed in recent decades. These include comparative typing methods that are based on electrophoretic banding patterns, library typing methods that are based on the sequence of selected genes, virulence gene arrays and whole genome sequencing projects. Although their routine implementation still faces challenges, for example in terms of cost recovery and turn-around times, it is inevitable that in the future they will contribute to improve the knowledge on the phenotypic character of pathogenic strains and even be among the routine tools used in diagnostic laboratories. 209,210

______Balbino M. Rocha, 2013 38 Cha pte r I: The Big Picture on Antibacterial Resistance

II. The Use of Antibacterials

8. The Use of Antibacterial Agents in Human Populations

Antibacterial agents have increasingly become among the most overused drugs in human medicine, being widely used nowadays to treat and prevent an assortment of infections. 211-213 The antibacterials used in human medicine belong to the same general classes as those used in animals, and in many cases even if they are not exactly the same compounds, their mode of action is the same. In most parts of the world, β-lactam agents (ranging from penicillin G to 4th-generation cephalosporins and carbapenems) play a major role, but sulfonamides (with or without trimethoprim), macrolides, lincosamides and streptogramins (the MLS group), quinolones (including fluoroquinolones), tetracyclines, aminoglycosides and glycopeptides are widely used, some mainly in the community and some mainly in hospitals. 10

With this wide selection of antibacterials available it is possible to treat infection with a high expectation of success. The benefits of use are clear both in hospitals and in the community, and failures of therapy – determinant for the evolution of acquired ABR – are likely to be due to factors linked to the misuse of these compounds by both practitioners and patients. 214 Factors such as misdiagnosis; inadequate antibacterial spectrum of the prescribed agent; insufficient control of the source of the infection; existence of a serious underlying disease; or use when clinical experience shows it to be inappropriate. 65 In the community, the unnecessary prescription of antibacterials is mainly for viral respiratory tract illnesses (VRTIs) and infantile diarrhea. 213,215,216 In children, from a cohort recruited at birth, it has been shown that at 200 days after birth, over 70% of the infants had been administered at least one antibacterial agent. 217 In older children, antibacterials have been shown to be prescribed in more than 40% of patients with common colds, upper respiratory infections (URIs) and bronchitis. 218 In adults, although antibacterials have little or no benefit for VRTIs, treatment for these diseases accounts for over 20% of all antibacterial prescriptions in developed countries. 213,215,219

8.1. The Relationship Between Antibacterial Use and Resistance

The first evidence for the relationship between antibacterial use and ABR is geographic 220 and time concordant. 221 However, in such analyses, even a very strong correlation does not enable us to estimate the risk, nor to distinguish the impact of antibacterial exposure on emergence and diffusion of ABR in bacterial populations, nor to conclude about the causality. From an epidemiological point of view, the main criteria required for the causal nature of an association are: 1) the coherence with existing information (biological plausibility); 2) the ______Balbino M. Rocha, 2013 39 Cha pte r I: The Big Picture on Antibacterial Resistance

consistency of the association in several studies; 3) time sequence; and 4) the specificity of the association and the strength of the association. 222

In the community, there is cumulative evidence that the antibacterial exposure of populations promotes acquired ABR in community pathogens such as Streptococcus pyogenes, Streptococcus pneumoniae, cutaneous staphylococci and propionibacteria. 223,224 In the hospital setting, an increased use of antibacterials is often associated with an increase in the frequency of ABR, 225 whereas a reduced consumption of antibacterials may be followed by a reduction of resistance to specific drugs, particularly in Enterobacteriaceae. 226 In addition, the relationship between antibacterial use and resistance is most evident when resistance is due to mutations selected during therapy, resulting in clinical failure. 227 In individuals on antibacterials, resistance tends to develop not only in the bacteria involved in the treated infection but also in the commensal bacterial flora. 228 This may explain the rapidity with which quinolone resistance has emerged in staphylococci 229,230 and pneumococci. 231 In most studies, however, it is not possible to distinguish between the risk of bacteria carried prior to treatment becoming resistant and the risk of acquiring resistant bacteria from somebody else. For S. pneumoniae, although the rate of penicillin G-resistant S. pneumoniae (PRSP) increases during β-lactam therapy, the risk of a previously carried pneumococci having an increased penicillin G MIC seems to be very low in comparison with carrying a new PRSP after the treatment. 232

Furthermore, the application of pharmacodynamic concepts as well as epidemiological studies suggest that bacterial exposure to low and prolonged concentrations of an antibacterial may have a role in the selection of resistance. 233,234

8.2. Critically Important Antibacterials for Human Medicine

Due to the established relationship between ABR and antibacterial human and non- human use, the WHO developed the Critically Important Antimicrobials for Human Medicine document – a classification listing of antibacterials ranked according to criteria that determine their relative importance in human medicine, with the main long-term goal of preserving the effectiveness of currently available antibacterials. 235

The criteria developed and by which the document in question is based on are: Criterion 1: Antibacterial agent is used as sole therapy or one of few alternatives to treat serious human disease; Criterion 2: Antibacterial agent is used to treat diseases caused by either: 1) organisms that may be transmitted via non-human sources or 2) diseases caused by organisms that may acquire resistance genes from non-human sources. 235

______Balbino M. Rocha, 2013 40 Cha pte r I: The Big Picture on Antibacterial Resistance

The resultant list consists of all antibacterial classes used in human medicine, categorized into three groups (Table 6):

 Category I: Critically Important. These antibacterials meet both criteria 1 and 2.

 Category II: Highly Important. Antibacterials in this category meet either criterion 1 or 2 and consist of those that can be used to treat a variety of infections including serious infections and for which alternatives are generally available. Bacteria resistant to drugs of this category are generally susceptible to Category I drugs which could be used as the alternatives.

 Category III: Important. Antibacterials in this category meet neither criterion 1 nor 2 and are used for treatment of bacterial infections for which alternatives are generally available. Infections caused by bacteria resistant to these drugs can, in general, be treated by Category II or I antibacterials. 235

Table 6: Summary of the antibacterial classes included in the three categories of Critically Important Antimicrobials for Human Medicine (Adapted from WHO - AGISAR, 2009). 235

 Carbapenems  Aminoglycosides (except topical agents)  Aminocyclitols (spectinomycin)  Cephalosporins – 3rd & 4th generations  Cephalosporins – 1st & 2nd generations  Aminoglycosides (topical agents)  Fluoroquinolones (including cephamycins)  Bacitracins  Glycopeptides  Fusidic acid  Fosfomycin

 Glycylcyclines  Lincosamides  Nitrofurans

 Ketolides  Macrolides  Phenicols (florfenicol)  Lipopeptides  Penicillins  Sulphonamides  Monobactams  Quinolones (except fluoroquinolones)  Tetracyclines  Nitroimidazoles (metronidazole)  Streptogramins  Trimethoprim

 Oxazolidinones  Trimethoprim/Sulfamethoxazole

Category I Category Category II Category  Penicillin-β-lactamase inhibitor III Category combinations  Polymyxins (colistin)  Therapeutic agents for tuberculosis (e.g., ethambutol, isoniazid, pyrazinamide and rifampin)

8.3. The Current Use of Antibacterials in Europe

In Europe, since 1999, comparable (using ATC/DDD classification), reliable and comprehensive antimicrobial consumption data have been collected from hospital and community settings. These data have been put out by a network of national surveillance systems called the European Surveillance of Antimicrobial Consumption Network (EASC- Net). A total (so far) of 35 countries are included in this network: 27 EU-MS, 3 EEA/EFTA countries (Iceland, Norway and Switzerland), 3 EU candidate countries (Croatia, Macedonia and Turkey) and 2 other countries (Russian Federation and Israel). 236

______Balbino M. Rocha, 2013 41 Cha pte r I: The Big Picture on Antibacterial Resistance

Figure 12: Total outpatient antibacterial use in 2009 in Europe (Adapted from ESAC, 2010). 236

Figure 13: Boxplotted distribution of outpatient antibacterial use between 1999 and 2009 among the participating European countries (Adapted from ESAC, 2010). 236

In the latest collected data, in 2009 (2010 and 2011 data are presently being processed), the outpatient consumption of antibacterials for systemic use (ATC group J01) varied from 10.19 Defined Daily Doses (DDD) per 1000 inhabitants per day (DID) in Romania to 38.64 DID in Greece (Figure 12; Table 7), with a median use of 18.97 DID and an inter- quartile range (25%-75%) of 15.15 to 23.10 DID. 236

______Balbino M. Rocha, 2013 42 Cha pte r I: The Big Picture on Antibacterial Resistance

Table 7: Outpatient antibacterial use in 2009 subdivided into the major antibacterial classes according to ATC classification (n=32 countries) (Adapted from ESAC, 2010). 236

Cephalosporins Sulfonamides Tetra- Other Total Country Penicillins and other MLS Quinolones and β-lactams cyclines Trimethoprim Classes (DID)

Greece 12.89 8.68 2.00 11.54 2.63 0.36 0,54 38.64 Cyprus * 16.01 6.45 2.87 3.98 4.13 0.46 0.55 34.44 France 16.08 2.92 3.39 4.15 2.00 0.42 0.62 29.58 Italy 15.18 2.78 0.52 5.33 3.61 0.47 0.77 28.66 Luxembourg 13.47 4.33 2.08 3.87 2.81 0.39 1.24 28.19 Belgium 15.13 1.82 2.14 2.96 2.61 0.37 2.49 27.52 Slovakia 9.56 4.12 1.50 6.09 2.03 0.43 0.05 23.78 Poland 10.68 2.89 2.47 3.88 1.25 0.95 1.48 23.59 Portugal 12.00 1.96 0.72 3.83 3.04 0.43 0.96 22.94 Israel 11.82 3.96 1.20 1.90 1.44 0.50 1.60 22.42 Malta 9.08 5.50 1.10 3.89 1.66 0.18 0.18 21.59 Croatia 9.69 3.70 1.57 3.24 1.33 0.98 0.70 21.21 Ireland 10.66 1.33 2.74 3.79 0.94 1.13 0.17 20.76 Lithuania * 10.08 1.27 2.00 1.93 1.23 0.01 3.21 19.72 Spain ** 12.31 1.56 0.60 1.90 2.42 0.30 0.59 19.68 Iceland 10.41 0.30 5.09 1.15 0.55 1.08 0.76 19.35 Bulgaria 8.40 2.30 1.62 3.20 1.97 0.86 0.25 18.59 Czech Rep. 7.73 1.55 2.39 3.66 1.27 0.89 0.95 18.44 Finland 6.14 2.33 4.01 1.46 0.87 1.05 2.10 17.96 UK 8.03 0.58 3.96 2.51 0.48 1.18 0.52 17.27 Hungary 7.06 1.98 1.35 3.00 1.79 0.65 0.14 15.98 Denmark 10.00 0.03 1.62 2.25 0.52 0.75 0.80 15.87 Austria 7.09 1.80 1,27 3.93 1.33 0.29 0.22 15.93 Norway 6.59 0.13 2.71 1.68 0.51 0.73 2.88 15.23 Germany 4.27 2.39 3.09 2.51 1.48 0.73 0.43 14.90 Slovenia 9.51 0.42 0.00 2.33 1.08 1.06 0.01 14.42 Sweden 6.98 0.24 3.03 0.63 0.79 0.54 1.75 13.93 Russia 4.23 0.47 1.46 1.72 2.01 0.89 1.42 12.20 Netherlands 4.48 0.04 2.68 1.46 0.89 0.56 1.27 11.39 Estonia 4.37 0.83 2.07 2.09 0.79 0.43 0.49 11.07 Latvia 4.80 0.43 2.10 0.87 0.85 1.09 0.33 10.48 Romania 4.31 2.47 0.11 1.84 1.26 0.16 0.04 10.19

* Cyprus, Greece, Lithuania: Total use, including the hospital sector. ** Spain: Reimbursement data, does not include over-the-counter sales without prescription.

The distribution of total outpatient antibacterial use between 1999 and 2009 is shown for all countries in Figure 13. The general distribution of the outpatient use among the reporting countries shows a general decrease from 1999 to 2004 followed by a gradual median increase up to 2008, only to decline in 2009. When comparing the trends of

______Balbino M. Rocha, 2013 43 Cha pte r I: The Big Picture on Antibacterial Resistance

outpatient antibacterial use per country (Figure 14), different complex temporal patterns were observed, including short-term increases or decreases and sudden changes. 236

Cyprus (CY), Lithuania (LT): total use, including the hospital sector. Greece (GR): total use for the years 2004-2008. Spain (ES): reimbursement data, does not include over-the-counter sales without prescription. Bulgaria (BG): total use until 2005, outpatient use as from 2006.

Figure 14: Trends of total outpatient antibacterial use in Europe from 1997 to 2009. Dark bars correspond to the year 2009 (Adapted from ESAC, 2010). 236

* Cyprus, Lithuania: total use, including the hospital sector. ** Spain: reimbursement data, does not include over-the-counter sales without prescription.

Figure 15: Outpatient antibacterial use in 2009 subdivided into the major antibacterial classes according to ATC classification (N=32 countries) (Adapted from ESAC, 2010). 236 ______Balbino M. Rocha, 2013 44 Cha pte r I: The Big Picture on Antibacterial Resistance

In 2009, penicillins represented the most frequently prescribed antibacterial group in all countries, ranging from 28.7% (Germany) to 66.0% (Slovenia) of the total outpatient antibacterial use (Table 7; Figure 15). The proportional usage for cephalosporins ranged from 0.2% in Denmark to 25.5% in Malta; from 0.02% in Slovenia to 26.3% in Iceland for tetracyclines; from 4.5% in Sweden to 29.9% in Greece for macrolides; from 2.8% in the UK to 16.5% in Russia for quinolones; from 0.03% in Lithuania to 10.4% in Latvia for sulfonamides and trimethoprim; and from 0.04% in Slovenia to 18.9% in Norway for the other classes (Table 7; Figure 15). 236

In 2009, the hospital consumption of antibacterials for systemic use varied from 1.26 DID in Hungary to 3.33 in Greece (Table 7). 236

Table 8: Hospital use of antibacterials for systemic use in 2009 (N= 22 countries) (Adapted from ESAC, 2010). 236

Cephalosporins Sulfonamides Tetra- Other Total Country Penicillins and other MLS Quinolones and β-lactams cyclines Trimethoprim Classes (DID)

Greece 1.58 0.67 0.05 0.29 0.31 0.03 0.39 3.33 Finland 0.64 0.98 0.24 0.17 0.38 0.15 0.61 3.17 Romania 1.36 0.49 0.03 0.07 0.33 0.04 0.29 2.62 Luxembourg 0.78 0.75 0.01 0.19 0.30 0.03 0.17 2.22 France 1.23 0.27 0.03 0.13 0.32 0.05 0.19 2.20 Latvia 0.57 0.70 0.08 0.09 0.33 0.06 0.35 2.18 Slovakia 0.72 0.52 0.02 0.11 0.33 0.03 0.12 1.85 Denmark 0.87 0.36 0.03 0.09 0.24 0.07 0.17 1.83 Russia 0.36 0.65 0.05 0.13 0.27 0.01 0.33 1.81 Slovenia 0.70 0.40 0.06 0.15 0.26 0.06 0.16 1.78 Belgium * 0.86 0.36 0.01 0.08 0.23 0.03 0.17 1.74 Estonia 0.54 0.43 0.10 0.12 0.25 0.05 0.15 1.64 Bulgaria 0.36 0.71 0.03 0.14 0.12 0.01 0.23 1.59 Sweden 0.69 0.21 0.17 0.06 0.15 0.08 0.12 1.47 Switzerland* 0.63 0.34 0.02 0.10 0.21 0.06 0.11 1.46 Norway 0.67 0.33 0.05 0.08 0.10 0.05 0.18 1.46 Israel 0.57 0.38 0.06 0.08 0.18 0.00 0.11 1.38 Portugal 0.48 0.42 0.02 0.15 0.08 0.06 0.17 1.38 Ireland 0.68 0.12 0.02 0.19 0.11 0.04 0.20 1.37 Malta 0.38 0.36 0.03 0.22 0.18 0.02 0.17 1.36 Croatia 0.28 0.39 0.06 0.12 0.21 0.06 0.21 1.32 Hungary 0.46 0.26 0.03 0.14 0.26 0.04 0.08 1.26

* Belgium and Switzerland: 2008 data

The mostly used subgroup in the hospital sector were the penicillins, followed by the cephalosporins and other β-lactams and the quinolones. Proportional use of penicillins

______Balbino M. Rocha, 2013 45 Cha pte r I: The Big Picture on Antibacterial Resistance

ranged from 19.7% in Russia to 55.7% in France. The proportion of cephalosporins use was highest in Bulgaria (44.5%), and low in Ireland (9.0%). Tetracycline use was the highest in Sweden (11.6%) and lowest in Luxembourg (0.7%). Macrolide use ranged from 2.8% in Romania to 16.4% in Malta; and quinolone use from 6.1% in Portugal to 20.4% in Hungary. Sulfonamide use was the highest in Sweden (5.2%) and lowest in Israel (0.1%). The use of other classes was highest in Finland (19.3%) and Russia (18.3%) (Table 8; Figure 16). 236

Figure 16: Hospital use of antibacterials for systemic use in 2009 (N= 22 countries) (Adapted from ESAC, 2010).236

8.4. The Current Use of Antibacterials in Portugal

In 2009, the use of antibacterials in ambulatory care reached 22.94 DID in Portugal, ranking it 9th of all 32 participating countries (Table 7; Figure 15). This corresponds to an increase of 1.5%, when compared to 2008 (Figure 18). In fact, since 2008 ambulatory antibacterial use has been increasing, contradicting the declining trend that had been ongoing since 2002 (the highest recorded yet). The most prescribed antibacterials in 2009 in ambulatory care were penicillins, representing 52.3% of total antibacterial use. Following are antibacterials from the MLS group (16.7%) and quinolones (13.2%) (Table 7; Figure 17A). In hospital care, use of antibacterial use represents 1.38 DID, setting Portugal as one of the countries with the lowest antibacterial hospital use. Proportional hospital use of penicillins and other β-lactams represented 34.8% and 30.4%, respectively (Table 8; Figure 17B). 236

______Balbino M. Rocha, 2013 46 Cha pte r I: The Big Picture on Antibacterial Resistance

Figure 17: Distribution of antibacterial classes in ambulatory (A) and hospital (B) care sectors in Portugal in 2009 (Adapted from ESAC, 2010). 236

Figure 18: Trends of antibacterial usage in ambulatory care sector in Portugal (Adapted from ESAC, 2010). 236

______Balbino M. Rocha, 2013 47 Cha pte r I: The Big Picture on Antibacterial Resistance

9. The Use of Antibacterials in Animal Husbandry

Before the major advances in animal science and veterinary medicine of the 19th and 20th centuries, animal rearing for food production was an uncertain venture overloaded by disastrous animal health risk. Today, without these developments, of which antibacterials are part of, high quality and low-cost food products for human consumption as we know it would not exist, especially with a world population that has just peaked seven billion people and still counting. In fact, modern intensive animal husbandry practices, such as livestock, poultry and fish farming, have expanded, along with their reliance on the various applications of antibacterials, in response to broad economic forces ultimately driven by the price elasticity of consumer demand for protein over the last century. 10

The impact of this practice may vary considerably between countries and regions, influenced by the interaction between human populations (social structure), land use, water sources, animal demography (species, distribution and density), national policies (production, animal health, food safety, etc.) and national/international trade. The production systems also vary between countries according to technological, social, and economic status. More than 50% of the world’s pork production and over 70% of poultry meat currently originate from industrialized countries, for example. 28

For companion animals such as cats, dogs and horses, antibacterial use is similar to that in general human medical practice, with individual treatment being the norm. The main disparity between antibacterial use in humans and animals is seen in the context of food production, with a synchronized mass administration of antimicrobials to numerous animals for the purposes of disease prevention and growth promotion. According to the available evidence, the total amount of antimicrobials used in animals, in which healthy food-producing animals correspond to the greater portion, accounts for well over 50% of overall antibacterial usage (Figure 19). 1,28,47

Figure 19: Annual antibacterial/antimicrobial use for human and veterinary practice in Denmark (Adapted from DANMAP, 2010). 4 ______Balbino M. Rocha, 2013 48 Cha pte r I: The Big Picture on Antibacterial Resistance

9.1. Definitions of Antibacterial Use

In animal husbandry, the main objective is to limit progression of disease in the population, since illness decreases animal performance. The CLSI has defined terms to describe antibacterial use: therapy, control, prophylaxis (and metaphylaxis), and growth promotion. 8 Therapy consists on ‘the administration of an antibacterial to an animal, or group of animals, which exhibit frank clinical disease’. While relatively practical for cattle and swine, individual animal therapy makes no sense in poultry, for instance. In these cases, flock therapy is indicated when illness is first recognized in a small proportion of the animals, being often administered in feed or water. 10 Control comprehends ‘the administration of an antibacterial to animals, usually as a herd or flock, in which morbidity and/or mortality has exceeded baseline norms’. 8

Prophylaxis, according to CLSI, is defined as ‘the administration of an antibacterial to exposed healthy animals considered to be at risk, but before expected onset of disease and for which no etiological agent has yet been cultured’. 8 An example of indications for the use of antibacterials as prophylaxis in animals is in the case of certain protozoan diseases, where the probability of clinical outbreaks or production losses due to subclinical disease is recurrently so high that treatment or prophylaxis with antiprotozoals (coccidiostats and histomonostats) is standard practice. Some antibacterial agents act against a number of these microorganisms and some coccidiostats also have antibacterial activity. 237 Another example of prophylactic use of antibacterials in animals is when physical stress is involved, for instance, in the movement of large numbers of animals. Whereas mass regimens can improve animal performance and the general welfare of the treated animals, such regimens do result in increased antibacterial usage. 238 Mass treatment programs generally err on the side of administering treatment to individuals that do not need it, whereas limitation of therapy to recognized clinical cases errs on the side of withholding treatment from some individuals that would benefit from it. Attempts to limit mass metaphylaxis to those individual animals most likely to benefit, using certain clinical indicators for treatment, have proven unsuccessful. 239 More sophisticated measures of disease status are being researched in order to improve treatment selection criteria in these cases. 10

As described by the CLSI, growth promotion is ‘the administration of an antimicrobial, usually as a feed additive, over a period of time, to growing animals that results in improved physiological performance’. 8 The growth promoting effects of antibacterials were first discovered in the 1940s when chickens fed by-products of tetracycline fermentation were found to grow faster than those that were not fed those by-products. 240 Since then, many antibacterials have been found to improve average daily weight gain and feed efficiency in livestock in a variety of applications. 241,242 Whereas some growth-promoting effects are

______Balbino M. Rocha, 2013 49 Cha pte r I: The Big Picture on Antibacterial Resistance

mediated through alterations of the normal intestinal microbiota, resulting in more efficient digestion of feed and metabolism of nutrients, others are mediated through pathogen and disease suppression and immune system release. 243,244 Although the precise mechanisms of growth promotion remain unknown, 242,245 the net benefit of antibacterial feeding to food- producing livestock is quantifiable. 246 Such measurable benefit coupled with provable target animal safety, edible tissue clearance and residue avoidance, and environmental safety is the basis for regulatory approval of growth promoting applications of antibacterials in animals intended for food production in some countries. 247

9.2. Pharmacodynamics and Pharmacokinetics of Antibacterial Use in Animal Husbandry

The basic pharmacodynamic principles can be applied to practices involving the use of antibacterials in food-producing animal operations. 248 It is though necessary to determine for each antibacterial use whether sufficient AUC/MIC ratios are obtained to achieve maximum effectiveness and prevent the development of resistance. 10

In the case of antibacterial therapy for treatment of infections in animals, it is likely that doses will be appropriate, with adequate AUC/MIC concentrations. As a result, therapeutic antibacterial use should lead to maximum eradication and prevention of the emergence of resistant microorganisms because the antibacterial concentration is high relative to the MIC of the organism. This, however, might not be the case when antibacterials are used to control/prevent infections or promote growth. In these situations, where the antibacterial is introduced into the feed or water, factors such as the given dose of antibacterial as well as the quantity of feed and water consumed by the animal must be considered as a function of the AUC/MIC. Again, the key antibacterial concentration is that where the bacteria reside and it may not be the blood. If the AUC/MIC is not maximized, these practices may lead to the emergence of resistance. 10

For orally administered antibacterials, little work has been done identifying whether sufficient AUC/MIC ratios have been achieved in the animal’s gut when these agents are used in food-producing animal operations. Complicating factors include the number of animals needed for such studies; intestinal content that makes analysis more difficult; dosing issues; duration of intake; site of sample acquisition; and differences in elimination for different animal species. Furthermore, the doses used must not cause toxicity in the animals. Finally, a withdrawal period is necessary and the impact of this on the development of resistant bacteria is not known. Considering the scarcity of data related to the actual concentrations over time that the animal’s gut flora is exposed to antimicrobials, it is obvious

______Balbino M. Rocha, 2013 50 Cha pte r I: The Big Picture on Antibacterial Resistance

that more work is needed before one can come to any scientific conclusion regarding the negative effect of the use of antimicrobials in animal feed or water. 10

Regrettably, except for the data from a few studies, we are left only with general principles that indicate that low doses of antibacterials tend to select for bacterial resistance and high doses tend to decimate the microorganisms rapidly. It is known, however, that the low doses of antimicrobials used for growth promotion continue to be effective, and that this includes the suppression of some infectious diseases. It thus seems possible that AUC/MIC ratios might be adequate in the gut. 10 Hence, it is inappropriate to conclude that the use of antibacterials in animal husbandry always results in the emergence of resistant bacteria. Sufficient data are not available to make a definitive conclusion about these issues. 145

9.3. Regulation and Authorization of Antibacterial Use in the EU

In Europe, antibacterials are regulated by the European Medicines Agency (EMA). 249 This agency, through its Committee for Medicinal Products for Veterinary Use (CVMP), cooperates closely with other relevant EU agencies such as the European Centre for Disease Prevention and Control (ECDC) 225 and the European Food Safety Authority (EFSA) 251 in order to provide scientific support, along with surveillance and monitoring activities regarding ABR throughout the entire food chain, health-care facilities and animal populations/operations. Only this way can decision makers, such as the Member States, European Commission and European Parliament, find sufficient evidence to put together policies and measures against human and animal health risks related to the possible emergence, spread and transfer of antibacterial resistance in the food chain and in human/animal populations. 251

Before veterinary medicinal products (VMP), including antibacterials, can be sold or supplied in the European Union (EU), premarketing evaluation by application of a coordinated procedure as established in the Commission Directive 2009/9/EC of 10 February, amending Directive 2001/82/EC is required. This directive provides exhaustive scientific and technical requirements regarding the testing of VMPs. Market authorization for a VMP is granted only after the product has undergone meticulous assessment on the criteria of safety, quality and efficacy. Safety includes the safety of the treated animals, the user of the product, the environment and the consumer of products from the treated animals. Applicants are required to address the microbiological properties of residues and the development of resistance, including resistance of relevance for clinical use in animals. 252

The EU requires by law that any food product derived from animals treated with VMPs must not contain any residue that might represent a hazard to the health of the consumer. Before an antibacterial of veterinary use intended for food-producing animals can be ______Balbino M. Rocha, 2013 51 Cha pte r I: The Big Picture on Antibacterial Resistance

authorized, the safety of its pharmacologically active substances and their residues must first be evaluated. The CVMP carries out the assessment of residue safety, including the possibility of a microbiological risk addressing both the development of antibacterial resistance in bacteria of the human gut flora and disruption of the colonization barrier. 1

9.4. The Current Use of Antibacterials in Europe

Until recently and contrasting with human medicine, important information concerning antimicrobial consumption in food-producing animals had been proven inadequate and insufficient worldwide, with most EU countries (including Portugal) being no exception. Monitoring of antibacterial usage used to be carried out in only a limited number of countries and, with very few exceptions, this was restricted to total amounts used, not categorized by animal species and antibacterial classes. As a result, in September 2009, EMA launched the European Surveillance of Veterinary Antimicrobial Consumption (ESVAC) project, following a request from the European Commission to develop a harmonized approach for the collection and reporting of data on the use of antimicrobial agents in animals from EU-MS. The ultimate objective was to collect usage data per animal species and per production category, taking into account the dosage and the treatment duration for each antibacterial agent. Only this way it is possible to relate the use of antibacterials to the encountered bacterial resistance, in order to structure and implement strategies to effectively fight resistance worldwide. 15

ESVAC is currently collecting detailed and standardized data for 2010 following a call for data sent to 22 European countries, including Portugal. However, EMA has already published the first report from this project in September of 2011. The report contains a harmonized analysis on antibacterial usage, per kg of animals and by country, in nine countries that had kept records between 2005 and 2009 (Table 10 & Figure 20): Czech Republic, Denmark, Finland, France, Netherlands, Norway, Sweden, UK and Switzerland. 15

In the 2011 report, for the eight countries for which data were available (Switzerland not included) for the years 2005-2009, the total sales (in tons of active ingredient) and the estimated population correction unit (PCU) of the animal population decreased by 11.2% and 3.1%, respectively (Table 9). The PCU is used as the term for the estimated weight of both livestock and slaughtered animals. The PCU (in 1000 tons) accounted for by the various food-producing animal species in the nine countries for 2009 is shown in Figure 20. 15

Table 9: Total sales of veterinary antibacterial agents (active ingredient) and PCU (1000 tons) in eight European countries (Switzerland not included) (Adapted from EMA, 2011). 15

2005 2006 2007 2008 2009

Total sales (t) 2 513 2 459 2 576 2 348 2 232 Total PCU (1.000 t) 22 288 22 323 22 493 22 020 21 579 ______Balbino M. Rocha, 2013 52 Cha pte r I: The Big Picture on Antibacterial Resistance

In addition, after standardizing the sales (in mg/PCU), significant differences between the nine countries were noted in the study period (Table 10). These differences are likely to be due in part to differences between countries in the composition of the animal population, the selection of antibacterial agents, and the dosing regimen. It should be emphasized that sales in mg/PCU are not indicators for the level of exposure. The main goal of calculating the amount of mg sold per PCU is to adjust trends in the sales within a country for possible changes in the size of animal livestock population and number of slaughtered animals. 15

Figure 20: PCU (in 1.000 tons) of the major food-producing animal species in 2009, by country (Adapted from EMA, 2011). 15

Table 10: Sales normalized by PCU (mg/PCU) for the years 2005-2009 (Adapted from EMA, 2011). 15

Country 2005 2006 2007 2008 2009 Czech Republic 103 113 101 114 106 Denmark 46 45 47 46 53 Finland 24 25 27 32 32 France 169 164 173 154 141 Netherlands 160 169 179 168 165 Norway 14 15 15 14 14 Sweden 20 21 21 20 19 Switzerland - 93 99 99 95 United Kingdom 72 65 64 63 68 Mean 112.8 110.2 114.5 106.6 103.4

Although in eight of the participating countries (Switzerland not included) antibacterial usage had decreased on average 8.3% from 2005 to 2009 (Tables 10 & 11), this apparent decrease in antibacterial consumption may prove to be misleading, with the actual decrease

______Balbino M. Rocha, 2013 53 Cha pte r I: The Big Picture on Antibacterial Resistance

in antibacterial usage not necessarily implying that the numbers of animals treated have declined. This is because the decrease reported by EMA was mainly in sales of tetracyclines, which require high usage dosages, while the sales of several other antibacterials that require lower doses did actually increase (Table 11). Fluoroquinolones and 3rd and 4th-generation cephalosporins were among the lower-dose antibacterials whose consumption increased between 2005 and 2009 (Table 11). For the eight EU countries, the use of 1st and 2nd- generation cephalosporins increased by 25.5%, the use of 3rd and 4th-generation cephalosporins by 18.8%, and the use of fluoroquinolones by 31.9% in 2009 in contrast to 2005 (Table 11). The use of pleuromutilins and penicillins also increased in 2009, when compared with 2005 (Table 11). 15

Table 11: Difference between 2009 and 2005 sales, expressed as tons of active ingredient and as mg/PCU, for eight European countries (Switzerland not included) (Adapted from EMA, 2011). 15

Antibacterial class Tons Proportion mg/PCU Proportion

Tetracyclines - 268,2 - 21,0% - 10,6 - 18,4%

Amphenicols - 2,5 - 18,4% - 0,1 - 15,7%

Penicillins 32,7 11,0% 1,9 14,6%

Cephalosporins (total) 3,1 19,2% 0,2 23,0%

1st and 2nd gen. cephalosporins 2,5 21,6% 0,1 25,5%

3rd and 4th gen. cephalosporins 0,5 15,1% 0,03 18,8%

Sulfonamides and trimethoprim (total) - 38,2 - 8,5% - 1,1 - 5,5%

Sulfonamides - 31,0 - 8,0% - 0,9 - 5,1%

Trimethoprim - 7,2 - 10,8% - 0,2 - 7,9%

Macrolides - 1,2 - 0,6% 0,2 2,6%

Lincosamides - 3,0 - 13,8% - 0,1 - 11,0%

Aminoglycosides - 14,5 - 12,6% - 0,5 - 9,7%

Quinolones (total) - 8,8 - 26,4% - 0,4 - 24,0%

Fluoroquinolones 1,9 27,8% 0,1 31,9%

Other quinolones - 10,7 - 42,0% - 0,5 - 40,1%

Polymyxins 1,1 1,6% 0,2 4,9%

Pleuromutilins 9,7 43,3% 0,5 48,0%

Others 3,5 32,3% 0,2 36,6%

Difference all classes - 281 - 11,2% - 9,4 - 8,3%

As the data presented in this report are aggregated per antibacterial class, they do not allow for more in-depth analysis. To identify the factors underlying the differences observed, there is a need for more detailed sales data. As a first step, the use of the standardized ESVAC template for the collection of the current ongoing data from the 22 participating countries will provide detailed data at package level, including information on administration ______Balbino M. Rocha, 2013 54 Cha pte r I: The Big Picture on Antibacterial Resistance

form and herd treatment versus individual treatment, allowing for more detailed analysis than can be done using the aggregated data. The next steps should be to collect data per animal species and to analyze the data taking into account variance in the dosing of the various agents. As some agents are administered in much higher dosages than others (e.g., tetracyclines vs. cephalosporins), there is a need to continue to refine the tools for analyzing the data on sales of antibacterial agents. 15

9.5. The Current Use of Antibacterials in Portugal

In Portugal, a national plan for the control and use of medicines (Plano Nacional de Controlo e Utilização de Medicamentos – PNCUM) has been put to action by the Direção Geral de Alimentação e Veterinária (DGAV) in recent years, with the intent to comply with the contents of paragraph 3 of Article 120th, of the Decree-Law no. 148/2008, of July 29th, amended and republished by Decree-Law no. 314/2009 of October 28th, in the Portuguese Diário da República (Official Gazette). 253

The PNCUM's main objective is to integrate an official surveillance and control system in the use of veterinary medicines in animal husbandry practices. An additional goal of this plan relies in taking part in the previously described ESVAC project. As a result, the first report has been put available in 2012, corresponding to data of total antimicrobial sales collected in 2010. These data should, however, due to their preliminary nature, be interpreted with caution since they have not been corrected yet for total biomass of treated animals, nor take into account the demographic differences of the various husbandry operations. 254

The total sales of VMPs containing antibacterial agents in their composition for 2010 in Portugal corresponded to 179.875 t of active ingredients (Tables 12 & 13). Oxytetracycline (46.481 t), followed by doxycycline (28.115 t), amoxicillin (24.866 t) and colistin (15.408 t) were the most sold active ingredients that year (Table 12). Among the different classes of antibacterials, tetracyclines were the most sold (46.61% of all sales), followed by penicillins (19.36%), polymyxins (8.57%) and macrolides (8.47%). These four classes together represent about 80% of total active ingredients released during that year (Table 12). 254

______Balbino M. Rocha, 2013 55 Cha pte r I: The Big Picture on Antibacterial Resistance

Table 12: Total 2010 sales of veterinary antibacterial agents (active ingredient, in tons and %) by antibacterial class in Portugal (Adapted from DGAV, 2010). 254

Rank Antibacterial Active AI 1 AI 2 AI 3 Total (#) Class Ingredient (t) (t) (t) (t) (%) Tetracycline 2.001 - - 2.001 1.11 1 Tetracyclines Oxytetracycline 46.481 0.058 - 46.539 25.87 Doxycycline 28.115 - - 28.115 15.63 Subtotal 76.655 42.61 Amoxicillin 24.866 0.002 - 24.868 13.83 Ampicillin 4.546 0.011 - 4.557 2.53 2 Penicillins Cloxacillin 0.263 0.097 0.101 0.360 0.20 Benzylpenicillin 3.494 1.314 - 4.909 2.73 Phenoxymethylpenicillin 0.144 - - 0.144 0.08 Subtotal 34.835 19.36 Colistin 15.408 - - 15.408 8.57 3 Polymyxins Subtotal 15.408 8.57 Erythromycin 0.013 - - 0.013 0.01 Gamythromycin 0.020 - - 0.020 0.01 Lincomycin 1.029 0.684 - 1.713 0.95 Spyramycin 0.252 0.026 - 0.278 0.15 4 Macrolides Tilmicosin 3.288 - - 3.288 1.83 Tulathromycin 0.014 - - 0.014 0.01 Tylosin 9.906 - - 9.906 5.51 Subtotal 15.232 8.47 Rifamycin 0.012 - - 0.012 0.01 Tiamulin 14.449 0.210 - 14.659 8.15 5 Others Valnemulin 0.113 - - 0.113 0.06 Subtotal 14.784 8.22 Sulfadiazine 8.714 1.764 - 10.478 5.83 Sulfaguanidine 0.014 0.003 - 0.017 0.01 Sulfonamides Sulfadoxine 0.025 0.005 - 0.03 0.02 6 & Trimethoprim Sulfaquinoxaline 0.007 0.015 - 0.022 0.01 Trimethoprim 0.257 1.288 - 1.545 0.86 Subtotal - 12.092 6.73 Danofloxacin 0.043 - - 0.043 0.02 Difloxacin 0.001 - - 0.001 0.00 Enrofloxacin 5.644 - - 5.644 3.14 7 Quinolones Flumequine 0.675 - - 0.675 0.38 Oxolinic acid 0.003 - - 0.003 0.00 Marbofloxacin 0.040 - - 0.040 0.02 Subtotal 6.406 3.56 Apramycin 1.020 - - 1.020 0.57 Aminosidine 0.252 - - 0.252 0.14 Clindamycin 0.001 - - 0.001 0.00 Streptomycin 0.332 0.193 - 0.525 0.29 8 Aminoglycosides Gentamicin 0.067 - - 0.067 0.04 Neomycin 0.264 0.010 - 0.274 0.15 Spectinomycin 0.019 - - 0.019 0.01 Subtotal 2.518 1.20 Florfenicol 1.491 - - 1.491 0.83 9 Amphenicols Subtotal 1.491 0.83 Cephalexin 0.477 0.004 - 0.481 0.27 Cefadroxil 0.003 - - 0.003 0.00 Cefalonium 0.009 - - 0.009 0.00 Cefapirin 0.005 - - 0.005 0.00 Cefazolin 0.004 - - 0.004 0.00 10 Cephalosporins Cefoperazone 0.012 - - 0.012 0.01 Cefovecin 0.002 - - 0.002 0.00 Cefquinome 0.099 - - 0.099 0.06 Ceftiofur 0.198 - - 0.198 0.11 Subtotal 0.813 0.44 Total 174.089 5.684 0.101 179.875 100.00

______Balbino M. Rocha, 2013 56 Cha pte r I: The Big Picture on Antibacterial Resistance

Table 13: Distribution of active ingredients (in tons) in each animal species, in Portugal in 2010 (Adapted from DGAV, 2010). 254

Animal Species (t) % Active Total within Ingredient Sw Po Bo Eq La Fe Ca All Ms (t) Total Amoxicillin 15.863 1.428 0.099 0.000 0.000 0.000 0.095 0.000 7.381 24.866 13.82 Ampicillin 0.000 0.000 0.003 0.000 0.000 0.000 0.000 0.039 4.514 4.557 2.53 Apramycin 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.020 1.020 0.57 Benzylpenicillin 0.000 0.000 0.038 0.032 0.000 0.000 0.000 0.000 4.838 4.908 2.73 Cefadroxil 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.003 0.003 0.00 Cephalexin 0.000 0.000 0.016 0.000 0.000 0.000 0.046 0.000 0.419 0.481 0.27 Cefalonium 0.000 0.000 0.009 0.000 0.000 0.000 0.000 0.000 0.000 0.009 0.00 Cefapirin 0.000 0.000 0.005 0.000 0.000 0.000 0.000 0.000 0.000 0.005 0.00 Cefazolin 0.000 0.000 0.004 0.000 0.000 0.000 0.000 0.000 0.000 0.004 0.00 Cefoperazone 0.000 0.000 0.012 0.000 0.000 0.000 0.000 0.000 0.000 0.012 0.01 Cefovecin 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.002 0.002 0.00 Cefquinome 0.000 0.000 0.084 0.000 0.000 0.000 0.000 0.000 0.015 0.099 0.05 Ceftiofur 0.036 0.000 0.033 0.000 0.000 0.000 0.000 0.000 0.129 0.198 0.11 Chloramphenicol 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.00 Clindamycin 0.000 0.000 0.000 0.000 0.000 0.000 0.001 0.000 0.000 0.001 0.00 Cloxacillin 0.000 0.000 0.354 0.000 0.000 0.000 0.000 0.000 0.005 0.359 0.20 Colistin 5.666 0.000 0.000 0.000 0.000 0.000 0.000 0.000 9.742 15.408 8.57 Danofloxacin 0.000 0.000 0.043 0.000 0.000 0.000 0.000 0.000 0.000 0.043 0.02 Difloxacin 0.000 0.000 0.000 0.000 0.000 0.000 0.001 0.000 0.000 0.001 0.00 Dihydrostreptom. 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.076 0.450 0.526 0.29 Doxycycline 13.835 0.000 0.000 0.000 0.000 0.000 0.000 0.000 14.280 28.115 15.63 Enrofloxacin 0.153 4.909 0.009 0.000 0.000 0.000 0.027 0.000 0.546 5.644 3.15 Erythromycin 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.013 0.013 0.01 Florfenicol 1.225 0.000 0.171 0.000 0.000 0.000 0.000 0.000 0.095 1.491 0.83 Flumequine 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.675 0.675 0.38 Gamythromycin 0.000 0.000 0.020 0.000 0.000 0.000 0.000 0.000 0.000 0.020 0.01 Gentamicin 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.068 0.068 0.04 Lincomycin 1.288 0.100 0.015 0.000 0.000 0.000 0.000 0.000 0.309 1.713 0.95 Marbofloxacin 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.039 0.040 0.02 Neomycin 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.274 0.274 0.15 Oxolinic acid 0.003 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.003 0.00 Oxytetracycline 0.584 0.001 0.079 0.000 0.000 0.000 0.000 0.023 45.852 46.539 25.87 Paromomycin 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.252 0.252 0.14 Phenoxymethylp. 0.000 0.144 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.144 0.08 Pirlimycin 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.00 Rifaximin 0.000 0.000 0.005 0.000 0.000 0.000 0.000 0.000 0.007 0.012 0.01 Spectinomycin 0.002 0.000 0.000 0.000 0.000 0.000 0.000 0.017 0.000 0.019 0.01 Spyramycin 0.000 0.000 0.029 0.000 0.000 0.000 0.000 0.000 0.249 0.278 0.16 Sulfadiazine 9.828 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.650 10.478 5.83 Sulfadoxine 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.030 0.030 0.02 Sulfaguanidine 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.017 0.000 0.017 0.01 Sulfaquinoxaline 0.000 0.000 0.000 0.000 0.007 0.000 0.000 0.000 0.015 0.022 0.01 Tetracycline 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 2.000 2.000 1.11 Tiamulin 5.859 0.220 0.000 0.000 0.000 0.000 0.000 0.000 8.580 14.659 8.15 Tilmicosin 0.238 0.000 0.021 0.000 0.000 0.000 0.000 0.000 3.028 3.288 1.83 Trimethoprim 0.000 0.530 0.093 0.003 0.000 0.000 0.000 0.000 0.919 1.545 0.86 Tulathromycin 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.014 0.014 0.01 Tylosin 0.012 0.254 0.000 0.000 0.000 0.000 0.000 0.000 9.640 9.906 5.51 Valnemulin 0.113 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.113 0.06 Total (t) 54.705 7.586 1.144 0.035 0.007 0.000 0.169 0.171 116.058 179.875 100.00 % within Total 30.41 4.22 0.80 0.02 0.00 0.00 0.09 0.10 64.52 100.00

Sw = Swine; Po = Poultry; Bo = Bovine; Eq = Equine; La = Lagomorphs; Fe = Feline; Ca = Canine; All = All species; Ms = Multispecies. ESVAC categorization criteria: All species - All target-species; Multispecies - More than one target-specie for the same active ingredient(s)

______Balbino M. Rocha, 2013 57 Cha pte r I: The Big Picture on Antibacterial Resistance

Table 14: Distribution of the antibacterial dosage forms in each animal species, in Portugal in 2010 (Adapted from DGAV, 2010). 254

Antibacterial Animal Species (t) Total % within Dosage form Sw Po Bo Eq La Ca All Ms (t) Total

Parenteral injection 0.550 0.147 0.407 0.032 0.000 0.000 0.147 8.532 9.815 5.46 IMM 0.000 0.000 0.581 0.000 0.000 0.000 0.000 0.048 0.629 0.35 IMM - DC 0.000 0.000 0.025 0.000 0.000 0.000 0.000 0.000 0.025 0.01 IU 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.018 0.018 0.01 PO - paste 0.000 0.000 0.000 0.003 0.000 0.000 0.000 0.004 0.007 0.00 PO - powder (indv.) 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.025 0.025 0.01 PO - powder (group) 0.320 0.015 0.000 0.000 0.000 0.000 0.017 7.246 7.597 4.22 PO - solution (indv.) 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.009 0.009 0.00 PO - solution (group) 0.559 7.169 0.000 0.000 0.007 0.000 0.008 15.945 23.687 13.17 PO - feed premix 53.276 0.254 0.131 0.000 0.000 0.000 0.000 84.092 137.753 76.58 PO - tablet 0.000 0.000 0.000 0.000 0.000 0.169 0.000 0.139 0.309 0.17 Total (t) 54.705 7.586 1.144 0.035 0.007 0.169 0.171 116.058 179.875 100.00 % within Total 30.41 4.22 0.80 0.02 0.00 0.09 0.10 64.52 100.00

Sw = Swine; Po = Poultry; Bo = Bovine; Eq = Equine; La = Lagomorphs; Ca = Canine; All = All species; Ms = Multispecies. * Values for felines correspond all to 0.000 t ESVAC categorization criteria: All species - All target-species; Multispecies - More than one target-specie for the same active ingredient(s)

In the sales of active ingredients by animal species (Table 13), swine and poultry are the single target-species to which consumption of these compounds is the highest (Tables 12 & 13). In fact swine contribute to 85.95% (54.705 t) of all antibacterial active ingredients sold in 2010, and when poultry consumption (7.586 t) is added, these two species constitute 97.87% of all single target-species consumption of antibacterials. Also, the most used antibacterial active ingredient in swine is amoxicillin (15.863 t), while in poultry is enrofloxacin (4.909 t), bovine cattle is cloxacillin (0.354 t), lagomorphs is sulfaquinoxaline (0.007 t), and equines is benzylpenicillin (0.032 t). In companion animals, amoxicillin is the mostly used active ingredient (0.095 t). In the sales of active ingredients by dosage form (Table 14), feed premixes corresponded to the highest dosage form sold in 2010 (137.753 t), comprising 76.58% of all sales, followed by other oral forms (17.57%) and parenteral forms (5.46%). 254

9.6. Applications of Antibacterials in Animal Husbandry Operations

The incidence of infectious diseases varies between animal species and consequently the choice of antimicrobials used in the management of these diseases. The antibacterial agents normally used in these practices are generally the same, or belong to the same classes, as those used in human medicine. 28 Worldwide, the pathologies requiring the most extensive use of antibacterial agents for treatment or prophylaxis in the animal husbandry ______Balbino M. Rocha, 2013 58 Cha pte r I: The Big Picture on Antibacterial Resistance

environment are respiratory and enteric diseases in swine and calves, mastitis in dairy cattle, and colibacillosis in poultry (Table 15). 1,40,255 Due to the nature of this dissertation, the following section will focus on the applications of antibacterials pertaining dairy production operations.

9.6.1. Antibacterial Use in Dairy Production Systems

On most dairy operations around the world, antimicrobial agents are primarily used for the prophylaxis (metaphylaxis) and treatment of a variety of pathologies affecting calves/heifers and adult females in the different rearing and milk production stages, respectively. Data from the USDA's National Animal Health Monitoring System (NAHMS) Dairy 2007 study, conducted in 17 major dairy US states (representing 79.5% of all US dairy operations and 82.5% of all US dairy cows), estimated that the percentage of farms that treated adult dairy cows with any antibacterial agent was 85.4% for mastitis, followed by lameness (58.6%); respiratory disorders (55.8%); reproductive system pathologies (52.9%); diarrhea/other digestive problems (25.0%); and 6.9% for all other categories (Figure 21). 32

Mastitis was the most commonly treated pathology in adult animals in these herds (16.4%), followed by reproductive system pathologies (e.g., metritis in fresh cows) (7.4%), lameness (e.g., footrot/footwarts) (7.1%), respiratory disorders (2.8%) and enteric disorders (e.g., diarrhea) (1.9%) (Figure 22). In preweaned and weaned calves it was estimated that 17.9% and 1.6% of these animals were treated with antibacterials for enteric disorders (e.g., diarrheas), respectively. About 1 of 10 preweaned heifers (11.4%) were treated for respiratory disorders, with this proportion declining in weaned animals (5.5%) (Figure 23). 32

Figure 21: Proportion of US dairy operations in 2007 that treated cows with any antibacterial for the main diseases/disorders (Adapted from USDA APHIS, 2008). 32

______Balbino M. Rocha, 2013 59 Cha pte r I: The Big Picture on Antibacterial Resistance

Figure 22: Proportion of US adult dairy cows treated with antibacterials for the main diseases/disorders in 2007 (Adapted from USDA APHIS, 2008). 32

Antibacterials are also used regularly on dairy operations for disease prevention, with over 90% of dairy farms carrying out antibacterial dry cow therapy – one of the more consistent uses of antibacterials in dairy herds, with the application of IMM antibacterials following the last milking of lactation in adult cattle. 32, 256 Furthermore, in countries like the USA, the majority of the almost 60% of dairy farms that use milk replacers, for some or entirety of the feeding program in preweaned heifers, employ medicated milk replacers (with ionophores) as metaphylaxis of diarrhea and other digestive disorders. 32

Figure 23: Proportion of preweaned and weaned heifers treated with antibacterials in the US in 2007 for the main diseases/disorders (Adapted from USDA APHIS, 2008). 32

The most widely used classes of antibacterials used in US dairy operations for disease prevention and treatment include: 1) Cephalosporins, lincosamides and non-cephalosporin β- lactams for mastitis; 2) Penicillin G/dihydrostreptomycin and cephalosporins for dry cow ______Balbino M. Rocha, 2013 60 Cha pte r I: The Big Picture on Antibacterial Resistance

therapy; 3) Tetracyclines, cephalosporins, and non-cephalosporin β-lactams for lameness and reproductive disorders; 4) Cephalosporins, and non-cephalosporin β-lactams for respiratory disorders; and 5) Sulfonamides and tetracyclines for respiratory and enteric disorders in calves. 32

In Europe, as mentioned in previous sections, studies like the NAHMS Dairy 2007 study are scarce. 2010 data from Denmark (one of the few countries with organized and reliable data) estimated that penicillins (mainly narrow spectrum) were the major class employed in mastitis therapy. Also in Denmark, tetracyclines (mainly oxytetracyclines) and macrolides comprised the major drugs of choice in the treatment of respiratory and enteric disorders in calves. 4,32

______Balbino M. Rocha, 2013 61 Cha pte r I: The Big Picture on Antibacterial Resistance

1

(Adapted from EMA, 2009). (AdaptedEMA, from

d type of treatment treatment type of d

producing animal species including organ, pathogen an pathogen speciesorgan, including producinganimal

-

Examples of diseases on the different food different the on diseases of Examples

:

Table 15Table ______Balbino M. Rocha, 2013 62 Cha pte r I: The Big Picture on Antibacterial Resistance

III. Dissemination and Transfer of Resistant Bacteria and Resistance Genes from Animals to Humans

In any ecosystem, the use of antibacterials may select for antibacterial-resistant bacteria. 257,258 As portrayed in the previous subchapter, antibacterial drugs used for food- producing animals are often administered in a way that increases resistance: subtherapeutic dosing, mass treatment, long-term administration, or by addition to food and water for prophylaxis. 259 By the use of antibacterials in food-producing animals, commensal bacteria and bacteria potentially pathogenic to humans become reservoirs of resistance genes. In their basic cellular biology, pathogenic bacteria differ little from commensal bacteria. The consequences of this are: 1) both the benign commensal flora and pathogens will be affected by the use of antibacterials; 2) genetic exchange of resistance genes in the commensal ecosystems; and 3) re-inoculation of resistant bacteria and their genes in other ecosystems, including the human gut. 237,260 Bacteria exchange resistance genes, and these genes may ultimately enter bacteria pathogenic to man or opportunistic bacterial pathogens. Routine diagnostics only look for resistance in specific pathogenic agents. Passive Public Health surveillance data are based on the aggregation of routine diagnostic data, and are thus inadequate to quantify the magnitude of resistance reservoirs in normal gut flora of animals or humans. Nonetheless, specific studies show that the prevalence of ABR in the commensal bacteria of humans and animals is a good indicator of the selective pressure and reflect the potential for resistance in future infecting agents. 261

Most of the research and evidence relating to the potential for transfer of a resistance problem from animals to humans comes from a consideration of the epidemiology of zoonoses, mainly of Gram-negative bacteria infections such as Salmonella spp. and Campylobacter spp., and of what have become known as ‘indicator organisms’ – enterococci and E. coli – which cause no disease in animals (animal-pathogenic E. coli excluded) but can cause disease in man and which might turn out to be zoonotic. 11 Although sophisticated methods of phenotyping and genotyping have made it possible to carry out particularly accurate epidemiological studies, the epidemiology of these diseases is, however, still far from simple since there are many possible sources other than food-producing animals and many routes of transmission other than food of animal origin (Figure 24). 10,29 The important antibacterial-resistant strains in the Public Health context are the multidrug-resistant Salmonella spp., macrolide- or fluoroquinolone-resistant Campylobacter spp., multidrug- resistant E. coli, glycopeptide- or streptogramin-resistant enterococci, S. pneumoniae and S. aureus – particularly MRSA. 10

______Balbino M. Rocha, 2013 63 Cha pte r I: The Big Picture on Antibacterial Resistance

10. Sources and Routes for ABR Dissemination and Transfer

Although an animal origin is likely or can be proved for many outbreaks of infection, in which genotypically and phenotypically indistinguishable bacterial species are found in animals and in patients or carriers, the route by which an infection can reach an individual is complex. Also, most of the studies of the food chain ignore the fact, already noted, that there are potential sources of antibacterial-resistant microorganisms other than food-producing animals (Figures 24 & 25). 262

Figure 24: Possible routes of transmission of antibacterial-susceptible or -resistant gastrointestinal pathogens or normal intestinal flora between animals and humans (adapted from Phillips et al., 2004). 10

The primary reservoirs in most of the referred microorganisms are the alimentary tracts of a wide range of wild and domestic fauna, including food-producing animals (swine, poultry and ruminants). This can result in a wide variety of foodstuffs, including foodstuffs of both animal and plant origin, becoming contaminated and acting as a source of infection for humans. 11

Furthermore, it is generally accepted that adequate cooking destroys bacteria. No evidence indicates however that ABR strains are more refractory to cooking than are the

______Balbino M. Rocha, 2013 64 Cha pte r I: The Big Picture on Antibacterial Resistance

largely susceptible strains and most research has been on Salmonella, with no specific work being conducted on ABR campylobacters or the indicator E. coli and enterococci. One must also assume that as with Salmonella, inadequate cooking fails to decontaminate food. It is also known that Salmonella cross-contamination between uncooked and cooked food may occur if hygiene measures are inadequate in food outlets, and it may be that such cross- contamination occurs with other bacteria as well, including resistant strains. Again, there is no direct information regarding this subject. There is no knowledge of the degree, if any, of contamination of food on the plate just before its ingestion, by any of these organisms. 10

Since commensal and pathogenic bacteria (including resistant strains) can reach the general environment via sewage, 263 wild fauna, particularly rodents and birds, can acquire these environmental contaminants and pass them on via their excreta to grazing land or to the foodstuffs of food-producing animals. Vegetables may also be contaminated from sewage, especially in countries in which human feces are used as fertilizer. Although diminishing in Europe, fish farming involves the use of antibacterials, and fish as food may be contaminated with resistant bacteria. 4 Also, antibacterials are widely used to prevent bacterial diseases in plants: tetracyclines and aminoglycosides are used to protect fruit trees from fire blight. 264 That the author is aware of, no rigorous epidemiological studies of these potential reservoirs have been conducted to date. Therefore, the assumption that they make minor contributions to human enteric pathogen resistance is unfounded.

Figure 25: Reservoirs of ABR bacteria causing human infections. Schematic overview of some of the most important ABR pathogens and the overlap between the different reservoirs. As indicated some pathogens are strictly confined within the human reservoir, whereas others have a mainly or partly animal reservoir (Adapted from WHO, 2009 and WHO, 2012). 28,265

______Balbino M. Rocha, 2013 65 Cha pte r I: The Big Picture on Antibacterial Resistance

Direct transfer is also possible, not only from farm animals in contact with farm workers or veterinarians but also from domestic animals and pets. 266-268 Even in these cases, one cannot exclude the possibility that both animals and humans acquired the strains from a common source, or even that the organisms were transferred from man to his animals. Direct human-to-human transfer may also occur, especially when hygiene measures are inadequate or in other contexts such as nursing homes. 269 Furthermore, certain bacteria can persist in biofilms in the domestic toilets of those who have gastroenteritis 270 and in the more general environment of infected children. 271

11. Bacteria of Public Health Concern 11.1. Foodborne Pathogens (Salmonella & Campylobacter)

Campylobacteriosis is the most frequently reported zoonotic disease in the EU and EEA/EFTA countries, followed by salmonellosis. The rate of confirmed cases of human campylobacteriosis has though remained stable during the most up-to-date reported period – 2006-2009 (Figure 26). Unlike campylobacteriosis, reported salmonellosis cases in humans have been steadily decreasing over the past years, with a trend that has been statistically significant over the same 2006–2009 period (Figure 27). This decline is assumed to be mainly due to the increasing implementation of national control measures by the EU- MS against Salmonella within the poultry industry, especially vaccination of laying hens and broilers. The large decrease observed (Table 16), especially in S. Enteritidis cases, supports this observation. 251 This serotype, along with S. Typhimurium, still correspond to the most commonly isolated serotypes from human infections (Table 16), as a result of their extensive dissemination among poultry since 1980. 29,272

Figure 26: Trend and number of reported confirmed human campylobacteriosis cases by month, in the EU and EEA/EFTA countries, 2006–09. (Adapted from ECDC, 2011). 272 ______Balbino M. Rocha, 2013 66 Cha pte r I: The Big Picture on Antibacterial Resistance

Figure 27: Trend and number of reported confirmed human salmonellosis cases by month, in the EU and EEA/EFTA countries, 2006–09. (Adapted from ECDC, 2011). 272

Table 16: Salmonella serotypes most frequently reported from human salmonellosis cases in the EU and EEA/EFTA countries and percentage change, 2008–09 (Adapted from ECDC, 2011).272

Serotype 2008 2009 % change

Enteritidis 70.936 53.951 - 24% Typhimurium 27.170 23.990 - 12% Infantis 1.378 1.632 18% Newport 838 788 -6% Virchow 935 774 - 17% Derby 662 675 2% Hadar 545 513 - 6% Saintpaul 444 473 7% Kentucky 518 469 -9% Stanley 619 456 -26%

Source: Country reports from Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Sweden, Spain and the UK.

In humans, the majority of Salmonella infections result in mild, self-limiting, gastrointestinal illness and usually do not require antibacterial treatment. However, on rare occasions, infection may cause severe enteric disease (with an associated life-threatening dehydration), or when invasive, bacteraemia or meningitis. In these cases, effective antibacterials are essential for treatment and can be life-saving. The treatment of choice for Salmonella infections is fluoroquinolones (ciprofloxacin) for adults and 3rd-generation cephalosporins for children. 272 In animals, particularly of certain species, subclinical infections can be common. Salmonella may spread rapidly and easily between animals in a herd or flock, without manifestation of any clinical signs in some cases and animals may become intermittent or persistent carriers. In other species, clinical disease may occur

______Balbino M. Rocha, 2013 67 Cha pte r I: The Big Picture on Antibacterial Resistance

following Salmonella infection and, in particular, cattle may succumb to fever, diarrhea and abortion following infection, particularly with some serovars. In calves, Salmonella can cause outbreaks of diarrhea with high mortality. Fever and diarrhea are less common in swine than in cattle and sheep, and poultry may also show no signs of infection. 11

As to Campylobacter infections, human patients may experience mild to severe symptoms, commonly including watery, sometimes bloody diarrhea, abdominal pain, fever, headache and nausea. Just like in salmonellosis, infections are usually self-limiting and short-lasting, usually not requiring antibacterial therapy. Extra-intestinal infections, invasive infections or post-infection complications such as reactive arthritis and neurological disorders can occur but these are sporadic. C. jejuni is a recognized antecedent cause of Guillain- Barré syndrome, a form of paralysis that can sometimes result in dysfunction of the respiratory and neurological systems and can even be fatal. Although the numbers of cases of invasive human campylobacteriosis are usually extremely low, resistance to antibacterials in Campylobacter isolates is of concern, owing to the high number of human cases of gastroenteritis they cause and because some of these cases require treatment. In the joint scientific opinion from ECDC, EFSA and EMA, resistance to quinolones (including fluoroquinolones such as ciprofloxacin) and macrolides in Campylobacter was regarded as being of major Public Health concern and relevance on the basis of current evidence of possible human health consequences. 1

In the latest (2010) data from the EU, resistance in Salmonella isolates from reported human salmonellosis cases was high for tetracyclines, ampicillin and sulfonamides. In contrast, resistance to the clinically important antibacterials ciprofloxacin and cefotaxime was relatively low. The highest resistance levels among S. Enteritidis from human isolates were to the quinolones nalidixic acid and ciprofloxacin, whereas in S. Typhimurium it was reported for ampicillin, tetracyclines, sulfonamides and streptomycin. 225 Information on ABR in Campylobacter isolates from human cases of campylobacteriosis reported the highest resistance levels in C. jejuni for ciprofloxacin and nalidixic acid. Levels of resistance to erythromycin, the first-choice drug for the treatment of campylobacteriosis in humans, were generally low but were higher in C. coli than in C. jejuni. 272

Also in 2010, moderate to high levels of resistance to many antibacterials were reported in Salmonella isolates from food-producing animals and meat derived from those animals by EU-MS, particularly to antibacterials such as ampicillin, tetracyclines and sulfonamides, which have been used therapeutically to treat the bacterial diseases of animals for many years. Resistance to ciprofloxacin was highest among Salmonella isolates from turkeys and broiler meat. Resistance to 3rd-generation cephalosporins was detected in Salmonella isolates from turkeys, broilers, swine, cattle and the meat derived from broilers and swine, but at levels considered to be low or very low. 11 ______Balbino M. Rocha, 2013 68 Cha pte r I: The Big Picture on Antibacterial Resistance

The observed levels of resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracyclines were much higher in S. Typhimurium than in S. Enteritidis isolates both from humans and from animals. This is likely because certain phage types of S. Typhimurium have an associated pattern of pentavalent resistance to these antibacterials. Foremost, among these, in recent years, has been S. Typhimurium DT104. In some EU-MS, S. Typhimurium DT104 is now declining in incidence and monophasic S. Typhimurium-like strains are emerging as the dominant serovars. These monophasic strains commonly, though not always, show resistance to ampicillin, streptomycin, sulfonamides and tetracyclines, and this is evident in the results for such isolates reported in 2010. 11

Among Campylobacter isolates from food-producing animals and meat, with the exception of some Nordic countries, very to extremely high levels of resistance to several antibacterials were reported by EU-MS in 2010. In particular, and similarly to the observed in human isolates, high levels of resistance to quinolones, especially ciprofloxacin, were also reported in C. jejuni isolates of poultry origin. This high level of resistance is of particular concern, since the EFSA BIOHAZ Panel, in its recent scientific opinion on the quantification of the risk of campylobacteriosis posed to humans by broiler meat, estimated that the handling, preparation and consumption of broiler meat may account for 20-30% of human campylobacteriosis cases, while 50-80% of cases may be attributed to the broiler reservoir as a whole. 1,11 In all reporting EU-MS, the level of resistance to erythromycin was highest in C. coli isolates from pigs and was lower in C. jejuni from bovine cattle and in C. coli and C. jejuni from broilers and broiler meat. These findings mirror those in many previous studies, in which macrolide-resistant isolates of C. coli from food-producing animals have mainly been of porcine origin. 273

11.2. Indicator (Commensal) Organisms

E. coli and Enterococcus spp., especially E. faecium and E. faecalis, are normally used in animal isolates as indicator organisms of, respectively, the Gram-negative and Gram- positive commensal intestinal flora. These three bacterial species are commonly isolated from animal feces, and most resistance phenotypes present in animal populations are present in these species. From an epidemiological perspective, ABR in indicator microorganisms can therefore be used to investigate the reservoir of resistance genes occurring in those bacteria and which could be transferred to bacteria that are pathogenic for humans or animals. In addition, the effects of use patterns of antibacterials in a given country and animal species, as well as trends in the occurrence of resistance can be studied more accurately in indicator organisms than in foodborne pathogens because all food-producing animals generally carry these indicator bacteria. 11

______Balbino M. Rocha, 2013 69 Cha pte r I: The Big Picture on Antibacterial Resistance

11.2.1. Enterococci

Enterococci belong to the normal bacterial flora of the gastrointestinal tract of humans, other mammals, birds and reptiles. Enterococci are regarded harmless commensals and are even believed to have positive effects on a number of gastrointestinal and systemic conditions. However, when the commensal relationship with the host is disrupted, enterococci can cause invasive diseases. Recently, the recognition of high risk clones such as those of the polyclonal subcluster CC17 in Enterococcus faecium suggests that some particular strains can act as true pathogens and not only as opportunistic commensals. Enterococci can cause a variety of clinical syndromes, including endocarditis, bacteraemia, meningitis, wound and urinary tract infections and are associated with peritonitis and intra-abdominal abscesses. 274

Table 17: Number of invasive E. faecalis and E. faecium isolates and proportion of high-level aminoglycoside- resistant E. faecalis and vancomycin-resistant E. faecium (%R), including 95% CI, reported per country in 2011 (Adapted from EARS-Net, 2012). 274

High-level Aminoglycoside-resistant E. faecalis Vancomycin-resistant E. faecium Country N %R (95%CI) N %R (95%CI) Austria 327 30.9 (26-36) 354 4.5 (3-7) Belgium 335 18.2 (14-23) 215 7.0 (4-11) Bulgaria 62 30.6 (20-44) 39 0.0 (0-9) Cyprus 54 18.5 (9-31) 17 0.0 (0-20) Czech Republic 556 46.2 (42-50) 211 7.6 (4-12) Denmark 45 31.1 (18-47) 615 1.3 (1-3) Estonia 32 19.1 (6-43) 15 0.0 (0-25) Finland 0 - 169 1.2 (0-4) France 955 20.0 (18-23) 569 1.4 (1-3) Germany 578 41.0 (37-45) 535 11.4 (9-14) Greece 653 37.4 (34-41) 424 23.1 (19-27) Hungary 461 48.6 (44-53) 120 0.8 (0-5) Iceland 19 0.0 (0-18) 13 0.0 (0-25) Ireland 244 29.9 (24-36) 347 34.9 (30-40) Italy 330 50.0 (44-56) 236 4.2 (2-8) Latvia 34 26.5 (13-44) 22 9.1 (1-29) Lithuania 48 43.8 (29-59) 26 7.7 (1-25) Luxembourg 27 44.4 (25-65) 24 4.2 (2-8) Malta 0 - 14 0.0 (0-23) Netherlands 363 33.3 (28-38) 481 1.0 (0-2) Norway 115 21.7 (15-30) 165 1.8 (0-5) Poland 190 48.4 (25-35) 202 8.4 (5-13) Portugal 403 29.8 (25-35) 208 20.2 (15-26) Romania 0 - 12 0.0 (0-26) Slovakia 188 49.5 (42-57) 101 4.0 (1-10) Slovenia 125 36.0 (28-45) 83 0.0 (0-4) Spain 917 39.3 (36-43) 542 1.5 (1-3) Sweden 707 19.2 (16-22) 353 0.0 (0-1) United Kingdom 75 16.0 (9-26) 302 8.9 (6-13)

The vast majority (around 80%) of clinical enterococcal infections in humans are caused by E. faecalis, while E. faecium accounts for the majority of the remaining 20%. ______Balbino M. Rocha, 2013 70 Cha pte r I: The Big Picture on Antibacterial Resistance

Epidemiological data collected over the last decades have documented the emergence of enterococci as important nosocomial pathogens, exemplified by the expansion of a major hospital-adapted polyclonal subcluster CC17 in E. faecium, and by CC2 and CC9 E. faecalis. The emergence of particular clones and clonal complexes of E. faecalis and E. faecium was paralleled by increases in resistance to glycopeptides (e.g., VRE) and high-level aminoglycosides. These two antibacterial groups represent the few remaining therapeutic options for treatment of human infections caused by E. faecium when resistance has emerged against penicillins. Besides the fact that infections caused by resistant enterococci are difficult to treat, they are highly tenacious and thus easily disseminate in the hospital setting. There is also the concern that the genes that encode for vancomycin resistance may spread to more virulent bacteria such as S. aureus. Fortunately, compared to ten years ago, there are more agents available to treat infections caused by VRE (e.g., linezolid). 274

Figure 28: (A) E. faecalis: trends of high-level resistance to aminoglycosides by country, 2008–2011. (B) E. faecium: Trends of resistance to vancomycin by country 2008–2011. Only countries that reported 20 isolates or more per year were included. The symbols ▲ and ▼ indicate significant increasing and decreasing trends, respectively. The asterisks indicate significant trends in the overall data that were not supported by data from laboratories consistently reporting for all five years (Adapted from EARS-Net, 2012). 274

The latest information on ABR in human clinical enterococcal isolates in Europe (29 countries) was reported in 2012 by the EARS-Net. High-level aminoglycoside resistance in E. faecalis seems stable in Europe but at a relatively high level of resistance. The majority of ______Balbino M. Rocha, 2013 71 Cha pte r I: The Big Picture on Antibacterial Resistance

countries reported proportions of resistant isolates between 25% and 50% (Table 17). However, a consistent decrease was reported by the UK, Greece, Portugal, Cyprus and Belgium; no significant increasing trends were observed (Figure 28A). 274

The prevalence in humans of vancomycin resistance in E. faecium (VRE) within Europe varies from <1 – 34.9% and seems to continue to decrease (Figure 28B). 275 In some countries (Greece, Slovenia, Sweden and the UK) the efforts to control glycopeptide- resistant enterococci are obviously successful and resulting in a continuous decrease of proportions of resistant isolates. Ireland, on the other hand, reported resistance around 35%, while most of the countries reported resistant proportions below 10% (Table 17). When considering data from laboratories reporting consistently for all four years, significant increasing trends were observed only for Germany, while decreasing trends were significant for Greece and the UK (Figure 28B). 274

Despite these stable trends, high levels of antibacterial-resistant enterococci remain a major infection control challenge in Europe. 274

The latest information on ABR in enterococcal isolates from food-producing animals and food in Europe was reported in 2010 by seven EU-MS and one non-MS (Switzerland). Most of the data related to isolates from broilers, swine and cattle and only two EU-MS (Denmark and Sweden) reported results from meat derived from those species. 11

Although there was a wide variation in the levels of resistance observed in reporting EU-MS, the resistance reported was at a high to extremely high level for many of the antibacterials. The highest resistance levels were observed among enterococcal isolates from broilers and swine, whereas resistance was at a lower level in isolates from cattle. However, given the small number of EU-MS reporting data, especially for cattle, the observed difference between the animal species was viewed with caution since it may not mirror a consistent situation throughout the EU. 239

Among both studied enterococci isolates from broilers, the level of resistance to tetracyclines and erythromycin was 56% and 47%, respectively, in E. faecium and 60% and 56%, respectively, in E. faecalis. In isolates from swine, tetracycline and erythromycin resistance levels were 53% and 35%, respectively, in E. faecium and 71% and 38%, respectively, in E. faecalis. In the case of E. faecium isolates from cattle, tetracycline and erythromycin resistance levels were 21% and 20%, respectively, while in E. faecalis isolates from cattle resistance levels were 26% for tetracyclines and 13% for erythromycin. 11

The observed high levels of resistance to macrolides are of particular importance because these substances have been defined as critically important antibacterials in human medicine. The differences, noted in the paragraph above, in the occurrence of macrolide resistance in enterococcal isolates from poultry, calves and pigs have been considered to ______Balbino M. Rocha, 2013 72 Cha pte r I: The Big Picture on Antibacterial Resistance

reflect the different levels and patterns of usage of antibacterials in those different species. This also probably accounts for the widespread occurrence of tetracycline resistance in broilers and swine, because these species have frequently received treatment with this antibacterial. 235,276,277

11.2.2. Escherichia coli

Escherichia coli is the most common Gram-negative rod isolated from human blood cultures in clinical settings. It is the most frequent cause of bacteraemia; community- and hospital-acquired urinary tract infections; spontaneous and surgical peritonitis; skin and soft tissue infections due to multiple microorganisms; neonatal meningitis; and is one of the leading causative agents in foodborne infections worldwide. 274

Worldwide, rapidly increasing rates of both multi-drug resistance and resistance of E. coli to the single antibacterial agents under surveillance have been observed over the years, with European countries not being spared to this reality. The EARS-Net 2012 surveillance report, relative to data from 2008 through 2011, emphasizes this worrisome fact. 274

The highest proportions of resistant E. coli were reported for aminopenicillins ranging up to 77.6% (Cyprus) (Table 20). The proportion of reported E. coli isolates resistant to 3rd- generation cephalosporins has also increased significantly during the last years in half of the reporting countries. Among the isolates resistant to 3rd-generation cephalosporins, a high proportion (71.1-100%) was identified as ESBL-positive (Table 18). These data indicate that ESBL production is highly prevalent in 3rd-generation cephalosporin-resistant E. coli in European hospitals. 274

Fluoroquinolone resistance in E. coli has also continued to increase over the years in Europe. The situation becomes progressively dire and more than half of the countries are reporting resistance proportions higher than 20% (Table 20), with only two countries (Sweden and the Czech Republic) reporting a decreasing resistance trend. Four countries (Portugal, Belgium, Hungary and Italy) had significant increases in the proportion of isolates resistant to aminoglycosides and eleven countries reported proportions higher than 10%. This indicates that resistance to aminoglycosides is increasing even among the countries already reporting high levels of resistance. Combined resistance to all four antibacterials was reported in 3.9% of the isolates (Table 19). 274

The latest information on ABR in E. coli isolates from food-producing animals and food in Europe, regarding 2010, was reported in 2012 by EFSA/ECDC. A high level of resistance to several antibacterials was verified, with very or extremely high levels reported by some individual EU-MS. This is of significance for both human and animal health since E. coli

______Balbino M. Rocha, 2013 73 Cha pte r I: The Big Picture on Antibacterial Resistance

bacteria from food-producing animals and food thereof can form a reservoir of ABR genes that may be transferred to bacteria pathogenic to humans or animals (Figure 25). Some E. coli isolates occurring in the intestinal flora of animals may also be directly pathogenic to humans. 11

rd Table 18: Number of invasive E. coli isolates resistant to 3 -generation cephalosporins (3GCREC) and proportion of ESBL-positive (%ESBL) among these isolates, as ascertained by the participating laboratories in 2011 (Adapted from EARS-Net, 2012). 274

Number of Number Country % ESBL Laboratories of 3GCREC Austria 27 237 91.1 Bulgaria 12 30 93.3 Czech Republic 42 305 89.5 Denmark 3 45 71.1 Estonia 5 11 100 France 16 99 83.8 Germany 12 190 92.1 Hungary 9 44 100 Ireland 26 189 82 Latvia 5 21 90.5 Lithuania 8 27 100 Luxembourg 5 29 96.6 Poland 33 98 85.7 Portugal 14 159 97.5 Slovakia 7 154 83.1 Slovenia 9 88 94.3 Spain 26 381 88.5

Only data from laboratories consistently reporting the ESBL test results for all isolates identified as resistant to 3rd-generation cephalosporins and from countries with at least 10 of such isolates were selected for the analysis.

Table 19: Overall resistance and resistance combinations among invasive E. coli isolates tested against aminopenicillins, fluoroquinolones, 3rd-generation cephalosporins and aminoglycosides (n=54338) in Europe, 2011 (Adapted from EARS-Net, 2012). 274

Number of % of Resistance Pattern Isolates Total Fully susceptible 22 586 41.6 Single resistance (to indicated drug classes) Aminopenicillins 17 954 33.0 Fluoroquinolones 1 236 2.3 Aminoglycosides 103 0.2 Resistance to 2 classes of antibacterial drugs Aminopenicillins + fluoroquinolones 4 610 8.5 Aminopenicillins + 3rd-generation cephalosporins 964 1.8 Aminopenicillins + aminoglycosides 797 1.5 Fluoroquinolones + aminoglycosides 86 0.2 Resistance to 3 classes of antibacterial drugs Aminopenicillins + fluoroquinolones + aminoglycosides 1 867 3.4 Aminopenicillins + 3rd-generation cephalosporins + fluoroquinolones 1 796 3.3 Aminopenicillins + 3rd-generation cephalosporins + aminoglycosides 201 0.4 Resistance to 4 classes of antibacterial drugs Aminopenicillins + 3rd-generation cephalosporins + fluoroquinolones + aminoglycosides 2 138 3.9 ______Balbino M. Rocha, 2013 74 Cha pte r I: The Big Picture on Antibacterial Resistance

Table 20: Number and proportion of invasive E. coli isolates resistant to aminopenicillins, 3rd-generation cephalosporins, fluoroquinolones, aminoglycosides and multi-drug resistant (%R), including 95% CI, reported per country in 2011 (Adapted from EARS-Net, 2012). 274

3rd-generation Multi-drug Aminopenicillins Fluoroquinolones Aminoglycosides Country Cephalosporins Resistance * %R %R %R %R %R N N N N N (95%CI) (95%CI) (95%CI) (95%CI) (95%CI) Austria 3148 50.3 3162 22.3 3160 9.1 3144 7.4 3121 2.6 (49-52) (21-24) (8-10) (7-8) (2-3) Belgium 3507 58.7 3549 21.5 3985 6.0 3831 9.3 3331 1.4 (57-60) (20-23) (5-7) (8-10) (1-2) Bulgaria 152 60.5 179 30.2 179 22.9 179 17.3 179 10.1 (52-68) (24-37) (17-30) (12-24) (6-15) Cyprus 134 77.6 137 47.4 138 36.2 138 23.9 137 18.2 (70-84) (39-56) (28-45) (17-32) (12-26) Czech Rep. 2683 60.7 2682 23.5 2684 11.4 2674 8.8 2667 3.7 (59-63) (22-25) (10-13) (8-10) (3-4) Denmark 3638 47.9 3583 14.1 2532 8.5 3638 6.4 2529 3.0 (46-50) (13-15) (7-10) (6-7) (2-4) 9.9 12.2 4.8 1.1 Estonia 0 - 312 90 314 89 (7-14) (6-21) (3-8) (0-6) Finland 1826 36.3 2420 10.8 2419 5.1 2420 5.3 2419 2.7 (34-39) (10-12) (4-6) (4-6) (2-3) France 8784 55.1 8694 17.9 8479 8.2 8742 7.9 8428 2.6 (54-56) (17-19) (8-9) (7-8) (2-3) Germany 3638 52.3 3636 23.7 3642 8.0 3645 7.6 3631 3.6 (51-54) (22-25) (7-9) (7-9) (3-4) Greece 1297 54.5 1433 26.6 1435 14.9 1434 16.8 1431 10.8 (52-57) (24-29) (13-17) (15-19) (9-13) Hungary 991 64.7 1213 31.2 1224 15.1 1226 14.8 1209 8.3 (62-68) (29-34) (13-17) (13-17) (7-10) Iceland 129 48.1 121 14.0 130 6.2 129 6.2 120 0.8 (39-57) (8-22) (3-12) (3-12) (0-5) Ireland 2118 69.5 2153 22.9 2166 9.0 2158 10.2 2148 3.6 (68-72) (21-25) (8-10) (9-12) (3-4) Italy 1530 67.1 1899 40.5 1870 19.8 1985 18.3 1745 10.3 (65-69) (38-43) (18-22) (17-20) (9-12) Latvia 130 54.6 131 16.8 132 15.9 132 11.4 131 9.2 (46-63) (11-24) (10-23) (7-18) (5-15) Lithuania 383 47.8 381 12.9 385 7.0 382 9.7 378 2.4 (43-53) (10-17) (5-10) (7-13) (1-4) Luxembourg 353 52.1 353 24.1 353 8.2 354 8.2 353 2.8 (47-57) (20-29) (6-12) (6-12) (1-5) Malta 219 53.0 219 32.0 219 12.8 219 15.5 219 9.6 (46-60) (26-39) (9-18) (11-21) (6-14) Netherlands 4425 48.5 4427 14.3 4408 5.7 4431 7.8 4400 2.2 (47-50) (13-15) (5-6) (7-9) (2-3) Norway 2617 39.1 2505 9.0 2523 3.6 2470 4.1 2259 1.2 (37-41) (8-10) (3-4) (3-5) (1-2) Poland 934 62.0 1141 27.3 938 11.7 1171 8.4 902 4.0 (59-65) (25-30) (10-14) (7-10) (3-5) Portugal 1963 56.5 1917 27.2 1901 11.3 1962 16.1 1891 7.5 (54-59) (25-29) (10-13) (14-18) (6-9) Romania 22 68.2 46 30.4 91 22.0 46 19.6 46 10.9 (45-86) (18-46) (14-32) (9-34) (4-24) Slovakia 608 68.6 737 41.9 738 31.0 738 17.9 737 12.9 (65-72) (38-46) (28-35) (15-21) (11-16) Slovenia 1002 53.9 1002 20.7 1002 8.8 1002 9.8 1002 4.1 (51-57) (18-23) (7-11) (8-12) (3-6) Spain 5592 65.6 5597 34.5 5600 12.0 5603 14.8 5594 4.9 (64-67) (33-36) (11-13) (14-16) (4-6) Sweden 1023 34.8 3295 7.9 3939 3.0 3203 3.7 2844 1.0 (32-38) (7-9) (3-4) (3-4) (1-1) UK 5074 62.8 5564 17.5 5182 9.6 5661 8.2 5005 3.6 (61-64) (17-19) (9-10) (7-9) (3-4)

* Multi-drug resistance defined as resistance to 3rd-generation cephalosporins, fluoroquinolones and aminoglycosides.

______Balbino M. Rocha, 2013 75 Cha pte r I: The Big Picture on Antibacterial Resistance

Resistance to antibacterials recognized as critically important in human medicine, such as fluoroquinolones and 3rd-generation cephalosporins, was also observed in the indicator E. coli isolates. Considering all reporting EU-MS, ciprofloxacin resistance was 29% in broilers, 2% in swine and 15% in cattle. Resistance to 3rd-generation cephalosporins was generally low. Similarly to ciprofloxacin was highest in broilers, at 5%, compared with 3% in cattle and 1% in swine. However, resistance to these compounds was still generally low. The occurrence of resistance to gentamicin was highest in cattle, at 9%, whereas it was 4% in broilers and 2% in swine. In most cases, the level of resistance to nalidixic acid was similar to that to ciprofloxacin, suggesting that mutation in the topoisomerase enzymes (gyrA or parC) may, in these cases, have been responsible for resistance. However, in some EU-MS, the level of resistance to ciprofloxacin was higher than that found for nalidixic acid, suggesting that mechanisms such as transferable fluoroquinolone resistance conferred by qnr genes may have been the responsible resistance mechanism, as such plasmid-mediated mechanisms can result in that phenotypic pattern of resistance. 11

11.3. Other Gram-negative Bacteria

In addition to Salmonella spp. and E. coli, there are many more Gram-negative bacteria that cause serious disease in health-care settings. 278 Examples include Enterobacter spp., P. aeruginosa, Serratia spp., Klebsiella spp. and Acinetobacter spp. Some may be untreatable with any antibacterial agent, including polymyxins. 279,280 Other examples include P. aeruginosa and Burkholderia spp., to which no effective antibacterials can be used in serious infections such as those acquired in intensive care units, patients with cystic fibrosis and complicated lung infections. Older and relatively toxic antibacterials (e.g., polymyxins) are increasingly being used as IV therapy as no other option may often be available to treat these resistant bacteria. 281 Fosfomycin is also being increasingly used in the treatment of multidrug-resistant Gram-negative infections. 27

Furthermore, as mentioned in the above sections, ESBL-producing bacteria (e.g., K. pneumoniae, P. aeruginosa and E. coli) are also a currently growing Public Health threat in hospital and community settings. Extensive empirical use of carbapenems, one of the few still effective groups of antibacterials, may lead to a large-scale resistance to these last resort antibacterials in a nearby future. In fact, the first carbapenem-resistant strains of K. pneumoniae and P. aeruginosa have already been identified in Europe. 274

11.4. Staphylococcus aureus

Staphylococcus aureus is a Gram-positive bacterium that colonizes the skin of about 30% of healthy humans. Although mainly an asymptomatic colonizer, S. aureus is one of the ______Balbino M. Rocha, 2013 76 Cha pte r I: The Big Picture on Antibacterial Resistance

most common, virulent bacteria that cause infections, especially health-care-associated infections. 272,282 Its oxacillin-resistant form – MRSA – is the most important cause of antibacterial-resistant health-care-associated infections worldwide. Since health-care- associated MRSA infections add to the number of infections caused by methicillin- susceptible S. aureus, a high incidence of MRSA adds to the overall burden of infections caused by this species in hospitals. 253 Moreover, infections with MRSA may result in prolonged hospital stays and up to 10% higher mortality rates, 283 owing mainly to the increased toxicity and limited effectiveness of alternative treatment regimens. In some cities, over 50% of community-acquired S. aureus infections are now due to MRSA. 27,274

Table 21: Number and proportion of invasive S. aureus isolates resistant to methicillin (MRSA) and rifampin (RIF), including 95% CI, reported per country in 2011 (Adapted from EARS-Net, 2012). 274

Methicillin Rifampin Country N %MRSA (95%CI) N %RIF (95%CI) Austria 1966 7.4 (6-9) 1850 0.3 (0-1) Belgium 1744 17.4 (16-19) 1014 0.6 (0-1) Bulgaria 214 22.4 (17-29) 162 14.2 (9-21) Cyprus 113 41.6 (32-51) 113 0.0 (0-3) Czech Republic 1555 14.5 (13-16) 782 1.8 (1-3) Denmark 1452 1.2 (1-2) 1452 0.1 (0-0) Estonia 116 1.7 (0-6) 3 0.0 (0-71) France 4716 20.1 (19-21) 4278 1.0 (1-1) Germany 2388 16.1 (15-18) 1656 0.7 (0-1) Greece 784 39.2 (36-43) 0 - Hungary 1156 26.2 (24-29) 570 0.4 (0-1) Iceland 71 2.8 (0-10) 3 0.0 (0-71) Ireland 1057 23.7 (21-26) 835 1.0 (0-2) Italy 1261 38.2 (36-41) 970 4.3 (3-6) Latvia 192 9.9 (6-15) 186 0.5 (0-3) Lithuania 278 5.4 (3-9) 158 0.6 (0-2) Luxembourg 127 20.5 (14-29) 90 0.0 (0-4) Malta 130 49.2 (41-59) 130 0.8 (0-4) Netherlands 1801 1.4 (1-2) 1581 0.4 (0-1) Norway 1223 0.3 (0-1) 446 0.0 (0-1) Poland 860 24.3 (21-27) 135 27.4 (20-36) Portugal 1307 54.6 (52-57) 1092 1.7 (1-3) Romania 107 50.5 (41-60) 101 7.9 (3-15) Slovakia 560 25.9 (22-30) 478 1.3 (0-3) Slovenia 464 7.1 (5-10) 443 0.5 (0-2) Spain 1950 22.5 (21-24) 1826 0.5 (0-1) Sweden 3099 0.8 (1-1) 2456 0.2 (0-1) United Kingdom 3408 13.6 (13-15) 1777 0.6 (0-1)

In Europe, the latest information (29 countries) on ABR in human clinical S. aureus isolates was recently reported (2012) by the EARS-Net. The proportion of S. aureus isolates found to be MRSA is stabilizing or decreasing in most European countries. Six countries (Belgium, Germany, Estonia, France, Ireland and the UK) reported sustained decreasing

______Balbino M. Rocha, 2013 77 Cha pte r I: The Big Picture on Antibacterial Resistance

trends while four (Luxembourg, Romania, Hungary and Poland) reported increasing trends (Figure 29). MRSA remains, though, a Public Health priority as the proportion of MRSA is still above 25% in eight of 29 countries (Table 21). Portugal leads this statistic with 54.6%. The occurrence of resistance to rifampin, which is recommended in combination with other antibacterials to treat various staphylococcal infections, remains, on the other hand, low (<5%) in most European countries, with only Bulgaria and Poland indicating worrisome resistance proportions – 27.4% and 14.2%, respectively (Table 21). 274

Figure 29: S. aureus: Trends of resistance to methicillin (MRSA) by country, 2008–2011. Only countries that reported 20 isolates or more per year were included. The symbols ▲ and ▼ indicate significant increasing and decreasing trends, respectively. The asterisks indicate significant trends in the overall data that were not supported by data from laboratories consistently reporting for all four years (Adapted from EARS-Net, 2012). 274

Up until recent years, reports on MRSA in livestock were mainly limited to occasional detections in dairy cattle mastitis. 284, 285 Since 2005 however, studies show the existence of a MRSA clone, CC398, which has been reported colonizing swine, 286-288 veal calves, 289 broilers 290 and dairy cattle. 291 This strain, also referred to as livestock-associated MRSA (LA-MRSA) or sequence type 398 (ST398), has also been detected in meat 292,293 but, like for other MRSA, the risk this might pose is rather unclear. For now, the most worrying aspect

______Balbino M. Rocha, 2013 78 Cha pte r I: The Big Picture on Antibacterial Resistance

seems to be its capacity to spread to humans. 294 Until recently, identifying the LA-MRSA strain resulted in classifying all individuals with close contact with swine or veal calves as a high-risk population for MRSA carrier status, consequently treated as such. In the meantime, more information has become available regarding the clinical picture associated with LA- MRSA, with or without comparison to hospital-acquired MRSA. LA-MRSA was found not to spread easily to other patients in the hospital. 295-298 The total number of patients with sepsis caused by LA-MRSA is minute, likely about 5 within a total of 30 cases of sepsis caused by MRSA per year. In recent years, patients with LA-MRSA infections have been found that cannot be related though to contact with food-producing animals. 299

The latest information on MRSA isolates from food-producing animals, pets and food in Europe (8 countries) was reported in 2012 by EFSA/ECDC, regarding 2010 data. MRSA and LA-MRSA were detected in a number of different animal species, including pigs, broilers, turkeys, cattle, dogs and solipeds, at levels ranging from 0% to 79% among reporting MS. 11

11.5. Streptococcus pneumoniae

Streptococcus pneumoniae is a common cause of disease, especially among young children, elderly people and patients with immunocompromised functions. The clinical spectrum ranges from upper airway infections, such as sinusitis, and otitis media to pneumonia and invasive bloodstream infections and meningitis. 300-302 In fact, S. pneumoniae is the most common cause of pneumonia worldwide and it is estimated that approximately three million people die of pneumococcal infections every year. 274 This bacteria does not have non-human reservoirs and around 80 different serotypes have been described, with the serotype distribution varying with age, disease and geographical region. Interestingly, serotypes most frequently involved in pneumococcal disease or colonization in infants are also most frequently associated with ABR. 27,274 Increasing levels of resistance are seen to all antibacterial agents, particularly to penicillins. Vancomycin is still very reliable in all circumstances to treat serious pneumococcal disease (including meningitis), although its penetration into CSF is relatively poor and it is not absorbed when given orally. Other agents such as linezolid appear to be effective as resistance in pneumococci is currently very low. Oral therapy is very important for the treatment of many infections other than meningitis. High dosages of oral amoxicillin appear to be effective when therapy is needed, even if intermediate penicillin resistance is present. Unfortunately increasing numbers of pneumococci are developing resistance to oral tetracyclines, co-trimoxazole and macrolides, limiting therapeutic options through this route. 300-302

______Balbino M. Rocha, 2013 79 Cha pte r I: The Big Picture on Antibacterial Resistance

IV. Response to the Increasing Burden of ABR: Control Strategies and Interventions – The One Health Approach

Clearly there is still so much to learn regarding the development and dissemination of bacterial ABR related to the animal production environment and potential human health implications. The emergence of ABR among human and veterinary bacterial pathogens is a serious crisis and cannot be solved in isolation. Hence the ‘‘One Health’’ concept. This concept is a worldwide strategy for expanding interdisciplinary collaborations and communications in all aspects of health care for humans, animals and the environment (Figure 30). Only this way can we better understand why the problem of ABR is currently so pervasive and how we should best intervene to improve the situation. An example of an interdisciplinary collaboration is the FAO-OIE-WHO Collaboration through the Tripartite Concept Note. This tripartite relationship envisages complementary work to develop normative standards and field programs to achieve One Health goals. 20 With this holistic approach in mind, several strategies have been proposed to approach and manage the ABR problem. 303

Figure 30: ECDC promotional One Health poster (Adapted from www.ecdc.europa.eu).

Pharmaceutical companies continue to make great advances in developing new classes of antibacterials. The actual implementation of these is still many years down the road. This may result in a “window of vulnerability” where bacterial pathogens of animal and human origin will become increasingly resistant to current available antibacterials, limiting therapeutic options. With this in mind, the first response to increasing levels of ABR must be to reduce the selective pressure generated by antibacterial usage with prevention seeking to be the ultimate goal. It is thus necessary to establish guidelines for the prudent use of antibacterials in human health-care facilities and animal husbandry operations. Advances like vaccines, competitive exclusion products and probiotics have also been suggested.

An additional approach is to increase our understanding of the complex ecological, biochemical and molecular origins of ABR mechanisms that could provide insight into new ______Balbino M. Rocha, 2013 80 Cha pte r I: The Big Picture on Antibacterial Resistance

preventive and therapeutic strategies for overcoming resistance development and dissemination. Studies that investigate optimal uses of antibacterials (dose, interval, duration, exposure, etc.) in animals in hopes of minimizing bacterial resistance development are needed as well. As mentioned in section 6, currently little information is available on the appropriate PK/PD relationships for antibacterials of veterinary use.

Improved surveillance of emerging ABR phenotypes is also critical to the development of new treatment guidelines and intervention strategies, helping shape national and international policies regarding the use of antibacterials. Additionally, human and veterinary diagnostic laboratories should continue to play a key role in the timely detection of resistant bacterial pathogens.

Furthermore, infection control is possibly the single most important control measure that can be applied to the containment of ABR in hospital settings. Money spent in this area almost invariably results not just in the control of ABR but also in reductions in death rates.

The following sections will provide a more detailed description on the numerous control strategies and interventions directed towards the increasing burden of ABR.

12. Surveillance Systems to Track Antibacterial Use & Resistance

Effective surveillance is the cornerstone of national and international efforts to control ABR in both humans and animals. The ultimate goal of surveillance of antibacterial use and ABR is to provide the information, insights and tools needed to guide policy on the appropriate use of these compounds and to inform and evaluate resistance containment interventions at local, national and global levels. Decisions on interventions have to balance the call to provide effective antibacterial therapy to patients today with the need to preserve the usefulness of medicines for future generations. 28,304

Surveillance systems involve the systematic collection and analysis of health-related data, and consequent dissemination to those who will use them in decision-making on Public Health issues. Ongoing and routine ABR surveillance enables analyses to be made of resistance rates to antibacterials among bacteria infecting or colonizing individuals in given locations during defined time periods. The surveillance of antibacterial usage tracks both how much antibacterials are being used and how they are used by human and animal patients and respective health-care providers. Local surveillance units may be linked at national and international levels to provide national, regional and global surveillance information. 28

At the local level, the data are used to formulate recommendations for rational antibacterial use and standard treatment guidelines as well as ensuring that health-care providers comply with recommendations. At sub-national or national levels, data on ______Balbino M. Rocha, 2013 81 Cha pte r I: The Big Picture on Antibacterial Resistance

resistance and usage both inform policy-makers to decide on the development or revision of essential medicines lists, and to identify priorities for Public Health actions such as education campaigns or regulatory measures. At regional and global levels, surveillance data have proved to be invaluable advocacy tools in stimulating politicians and health-care providers into urgent action. 28

Efforts to establish surveillance of antibacterial use and ABR have been made in different parts of the world, with varying degrees of success, depending on the surveillance capacity and performance. 305,306

12.1. Surveillance of Antibacterial Resistance

Existing surveillance networks vary widely in scope. They range from networks covering sentinel laboratories to those that include all patient-care laboratories. For ABR surveillance, routine diagnostic laboratories, often within hospitals, are the primary source of data. They may be selective for only some bacteria or specimen types, or comprehensive covering all species and specimen types. Outputs can also vary from summaries to full reports on all isolates. Networking may be local, multi-centered, national or international. A number of regional surveillance initiatives have been launched in all WHO regions (Table 22). 307

Table 22: ABR surveillance networks for common bacterial pathogens in the WHO Regions (Adapted from Grundmann et al., 2011). 307

Years of Microorganisms Region Program Name Participants Activity under Surveillance 8 epidemic-prone AFR Integrated Disease Surveillance and Response (IDSR) 2002–present 43 countries pathogens Red Latinoamericana de Vigilancia a las Resistencias 21 countries 16 pathogens ABR 1996–present Antimicrobianas (Re-LAVRA) 519 laboratories All sample types 9 countries 7 pathogens Antibacterial Resistance in the Mediterranean (ARMed) 2001–2005 27 laboratories Blood and CSF EMR 28 species Regional Program for Surveillance of ABR Proposed All sample types 33 countries European Antibacterial Resistance Surveillance (EARSS) 1999–2009 917 laboratories EUR European Antibacterial Resistance Surveillance Network 28 countries 7 pathogens 2010–present (EARS-Net) 886 laboratories Blood and CSF SEAR National and regional surveillance system Proposed in 2010 22 species WPR Regional Program for Surveillance of ABR 1990–2000 13 countries All sample types

AFR: African Region; ABR: Region of the Americas; EMR: Eastern Mediterranean Region; EUR: European Region; SEAR: South-East Asia Region; WPR: Western Pacific Region.

In addition to the ABR data from routine clinical laboratories, reference laboratories produce more detailed information on selected specific isolates (e.g., for serotyping Salmonella isolates). ______Balbino M. Rocha, 2013 82 Cha pte r I: The Big Picture on Antibacterial Resistance

There are also large parts of the world where little, if any, surveillance is undertaken, and privately funded initiatives, such as the Asian Network of Surveillance of Resistant Pathogens (ANSORP), 308 the SENTRY Antibacterial Surveillance Program 309 and the Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) 310 have also contributed with data on important resistant bacteria. Integrating such data generates additional information and could also help in cross-validating clinical laboratory results. 28

Other initiatives such as the International Surveillance of Reservoirs of Antibiotic Resistance (ISRAR), coordinated by the Alliance for the Prudent Use of Antibiotics (APUA), collect and analyze environmental and veterinary commensal organisms which may serve as reservoirs for ABR. APUA Global Chapters, together with local laboratories in India, Republic of Korea, Turkey, Thailand, Vietnam, Bangladesh, Georgia and Uganda, collect bacteria from soil, water and animals, and carry out preliminary characterization and resistance analyses. 311 The WHO Advisory Group on Integrated Surveillance of Antibacterial Resistance (AGISAR) attempts to integrate surveillance of ABR in food-producing animals worldwide. 312

12.2. Surveillance of Antibacterial Usage

For the surveillance of antibacterial usage the situation is less clear-cut as it is not carried out within a single clinical discipline. Data on the usage of antibacterials may be obtained from many sources such as health-care facilities, pharmacies and drug procurement/sales services. This type of data has proved valuable in comparing usage in different countries in the same region over a period of time. 28

12.3. Combined Surveillance

In countries with functioning health systems, combined surveillance of antibacterial usage and resistance has been shown to be feasible and beneficial, contributing to a better understanding of the relationship between consumption and resistance and supporting important policy changes which modify ABR trends. An initiative of this type, involving several European countries over the past decade, has led to significant improvements in this field. An important element contributing to these achievements has been the collaborative efforts of two EU-funded projects currently managed by the ECDC: 1) The European Surveillance of Antibacterial Consumption Network (ESAC-Net), collects data from national statistics on antibacterial consumption in hospital and community settings from 34 European countries. ESAC-Net has developed and validated protocols for quantitative measurement and qualitative description of antibacterial use patterns, and has been a forceful advocate with national authorities and the European Commission to improve the use of antibacterials

______Balbino M. Rocha, 2013 83 Cha pte r I: The Big Picture on Antibacterial Resistance

in Europe; and 2) The European Antibacterial Resistance Surveillance Network (EARS-Net), already mentioned previously, collects data on seven pathogens of Public Health importance from blood and cerebrospinal fluid samples from over 1400 health-care facilities in over 28 European countries. ESAC-Net and EARS-Net findings are highlighted each year by the ECDC on European Antibiotic Awareness Day (November 18th), an annual campaign targeting national authorities, health-care providers, the media and the general public to raise awareness of the threat posed by the misuse of antibacterials and the challenges posed by resistant organisms. 250

13. Reducing Antibacterial Use in Humans

13.1. Promoting Rational Antibacterial UseRational use of medicines requires that patients receive medications appropriate to their clinical needs, in doses that meet their own individual requirements for an adequate period of time, and at the lowest cost to them and their community. However, this condition is, in many nations, far from achievement. On the other hand, irrational use takes into account actions like over-prescription, under- prescription, and prescription and dispensing of unnecessary antibacterial combinations: Physicians may prescribe too many drugs, expensive drugs or inappropriate drugs because of fear of treatment failure, lack of knowledge of the local ABR situation, real or perceived patients’ expectations, drug company promotional efforts, or for personal financial gain; Commercial outlets may also seek to maximize their income by dispensing medicines without prescriptions; Consumers may practice self-medication using unnecessary or ineffective antibacterials, or insufficient quantities of an appropriate antibacterial. Apart from lack of knowledge, other reasons for antibacterial misuse include financial motivation on the part of prescribers, demand by patients for a variety of cultural, social and economic reasons, fear of litigation, lack of unbiased information on medicines, heavy workload with short consultation times that preclude making a proper diagnosis, and junior prescribers following the poor example of their senior colleagues. 28

Hence, many recommendations from several entities to promote rational use of antibacterials include: educating prescribers and dispensers on appropriate use of antibacterials; supporting treatment decisions through improved diagnostic services and treatment guidelines; encouraging restrictions in prescriptions to a selected range of antibacterials; instituting prescription audits and feedback; and establishing and implementing regulations on quality, dispensing and promotion of antibacterials. Including rational use as part of the curriculum for professional courses and educating patients on antibacterials use have also been proposed. 28

______Balbino M. Rocha, 2013 84 Cha pte r I: The Big Picture on Antibacterial Resistance

13.2. Infection Prevention and Control in Health-Care Facilities

The hospital environment favors the emergence and spread of resistant bacteria. In Europe alone, the death toll from health-care associated infections caused by multidrug- resistant bacteria is estimated to exceed 25 000 per year (Table 1, Subchapter I) and the death rate may be higher in other parts of the world. 28

In addition to human suffering, the consequences of ABR also result in higher direct and indirect financial costs. Infection prevention and control (IPC) measures are designed to prevent the spread of pathogens, including those with ABR, within and between health-care facilities, and from facilities to the community, and also vice versa. Yet, in many parts of the world, implementation of even the most basic recommendations poses tremendous challenges. Differences between countries in the existence of effective IPC practices within their facilities contribute to glaring inequities related to health-care delivery. These differences extend as far as IPC measures related to environmental hygiene and sanitation, which are proven to be important in reducing ABR spread and infections. 313

Overcrowding, inadequate infrastructures, insufficient trained personnel, limited access to commodities needed for IPC and limitations in financial resources are all barriers to the implementation of IPC recommendations. With such wide variations in the levels of IPC implementation, situation analyses at national and facility levels would help attain an overview of the current situation, so that realistic goals could be set according to the needs and opportunities within the local context, with strategies for progressive improvement. 28

Education of health-care workers in IPC is also being carried out in many countries with positive results. Another positive measure is the education of patients on infection prevention, which is being undertaken in some countries. Many national and international professional societies also play an important role in knowledge sharing and in promoting IPC as part of medical and nursing curricula. 28

13.3. Fostering Innovation

By undertaking the treatment of a broad range of common infections, antibacterials ensure the successful application of modern medical advances, from organ transplants to cancer chemotherapy. Logically, as a result, for effective treatment, antibacterials should keep a step ahead of resistant pathogens. Ever since ABR developed to the first antibacterials introduced, the pharmaceutical industry responded by producing synthetic derivatives and a range of new compounds to deal with the problem, hence keeping that step ahead. However, the flow of truly new agents has slowed down during the last three decades and a growing range of bacteria are rapidly developing resistance to more and more

______Balbino M. Rocha, 2013 85 Cha pte r I: The Big Picture on Antibacterial Resistance

antibacterials, rendering them useless in the ability to treat serious nosocomial infections caused by Gram-negative and -positive pathogens (see Subchapter III). In reality, only two truly novel classes of antibacterials have been developed over this period (Figure 31) and both are for the treatment of Gram-positive bacterial infections, which represent only a part of the whole spectrum of emerging resistant bacterial pathogens. 314

Innovative technologies and strategies are therefore needed in order to alleviate the dearth of new antibacterials and other products for limiting ABR, ranging from scientific to financial and regulatory aspects.

Figure 31: Discovery timeline of new antibacterial classes (1930s to 2000s) (Adapted from WHO, 2012). 28

While antibacterial agents are the mainstay of treatment for bacterial infections, alternatives like efficient diagnostic tools and vaccines play important complementary roles by promoting rational use of such medicines and preventing infections that would require antibacterial treatment.

Several studies have shown a significant reduction in resistant S. pneumoniae following the introduction of multivalent pneumococcal conjugate vaccines in infants and children, not only in the vaccinated children but also in the population as a whole, due to reduced transmission of infection. Along with the example in subsection 14.4, this illustrates how developments in vaccines and the strengthening of immunization programs contribute indirectly to the control of ABR. Also, rapid point-of-care diagnostic tools for case- management of individual patients could play a valuable role by removing clinical uncertainty and reassuring patients that some conditions do not require antibacterials. Without such tools, patients may be under-diagnosed but over-treated. For example, an improved diagnostic tool for acute lower respiratory infection could theoretically save over 400 000 unnecessary antibacterial treatments per year in developing countries. 315 Diagnostic tools could also assist in the selection of an effective antibacterial in cases where resistance has

______Balbino M. Rocha, 2013 86 Cha pte r I: The Big Picture on Antibacterial Resistance

rendered first-line treatment ineffective, along with surveillance and infection prevention/control. 28

Furthermore, setting priorities for research and development (R&D) involves making strategic choices and identifying complementary technologies. Carefully weighed financing mechanisms  push and pull incentives  are being used to spur R&D to new technologies. Push incentives that de-link the return on investment from volume-based sales, such as the public funding of clinical trials and providing services that help bring promising compounds to trials are being explored. Such incentives could also reduce the inappropriate use of antibacterials by preventing the need to sell large volumes to improve return on investment. Strategies such as pooled procurement and Advanced Market Commitments can help to create markets that reassure the private sector of returns on investment. Target Product Profiles are also being increasingly used to help align Public Health goals with economic 28 incentives, especially in pharmaceutical R&D, facilitating public sector return on investment.

14. Reducing Antibacterial Use in Animal Husbandry 14.1. Regulations to Restrict the Use of Antibacterials in Food-Producing Animals

National and international efforts to control ABR require a firm legal and regulatory foundation on which measures can be introduced and enforced. Regulations can contribute at many levels, from licensing to ending use of antibacterials. While regulatory frameworks exist in most countries, there are differences in the extent to which regulations are implemented. In most countries, veterinary pharmaceutical products undergo a licensing process that assesses the risk/benefit balance of the proposed products, similar to the process followed for human use products (see subsection 9.3). For antibacterials, an evaluation of the potential impact on human health is also included in many countries. Initially this evaluation focused on avoiding antibacterial residues in food products, but more recently it has been extended to include effects on ABR in bacterial populations in slaughter-ready animals. The approval process may also include consideration as to whether specific antibacterials are of critical importance for human health, 235,316 often with measurable impact on ABR. An example of this occurred in the USA, in 2005, when the USDA successfully withdrew the approval of fluoroquinolones for use in poultry. 317 To achieve the withdrawal, the agency had to demonstrate that the use of enrofloxacin in poultry causes the development of fluoroquinolone-resistant Campylobacter in this animal species; that these fluoroquinolone-resistant organisms are transferred to humans; that they may cause the development of fluoroquinolone-resistant Campylobacter in humans; and that fluoroquinolone-resistant Campylobacter infections in humans are a health hazard. The ______Balbino M. Rocha, 2013 87 Cha pte r I: The Big Picture on Antibacterial Resistance

process began in 2000, involved the collection and evaluation of thousands of studies, expert testimonies, oral hearings and a complex risk assessment. Yet, current national legislations do not always restrict the use of such critical antibacterials in animals. 28

In most countries it can be difficult to withdraw approval for an already licensed pharmaceutical product. However, it is often possible within the existing legislation to implement restrictions on the approved usages of licensed antibacterials. For instance, it is possible to limit off-label/extra-label use or to restrict use to individual animals. Restrictions on the mode of administration could be another useful means of limiting usage in animals. However, this type of restriction is applicable in individual animal treatment but may not always be feasible for large numbers, e.g. in poultry flocks. 28

Growth promoters are considered to be a major contributor to ABR, mainly due to their large consumption when compared to other veterinary and human antibacterials (Figure 19, Subchapter II). Although the use of growth promoters remains widespread worldwide, increasing numbers of countries are starting to launch banning on their use. In Europe, an EU-wide ban on the use of all these compounds in animal feed entered into effect between 1999 and 2006. The EU banned the use of nine antibacterials: virginiamycin, avoparcin, tylosin, bacitracin, spyramycin, avilamycin, flavophospholipol, monensin and salinomycin, plus the drugs carbadox and olaquindox. 318,319 Although, initially, the advice of the European Union’s own Scientific Committee on Animal Nutrition (SCAN) was that there were insufficient data to support a ban, research subsequently showed that the ban did actually reduce substantially some types of antibacterial resistance. 320-322

Figure 32: Macrolide use and resistance among enterococci in swine, Denmark (Adapted from DANMAP, 2010 and WHO, 2012). 4, 28

Denmark, for instance, began voluntarily cessation of some antibacterials as growth promoters in 1995. Initially, before the ban, about 75% of E. faecium isolates from broilers were resistant to avoparcin (and thus also to vancomycin) and 65% to virginiamycin (and thus to quinupristin–dalfopristin). In addition, 75% were resistant to avilamycin which has no current counterpart used in human medicine. In 2000, after the EU-wide ban, the resistance ______Balbino M. Rocha, 2013 88 Cha pte r I: The Big Picture on Antibacterial Resistance

rates were less than 5% for avoparcin and avilamycin. 323 Another example, still in Denmark, is the temporal association between the reduction of macrolide use and the prevalence of ABR among enterococci isolated from swine (Figure 32). The relatively rapid incidence, verified in both examples, happens mainly since, although individual strains may retain resistance genes, they are often replaced by susceptible strains when the selective pressure is removed. 324,325 On the other hand, the resistance rates for virginiamycin did not drop so rapidly, remaining at approximately 30%. 323 There is in fact evidence that some resistance may persist long after the use of an antibacterial has been discontinued. 11,326,327 The persistence of virginiamycin resistance after its ban has been attributed to the use of penicillin selecting for associated resistance to virginiamycin. 323 It has been recently suggested that the use of copper as a feed supplement might also co-select ABR in E. faecium. 328 Such associated resistance is of general importance since the use of one antibacterial can select for resistance to another that is unrelated since the two resistance determinants are genetically linked on the same plasmid or transposon. The complex relationship between reducing usage of antibacterials and the levels of resistance is still currently under research. 329-331

Figure 33: Cephalosporin resistance in poultry industry in Quebec, Canada (Adapted from Dutil et al., 2010 and WHO, 2012). 28,332

Experience has shown that any negative effects due to the prohibition of growth promoters are minimal in the long term once industry adapts to the changes. 333 Apart from prohibitions on the use of antibacterials in food-producing animals, there have also been a number of voluntary withdrawals.

A survey of antibacterial use in hatcheries in Quebec confirmed that in 2004 all chicken hatcheries had switched to exclusive use of ceftiofur. This was after 3rd-generation cephalosporins were legally acceptable in an extra-label manner for routine prophylaxis in eggs or 1-day-old chicks in hatcheries in Canada and the U.S.A. In early 2005, surveillance ______Balbino M. Rocha, 2013 89 Cha pte r I: The Big Picture on Antibacterial Resistance

demonstrated a marked increase in the prevalence of resistance to 3rd-generation cephalosporins and penicillins among S. enterica serotype Heidelberg isolates from humans and chickens in the province of Quebec, Canada. As a result, Quebec hatcheries stopped this use voluntarily, after which there was a dramatic decline in the prevalence of ceftiofur resistance (Figure 33). Unreliable reports indicate that the industry has subsequently re- introduced alternating use of ceftiofur with other antibacterials, and that this has been followed by a resurgence of resistance. 332

Unfortunately there are few incentives to encourage voluntary withdrawal of growth promoters and no barriers or sanctions for re-introducing them. Furthermore, easy access to antibacterials through sources such as online pharmacies, animal feed outlets and pet shops contributes to their overall excessive use and makes it increasingly difficult to enforce regulations on the use of these products. 28

14.2. Financial Incentives

Ideally, sales of an antibacterial should never involve financial benefit for the prescriber. Limitations on the sales profits obtained by veterinarians in Denmark from 1994 to 1995 led to major reductions in the therapeutic use of antibacterials, especially tetracyclines, with no apparent overall harm to animal health. 28

14.3. Prudent use Guidelines and Education

In order to promote prudent use of antibacterials, developing treatment guidelines and popularizing them among veterinarians and farmers is likely to be helpful. Prudent use guidelines have been issued in the Netherlands (1986), Denmark (1998), USA (1999/2000), Germany (2000) and in many other countries more recently. 28 The influence of these guidelines has not yet been adequately monitored, with countries like the Netherlands, for example, still among the highest users of antibacterials in food-producing animals (see subsection 9.4). 15

14.4. Improving Animal Health

The most effective means to reduce the use of antibacterials and prevent ABR is to reduce the need for antibacterial treatment. This could be achieved by improving animal health through measures such as immunization against prevalent infections. In Norway, the introduction of effective vaccines in farmed salmon and trout and improved health management reduced the annual use of antibacterials in farmed fish by 98% between 1987

______Balbino M. Rocha, 2013 90 Cha pte r I: The Big Picture on Antibacterial Resistance

and 2004 (Figure 34). 334 The EU and many other countries already have regulations in place to enforce and promote vaccination as a method of reducing infections in food-producing animals (see section 11.). But, even if health improves, it is not certain that established practices and consumption will change, since most antibacterial agents for growth promotion and prophylaxis are used without any evidence of the need for or benefit from their use in the first place. 28

14.5. Improving Hygiene in Food Production

The FAO/WHO Codex Alimentarius© provides recommendations for many aspects of food-production from primary production to final consumption, highlighting the key controls at each stage ("HACCP approach"). 335 Good agriculture practices particularly at the farm level have also been defined. The Codex Task Force on Antibacterial Resistance recently developed a risk analysis and management tool to assess the risks to human health associated with foodborne ABR. 28,335 Microbiological criteria for a maximum acceptance level for certain types of ABR S. enterica in food-producing animals have been implemented in countries like Denmark. The impact of these interventions has not yet been fully evaluated but Denmark has confirmed low rates of domestically-acquired Salmonella infections. 28

Figure 34: Reduction in antibacterial use after the introduction of vaccination in aquaculture in Norway (Adapted from FAO & OIE, 2006). 334

14.6. Applying Advances in Data Management Technology

Herd Health and Production Management (HHPM) programs have been used to improve productivity incrementally, mainly in intensive production systems. HHPM monitors the interaction between farm management, herd health and production, integrating these components in order to obtain optimal results. These programs use computer-based Management Information Systems (MIS) and the developed databases allow recognition of ABR development and dissemination by local management entities. 28 ______Balbino M. Rocha, 2013 91 CHAPTER II:

Antibacterial Resistance of Mastitis Pathogens Cha pte r II: Antibacterial Resistance of Mastitis Pathogens

I. Mastitis in Dairy Production Operations 1. Introduction

Bovine mastitis (mast = breast; itis = inflammation) is defined as an inflammatory reaction of the mammary gland and is the most common and costly disease in dairy production worldwide, related with both direct (e.g., veterinary treatments, increased labor, production loss, etc.) and indirect costs (e.g., subsequent disorders, reduced fertility, increased risk of premature culling and/or mortality, reduced milk price due to increased bulk- tank milk SCC, etc.). 336-338 Mastitis differs from most other animal pathologies in that is primarily caused by a vast assortment of microorganisms such as bacteria, yeasts, algae and mycoplasmas. In general, these pathogenic microorganisms invade the udder through the teat canal, overcome the cow’s defense mechanisms, multiply and produce toxins that are harmful to the mammary gland. Mammary tissue is subsequently damaged, which causes an increase in vascular permeability. As a result, milk composition is altered: 1) leakage of blood constituents, serum proteins, enzymes, and salts into milk; 2) decreased synthesis of casein and lactose; and 3) decreased milk fat quality. 18,339 The extent of these changes is determined by the severity of the infection. 18,336,340

The severity of the inflammation can be classified into clinical and subclinical forms. In practice, whether a case of mastitis is classified as clinical or subclinical, often depends on how carefully the animal is observed at the time of diagnosis. 336

Clinical mastitis (CM) gives rise to visibly abnormal milk (e.g., color, fibrin clots). As the extent of the inflammation increases, changes in the udder (e.g., swelling, heat, pain, redness) may also become apparent. Clinical cases that only include local signs are referred to as mild or moderate. If the inflammatory response includes systemic involvement (e.g., fever, loss of appetite, shock), the case is termed severe. 341

When the only evidence of disease is an increase in milk SCC of an individual cow, in the absence of apparent visible signs of local inflammation, systemic involvement or abnormal milk appearance, mastitis is referred to as subclinical mastitis (SCM) – the most prevalent form of mastitis. An infection-free animal normally maintains SCC values of less than 100×103 cells/mL, and values above 200×103 cells/mL is a strong indicator of SCM. Although transient episodes of abnormal milk or udder inflammation may appear, these infections are for the most part asymptomatic. 342,343

The duration of infection further classifies mastitis as acute (sudden and severe onset) or chronic – characterized by an inflammatory process that persists for at least two months and may result in progressive development of fibrous tissue. 336,341

______Balbino M. Rocha, 2013 93 Cha pte r II: Antibacterial Resistance of Mastitis Pathogens

Mastitis is a complex multifactorial disease. As such, its incidence depends on exposure to pathogenic microorganisms, effectiveness of udder defense mechanisms and presence of environmental risk factors, as well as interactions between these factors. 344, 345

Several individual animal features can be identified which might indicate an increased risk of mastitis development. When compared to primiparous animals, multiparous cows are generally at higher risk of developing mastitis, 346,347 except in the very early stages of lactation where the relationship is the opposite. 348,349 The risk of developing CM is highest in early lactation, 348-350 whereas the risk of SCM increases with increasing days in milk. 351 Mastitic cows tend to have higher milk yield than non-mastitic cows before they develop mastitis, indicating that high milk yield can be a risk factor for this pathology. 347,352,353 Previous mastitis episodes or high SCC substantially increased the risk of a cow developing a new case of mastitis. 349 Other disorders such as dystocia, milk fever, retained placenta, metritis, ketosis and lameness are also known to increase the risk of mastitis. 346,354 Breed has also been described as a mastitis risk factor. 350

Management practices and the surrounding environment also influence the incidence of mastitis with factors such as housing, milking equipment, feeding regime, hygienic quality of feed and water, udder cleanliness, implementation of preventive measures, etc. 355-358 Season also affects the incidence of mastitis, which has been reported to be highest during the winter months. 349,359

Effective and economical mastitis control programs aim to rely on implementation of preventive measures rather than purely treatment protocols. The consideration of factors such as improved management and housing conditions, use of teat disinfectants, culling and segregation, among many others, should decrease the incidence of new intramammary infections (IMI). These programs are associated with extra costs for the farmer in terms of investments and labor, with interventions normally made if the resulting increase in revenue can be expected to offset the incurred costs. Herds with these programs produce higher quality milk at less cost. Nonetheless, therapeutic interventions are an important part of a mastitis control program, with clearly established benefits; one must yet keep in mind that improper or overuse could tip the scales, favoring selection of ABR bacterial pathogens. 31,336,341

The top goal of modern dairy operations is to produce maximum quantities of high quality milk, which is more flavorsome, nutritious and with a longer shelf-life. Conversely, reduced-quality milk affects all segments of this industry due to its negative impact on several important aspects of cow and herd performance, ultimately leading to milk with decreased manufacturing properties and dairy products with reduced shelf-life. Its prevention and control have, thus, proven to be key challenges in today's dairy farming throughout the

______Balbino M. Rocha, 2013 94 Cha pte r II: Antibacterial Resistance of Mastitis Pathogens

world, relying on increasingly advanced veterinary diagnostic methods, treatment protocols and herd management strategies. 31

2. Mastitis Pathogens

From the 137 different microorganisms that have been identified as possible etiological mastitis pathogens, the majority are bacteria. 360 These have traditionally been categorized into major or minor pathogens, depending on the magnitude of inflammatory response and subsequent damage associated with infection. Major pathogens often cause CM and give rise to the most extensive changes of milk composition. These infections are due to S. aureus, streptococci (S. agalactiae, S. dysgalactiae subs dysgalactiae and S. uberis), E. coli and Klebsiella spp. Minor pathogens, including Corynebacterium bovis and Coagulase- negative staphylococci (CNS) are generally the cause of moderate infections, frequently associated with SCM. 18,361

ECO: E coli; SAG: S. agalactiae; SAU: S. aureus; SDY: S. dysgalactiae; SUB: S. uberis

Figure 35: Sliding scale for contagious and environmental origin of mastitis pathogens, based on insights from molecular epidemiology. Vertical axis indicates to what extent species behave as contagious (black) or as environmental (white) pathogens (Adapted from Zadoks & Schukken, 2006). 210

Until recently, bacteria were considered either as contagious (host adapted) or environmental (opportunistic) pathogens, depending on the primary reservoir and mode of transmission. Contagious mastitis pathogens are considered to be found in the udders of infected cows (major reservoir) and are commonly transmitted among animals, primarily during milking, with the tendency to result in chronic subclinical infections with flare-ups of

______Balbino M. Rocha, 2013 95 Cha pte r II: Antibacterial Resistance of Mastitis Pathogens

clinical episodes. The most important contagious mastitis pathogens include S. agalactiae, S. aureus, C. bovis and Mycoplasma spp. Central environmental pathogens include E. coli, Klebsiella spp., S. dysgalactiae subs dysgalactiae and S. uberis. Cows are continuously exposed to environmental mastitis pathogens since bedding, manure and soil present as their primary sources. The majority of infections caused by environmental pathogens are clinical and of short duration. Unlike mastitis caused by contagious pathogens, environmental mastitis cannot be eradicated from a dairy herd. 18

Nowadays, the niche adaptation of mastitis pathogens implies that these two traditional classifications are too simplistic. Some species such as S. aureus tend to be contagious, whereas other species such as S. uberis are commonly of environmental origin. Depending on management conditions and strains, however, environmental S. aureus 362-365 and contagious S. uberis may occur. 366 Even S. agalactiae, which can be considered the prototype of contagious pathogens, can on rare occasions originate from environmental sources (human, companion animal). 367 At the other end of the spectrum, E. coli, the prototype of environmental pathogens, appears to be adapting to long-term survival in the bovine host. 368 Thus, a black-&-white dichotomy does not do the epidemiology of mastitis justice and fails to provide dairy producers with adequate management advice in all circumstances. Rather, Zadoks & Shukken (2006) suggest that a sliding scale with S. agalactiae at the contagious end and E. coli at the environmental end should be used to represent the epidemiology of mastitis (Figure 35). Molecular typing data from milk isolates has been used to differentiate between contagious and environmental transmission. 210

Table 23: Prevalence of mastitis pathogens in dairy herds from Northwestern Portugal, between 2005 and 2008 (Adapted from Pinho et al., 2008). 369

Number of % of Positive Isolate % of Results Culture Isolates Samples

'No growth' 2009 10.59 - Contaminated 2209 11.64 - Streptococcus spp.1,* 3053 16.09 20.68 Corynebacterium spp. † 2438 12.85 16.52 CNS * 2208 11.63 14.96 Coliforms * 2112 11.13 14.31 S. aureus † 1899 10.01 12.86 Yeast * 1391 7.33 9.42 Enterococcus spp. * 631 3.32 4.27 Bacillus spp. * 514 2.71 3.48 Prototheca spp. * 155 0.82 1.05 A. pyogenes * 145 0.76 0.98 S. agalactiae † 138 0.73 0.93 Others 40 0.21 0.27 Fungi * 37 0.19 0.25

1 Not including S. agalactiae. * Environmental pathogens † Contagious pathogens ______Balbino M. Rocha, 2013 96 Cha pte r II: Antibacterial Resistance of Mastitis Pathogens

In Portugal, information about the prevalence of mastitis pathogens is scarce. The latest known survey determining the prevalence of mastitis pathogens covered dairy herds from Northwestern Portugal between 2005 and 2008. Environmental pathogens were the most common isolated pathogens during that period (69.4%). Non-S. agalactiae streptococci were the most frequent agents of this group, followed by CNS, coliform bacteria, yeast, Enterococcus spp., Bacillus spp., Prototheca spp., A. pyogenes and fungi (Table 23). Contagious agents, comprised 30.3% of all isolated bacteria, with Corynebacterium spp. being the most prevalent pathogens, followed by S. aureus and S. agalactiae (Table 23). 369

3. Current Approaches for Mastitis Diagnosis

Early diagnosis is of the utmost importance due to the high costs of mastitis. EU legislation, through the EC Regulation No. 853/2004, stresses that milk selected for human consumption must originate from healthy animals. 370

Diagnostic methods have been developed to check milk quality through detection of mammary gland inflammation and diagnosis of the infection and its causative pathogens. At present, frequently used assays include measurement of SCC, enzymatic analysis, microbiologic culture techniques, electrical conductivity, pH tests, among others (Table 24). 340 Colorimetric and fluorometric assays have been developed for measuring the concentrations of enzymes elevated in milk during mastitis (e.g., NAGase or LDH). Use of culturing techniques for the detection of mastitis-causing microorganisms is still the 'gold standard', despite very labor-intensive and expensive. Mastitis can also be detected using ‘cow-side’ or ‘on-site’ tests, which can be used by both farmers and veterinarians and which require relatively little training. 371 One of the oldest and best known is the California Mastitis Test (CMT), which indirectly measures SCC. It is based on the principle that the addition of a detergent to a milk sample with a high cell count will lyse the cells, release nucleic acids and other constituents and lead to the formation of a ‘gel-like’ matrix consistency. The higher the cell count, the more jellified is the consistency of the formed product. Interpretation can, nevertheless, be subjective and this might result in false positives/negatives. 372 Mastitis can also be detected via changes in conductivity or pH. Although these effects are easy to monitor, they provide relatively low sensitivities. There is, therefore, a major need for new specific biomarkers for mastitis that are easy to detect and measured ‘on-site’. 371

Technological advances, together with increased proteomic and genomic information, have resulted in improvements in the sensitivity of assays used for the detection of mastitis. Immunoassays, such as ELISA, can provide a reliable and inexpensive approach provided that suitable antibodies are available against specific inflammation-related biomarkers or the

______Balbino M. Rocha, 2013 97 Cha pte r II: Antibacterial Resistance of Mastitis Pathogens

causative microorganisms. There have also been significant developments in nucleic-acid- based testing for the identification of the latter. 210,371

Table 24: Current SCC measuring methods and alternatives for mastitis detection (Adapted from Viguier et al., 2009). 371

 California Mastitis Test (CMT) . Advantages: rapid and the device is easily transportable. . Disadvantage: relatively expensive. This assay indirectly measures the SCC in milk samples. A bromocresol-purple-containing detergent is used to break down the  Electrical conductivity (EC) test cell membrane of somatic cells, and the subsequent release and This test measures the increase in conductance in milk caused by the aggregation of nucleic acid forms a gel-like matrix with a viscosity elevation in levels of ions such as sodium, potassium, calcium, that is proportional to the leukocyte number. magnesium and chloride during inflammation. . Advantages: cost effective (~ €10 for 350 tests), rapid, user friendly and can be used ‘on-site’ or in the laboratory. . Advantage: can be used ‘on-site’. . Disadvantages: can be difficult to interpret and has low sensitivity. . Disadvantage: non-mastitis-related variations in EC can present problems in diagnosis.  Portacheck PortaSCC® milk test  Culture tests This assay uses an esterase-catalyzed enzymatic reaction to determine the SCC in milk. Laboratory-based tests use selective culture to identify different microorganisms involved in causing mastitis. . Advantages: cost effective (~€2 per test), rapid and user friendly. . Disadvantage: low sensitivity at low SCCs. . Advantage: identifies specific pathogens causing mastitis. . Disadvantages: cannot be used ‘on-site’ and long waiting times (days).  FossomaticTM SCC This counter operates on the principle of optical fluorescence.  pH tests Ethidium bromide penetrates and intercalates with nuclear DNA, and The rise in milk pH, due to mastitis, is detected using bromothymol the fluorescent signal generated is used to estimate the SCC in milk. blue. . Advantages: rapid and automated. . Disadvantages: expensive deice (~€5500) and complex to use. . Advantage: user friendly, cost effective and rapid. . Disadvantages: not as sensitive as other tests.  DeLaval Cell Counter DCC  Enzymes This counter operates on the principle of optical fluorescence, whereby propidium iodide is used to stain nuclear DNA to estimate Assays are used to detect enzymes, such as NAGase and LDH. the SCC in milk. . Advantage: assays are rapid. . Disadvantages: assays might be laboratory-based.

II. Mastitis Antibacterial Therapy and the Use of Susceptibility Profiles for Treatment Decisions

4. Assessing Efficacy

Therapy of infectious pathologies should either assist host defenses in eliminating invading pathogens and/or reduce the pathophysiologic consequences of infection. Logically, research emphasis and clinical application of antibacterials for therapy of mastitis has focused on the elimination of infectious agents. However, therapeutic success for some IMI may be better measured by evaluating reduction of clinical symptoms rather than total elimination of the pathogen from the gland. Ultimately, the best outcome of mastitis therapy is a positive effect on the amount of marketed milk produced and long-term cow survival. 12

Determination of IMI status and definition of cures is dependent on bacteriologic culture of milk samples and the sensitivity and specificity of this technique to correctly assess infection status. The conventional definition of an IMI is either the presence of the same microorganism in two of three consecutive cultures (different sampling dates) or the ______Balbino M. Rocha, 2013 98 Cha pte r II: Antibacterial Resistance of Mastitis Pathogens

presence of the pathogen in both samples of duplicate samples (collected at the same time). By following these guidelines, the chance of determining an IMI based on false-positive isolations from contamination is relatively low. Isolation from a single sample of the contagious pathogens S. agalactiae and S. aureus is probably sufficiently indicative of an IMI due to the low rate of environmental contamination of milk samples with these pathogens. Mammary gland quarters that have an IMI caused by a particular pathogen before treatment but do not have an IMI caused by the same pathogen after treatment would be defined as cured. Conversely, quarters that remain bacteriologically positive after treatment are not cured. Although this is a rather simple premise of efficacy, a survey of mastitis therapy trials can result in numerous and perhaps misleading methods in determining bacteriologic cures. The number of times a quarter is sampled before and after therapy, the volume of milk that is inoculated for culture, the time-period after therapy when sampling occurs and elapsed time between collection of consecutive samples is dissimilar between many reports. Belief in bacteriologic cures that in reality are false-negative culture results can be readily attained if care is not taken in data analysis, particularly when assessing therapeutic outcomes for IMI caused by such invasive pathogens as S. aureus and S. uberis and Gram-negative rods such as Pseudomonas spp. and Klebsiella spp. Bacteria exposed to antibacterials may be inhibited from growth and can remain so for some time after the termination of therapy. Intracellular survival (within phagocytes), abscess formation and S. aureus L-forms can reduce the probability of successful isolation of bacteria following routine aerobic culture of milk samples. 373 A 30-day refractory period of decreased probability to isolate bacteria in milk has been demonstrated for S. aureus IMI, and P. aeruginosa can be isolated from affected quarters subsequent to a case of clinical mastitis over 12 months after initial therapy, despite frequently collected negative cultures. 374,375 Additionally, many chronic IMI result in intermittent shedding of bacteria in milk and one or even two samples collected after treatment may not be adequate to insure the absence of bacteria in the affected quarter. 376 This being said, the underlying message is that bacteriologic cures should be reviewed critically either in a research or clinical setting before success of therapy can be affirmed.

The other potential goal of therapy is to attain clinical cures, with or without bacteriologic cures. This may be desirable to promote the marketing of an affected cow’s milk or to improve the effects of a severe or life-threatening IMI. Clinical cures attained by antibacterial therapy can be more inherently obvious than bacteriologic cures, but assessment can be tainted by subjective outcomes. Clinical mastitis, as described in the beginning of this chapter, is defined as ‘abnormal milk, with or without quarter involvement and systemic signs’. Return to normal appearance is accepted as a clinical cure. Relapses and recurrences should, nonetheless, be noted as part of the therapeutic evaluation. For systemic (severe) CM cases, clinical pannels and/or objective measures such as heart rate

______Balbino M. Rocha, 2013 99 Cha pte r II: Antibacterial Resistance of Mastitis Pathogens

and rectal temperature can be measured. The best indicators for clinical efficacy, however, are dry-matter intake, milk production and post-treatment culling/death rates. 12

Another way of predicting IMI is using SCC thresholds at either the quarter or cow level. There are some obvious problems with using composite milk SCC to identify infected cows because of dilution of SCC values with milk from uninfected quarters. Considering a hypothetical situation with a cow producing 20 kg of milk per milking, evenly distributed between 4 quarters (5 kg per quarter) but only 1 quarter is infected with SCM. If the SCC of the milk from the 3 uninfected quarters is 100×103 cells/mL, the composite SCC value will not reach a threshold of 250×103 cells/mL until the SCC from the infected quarter exceeds 700×103 cells/mL. 377

The sensitivity and specificity of using a SCC threshold of 200×103 cells/mL as the cut point for IMI have been evaluated in several studies. 378-380 Reported sensitivities range from 73-89% with corresponding specificities of 75-85%. The sensitivities are relative sensitivities because the “gold standard” was bacterial culture, which is not a perfect test. A SCC threshold of 100×103 cells/mL for quarter samples had the maximal sensitivity and specificity for detecting IMI in fresh cows that were tested on day 5 post-calving. 381 The probability that a cow over the threshold will actually be infected (the positive predictive value) or the probability that a cow under the threshold is actually uninfected (the negative predictive value) are useful values for on-farm problem solving. Positive and negative predictive values are a function of the underlying prevalence of disease in the tested herd. This concept is somewhat self evident in that 100% of test positive animals are truly positive in a herd with 100% prevalence, whereas 100% of test negative animals are truly negative in a herd with zero prevalence. 377

Bulk tank somatic cell count (BTSCC) is the most frequent reference point for milk quality. Normally, most dairy farms around the world have periodic BTSCC and bacterial count data supplied by their milk purchaser. BTSCC vary regionally, seasonally and with herd size. Many dairy farmers consistently produce high quality milk. In the USA, official regulatory records of all Wisconsin dairy farms in 1998 revealed that more than 1,800 WI dairy farms had average BTSCC of <130×103 cells/mL and over 4,500 dairy farms obtained annual average BTSCC of <200×103 cells/mL. 382 The median BTSCC was 290×103 cells/mL for grade A dairy farms and farms with average BTSCC values that exceeded 400×103 cells/mL were ranked in the bottom 25% of herds. The risk of having a violative antibacterial residue increases after BTSCC levels exceed 400×103 cells/mL. 382 BTSCC values verify the existence of a mastitis problem but individual cow SCC values are needed to define the problem on a herd basis. BTSCC values often differ considerably from herd SCC values estimated by official regulatory entities. These entities usually estimate SCC values as a weighted average of the milk sample SCC multiplied by the individual cow milk yield. The ______Balbino M. Rocha, 2013 100 Cha pte r II: Antibacterial Resistance of Mastitis Pathogens

error associated with both measures contributes to error in estimating BTSCC. Additional reasons for the disparity include differences in methodology and sampling and differences in animals contributing to the bulk tank versus official reports. There is no simple way to estimate the prevalence, incidence or effect of mastitis control procedures without individual cow SCC values. Common industry goals for subclinical mastitis are: 85% cows with SCC <250×103 cells/mL and less than <5% of cows developing new SCM infections per month. 383

5. Pharmacological Considerations

The goal of antibacterial therapy is to attain effective concentrations of the drug at the site of infection. For bovine mastitis, there are three potential therapeutic targets, or pharmacologic compartments (Table 25).

12 Table 25: Summary of the 3-compartment model to target mastitis pathogens (Adapted from Erskine et al., 2003).

Pharmacologic Compartment Mastitis Pathogens Milk and ducts Parenchyma Cow

S. agalactiae +++ - -

Streptococcus spp. +++ + - S. aureus + +++ - Staphylococcus spp. +++ - - Coliforms * + - +++ Mycoplasma spp., other Gram-negatives * - - +++

* Severe CM: Supportive care and prevention of secondary bacteremia are primary concerns. +++ primary target; ++ some benefit; - of little value.

The first (and most commonly targeted compartment) consists of the milk and the epithelial lining of the ducts and alveoli of the mammary gland. Pathogens that typically reside in this compartment are generally non-invasive and are not believed to cause abscess formation in the parenchyma. IMI caused by organisms like S. agalactiae, S. dysgalactiae, CNS and other Gram-positive cocci of short duration, would be appropriately targeted with antibacterial therapy that attain effective concentrations in milk. The most simple and effective method of obtaining this outcome would be to administer antibacterials by IMM administration. 12 However, when in the presence of chronic IMI, this route of administration points out some limitations. Formation of fibrin casts and micro-abscess (e.g., S. aureus) interfere with the distribution of infused drugs to the site of infection in the terminal alveoli. Moreover, the typical 24-hour to 36-hour duration of therapy for IMM infusions limits the time period of effective concentration in the gland required to eliminate more chronic or invasive

______Balbino M. Rocha, 2013 101 Cha pte r II: Antibacterial Resistance of Mastitis Pathogens

IMI. As a result, systemic administration of antibacterials has received attention as an adjunct therapy to IMM therapy. This route of administration is appropriate when the therapeutic target includes the second compartment for pharmacologic consideration: the deep tissue of the gland. 12

As a basis for practical PK of mastitis therapy, the ideal antibacterial for parenteral mastitis therapy would: 1) have a low MIC against the majority of udder pathogens; 2) have high bioavailability from intramuscular injection sites; 3) be weakly alkaline or otherwise non- ionized in serum; 4) be sufficiently lipid soluble; 5) have a low degree of protein binding; 6)

have a long half-life (t1/2) in the body; 7) retain activity in inflammatory secretions; and 8) have clearance from body organs and tissues similar to the clearance of the drug from the blood. 53 Systemically administered sulfonamides, penicillins, aminoglycosides and early-generation cephalosporins do not readily penetrate the mammary gland. Conversely, macrolides, trimethoprim, tetracyclines and fluoroquinolones cover a good distribution in the mammary gland. Systemic use of antibacterials has been moderately successful for improving cure rates compared with IMM infusions for chronic S. aureus IMI in dry and lactating cows. 385,386

Recent evidence has suggested that the primary target for the treatment of severe coliform mastitis should be the third compartment of mastitis therapy: the cow. Bacteremia can occur as a consequence of coliform mastitis in ≥40% cases and beneficial clinical outcomes have been reported for cows treated with systemic antibacterials and supportive therapy. 387-390 Systemic administration of antibacterials for mastitis involves, for most cases, extra-label drug use, enhancing the risk of antibacterial residues in milk and meat, and 12 consequently increasing the need to develop longer withholding periods for the treated cows.

6. Susceptibility Testing for Mastitis Pathogens 6.1. Determination and Validation of Susceptibility Breakpoints for Mastitis Pathogens

Ideally, accurate antibacterial susceptibility test breakpoints should derive from: 1) MIC data for bovine mastitis bacterial pathogens; 2) PK/PD data for lactating dairy cows; and 3) the results of field studies that measure the rates of clinical and bacteriologic cure. Clinical and bacteriologic cure rates may provide a clear breakpoint or, in other situations, can be used in conjunction with PK/PD data to suggest the most appropriate breakpoint. 13 Unfortunately, the ideal approach to determine accurate susceptibility breakpoints is hampered by three main difficulties: 1) limited availability of MIC values for bovine mastitis bacterial pathogens; 2) incomplete PK/PD data for lactating dairy cows; and 3) inadequate number of field studies validating susceptibility breakpoints. 6

______Balbino M. Rocha, 2013 102 Cha pte r II: Antibacterial Resistance of Mastitis Pathogens

6.1.1. Limited Availability of MIC Values for Mastitis Pathogens

Currently there are no adequate databases of in vitro MIC values for CM pathogens, although adequate databases are available for SCM isolates. It is recommended that MIC values be determined on 300 to 600 isolates from representative clinical cases from a large geographic area. 7,13 The existence of such a contemporary dataset was though not revealed in a literature search. MIC values have been published for 1063 Staphylococcus spp. isolates from cows with SCM in Germany; 391 107 S. aureus isolates from cows with CM in the USA; 392 151 S. aureus isolates from cows with CM/SCM in the Czech Republic; 393 71 Streptococcus spp. isolates from cows with CM in the USA; 394 358 395 and 362 396 Streptococcus spp. isolates from cows with CM and SCM in the USA, respectively; and 131 E. coli isolates from cows with CM/SCM in Denmark. 397

There are a number of datasets reporting MIC values for some antibacterials. A large dataset containing MIC values for enrofloxacin has been published for 200 E. coli, 206 S. aureus and 309 Streptococcus spp. isolates from cows with CM in Germany. 398 Another large dataset of MIC values for penicillin G has been published for 505 Streptococcus spp. isolates from cows with SCM in Germany. 399 Two large datasets of 530 and 225 isolates have been published containing MIC values for pirlimycin 400 and penicillin/novobiocin, respectively. 401

6.1.2. Incomplete PK/PD Data for Lactating Dairy Cows

Although the pharmacokinetics of many parenteral antibacterials used to treat CM are well known, most PK data has been obtained from healthy cattle. It has not though been determined whether PK values in healthy cows are the same as those in cows with mastitis. In addition, many of the IMM antibacterials used to treat CM have unknown PK values, with limited understanding of the PD of these compounds in treating mastitis. More important, the breakpoints currently recommended for most antibacterials are based on achievable serum and interstitial fluid concentrations in humans after oral or IV antibacterial administration. The relevance of these breakpoints for achievable milk concentrations in lactating dairy cows after IMM, SC, IM or IV administration is dubious at best. 6, 190

6.1.3. Inadequate Number of Field Studies Validating Susceptibility Breakpoints

Antibacterial susceptibility test results must be correlated with clinical and bacteriologic cure rates to confirm the validity of the assigned breakpoints. 13 In vitro antibacterial susceptibility test results have been suspected to be poorly correlated with treatment outcome for CM by many researchers 402-409 and the value of the agar disk diffusion method to guide mastitis treatment decisions has been questioned. 402,410 Myllys et al. (1992) came to ______Balbino M. Rocha, 2013 103 Cha pte r II: Antibacterial Resistance of Mastitis Pathogens

the conclusion that ‘‘susceptibility testing in artificial media cannot simulate what happens in the mammary gland milk phase and as themselves cannot predict the outcome of therapy.’’ 411 Rossitto et al. (2002) concluded ‘‘use of human interpretive criteria to categorize isolates as susceptible or resistant can be misleading and is inappropriate.’’ 396

With the exception of pirlimycin 400 and penicillin/novobiocin, 401 zone diameters in the agar disk diffusion test have not been related to antibacterial concentrations achieved in the bovine mammary gland with dosage regimens used by veterinarians and dairy producers. 6

Results from six field studies 412-417 are available to evaluate the validity of susceptibility breakpoints in guiding treatment of cows with mastitis. These results suggest that the following antibacterials may have valid (but not necessarily optimal) breakpoints for treating CM or SCM caused by specific bacteria: parenteral penicillin G for subclinical S. aureus infections, IMM cephapirin for clinical streptococcal infections and parenteral TMP-SMX for clinical E. coli infections. Of these three antibacterials, the breakpoints for penicillin G and cephapirin have only been validated for bacteriologic cure, whereas the breakpoint for TMP- SMX is validated for clinical cure. Because treatment outcomes may be influenced by factors such as the duration of infection before treatment, antibacterial dosage, dosage interval and extent of treatment, many more field studies must be completed to validate the currently assigned antibacterial breakpoints for mastitis-causing pathogens. 6

6.2. Test Methods

Antibacterial susceptibility testing has been used as a basis for therapy selection for nearly more than half a century. 12 Several methods have been used to determine the susceptibility of bovine mastitis pathogens to antibacterial agents: broth dilution, milk dilution, agar dilution, MBC determination, determination of killing kinetics and the agar disk diffusion. The first five methods are quantitative, while the agar disk diffusion method is qualitative. Because of issues related to cost and complexity of each testing procedure, the broth microdilution method is the recommended ‘‘gold standard’’ method for in vitro susceptibility testing, whereas agar disk diffusion method provides an alternate crude, inexpensive and clinically practical method. 6 Since most of these methods have already been described in section 7 of Chapter I, the following subsection will describe the milk dilution method – specific when determining the susceptibility of bovine mastitis pathogens.

6.2.1. Milk Dilution Method

The milk dilution method provides a direct measurement of MIC and determines the ability of the mastitis pathogen to grow in mastitic milk containing a known antibacterial ______Balbino M. Rocha, 2013 104 Cha pte r II: Antibacterial Resistance of Mastitis Pathogens

concentration. A commercially available test procedure developed for bovine milk is available (MASTiK®, ImmuCell Corp., Portland, ME - USA). A mL of mastitic milk is mixed with 3 mL of reagent (a combination of sterile milk and bromcresol purple) and the mixture is incubated for 1-3 hours (CM) or 6 hours (SCM) at 35-37ºC. Two drops (500 µL) of the incubated mixture are placed in each well of a 32-well microtiter plate containing a geometric progression of three to four concentrations of eight dehydrated antibacterial agents (ampicillin, cephalothin, erythromycin, oxacillin, oxytetracycline, penicillin, pirlimycin and sulfadimethoxine). Growth in each well is recorded after 4-12 hours of incubation at 35-37ºC by examining the color of the media in each well. A change in color from purple to yellow means the pH is lower and growth has occurred, indicating that the bacteria is resistant to the antibacterial at the concentration in the well. The color change occurs in the presence of lactose-fermenting bacteria because lactose is fermented to lactic acid, with an accompanying decrease in pH. Most mastitis pathogens are lactose fermenters. Non–lactose-fermenting pathogens (yeast, Mycoplasma spp., Prototheca spp. and P. aeruginosa) do not change pH when growing and, therefore, cannot be detected by the milk dilution test.

The advantages of milk dilution over broth dilution or agar disk diffusion are cost, rapidity of results and use of milk as the test medium. There are, however, technical issues to be considered when determining bacterial growth inhibition in milk due to its turbidity. These issues have been addressed and assays such as reduction of triphenyltetrazolium chloride 418,419 and the fluorometric resazurin and b-glucuronidase assays 418,420 offer promise as a means of providing accurate MIC values for pathogens from mastitic quarters. It is also important to remember that mastitic milk may contain more than one type of bacteria and that an important goal of mastitis diagnosis is to identify the causative agent. 6 The main theoretic disadvantages are that the mastitis pathogen is not identified (unless separate culturing is performed) and that a standardized inoculum is not used, which may result in altered growth kinetics. 6,421

The susceptibility test results of the milk dilution method have been compared to agar disk diffusion 421 and broth dilution. 422 The susceptibility test results of the commercially available milk dilution method (MASTiK®) varied in their agreement (27-100%) with those of agar disk diffusion, depending on the pathogen and cutoff selected for each antibacterial. Moreover, only 91% of milk samples tested using the MASTiK® assay were positive for growth, 421 relative to growth on sheep blood agar. This result indicates that the MASTiK® test does not identify all bacteria associated with a mastitis episode. For 107 S. aureus isolates from bovine mammary glands, the MASTiK® susceptibility test results also varied in their agreement (66-96%) with those of broth dilution, depending on the antibacterial used. 422

______Balbino M. Rocha, 2013 105 Cha pte r II: Antibacterial Resistance of Mastitis Pathogens

6.3. Guidance for Antibacterial Selection Using Susceptibility Test Results and MIC Values

Evidence-based principles should be used to select the most appropriate antibacterial for treating mastitis in a herd. Antibacterials have been historically selected based on the ‘‘pathogen profile’’ for the herd and low in vitro MIC values. Use of these selection criteria appears flawed though. In vitro MIC values may not predict in vivo antibacterial efficacy because: 1) the in vitro MIC test may have used a different antibacterial to the active ingredient present in commercially available products; 2) specific antibacterials can depress normal mammary defense mechanisms; and 3) antibacterials may be distributed unevenly in an inflamed gland. 61 The following subsections consist of detailed discussions of these issues, in order to evaluate the role that antibacterial susceptibility testing should play in guiding treatment of mastitis cases.

6.3.1. Validity of Developing a ‘‘Herd Profile’’ for Susceptibility

Antibacterial susceptibility testing has been promoted on the basis of developing a ‘‘herd profile’’ for mastitis pathogens, thereby facilitating future treatment decisions. 423 Although this is a useful approach for detecting β-lactamase production by S. aureus isolates and, therefore, predicting treatment outcomes in herds with contagious mastitis, 424 the herd- profile approach has little merit in herds where infection comes from diverse sources (e.g., environmental mastitis). 6

6.3.2. Validity of Selecting the Antibacterial with the Lowest In Vitro MIC Value

Some veterinarians select antibacterials for treatment on the basis of the lowest in vitro MIC value against common mastitis pathogens. This approach is inconsistent, since antibacterial selection based on low MIC values incorrectly assumes that: 1) in vitro MIC values are identical to in vivo MIC values; 2) bovine mastitis pathogens are similar to those causing systemic illness in humans; 3) dosage protocols and PK of antibacterials used to treat dairy cows with mastitis are similar to those used to treat humans with systemic infections; 4) all antibacterials have identical dosage protocols, bioavailability, and pharmacodynamics; 5) all antibacterials penetrate equally to the site of infection; 6) all antibacterials have similar mechanisms of action and duration of post-antibacterial effect; and 7) all antibacterials have no deleterious effects on mammary defense systems such as neutrophil phagocytosis. Also, a long-held view has been that the results of susceptibility testing can guide the practitioner as to which antibacterials should not be used. 395,407 This belief has not been validated though. If the argument is accepted that bacteria categorized

______Balbino M. Rocha, 2013 106 Cha pte r II: Antibacterial Resistance of Mastitis Pathogens

as susceptible in vitro may be resistant in vivo, then the alternative statement is also true – bacteria categorized as resistant in vitro may be susceptible in vivo. 6

6.3.3. Validity of Assumption that all Antibacterials Within a Class have Identical MIC Values

Specific antibacterials that are considered representative of their class are routinely used in susceptibility testing. For instance, in countries like the USA, cephapirin is a 1st-generation (narrow spectrum) cephalosporin that is used to treat mastitis in dairy cows; susceptibility testing does not, though, use cephapirin as the test antibacterial. Instead, cephalothin is used because it is the recommended representative of 1st-generation compounds. 190 This recommendation ignores the results of an in vitro study indicating one

dilution difference in MIC50 values between cephapirin and cephalothin for S. aureus, P. mirabilis and Citrobacter spp. 425 and ignores recommendations that the antibacterial to be used clinically should be tested. 7

Since other antibacterials are used as class representatives, it is uncommon for susceptibility testing to employ the active antibacterial agents present in commercially available antibacterial products for cattle. It is, therefore, likely that susceptibility test results based on class representatives rather than the active antibacterial agent will lead to erroneous results. 6

6.3.4. Effect of Milk on MIC Values

When tested in the Mueller-Hinton medium, milk markedly decreases the activity of many antibacterials, particularly when they are highly lipid or protein bound. 410,418-420 Antibacterials such as erythromycin, 410 neomycin, 404 oxytetracycline, 404,415,418 spyramycin, 419 streptomycin, 404,410 tetracycline, 404,410,420 TMP-SMX 418-420 and vancomycin have decreased in vitro activity in this medium. 410,418 In contrast, milk has a variable effect on the in vitro activity of β-lactam antibacterials (ampicillin, ceftiofur, cephalothin, oxacillin and penicillin G), gentamicin and enrofloxacin. 404,410,418-420 In mastitic milk, an increased in vitro activity of enrofloxacin, gentamicin, spyramycin and TMP-SMX has been reported when compared with normal milk. 419,420

Casein is suspected to be the major milk constituent responsible for decrease in antibacterial activity. The pH of the medium may also alter the MIC value because the pH of Mueller-Hinton medium is 7.4 (corresponding to that of human extracellular fluid), whereas the pH of normal bovine milk is 6.6 and the pH of mastitic bovine milk ranges from 6.8 to 7.2. Other factors such as electrolyte concentration, lipid solubility, leukocyte concentration,

______Balbino M. Rocha, 2013 107 Cha pte r II: Antibacterial Resistance of Mastitis Pathogens

degree of protein binding, molecular size and lactoferrin concentration can also influence antibacterial activity in Mueller-Hinton medium. 419,420,426,427

Milk is therefore preferable to Mueller-Hinton broth as a test medium for determining susceptibility of mastitis pathogens because it simulates the actual environment in which the antibacterial and bacteria interact in vivo. 420 A logical extension of this argument is that mastitic milk from the affected quarter would be the best theoretic test medium, and this is the basis for the commercially available MASTiK® test, already described in subsection 6.2.1.

6.3.5. Deleterious Effects of Antibacterials on Normal Mammary Defense Mechanisms

It has been known for many years that some antibacterials decrease the animal’s ability to eliminate pathogenic bacteria. Infusion of commercially available IMM formulations of benzathine cephapirin and sodium novobiocin, for instance, into non-infected quarters at dry-off transiently increased the neutrophil concentration in glandular secretion and caused a more sustained decrease in the ability of neutrophils to phagocytose and destroy S. aureus. 428 Sodium novobiocin had a more pronounced effect on neutrophil phagocytosis and killing than did benzathine cephapirin. 428 Clinically relevant concentrations of novobiocin/penicillin, chloramphenicol and tiamulin decreased bovine neutrophil viability and novobiocin/penicillin, amikacin, rifampin, chloramphenicol and tiamulin decreased phagocytosis of S. aureus by bovine neutrophils. 429 A clinically high concentration of oxytetracycline (≥15 µg/mL) caused a reversible decrease in peroxidase activity by bovine neutrophils but did not affect killing ability; 430 these high oxytetracycline concentrations are not achievable in the mammary gland using current dosage protocol for parenteral administration. In summary, because some antibacterials alter neutrophil function at clinically relevant concentrations, MIC values determined in vitro may not reflect the in vivo response to therapy. 6

6.3.6. Distribution of Antibacterials in an Inflamed Mammary Gland

IMM antibacterials are widely used in the treatment of CM; these are, nonetheless, distributed unevenly in an inflamed gland due to swelling and fibrosis that can block milk ducts, thereby preventing antibacterial diffusion throughout the gland. In addition, the intracellular location of some bacteria such as S. aureus means that estimates of in vivo milk phase concentrations may not accurately reflect intracellular concentrations. Because antibacterials have variable penetration to the site of infection in mastitis (particularly in chronic infections), it is likely that MIC values determined in vitro do not accurately reflect the in vivo response to therapy. 431

______Balbino M. Rocha, 2013 108 Cha pte r II: Antibacterial Resistance of Mastitis Pathogens

7. Calculation of Antibacterial Dosage

In bovine practice, the constraints in calculating a dose of an antibacterial agent are the dose interval the client prefers for practicality; the MIC of the drug for the pathogen; the preferred route of administration; the compartment of the cow where the drug must act; the PK parameters of the selected drug; the possible toxicity of the drug; and the withholding periods. IMM administration is generally best for achieving effective concentrations in the milk compartment. Thus, achievement of effective antibacterial concentrations in deep udder tissue (parenchyma) or plasma (cow) compartments should be considered of equal primary importance. 12

Figure 36: Concentration-versus-time curve for drug concentration in milk and plasma (Adapted from Erskine et al., 2003). 12

Similarly to Figure 6 (Chapter I), Figure 36 is a simplified schematic of drug concentration in plasma following IV administration. After initially attaining maximum drug concentration and rapid distribution to other tissues, a stable elimination rate is achieved

from which the t1/2 for PK purposes can be estimated. Thus, for a time-dependent antibacterial such as oxytetracycline, the dose to be administered can be estimated if the

preferred dose interval, the MIC of the pathogen, the t1/2 and volume of distribution (Vd) are

known for the drug. Initial maximum serum concentration (Cmax) that would be required can be estimated from calculating the number of elimination half-lives that occur between the

initial dose and the second dose. The formula: Dose = Vd x Cmax is then applied to calculate

dose in mg/kg. Consideration for changes in both Cmax and t1/2 must be given if the antibacterial is administered by way of a route other than IV. Because of slower absorption

into plasma from the injection site and simultaneous distribution into tissue, the Cmax is 12 usually lower and the t1/2 longer for IM and SC administrations.

The mammary gland poses a significant barrier to drug distribution from plasma. As

might be expected from their relatively high Vd, lipophilic antibacterials such as macrolides ______Balbino M. Rocha, 2013 109 Cha pte r II: Antibacterial Resistance of Mastitis Pathogens

and oxytetracycline penetrate the mammary gland and milk at concentrations equal to or perhaps higher than concurrent concentrations in plasma. On the other hand, β-lactams, sulfonamides and aminoglycosides penetrate mammary tissue poorly and do not achieve concentrations in the mammary gland as high as those achieved in plasma (Figure 36). As a result, dosages of the drugs may have to be increased to compensate for poor distribution to the mammary gland. It is important to keep in mind the increased risk of toxicity with increased dose, particularly where extra-label drug use is employed. Alternatively, more frequent dosing may be indicated to maintain drug concentrations in tissues. It is suggested that a realistic basis for therapeutic protocols should be based on assurance that the

concentration of the drug for which 90% of bacterial isolates will be inhibited (MIC90) determined from multiple isolates of the same pathogen among dairy herds (or preferably the same herd) are below the clinical cutoff. Table 26 provides MIC information from mastitis isolates cultured and tested in the Michigan Animal Health Diagnostic Laboratory, collected from Michigan dairy farms between 1999 and 2000. These data (when not available from a diagnostic laboratory often used for clients) offer a starting point to estimate MIC of typical pathogens but can vary geographically, depending on strains of pathogen and previous exposure to antibacterials on a farm. 12

Although these data can be a useful means for designing an antibacterial protocol for a dairy, some caution should apply. PK parameters used in calculations are not absolute constants and may be extrapolated from species other than bovine or in steers rather than

cows. In addition, even within single studies, mean values that are calculated for t1/2 and other parameters can range by greater than 100% between animals. Almost entirely, studies to determine PK parameters used in determining label doses have been performed in clinically normal healthy animals. Thus, estimates of drug distribution and elimination used in dose determination may not account for alterations imposed by endotoxic shock, for example. In addition, subsequent doses following the initial dose may cause accumulation of drug in the body and there may be residual effect of drug that remains in plasma.

The following are some general principles to apply for pharmacologic considerations of mastitis therapy: 12

 The major factors that have the most influence on therapeutic regimens are the

elimination t1/2 and MIC of the target pathogen for the drug.

 Especially for antibacterial drugs that have a relatively short t1/2 (<10-12 hours), it is probably better to increase frequency of dosing rather than raise of dosage.  To attain effective time-dependent killing of bacteria, initial therapy should be maintained without switching of antibacterial drugs unless susceptibility testing suggests otherwise.

______Balbino M. Rocha, 2013 110 Cha pte r II: Antibacterial Resistance of Mastitis Pathogens

 When drugs are administered through other routes other than intravenously, a larger

dose may be indicated; t1/2 may be extended, which may impact withholding periods and therapeutic regimens.

Table 26: MIC data for several bacterial isolates from mastitic milk samples from the Michigan Animal Health Diagnostic Laboratory, 1999-2001 (Adapted from Erskine et al., 2003). 12

Antibacterial MIC50 MIC75 MIC90 Range

Ampicillin 2 2 16 1-16 Cephapirin 8 8 16 2-32

Gentamicin 0,5 0.5 1 0.25-8

TMP-SMX 0.5/9.5 0.5/9.5 0.5/9.5 0.5/9.5-4/76 E. E. coli Tetracycline 2 > 16 > 16 0.25 - >16 Ceftiofur 0.25 0.5 0.5 0.12-1

Ampicillin >16 >16 >16 4 - >16

Cephapirin 2 2 4 1-16 spp. Gentamicin 0.25 0.25 0.5 0.12-4 TMP-SMX 0.5/9.5 0.5/9.5 0.5/9.5 0.5/9.5 Tetracycline 1 1 >16 0.5-16

Klebsiella Ceftiofur 0.5 0.5 0.5 0.12-1

Ampicillin 0.12 0.5 2 0.12-4 Cephapirin <1 <1 <1 <1

Ceftiofur 0.5 0.5 1 0.25-1 Erythromycin 0.25 0.25 0.25 0.25-8

aureus Penicillin 0.12 0.25 2 0.12-4

S. S. Pirlimycin 0.25 0.25 0.5 0.06-4 TMP-SMX 0.5/9.5 0.5/9.5 0.5/9.5 0.5/9.5 Tetracycline 0.12 0.25 0.25 0.12-16

Ampicillin 0.12 0.12 0.12 0.12-0.25 Cephapirin <1 <1 <1 <1

Ceftiofur 0.25 0.25 0.25 0.25 Erythromycin 0.25 2 8 0.25-8 Penicillin 0.12 0.25 2 0.12-2

S. S. uberis Pirlimycin 0.12 0.12 1 0.12-4 TMP-SMX 0.5/9.5 0.5/9.5 0.5/9.5 0.5/9.5 Tetracycline 0.5 16 >16 0.12->16

Ampicillin 0.12 0.12 0.12 0.12

Cephapirin <1 <1 <1 <1 Ceftiofur 0.25 0.25 0.25 0.25-0.5 Erythromycin 0.25 0.25 2 0.25-8 Penicillin 0.12 0.12 0.12 0.12-0.25 Pirlimycin 0.12 0.25 0.5 0.12-4

S. S. dysgalactiae TMP-SMX 0.5/9.5 0.5/9.5 0.5/9.5 0.5/9.5 Tetracycline 1 2 >16 0.12->16

MIC data are reported as µg/mL and represent the concentration of drug for which 50%, 75% and 90% of bacterial isolates will be inhibited.

______Balbino M. Rocha, 2013 111 Cha pte r II: Antibacterial Resistance of Mastitis Pathogens

III. Resistance Patterns of Mastitis Pathogens

Resistance to antibacterial agents in mastitis pathogens discloses three relevant aspects: 1) Reduction in cure rates after treatment of mastitis cases; 33,34 2) Potential risk of transmission of resistant bacteria to humans via the food chain. 35 This is, however, not likely to occur with milk from clinical mastitis cases, since this milk is banned from human consumption. Nonetheless, clinical cases may turn into subclinical cases or latent infections. Resistant bacteria from these infections are present in the bulk tank milk and may therefore be transmitted to humans via raw milk products; and 3) Potential risk of transmission of resistance genes between mastitis pathogens and other environmental pathogens, which may consequently, through other routes, affect humans.

ABR among mastitis pathogens has been well documented over the years, with the publication of massive tables documenting ABR patterns of a large number of mastitis isolates based on the results of the agar disk diffusion method. Although such data has minimal clinical relevance in guiding the treatment of clinical mastitis in individual cows, because the results of susceptibility testing are repeatable, the outcomes of population susceptibility testing do provide useful information on the development or loss of ABR characteristics for mastitis pathogens in a population over time. 6 A summary of some relevant literature on the ABR trend patterns of major mastitis pathogens isolated in milk from cows with mastitis worldwide (Tables 28-34 of Appendix 1) is presented in the following section. Of notice, only data from studies conducted over a six-month period or greater were considered. 432-441

8. Trends on Resistance Patterns Over Time in Response to Antibacterial Usage

Very few studies have thoroughly demonstrated the long-term effects or trends regarding the use of antibacterials on antibacterial susceptibility of mastitis pathogens from dairy cows. The most extensive available data derive from a 7-year study by Erskine et al. 108 and a 6-year study by Nam et. al. 433 The majority of studies have conducted their research over shorter time-frames (e.g., 6 months to 3 years). 434-441

In the longest trends reported, from a 7-year study of US (Michigan) dairy herds that included Gram-positive (Tables 28 & 33 - Appendix 1) and Gram-negative (Tables 31 & 32 - Appendix 1) mastitis pathogens, the percentage of bacterial isolates susceptible to antibacterials did not change for the majority of the tests. 432 On the other hand, Rajala- Schultz et al. (2004) conducted a 16-month study on antibacterial susceptibility of mastitis pathogens isolated from first lactation and older dairy cows (Tables 28, 29, 32 and 34 - ______Balbino M. Rocha, 2013 112 Cha pte r II: Antibacterial Resistance of Mastitis Pathogens

Appendix 1). The study targeted CNS, esculin-positive streptococci and Gram-negative pathogens (E. coli, Serratia spp., Klebsiella spp., Citrobacter spp. and Enterobacter spp.). Resistance was mainly observed against penicillin with 39% and 26% of CNS isolates from older and first lactation cows, respectively, demonstrating resistance to this antibacterial. Although resistance to penicillin and tetracycline was higher in older and first lactation cows, respectively, differences in proportions of resistant isolates between the two groups were not statistically significant. 439

Table 27: Conclusions from short- to long-term studies on the effect of antibacterials on resistance of mastitis pathogens worldwide (Adapted from Oliver & Murinda, 2012). 31

Reference, Country, Year Comment

Bengtsson et al. 434, Sweden, 2009 Bacteria associated with acute mastitis for the most part were susceptible to antibiotics used in therapy, but resistance to penicillin in S. aureus is not uncommon.

Botrel et al. 435, France, 2010 The overall proportion of antibiotic resistance was low, except for penicillin G in staphylococci, as well as for macrolides and tetracycline in streptococci.

Ebrahimi et al. 442, Iran, 2007 Results indicated the world hazard of increased resistance by environmental mastitis pathogens. E. coli resistance (71%-88% for 5 of 11 antibiotics) was most pronounced.

Analysis for linear trends indicated increased susceptibility by some pathogens to some 432 Erskine et al. , USA, 2002 antibiotics. Overall there was no indication of increased resistance of mastitis isolates to antibacterials that are commonly used in dairy cattle mastitis.

Kalmus et al. 436, Estonia, 2011 Antimicrobial resistance was highly prevalent, especially penicillin resistance in 5 aureus and CNS.

Wide differences in the prevalence of resistance were apparent among individual 443 Nam et al. , PRK, 2009 Streptococcus spp. Some were 100% susceptible, but others showed varying rates of resistance. There was no significant change in the prevalence of bacterial and the proportion of 433 Nam et al. , PRK, 2009 antimicrobial resistance among gram-negative bacteria isolates during a 6-y period. A relatively high resistance to tetracycline was observed. S. aureus and CNS were the most frequently isolated pathogens. Whereas, 45% of S. 437 Persson , Sweden, 2011 aureus isolates and 35% of the CNS isolates were resistant to penicillin G. Resistance to other antimicrobials was uncommon. Most isolates of S aureus, CNS, and Streptococcus spp. were inhibited at the lowest Pol & Ruegg 438, USA, 2007 dilution of most antimicrobial drugs tested. Exposure to most antimicrobial drugs commonly used for prevention and treatment of mastitis was not associated with resistance. Differences in the proportions of resistant isolates of CNS between first lactation and older 439 Rajala-Schultz et al. , USA, 2004 cows were not statistically significant. Resistance patterns of the CNS isolated during the study were concordant with antimicrobial usage in the study herd.

Roesch et al. 440, Switzerland, 2006 Antibiotic resistance in mastitis pathogens (S. aureus, non-aureus staphylococci, S. dysgalactiae, S uberis) from organic and conventional dairy farms was not different.

Enterococcus spp. were the most resistant organisms tested. Environmental streptococci 396 Rossitto et al. , USA, 2002 are a diverse group of organisms composed of several different genera and species and their identification to species level is needed for targeted control methods. All S. aureus isolates were susceptible to ciprofloxacin, gentamicin, imipenem. 444 Sahebekhtiari et al. , Iran, 2011 minocycline, oxacillin, and vancomycin and demonstrated highest resistance to ampicillin (64%) and penicillin (56%), and median resistance to other antimicrobials. E. coli was sensitive to most antimicrobials. CNS demonstrated greatest resistance San Martin et al. 441, Chile, 2012 (26.8%-56.9%) to antibiotics. S. aureus showed the highest level of resistance (24%- 38.9%) to five antibiotics. Streptococcal strains were highly resistant to lincomycin (61.9%).

Percentages of antimicrobial-resistant bacteria (CNS, environmental streptococci, A 445 Suriyasathaporn , Thailand, 2010 pyogenes, C. bovis) at a former organic farm decreased after 6 months operating as an organic farm system.

______Balbino M. Rocha, 2013 113 Cha pte r II: Antibacterial Resistance of Mastitis Pathogens

Table 27 summarizes the conclusions from fifteen short- to long-term studies that reported resistance in mastitis pathogens over a period of 6 months to 7 years. These studies suggest that most mastitis pathogens are generally susceptible to antibacterials used for treatment of mastitis. 396,432-445 Some of these studies pointed towards increased resistance. For example, S. aureus has revealed heightened resistance particularly to penicillin 434,436,437,441,444 and ampicillin. 441,444 Even though generally susceptible to most antibacterial agents, 441 E. coli has demonstrated increased resistance to some antibacterials, such as tetracycline, β-lactams and lincomycin. 433,435,441

Over a 1-year study during a dry cow mastitis program – one of the more consistent uses of antibacterials in dairy operations – indicated that therapeutic antibacterial treatment with IMM administration of large doses of penicillin/dihydrostreptomycin had little or no effect on drug resistance to E. coli in the dairy herd and its immediate environment. 446

One way to assess the effects of antibacterial use on resistance is to compare and contrast systems that employ different production strategies – such as organic dairies, that use little to no antibacterials, and conventional dairies where antibacterials are used in all categories of dairy animals. 447 Pol & Ruegg (2007) analyzed relationships between antibacterial usage at the farm level, comparing organic versus conventional US dairies and antibacterial susceptibility of S. aureus (Table 28 - Appendix 1), CNS (Table 29 - Appendix 1), esculin-positive streptococci and Enterococcus spp. isolates (Table 33 - Appendix 1), collected from 1994 to 2000. Contrary to expected, more IMIs were present in organic than in conventional herds and all isolates (except coliforms) were more prevalent on organic herds. 438 Moreover, Roesch et al. (2006) indicated that antibacterial resistance in mastitis pathogens from organic and conventional dairy herds was not different, with the authors suggesting that this discrepancy needs a study of the factors accounting for the absence of reduced resistance in organic farms. 440 Another study, that researched antibacterial susceptibility of S. aureus in bulk tank milk in organic and conventional dairies in Demark and in the USA reported small differences between them. 448

______Balbino M. Rocha, 2013 114 MATERIALS & METHODS Mat eri als & Met hods

I. Criteria for Selection of Cases

This retrospective study consisted in reviewing records of all bacteriological outcomes obtained from clinical and subclinical mastitis milk samples from dairy cattle of Portuguese northwestern, central and southern herds. Milk samples were forwarded to an Animal Health and Food Safety Laboratory (Segalab, S.A. - Matosinhos, Portugal) between January 2004 and September 2012. Data results from antibacterial susceptibility testing were included in the study.

II. Sample Collection and Microbiology

In addition to formal quality control procedures, Segalab has ISO 17025 accreditation by the Instituto Português de Acreditação (IPAC) and is supported by interlaboratory proficiency testing (Vetqas®, provided by the AHVLA). This norm represents the basic requirements for a quality management system as illustrated by the ISO 9001 model but adds additional technical requirements needed to demonstrate competence in testing and/or calibration activities. The laboratory's commercial nature has allowed, over the years, the reception of clinical and subclinical mastitis milk samples as part of either occasional private individual initiatives by farmers or a herd's assisting veterinarian, or by means of milk quality programs established and performed by the lab's technical services to herds that have taken part in these programs. Mastitic milk samples are collected by aseptic technique and submitted to the laboratory under refrigerated conditions and short time-frames, under NMC guidelines. At the laboratory, milk samples are cultured and mastitis pathogens are identified using standard microbiologic methods. Briefly, 0.01 mL of the milk sample is streaked on a portion of a Columbia nalidixic acid blood agar plate (CNA; bioMérieux®) and a portion of a McConkey agar plate (MCK; bioMérieux®). Another portion of the sample is added to a Brain Heart Infusion tube (BHI; Biokar Diagnostics®). All plates and tubes are incubated at 35 to 37°C and examined for growth at 4 (BHI only), 24 and 48 h. Bacteria are identified either by genus (using colony morphology, Gram staining and biochemical tests), or by species (via the automated Vitek® 2 Compact system; bioMérieux®). By default, the laboratory identifies all isolates by genus. Speciation is only determined when requested by the submitting farmer/veterinarian, or when in cases of uncertainty by the lab technician, particularly when it comes to major mastitis pathogens. Contaminated samples are defined, by NMC guidelines, as a combination of three or more isolated dissimilar colony types. Once identified, pure cultures of mastitis pathogens were tested for in vitro antibacterial susceptibility by the Kirby-Bauer disk diffusion methodology.

______Balbino M. Rocha, 2013 116 Mat eri als & Met hods

III. In vitro Antibacterial Susceptibility Testing

In vitro antibacterial susceptibility testing was conducted by the Kirby-Bauer disk diffusion test method in accordance with the standards described in the CLSI M31-A3 document, including suggested breakpoints to determine susceptibility and resistance. 190 Each isolate was added to sterile diluents to contain approximately 108 CFU/mL (0,5 on McFarland scale) and plated on Mueller-Hinton agar (bioMérieux®), with or without supplementation of 5% defibrinated sheep blood, depending on the isolate's genera. Disks impregnated with the tested antibacterial agents were placed over the agar and incubated at 37ºC for 24 hours. Susceptibility data was determined by measurement of zone of inhibition around the antibacterial disks, according to the zone diameter interpretative CLSI standards, 190 and when not available, according to the disk manufacturers’ instructions. E. coli - ATCC isolate 25922, and S. aureus - ATCC isolate 33862, were used as the quality control organisms. Only quality controlled results were reported. Isolates were classified as susceptible, of intermediate susceptibility, or resistant on the basis of CLSI standards. 190 Laboratory protocols (using NMC guidelines) remained practically unchanged during the study period. Antibacterial disks and/or manufacturers changed sporadically, depending on stock/market availability.

IV. Tested Antibacterials

The tested antibacterials considered for analysis were: Amoxicillin/Clavulanic acid (AUG), 30 g (20g + 10g); Cloxacillin (OB/CX), 5 g; Penicillin G (P), 10 IU; Cefazolin (KZ), 30 g; Cefquinome (CEQ), 10 g; Gentamicin (CN), 10 g; and Trimethoprim/Sulfamethoxazole (SXT), 25 g (1,25 g + 23,75 g). The selection of the tested antibacterials was based on the following criteria: 1) License and availability on the Portuguese market for mastitis intramammary therapy in lactating cows; 449 2) Sales and use in mastitis intramammary therapy in lactating cows; 254 3) Use in human medicine (Cefquinome as exception); 4) Listed as "critically important" and "highly important" antibacterials in human medicine (Cefquinome as exception). 235

V. Selection of Pathogens

Only tested pure cultures of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Streptococcus dysgalactiae, Enterococcus spp. (E. faecium and E. faecalis only), Escherichia coli and Klebsiella pneumoniae, were considered for analysis. The selection of these pathogens was based on the following criteria: 1) Classification as major ______Balbino M. Rocha, 2013 117 Mat eri als & Met hods

mastitis pathogens; 2) Importance for veterinary medicine and dairy industry; 3) Importance for Public Health.

VI. Data Analysis

For purposes of statistical analysis, isolates classified as being of intermediate susceptibility were not included in the study. Also, E. faecium and E. faecalis isolates were sorted, forming the Enterococcus spp. group. The proportion of tests that were resistant to an individual antibacterial agent was summarized for each year, for the study's nine-year period. The terms used to describe the antibacterial resistance levels were based on EFSA standards: 1) Rare: <0.1%; 2) Very low: 0.1% to 1%; 3) Low: >1% to 10%; 4) Moderate: >10% to 20%; 5) High: >20% to 50%; 6) Very high: >50% to 70%; and 7) Extremely high: >70%. 11 Logistic regression was performed to determine the probability of antibacterial resistance by year. The logistic regression model for the levels of resistance by year included resistance as a response variable ("yes" vs. "no") and year as a continuous variable [coded from "0" (2004) to "8" (2012)]. Additionally, a second logistic regression model was performed. This model included resistance as a response variable ("yes" vs. "no") and year as a ordinal variable ("2004" to "2012"). Of the available contrast methods in SPSS, the repeated method ("Each category of the predictor variable except the first category is compared to the category that precedes it") was considered to be the most adjusted for this last model, since the goal was to compare each year's probability of antibacterial resistance with the one that precedes it. The difference between the two models and the reasons to apply both is that, while the first model reflects the overall probability of observing resistance during any given year, compared with the year before and therefore allowing to identify overall trends throughout the nine years of the study; the second model compares the probability of antibacterial resistance of a certain specific year with the one that precedes it and, as a result, allows a more punctual approach, determining significant changes between two successive years. For all analyses, values of p<0.05 were considered significant. The statistical analysis was performed using IBM SPSS Statistics, version 21.0 (New York, U.S.A.).

______Balbino M. Rocha, 2013 118 RESULTS Results

Throughout the study period, antibacterial agents used in susceptibility testing varied on the basis of isolated pathogen, year, laboratory protocol and stock/market availability of diffusion disks (Table 28). A total of 47,413 antibacterial susceptibility testing results were included in the present study. Overall, 28,136 antibacterial susceptibility tests were performed on Staphylococcus aureus isolates; 5,916 on Escherichia coli isolates; 5,799 on Streptococcus uberis isolates; 4,589 on Streptococcus agalactiae isolates; 1,231 on Streptococcus dysgalactiae isolates; 979 on Enterococcus spp. (E. faecium and E. faecalis only) isolates; and 773 on Klebsiella pneumonia isolates (Tables 29 and 30).

Table 28: Antibacterial agents used in 47,413 susceptibility tests of bacterial pathogen isolates obtained from dairy cow milk samples and submitted for bacterial culture between January 2004 and September 2012.

Pathogens isolated Antibacterial Agents tested

Staphylococcus aureus AUG, OB/CX, P, KZ, CEQ, CN and SXT

Escherichia coli AUG, OB/CX, P, KZ, CEQ, CN and SXT

Streptococcus uberis AUG, OB/CX, P, KZ, CEQ, CN and SXT

Streptococcus agalactiae AUG, OB/CX, P, KZ, CEQ and SXT

Streptococcus dysgalactiae AUG, OB/CX, P, KZ and SXT

Enterococcus spp. * AUG, OB/CX, P, KZ and SXT

Klebsiella pneumoniae AUG, OB/CX, P, KZ, CN and SXT

* includes only Enterococcus faecium and Enterococcus faecalis species. AUG - Amoxicillin/Clavulanic acid; OB/CX - Cloxacillin; P - Penicillin G; KZ - Cefazolin; CEQ - Cefquinome; CN - Gentamicin; SXT - Trimethoprim/Sulfamethoxazole.

Table 29: Number of antibacterial susceptibility tests along the study period (2004 - 2012).

Staphylococcus Streptococcus Streptococcus Streptococcus Enterococcus Escherichia Klebsiella Total Total aureus uberis agalactiae dysgalactiae spp. * coli pneumoniae (%)

2004 1,491 604 159 137 199 127 56 2,773 5.9

2005 2,715 426 278 132 205 150 15 3,921 8.3 2006 3,766 298 620 87 84 364 131 5,350 11.3 2007 4,866 633 792 124 199 805 137 7,556 15.9 2008 5,070 925 586 323 119 935 76 8,034 16.9 2009 4,051 983 456 155 NT 978 138 6,761 14.3 2010 2,602 874 894 273 173 1,086 134 6,036 12.7 2011 2,138 580 376 NT NT 846 65 4,005 8.4 2012 1,427 476 428 NT NT 625 21 2,977 6.3

Total 28,126 5,799 4,589 1,231 979 5,916 773 47,413

Total (%) 59.3 9.7 2.6 2.1 12.5 12.2 1.6 100.0

* includes only Enterococcus faecium and Enterococcus faecalis species.

______

Balbino M. Rocha, 2013 120 Results

Table 30: Overall resistance patterns of major mastitis bacterial pathogens obtained from submitted dairy cow milk samples, sorted by antibacterial agent.

S. aureus S. uberis S. agalactiae S. dysgalactiae Enterococcus E. coli K. pneumoniae (n = 28,126) (n = 5,799) (n = 4,589) (n = 1,231) spp. (n = 979) (n = 5,916) (n = 773)

N.º R N.º R N.º R N.º R N.º R N.º R N.º R tested (%) tested (%) tested (%) tested (%) tested (%) tested (%) tested (%)

AUG 5,387 12.3 1,309 0 1,108 0 307 0 257 3.9 1,292 30.3 198 37.4

OB/CX 5,348 2.9 1,257 22.1 1,093 11.4 307 5.5 263 96.2 214 99.5 58 100

P 4,834 44.7 517 0.4 404 0.2 130 0.8 50 70.0 333 99.4 73 100

KZ 3,844 0.5 1,225 1.0 1,029 0.1 307 0.7 254 79.9 1,224 9.6 189 8.5

CEQ 2,450 1.3 441 0.2 460 0.7 NT NT 707 8.2 NT

CN 5,242 2.2 169 71.0 NT NT NT 1,179 3.6 157 1.3

SXT 1,021 0.7 881 3.1 495 1.4 180 3.8 155 23.9 967 13.1 98 6.1

AUG - Amoxicillin/Clavulanic acid; OB/CX - Cloxacillin; P - Penicillin G; KZ - Cefazolin; CEQ - Cefquinome; CN - Gentamicin; SXT - Trimethoprim/Sulfamethoxazole; NT - Not tested; R - Resistance; Enterococcus spp. includes only Enterococcus faecium and Enterococcus faecalis species.

The resistance patterns of the major mastitis pathogens tested revealed a wide variation in the levels of resistance, ranging from 0 to 100% (Table 30). Generally and in average, the proportion of tests that were found to be resistant to the selected antibacterial agents was below 15% (Table 30). S. agalactiae and S. dysgalactiae were the pathogens with the lowest resistance proportions (0 to 11.4%, between both). Nonetheless, antibacterial resistance was prevalent, at high to extremely high levels, among some pathogens (Table 30): S. uberis for cloxacillin (22.1%) and gentamicin (71.0%); S. aureus for penicillin (44.7%); E. coli for amoxicillin/clavulanic acid (30.3%), penicillin (99.4%) and cloxacillin (99.5%); Enterococcus spp. for trimethoprim/sulfamethoxazole (23.9%), penicillin (70.0%), cefazolin (79.9%) and cloxacillin (96.2%); and K. pneumoniae for amoxicillin/clavulanic acid (37.4%), cloxacillin (100%) and penicillin (100%). Penicillin and cloxacillin were the antibacterials to which more pathogens presented the highest resistance proportions. On the other hand, cefquinome, amoxicillin/clavulanic acid and trimethoprim/sulfamethoxazole were the antibacterials to which pathogens presented the lowest resistance proportions (Table 30).

In general, for all the antibacterial susceptibility tests, the percentage of pathogen isolates resistant to the tested antibacterial agents did not have a significant change over time (Table 31). Still, some isolates' resistance proportions to the tested antibacterial agents did have a significant increase over time (Table 31), namely: S. aureus (amoxicillin/clavulanic acid, cloxacillin, cefquinome and gentamicin); S. agalactiae (cloxacillin); S. uberis (cloxacillin and gentamicin); S. dysgalactiae (cloxacillin); E. coli (amoxicillin/clavulanic acid, cefazolin and trimethoprim/sulfamethoxazole); K. pneumoniae (amoxicillin/clavulanic acid). Enterococcus spp. did not exhibit any significant change over time for any of the tested antibacterials. Also, penicillin was the only antibacterial agent that did not present any significant change over time for any of the isolated pathogen resistance proportions.

______

Balbino M. Rocha, 2013 121 Results

Table 31: Results of logistic regression analysis (model 1) to determine, for the antibacterial susceptibility tests, whether the resistance levels to the various antibacterial agents changed with year.

AUG OB/CX P KZ CEQ CN SXT

OR p OR p OR p OR p OR p OR p OR p (95% CI) value (95% CI) value (95% CI) value (95% CI) value (95% CI) value (95% CI) value (95% CI) value

1.42 1.19 0.99 0.99 1.29 1.15 0.80 < 0.001 < 0.001 0.797 0.993 0.004 0.001 0.453 S. aureus (1.36-1.48)1 (1.10-1.28)1 (0.97-1.03)1 (0.76-1.32)2 (1.09-1.54)1 (1.06-1.26)1 (0.45-1.43)3 1.19 1.09 2.86 1.58 0.93 S. agalactiae 1.00 1 1.000 < 0.001 0.842 0.265 0.264 NT NT 0.709 (1.08-1.30)1 (0.46-2.58)4 (0.45-18.22)1 (0.71-3.54)1 (0.65-1.35)6 1.09 0.76 1.13 3.15 0.98 S uberis 1.00 1 1.000 0.008 0.490 0.416 0 8 0.988 < 0.001 0.825 (1.02-1.15)1 (0.35-1.66)7 (0.85-1.50)1 (2.09-4.73)9 (0.82-1.17)1 1.45 0.62 0.83 1.03 S. dysgalactiae 1.00 10 1.000 0.028 0.548 0.562 NT NT NT NT 0.906 (1.04-2.00) 10 (0.13-3.00)11 (0.43-1.58)10 (0.65-1.63)10

Enterococcus 0.89 1.04 1.08 1.10 1.19 12 0.475 12 0.823 11 0.741 12 0.180 NT NT NT NT 12 0.126 spp. * (0.66-1.22) (0.77-1.40) (0.68-1.71) (0.96-1.27) (0.95-1.49)

1.50 9 0.32 1.18 0.83 0.99 1.13 E. coli < 0.001 0 0.994 14 0.281 0.001 15 0.064 16 0.939 15 0.034 (1.39-1.62)13 (0.04-2.51) (1.07-1.31)1 (0.67-1.01) (0.85-1.17) (1.01-1.26) 1.35 9 14 1.14 0.83 1.12 K. pneumonia < 0.001 0 1.000 0 1.000 1 0.315 NT NT 18 0.545 19 0.749 (1.15-1.58)17 (0.88-1.47) (0.46-1.51) (0.57-2.19)

AUG - Amoxicillin/Clavulanic acid; OB/CX - Cloxacillin; P - Penicillin G; KZ - Cefazolin; CEQ - Cefquinome; CN - Gentamicin; SXT - Trimethoprim/Sulfamethoxazole; NT - Not tested; OR - Odds ratio; CI - Confidence Interval; * includes only Enterococcus faecium and Enterococcus faecalis species; 1 All years included (2004-2012); 2 2004-2011; 3 All years included, except 2006, 2009, 2010 and 2012; 4 All years included, except 2008-2011; 5 All years included, except 2008; 6 All years included, except 2004, 2009 and 2011; 7 All years included, except 2009-2011; 8 All years included, except 2005, 2008 and 2009; 9 2004-2007; 10 2004-2010; 11 2004-2008; 12 All years included, except 2009, 2011 and 2012; 13 2005-2012; 14 2005-2008; 15 2007-2012; 16 All years included, except 2006; 17 All years included, except 2005; 18 All years included, except 2005 and 2006; 19 2006-2010.

The percentage of S. aureus tests resistant to amoxicillin/clavulanic acid had an overall significant increase (p<0.001), from 0.7% in 2004 to 26.6% in 2012 (Tables 31 and 32). Along the years (2nd LR model), statistically significant changes were verified in the percentage of S. aureus tests resistant to this antibacterial: 1) An increase, from 0.7% in 2004 to 10.1% in 2005 (p<0.001); 2) A decrease, from 10.1% in 2005 to 0.7% in 2006 (p<0.001); 3) An increase, from 1.0% in 2007 to 13.7% in 2008 (p<0.001); 4) An increase, from 13.7% in 2008 to 25.0% in 2009 (p<0.001); 5) An increase, from 18.4% in 2011 to 26.6% in 2012 (p=0.005) (Table 32. See also Figure 37 and Table 46 of Appendix 2). The percentage of S. aureus tests resistant to cloxacillin had an overall significant increase (p<0.001), from 1.1% in 2004 to 3.0% in 2012 (Tables 31 and 32). Along the years (2nd LR model), statistically significant changes were verified in the percentage of S. aureus tests resistant to this antibacterial: 1) A decrease, from 2.7% in 2006 to 1.0% in 2007 (p=0.020); 2) An increase, from 2.0% in 2008 to 6.8% in 2009 (p<0.001) (Table 32. See also Figure 37 and Table 46 of Appendix 2). The percentage of S. aureus tests resistant to cefquinome had an overall significant increase (p=0.004), from 0% in 2004 to 3.1% in 2012 (Tables 31 and 32). Along the years (2nd LR model), statistically significant changes were verified in the percentage of S. aureus tests resistant to this antibacterial: 1) An increase, from 0.6% in 2010 to 4.3% in 2011 (p=0.003) (Table 32. See also Figure 37 and Table 46 of Appendix 2). The percentage of S. aureus tests resistant to gentamicin had an overall significant increase (p=0.001), from 1.2% in 2004 to 2.9% in 2012 (Tables 31 and 32). Along the years (2nd LR model), statistically significant changes were verified in the percentage of S. aureus tests resistant to this antibacterial: 1) An increase, from 0.5% in 2009 to 3.6% in 2010 (p<0.001) (Table 32. See also Figure 37 and Table 46 of Appendix 2). No other antibacterials had a significant change in the percentage of resistant S. aureus tests (Table 31). Despite this, ______

Balbino M. Rocha, 2013 122 Results

when comparing the probability of antibacterial resistance of specific years with the preceding ones (2nd LR model), penicillin revealed statistically significant changes in the percentage of resistant S. aureus tests, to be exact: 1) A decrease, from 51.3% in 2004 to 17.7% in 2005 (p<0.001); 2) An increase, from 17.7% in 2005 to 47.5% in 2006 (p<0.001) (Table 32. See also Figure 37 and Table 46 of Appendix 2).

Table 32: Staphylococcus aureus resistance proportions, among each tested antibacterial agent, along each tested year (n = 28,126 tests).

Antibacterial Year Agent 2004 2005 2006 2007 2008 2009 2010 2011 2012 Total

2/269 49/487 * 5/727 * 9/931 123/896 * 189/757 * 110/519 85/463 90/338 * 662/5387 AUG (0.7%) (10.1%) (0.7%) (1.0%) (13.7%) (25.0%) (21.2%) (18.4%) (26.6%) (12.3%) 3/271 7/487 14/527 10/979 * 20/979 54/795 * 23/505 16/476 10/329 157/5348 OB/CX (1.1%) (1.4%) (2.7%) (1.0%) (2.0%) (6.8%) (4.6%) (3.4%) (3.0%) (2.9%) 139/271 47/266 * 320/673 * 388/775 441/942 352/768 222/532 188/479 62/128 2159/4834 P (51.3%) (17.7%) (47.5%) (50.1%) (46.8%) (45.8%) (41.7%) (39.2%) (48.4%) (44.7%) 0/262 3/437 5/757 3/737 9/944 1/660 0/23 0/24 21/3844 NT KZ (0%) (0.7%) (0.7%) (0.4%) (1.0%) (0.2%) (0%) (0%) (0.5%) 0/70 1/189 4/359 1/158 6/355 0/317 3/500 9/209 * 9/293 33/2450 CEQ (0%) (0.5%) (1.1%) (0.6%) (1.7%) (0%) (0.6%) (4.3%) (3.1%) (1.3%) 3/255 6/459 21/723 14/823 13/907 4/754 19/523 * 25/459 10/339 115/5242 CN (1.2%) (1.3%) (2.9%) (1.7%) (1.4%) (0.5%) (3.6%) (5.4%) (2.9%) (2.2%) 1/93 5/390 0/463 0/47 1/28 7/1021 NT NT NT NT SXT (1.1%) (1.3%) (0%) (0%) (3.6%) (0.7%)

AUG - Amoxicillin/Clavulanic acid; OB/CX - Cloxacillin; P - Penicillin G; KZ - Cefazolin; CEQ - Cefquinome; CN - Gentamicin; SXT - Trimethoprim/Sulfamethoxazole; NT - Not tested; * p<0.05.

Table 33: Streptococcus agalactiae resistance proportions, among each tested antibacterial agent, along each tested year (n = 4,589 tests).

Year Antibacterial Agent 2004 2005 2006 2007 2008 2009 2010 2011 2012 Total

0/36 0/57 0/132 0/167 0/149 0/139 0/221 0/119 0/88 0/1108 AUG (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%)

0/36 0/57 0/128 5/167 10/162 81/145 * 24/194 * 5/115 * 0/89 125/1093 OB/CX (0%) (0%) (0%) (3.0%) (6.2%) (55.9%) (12.4%) (4.3%) (0%) (11.4%) 0/36 0/57 0/134 1/138 0/39 1/404 NT NT NT NT P (0%) (0%) (0%) (0.7%) (0%) (0.2%) 0/36 0/50 0/133 0/119 0/162 0/145 0/222 1/111 0/51 1/1029 KZ (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0.9%) (0%) (0.1%) 0/15 0/14 0/77 0/30 0/27 1/193 2/31 * 0/73 * 3/460 NT CEQ (0%) (0%) (0%) (0%) (0%) (0.5%) (6.5%) (0%) (0.7%) 0/43 0/16 4/171 2/113 0/64 1/88 7/495 NT NT NT SXT (0%) (0%) (2.3%) (1.8%) (0%) (1.1%) (1.4%)

AUG - Amoxicillin/Clavulanic acid; OB/CX - Cloxacillin; P - Penicillin G; KZ - Cefazolin; CEQ - Cefquinome; SXT - Trimethoprim/Sulfamethoxazole; NT - Not tested; * p<0.05.

The percentage of S. agalactiae tests resistant to cloxacillin had an overall significant increase (p<0.001) along the tested years (Tables 31 and 33). Along the years (2nd LR model), statistically significant changes were verified in the percentage of S. agalactiae tests resistant to this antibacterial: 1) An increase, from 6.2% in 2008 to 55.9% in 2009 (p<0.001);

______

Balbino M. Rocha, 2013 123 Results

2) A decrease, from 55.9% in 2009 to 12.4% in 2010 (p<0.001); 3) A decrease, from 12.4% in 2010 to 4.3% in 2011 (p=0.025) (Table 33. See also Figure 39 and Table 47 of Appendix 2). No other antibacterials had a significant change in the percentage of resistant S. agalactiae tests (Table 31). Despite this, when comparing the probability of antibacterial resistance of specific years with the preceding ones (2nd LR model), cefquinome revealed statistically significant changes in the percentage of resistant S. agalactiae tests, to be exact: 1) An increase, from 0.5% in 2010 to 6.5% in 2011 (p=0.037) (Table 33. See also Figure 39 and Table 47 of Appendix 2).

The percentage of S. uberis tests resistant to cloxacillin had an overall significant increase (p=0.008) along the tested years (Tables 31 and 34). Along the years (2nd LR model), statistically significant changes were verified in the percentage of S. uberis tests resistant to this antibacterial: 1) An increase, from 8.1% in 2007 to 17.3% in 2008 (p=0.022); 2) An increase, from 17.3% in 2008 to 72.3% in 2009 (p<0.001); 3) A decrease, from 72.3% in 2009 to 0% in 2010 (p<0.001); 4) An increase, from 0% in 2010 to 15.3% in 2011 (p=0.001); 5) A decrease, from 15.3% in 2011 to 5.9% in 2012 (p=0.031) (Table 34. See also Figure 41 and Table 48 of Appendix 2). The percentage of S. uberis tests resistant to gentamicin had an overall significant increase (p<0.001), from 41.5% in 2004 to 88.2% in 2007 (Tables 31 and 34). Along the years (2nd LR model), statistically significant changes were verified in the percentage of S. uberis tests resistant to this antibacterial: 1) An increase, from 60.6% in 2005 to 98.0% in 2006 (p=0.001) (Table 34. See also Figure 41 and Table 48 of Appendix 2). No other antibacterials had a significant change in the percentage of resistant S. uberis tests (Table 31).

Table 34: Streptococcus uberis resistance proportions, among each tested antibacterial agent, along each tested year (n = 5,799 tests).

Year Antibacterial Agent 2004 2005 2006 2007 2008 2009 2010 2011 2012 Total

0/121 0/84 0/52 0/121 0/216 0/254 0/228 0/130 0/103 0/1309 AUG (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) 11/119 7/80 1/51 10/123 38/220 * 185/256 * 0/176 * 20/131 * 6/101 * 278/1257 OB/CX (9,2%) (8.8%) (2.0%) (8.1%) (17.3%) (72.3%) (0%) (15.3%) (5.9%) (22.1%) 1/119 0/79 0/54 1/106 0/85 0/74 2/517 NT NT NT P (0.8%) (0%) (0%) (0.9%) (0%) (0%) (0.4%) 0/119 0/80 0/49 1/100 2/235 8/260 1/220 0/133 0/29 12/1225 KZ (0%) (0%) (0%) (1.0%) (0.9%) (3.1%) (0.5%) (0%) (0%) (1.0%) 1/32 0/24 0/21 0/225 0/71 0/68 1/441 NT NT NT CEQ (3.1%) (0%) (0%) (0%) (0%) (0%) (0.2%) 22/53 20/33 48/49 * 30/34 120/169 NT NT NT NT NT CN (41.5%) (60.6%) (98.0%) (88.2%) (71.0%)

1/41 1/70 0/19 7/128 2/169 11/213 0/25 5/115 0/101 27/881 SXT (2.4%) (1.4%) (0%) (5.5%) (1.2%) (5.2%) (0%) (4.3%) (0%) (3.1%)

AUG - Amoxicillin/Clavulanic acid; OB/CX - Cloxacillin; P - Penicillin G; KZ - Cefazolin; CEQ - Cefquinome; CN - Gentamicin; SXT - Trimethoprim/Sulfamethoxazole; NT - Not tested; * p<0.05.

______

Balbino M. Rocha, 2013 124 Results

The percentage of S. dysgalactiae tests resistant to cloxacillin had an overall significant increase (p=0.028), from 3.2% in 2004 to 11.8% in 2010 (Tables 31 and 35). No other antibacterials had a significant change in the percentage of resistant S. dysgalactiae tests (Table 31).

Table 35: Streptococcus dysgalactiae resistance proportions, among each tested antibacterial agent, along each tested year (n = 1,231 tests).

Year Antibacterial Agent 2004 2005 2006 2007 2008 2009 2010 Total

0/31 0/27 0/18 0/26 0/76 0/40 0/89 0/307 AUG (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) 1/31 0/27 1/19 1/27 1/78 3/40 10/85 17/307 OB/CX (3.2%) (0%) (5.3%) (3.7%) (1.3%) (7.5%) (11.8%) (5.5%) 0/31 1/27 0/20 0/19 0/33 1/130 NT NT P (0%) (3.7%) (0%) (0%) (0%) (0.8%) 0/31 0/27 1/20 0/23 1/78 0/40 0/88 2/307 KZ (0%) (0%) (5.0%) (0%) (1.3%) (0%) (0%) (0.7%) 1/13 0/24 1/10 0/29 4/58 0/35 1/11 7/180 SXT (7.7%) (0%) (10.0%) (0%) (6.9%) (0%) (9.1%) (3.9%)

AUG - Amoxicillin/Clavulanic acid; OB/CX - Cloxacillin; P - Penicillin G; KZ - Cefazolin; SXT - Trimethoprim/Sulfamethoxazole; NT - Not tested.

Table 36: Enterococcus spp. (E. faecalis and E. faecium) resistance proportions, among each tested antibacterial agent, along each tested year (n = 979 tests).

Year Antibacterial Agent 2004 2005 2006 2007 2008 2009 2010 Total

4/58 1/52 1/20 2/50 0/19 2/58 10/257 NT AUG (6.9%) (1.9%) (5.0%) (4.0%) (0%) (3.4%) (3.9%) 57/58 49/53 20/21 47/49 32/32 48/50 253/263 NT OB/CX (98.3%) (92.5%) (95.2%) (95.9%) (100%) (96.0%) (96.2%) 4/6 5/8 8/9 9/16 9/11 35/50 NT NT P (66.7%) (62.5%) (88.9%) (56.3%) (81.8%) (70.0%) 44/59 39/49 18/22 24/35 31/32 47/57 203/254 NT KZ (74.6%) (79.6%) (81.8%) (68.6%) (96.9%) (82.5%) (79.9%) 0/18 11/43 4/12 11/49 8/25 3/8 37/155 NT SXT (0%) (25.6%) (33.3%) (22.4%) (32.0%) (37.5%) (23.9%)

AUG - Amoxicillin/Clavulanic acid; OB/CX - Cloxacillin; P - Penicillin G; KZ - Cefazolin; SXT - Trimethoprim/Sulfamethoxazole; NT - Not tested.

The percentage of E. coli tests resistant to amoxicillin/clavulanic acid had an overall significant increase (p<0.001), from 22.6% in 2005 to 55.0% in 2012 (Tables 31 and 37). Along the years (2nd LR model), statistically significant changes were verified in the percentage of E. coli tests resistant to this antibacterial: 1) An increase, from 23.8% in 2009 to 34.7% in 2010 (p=0.012); 2) An increase, from 34.7% in 2010 to 51.9% in 2011 (p<0.001) (Table 37. See also Figure 47 and Table 51 of Appendix 2). The percentage of E. coli tests resistant to cefazolin had an overall significant increase (p=0.001), from 4.9% in 2004 to 24.4% in 2012 (Tables 31 and 37). Along the years (2nd LR model), statistically significant

______

Balbino M. Rocha, 2013 125 Results

changes were verified in the percentage of E. coli tests resistant to this antibacterial: 1) An increase, from 5.1% in 2010 to 11.4% in 2011 (p=0.025); 2) An increase, from 11.4% in 2011 to 24.4% in 2012 (p=0.003) (Table 37. See also Figure 47 and Table 51 of Appendix 2). The percentage of E. coli tests resistant to trimethoprim/sulfamethoxazole had an overall significant increase (p=0.034), from 9.6% in 2007 to 14.3% in 2012 (Tables 31 and 37). Along the years (2nd LR model), statistically significant changes were verified in the percentage of E. coli tests resistant to this antibacterial: 1) An increase, from 8.7% in 2010 to 20.5% in 2011 (p=0.004) (Table 37. See also Figure 47 and Table 51 of Appendix 2). No other antibacterials had a significant change in the percentage of resistant E. coli tests (Table 31). Despite this, when comparing the probability of antibacterial resistance of specific years with the preceding ones (2nd LR model), cefquinome revealed statistically significant changes in the percentage of resistant E. coli tests, to be exact: 1) An increase, from 0% in 2008 to 27.7% in 2009 (p<0.001); 2) A decrease, from 27.7% in 2009 to 2.5% in 2010 (p<0.001) (Table 33. See also Figure 39 and Table 47 of Appendix 2).

Table 37: Escherichia coli resistance proportions, among each tested antibacterial agent, along each tested year (n = 5,916 tests).

Antibacterial Year Agent 2004 2005 2006 2007 2008 2009 2010 2011 2012 Total

7/31 13/86 22/181 35/206 49/206 83/239 * 110/212 * 72/131 391/1292 NT AUG (22.6%) (15.1%) (12.2%) (17.0%) (23.8%) (34.7%) (51.9%) (55.0%) (30.3%) 43/43 31/31 94/94 45/46 213/214 NT NT NT NT NT OB/CX (100%) (100%) (100%) (97.8%) (99.5%) 31/31 99/99 124/125 77/78 331/333 NT NT NT NT NT P (100%) (100%) (99.2%) (98.7%) (99.4%) 2/41 4/29 10/85 8/124 14/213 17/211 11/215 20/175 * 32/131 * 118/1224 KZ (4.9%) (13.8%) (11.8%) (6.5%) (6.6%) (8.1%) (5.1%) (11.4%) (24.4%) (9.6%) 0/28 0/94 43/155 * 6/240 * 4/85 5/105 58/707 NT NT NT CEQ (0%) (0%) (27.7%) (2.5%) (4.7%) (4.8%) (8.2%) 0/43 2/28 3/114 14/211 3/205* 9/243 5/203 6/132 42/1179 NT CN (0%) (7.1%) (2.6%) (6.6%) (1.5%) (3.7%) (2.5%) (4.5%) (3.6%)

18/187 16/133 27/201 13/149 35/171 * 18/126 127/967 NT NT NT SXT (9.6%) (12.0%) (13.4%) (8.7%) (20.5%) (14.3%) (13.1%)

AUG - Amoxicillin/Clavulanic acid; OB/CX - Cloxacillin; P - Penicillin G; KZ - Cefazolin; CEQ - Cefquinome; CN - Gentamicin; SXT - Trimethoprim/Sulfamethoxazole; NT - Not tested; * p<0.05.

The percentage of K. pneumoniae tests resistant to amoxicillin/clavulanic acid had an overall significant increase (p<0.001), from 57.1% in 2004 to 71.4% in 2012 (Tables 31 and 38). Along the years (2nd LR model), statistically significant changes were verified in the percentage of K. pneumoniae tests resistant to this antibacterial: 1) A decrease, from 57.1% in 2004 to 3.4% in 2006 (p=0.002); 2) An increase, from 26.3% in 2008 to 64.7% in 2009 (p=0.010); 3) A decrease, from 64.7% in 2009 to 29.3% in 2010 (p=0.003); 4) An increase, from 29.3% in 2010 to 77.3% in 2011 (p=0.001) (Table 38. See also Figure 49 and Table 52 of Appendix 2). No other antibacterials had a significant change in the percentage of resistant K. pneumoniae tests (Table 31). Despite this, when comparing the probability of ______

Balbino M. Rocha, 2013 126 Results

antibacterial resistance of specific years with the preceding ones (2nd LR model), cefazolin revealed statistically significant changes in the percentage of resistant K. pneumoniae tests, to be exact: 1) An increase, from 2.5% in 2010 to 27.3% in 2011 (p=0.017) (Table 33. See also Figure 39 and Table 47 of Appendix 2).

Table 38: Klebsiella pneumoniae resistance proportions, among each tested antibacterial agent, along each tested year (n = 773 tests).

Year Antibacterial Agent 2004 2005 2006 2007 2008 2009 2010 2011 2012 Total

8/14 1/29 * 4/32 5/19 22/34 * 12/41 * 17/22 * 5/7 74/198 NT AUG (57.1%) (3.4%) (12.5%) (26.3%) (64.7%) (29.3%) (77.3%) (71.4%) (37.4%) 14/14 5/5 31/31 8/8 58/58 NT NT NT NT NT OB/CX (100%) (100%) (100%) (100%) (100%) 5/5 34/34 27/27 7/7 73/73 NT NT NT NT NT P (100%) (100%) (100%) (100%) (100%) 1/14 0/5 2/26 3/20 1/19 1/36 1/40 6/22 * 1/7 16/189 KZ (7.1%) (0%) (7.7%) (15.0%) (5.3%) (2.8%) (2.5%) (27.3%) (14.3%) (8.5%) 0/14 1/19 0/19 1/34 0/43 0/21 0/7 2/157 NT NT CN (0%) (5.3%) (0%) (2.9%) (0%) (0%) (0%) (1.3%) 0/11 2/31 2/12 1/34 1/10 6/98 NT NT NT NT SXT (0%) (6.5%) (16.7%) (2.9%) (10.0%) (6.1%)

AUG - Amoxicillin/Clavulanic acid; OB/CX - Cloxacillin; P - Penicillin G; KZ - Cefazolin; CN - Gentamicin; SXT - Trimethoprim/Sulfamethoxazole; NT - Not tested; * p<0.05.

______

Balbino M. Rocha, 2013 127 DISCUSSION Discussion

I. Novelty aspects of this study

1. Very few studies have thoroughly demonstrated the long-term effects or trends regarding the use of antibacterials on antibacterial resistance of mastitis pathogens from dairy cattle. To the author's knowledge, this research is among the studies with the largest assembled data available and with the most extensive time-frame, compiling nine years of information (2004 - 2012). To date, the most widespread available data derived from a 7-year study by Erskine et al. 432 and a 6-year study by Nam et. al. 433 The majority of studies have conducted their research over shorter time-frames (e.g., 6 months to 3 years). 434-441,451 2. In Portugal, although some studies have determined antibacterial susceptibility patterns for mastitis pathogens, they all have used reduced sample sizes as well as shorter time-frames. Moreover, none have determined trends for those patterns over the respective study period. 451-453

II. Antibacterial Resistance Pattern and Trend Analysis

It is the author's opinion that individual antibacterial susceptibility tests of mastitis pathogens, based on the results of the agar disk diffusion method, may have minimal clinical relevance in guiding individual cow therapy. However, since the results of susceptibility testing are repeatable, large number of mastitis isolates may be processed to produce useful information of antibacterial susceptibility/resistance traits and long-term trends of those traits over time in a certain population or region. Deliberations may be, therefore, made in the direction of guiding local clinicians to make more grounded choices when selecting for the most suitable antibacterial. As a result, this may contribute to a more successful therapeutic outcome, thereby reducing the use of antibacterials and, consequently, the selection pressure among mastitis pathogens and their dissemination to the environment. Even so, one must have in mind that this strategy does not decrease the incidence of new IMI and, at most, may influence the incidence of recurrent cases.

Available relevant research worldwide has suggested that most mastitis pathogens are generally susceptible in vitro to antibacterials used for mastitis treatment (Table 27). 396,432-445 When comparing the resistance patterns and trends of major mastitis bacterial pathogens from submitted samples in this study (Tables 30 and 31) with those studies (Tables 39-45 of Appendix 1), some parallelisms may be established. 432-441,451 However, a critical approach should be directed when comparing and/or extrapolating any similarities or discrepancies among them all, due to differences in the origin of isolates, laboratory procedures, interpretive guidelines (e.g., CLSI vs. EUCAST), among other factors. 284,432,454

______Balbino M. Rocha, 2013 129 Discussion

With cloxacillin as an exception, when analyzing the resistance patterns of the major mastitis pathogens for the different tested β-lactam antibacterials, all three Streptococcus species (S. agalactiae, S. uberis and S. dysgalactiae) exhibited low resistance proportions, ranging from rare (0%) to very low levels (1.0%) (Table 30). This information is consistent with the Gram-positive spectrum of these compounds, especially in these species, and is also in agreement with what has been described in literature. 33,395,396,432,436,440,443,455 Cloxacillin was, in fact and on the other hand, the β-lactam that displayed the highest resistance proportions among these species, ranging from 5.5% in S. dysgalactiae to 22.1% in S. uberis (Table 30). In addition, this antibacterial was the only tested β-lactam that displayed evidence of a significant increasing trend, throughout the study, among all these three species (Table 31). Possible explanations for these trends may be that, since: 1) this β- Lactam is among the most frequently used IMM antibacterials in lactating- and dry-cow therapy in Portugal; 2) these streptococci and Enterococcus spp. are environmental pathogens with similar biochemical and structural characteristics; and 3) Enterococcus spp. are known to be key reservoirs of antibacterial resistance genes In response to the selective pressure from the use of this antibacterial, genes expressing resistance to cloxacillin in Enterococcus spp. may have been exchanged and consequently disseminated amongst these streptococci over time.

As for the tests including Enterococcus spp. (E. faecium and E. faecalis), resistance was generally extremely high for the selected β-lactam antibacterials: 70.0%, 79.9% and 96.2% for penicillin, cefazolin and cloxacillin, respectively (Table 30). This outcome is in agreement with available literature, that states that Enterococcus spp. are among the most resistant organisms tested. 396 Amoxicillin/clavulanic acid was, on the other hand, the only β- lactam with the lowest resistance proportion (3.9%), an outcome consistent with data from other reports. 455 Furthermore, Enterococcus spp. were the only pathogens that did not exhibit any significant trend over time (Table 31) for any of the tested antibacterials (Table 34). This is all explained because enterococci exhibit intrinsic resistance to penicillinase- susceptible penicillin (low level), penicillinase-resistant penicillins and cephalosporins. 456 This is due to low affinity penicillin binding proteins (PBPs), which enable them to synthesize cell wall components even in the presence of modest concentrations of most β-lactam antibacterials. In addition, enterococci are tolerant to the activity of β-lactams, that is, they are inhibited but not killed by these agents. This property is an acquired characteristic. 456 Lastly, enterococci, exclusively strains of E. faecalis expressing β-lactamase enzymes and having high level resistance to penicillin, have been reported. 457-460 These E. faecalis are not susceptible to anti-staphylococcal penicillins but are susceptible to ampicillin, amoxicillin and piperacillin combined with drugs that inhibit penicillinase such as clavulanic acid, sulbactam and tazobactam. 456,457 This may explain the low resistance proportions to

______Balbino M. Rocha, 2013 130 Discussion

amoxicillin/clavulanic acid. On the other hand, one should take notice that isolates of E. faecium do not produce penicillinase, yet confer high level resistance. 461,462 Thus the importance of having both species analyzed separately in future similar studies.

A wide variation in the levels of resistance was observed in the S. aureus isolates tested for β-lactams, ranging from very low (0.5%, cefazolin) to high levels (44.7%, penicillin) (Table 30). Similar variations in resistance for S. aureus isolates can also be observed in other reports, with S. aureus also exhibiting generally high in vitro susceptibility patterns in some of them. 432,434,436,437,439,441,444 However, and as known, this in vitro susceptibility does not guarantee nor reflect the in vivo treatment success rates. Several factors including the ability of S. aureus to survive inside neutrophils, 463, 464 to form small-colony variants or L- forms, 465 to induce fibrosis and formation of microabscesses, 12,417,466 and to invade into mammary epithelial cells 467,468 are potential contributors to the poor response of chronic S. aureus to antibacterial treatment.

When analyzing the long-term effects of the resistance patterns of S. aureus for the different tested β-lactam antibacterials, a significant increase was verified throughout the study period for amoxicillin/clavulanic acid, cloxacillin and cefquinome (Table 31). Our results are not in agreement with results of other studies, in which resistance of S. aureus to cloxacillin decreased, for example. 469 The author considers this fact to be of paramount importance from both a public health and an epidemiological points-of-view and the reasons for these increases and respective points of origin have to be determined. In fact, these outcomes, in addition to the previous results from the Streptococcus spp., do indeed suggest dissemination of antibacterial resistance genes. Other theories should be, however, put to consideration, due to this pathogen's importance in veterinary and human medicine.

For both of the tested Gram-negative pathogens, levels of resistance ranged from 8.2% (E. coli) to 100% (K. pneumoniae) towards the tested β-lactams (Table 30). This information seems to make sense, taking into account that these compounds are considered to be more effective against Gram-positive bacteria. Still, some molecules such as cefquinome (4th- generation cephalosporin), are broad spectrum agents with greater activity against Gram- negative bacteria and this is supported by the fact that this antibacterial presents the lowest resistance proportions for these pathogens. Actually, among all β-lactams, both tested cephalosporins showed the lowest resistance proportions. This fact was not only true for both Gram-negative pathogens, but for all pathogens in general.

Still in regard to E. coli and K. pneumoniae, both pathogens also exhibited increases in resistance proportions that were significant for amoxicillin/clavulanic acid: From 22.6% in 2005 to 55.0% in 2012 (Tables 31 and 37) for the E. coli isolates; and from 57.1% in 2004 to 71.4% in 2012 (Tables 31 and 38) for K. pneumoniae isolates. E. coli also displayed a

______Balbino M. Rocha, 2013 131 Discussion

significant increase for cefazolin, from 4.9% in 2004 to 24.4% in 2012 (Tables 31 and 37). Possible explanations for these facts may be the acquisition of plasmids containing genes that encode for extended-spectrum β-lactamases (ESBLs) in these species. 45,46,171

Aminoglycosides and sulphonamides are regarded as broad-spectrum antibacterials with great activity against Gram-negative pathogens. This information is in conformity with our results and similar to other studies. 432,434-436,441,444 When analyzing the resistance patterns of the major mastitis pathogens for gentamicin and trimethoprim/sulfamethoxazole, with the exception of S. uberis (for gentamicin) and Enterococcus spp. (for trimethoprim/sulfamethoxazole), very low to moderate resistance proportions were verified among all isolates, ranging from 0.7% to 13.1% (Table 30).

As mentioned in the previous paragraph, S. uberis revealed the highest resistance proportions (71.0%) to gentamicin. In fact, S. uberis was, together with S. aureus, the two pathogens to which resistance proportions had an overall significant increase, from 41.5% in 2004 to 88.2% in 2007 for S. uberis (Tables 31 and 34); and from 1.2% in 2004 to 2.9% in 2012 for S. aureus (despite its low levels of resistance) (Tables 31 and 32). Regarding trimethoprim/sulfamethoxazole, the tests regarding Enterococcus spp. were the ones that exhibited the highest proportions (23.9% - Table 30). E. coli was the only pathogen, though, that displayed a significant increase in the resistance proportions to trimethoprim/sulfamethoxazole (Tables 31 and 37). Possible explanations for these facts may be the widespread use of gentamicin and trimethoprim/sulfamethoxazole in therapy protocols for gastrointestinal and other pathologies in cattle.

The observed levels of resistance to all tested antibacterials are of extreme importance since these substances have been defined by the WHO as "critically important" (i.e., amoxicillin/clavulanic acid, penicillin and gentamicin) and "highly important" (cloxacillin, cefazolin and trimethoprim/sulfamethoxazole) antibacterials in human medicine. 235

III. Data Analysis

The odds ratio (OR) from the logistic regression analysis (Table 31) can be used to determine the rate at which the prevalence of resistance is increasing or decreasing each year. Statistically significant (p<0.05) OR < 1.0 reflect a reduced odds of observing resistance during any given year, compared with the year before. Statistically significant (p<0.05) OR > 1.0 reflect an increased odds of observing resistance during any given year, compared with the year before. For instance, the OR of 1.15 for resistance of S. aureus isolates to gentamicin can be interpreted to mean that the likelihood that S. aureus would be resistant to gentamicin was 1.15 that of a previous year (Table 31).

______Balbino M. Rocha, 2013 132 Discussion

IV. Limitations of the Study

Although laboratory protocols changed very little during the study period, allowing the analysis of changes in antibacterial resistance patterns, the results of this study were still limited by the nature of the available data:

1. The variable year was the only factor to be included in the logistic regression models to explain the changes in the percentage of bacterial isolates resistant to the tested antibacterials. It is quite obvious to the author that factors other than year influenced those changes and would therefore need to be considered in order to create a model that completely describes the data. 2. Several of the isolated pathogens were not tested for all antibacterial agents in a consistent manner throughout the study period. This was because the antibacterial agents were employed depending mainly on the type of pathogen isolated, the client's request, herd representativity, and on the laboratory/market availability of respective diffusion disks. This explains some of the missing data along the study´s statistics. 3. The Kirby-Bauer disk diffusion method, due to its inexpensive and clinically practical methodology, was used to determine antibacterial susceptibility of isolates in the present study. The primary disadvantage of using this method when monitoring development of resistance is that outcomes are reported on a qualitative basis (susceptible, intermediate, or resistant) rather than MIC values (quantitative basis).

Additionally and similarly to what happens in all laboratories worldwide, human interpretive criteria were used to categorize these isolates, providing inappropriate and potentially misleading conclusions. The validity of applying these breakpoints to the treatment of bovine mastitis has not been established and is questionable because: 1) bovine milk pH and electrolyte, fat, protein, and leukocyte concentrations, growth factor composition, and pharmacokinetic profiles are different than those for human plasma; and 2) human bacterial pathogens are often different from bovine mastitis pathogens. Also, antibacterials are distributed unevenly in an inflamed mammary gland, and high antibacterial concentrations can alter neutrophil morphology or function in vitro and thereby inhibit bacterial clearance in vivo. 396

Furthermore, and as already mentioned before, it is difficult to compare outcomes of antibacterial susceptibility testing among studies because of differences in the origin of isolates, laboratory procedures, and interpretive guidelines (e.g., CLSI vs. EUCAST) , and percentages of isolates resistant to particular antibacterials may vary from one study to the next. 284,432,454

______Balbino M. Rocha, 2013 133 Discussion

V. Improvement Suggestions for Future Similar Research

The following points disclose several of the author's thoughts and suggestions as to what may be improved if a similar study was to be repeated, in order to reduce all previously mentioned errors, limitations and bias:

1. Other factors influencing resistance patterns have to be included in the logistic regression model to accurately explain the changes in the percentage of bacterial isolates resistant to the tested antibacterials. Some examples of variables/data that may be collected from farms submitting milk samples for susceptibility testing are:  Herd information: geographical location; treatment protocols employed (antibacterials administered, routes, dosages, frequency and duration of administration); history of antibacterial use and past susceptibility testing results; cure rates; heifer raring routines; milking routines; etc.  Sample/individual animal information: parity; stage of lactation; type of mastitis (clinical vs. subclinical); mastitis history (reoccurrences, severity, onset, treatments, treatment success, past susceptibility testing results, SCC, etc.); history of other pathologies (concurrences, reoccurrences, severity, onset, treatments, etc.); repetition of submission of samples; etc.

2. The use of antibacterial susceptibility tests that document quantitative data (MIC), such as broth dilution or milk dilution methods, is recommended: 6 Ideally, accurate antibacterial susceptibility test breakpoints should be resultant from: 1) MIC data for mastitis pathogens; 2) PK/PD data for lactating dairy cows; and 3) the results of field studies that measure the rates of clinical and bacteriologic cure. Clinical and bacteriologic cure rates may provide a clear breakpoint or, in other situations, these data can be used in conjunction with PK/PD data to suggest the most appropriate breakpoint. 13

3. Science-based evidence could also involve the use of comparative antibacterial susceptibility tests, before and after antibacterial administration, for example. 172

VI. Further Research Ideas and Recommendations

Despite the pleasing results achieved from this study, there are still numerous questions that need to be answered relatively to this topic. For this reason, future research projects could:

1. Focus on gathering evidence on the potential risk of transmission of antibacterial resistance genes between mastitis pathogens and other environmental pathogens, which ______Balbino M. Rocha, 2013 134 Discussion

may consequently, through other routes, be transferred to humans either directly via the food chain or indirectly as a result of spread of animal waste on cropland.

2. Focus on understanding the complete effects of antibacterials in the specific dairy production environment's microbiome, ultimately reducing the appropriate selection pressure(s). The dairy production setting is substantially different from all other food-animal production settings. As a result, the consideration of factors such as improved management and housing conditions, use of teat disinfectants, culling and segregation pressures, distinct administration routes (IMM therapy - bovine udder as a unique body compartment), and dry cow therapy should decrease the incidence of new IMI, thereby reducing the use of antibacterials and, consequently, the selection pressure among mastitis pathogens. Clearly, there are established benefits from antibacterial treatment of mastitis cases; however, one must always keep in mind that overuse or improper use could tip the scales, favoring selection of antibacterial-resistant bacterial pathogens.

3. Focus on gathering evidence that may determine that alternatives like efficient diagnostic tools and vaccines may indeed play an important complementary role to antibacterial use. In fact, a preventive vaccine for bovine mastitis (StartVac® from Hipra® - Girona, Spain) is already registered (EMA) and available for commercial use in Europe with promising results. 254 A substitute like this will definitely aid in promoting rational use of such compounds, preventing infections that would require antibacterial therapy.

4. The recommended antibacterial susceptibility testing methods provide reliable results when used according to the procedures defined by the CLSI/EUCAST or by the manufacturers of the commercial products. There is, though, considerable opportunity for improvement in the area of rapid and accurate recognition of ABR. There is a need for development of new automated instruments that could provide faster results and also save money by virtue of lower reagent costs and reduced labor requirements. To accomplish this, it will likely be necessary to explore different methodological approaches for detection of bacterial growth. The direct detection of resistance genes by PCR or similar techniques has limited utility, because only a few resistance genes are firmly associated with phenotypic resistance (e.g., mecA, vanA, and vanB). 207

5. Despite the ongoing controversial debate concerning antibacterial resistance and the use of such compounds in the different food-producing animal settings, focus on research that would measure the consequences of what would happen if all antibacterials were to be banned or limited for use in the dairy industry. Although it is clear that use of antibacterials in adult dairy cows and other food-producing animals does contribute to increased antibacterial resistance, it is the author's opinion that the advantages of using antibacterials in adult dairy cows far outweigh the disadvantages.

______Balbino M. Rocha, 2013 135 CONCLUSIONS Conclusions

In summary, the main conclusions to be drawn from this dissertation are:

 Antibacterial susceptibility patterns were determined for major bovine mastitis pathogens isolated from submitted milk samples from dairy herds in northwestern, central and southern Portugal. A wide variation in resistance levels was verified, ranging from rare (0%) to extremely high (100%) for the tested antibacterial agents/mastitis pathogens.

 When assessing the evolution of the resistance patterns over the nine years of data, results did indicate a trend towards increased antibacterial resistance for the following antibacterial agents/mastitis pathogens:

 The percentage of resistance to amoxicillin/clavulanic acid increased for S. aureus (from 0.7% to 26.6%), for E. coli (from 22.6% to 55.0%) and for K. pneumoniae (from 57.1% to 71.4%);  The percentage of resistance to cefazolin increased for E. coli (from 4,9% to 24.4%);  The percentage of resistance to cefquinome increased for S. aureus (from 0% to 3.1%);  The percentage of resistance to gentamicin increased for S. aureus (from 1.2% to 2.9%) and for S. uberis (from 41.5% to 88.2%);  The percentage of isolates resistant to trimethoprim/sulfamethoxazole increased for E. coli (from 9.6% to 14.3%);  The percentage of resistance to cloxacillin increased for S. aureus and all three streptococci species included for analysis;  No significant changes were verified for resistance percentages of Enterococcus spp. (E. faecium and E. faecalis) to all selected antibacterials;  No pathogens had a significant change in the percentage of resistance to penicillin.

 Further research should be addressed towards finding proper basis to justify the increases and non-increases of antibacterial resistance in the region.

 Since data is representative of the northwestern, central and southern Portuguese populations, inferences can be useful to guide local clinicians in making more grounded choices when selecting for the most suitable antibacterial. This may contribute to a more successful therapeutic outcome, thereby reducing the use of antibacterials and, consequently, the selection pressure among mastitis pathogens and their dissemination to the environment.

 Similar research should be developed in other parts of the country. Once antibacterial resistance traits and time trends from other regions are determined, nationwide

______

Balbino M. Rocha, 2013 137 Conclusions

programs may be prepared and established, contributing to a broader approach in the lowering of the antibacterial resistance burden.

 Taking into account the overall epidemiological aspects of the use of antibacterial agents and their linkage to the development of antibacterial resistances in bacterial populations and resultant public health risks, further research should be addressed in the direction of determining the unique singularities of the bovine dairy industry, in comparison to other food-producing animal settings.

______

Balbino M. Rocha, 2013 138 REFERENCES Refe re nces

1. EMA - European Medicines Agency, EFSA - European Food Safety Authority, ECDC - European Center for Disease Control. (2009) Joint Opinion on antimicrobial resistance (AMR) focused on zoonotic infections. EFSA Journal. 11 (7): 1372. 2. Norwegian Institute of Public Health. (2012) WHO Collaborating Centre for Drug Statistics Methodology (WHOCC). In http://www.whocc.no/use_of_atc_ddd (accessed October, 12th, 2012). 3. Dorland’s Illustrated Medical Dictionary. (2012) In http://www.dorlands.com. (accessed August 8th, 2012). 4. Agersø, Y., Hald, T., Høg, B. (2011) DANMAP 2010 - The Danish Integrated Antimicrobial Resistance Monitoring and Research Programme: Use of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, food and humans in Denmark. In http://www.danmap.org. Copenhagen, Denmark. (accessed August 8th, 2012). 5. Biology online (2013). In http://www.biology-online.org (accessed June 6th 2013). 6. Constable, P.D., Morin, D.E. (2003) Treatment of Clin mastitis: Using antimicrobial susceptibility profiles for treatment decisions. Vet Clin N Am-Food A. 19(1):139-55. 7. EUCAST - European Committee for Antimicrobial Susceptibility Testing. (2000) Definitive Document E. Def 1.2, May 2000: Terminology relating to methods for the determination of susceptibility of bacteria to antimicrobial agents. Clin Microbiol Infec. 6 (9): 503-8. 8. CLSI - The Clin and Laboratory Standards Institute. (2011) The CLSI Harmonized Terminology Database. In http://www.clsi.org (accessed December 20th 2012). 9. US Environmental Protection Agency. (2013) Dairy Glossary. In http://www.epa.gov/agriculture/ag101/dairyglossary.html (last update 27th June, 2012) (accessed June 6th 2013). 10.Phillips, I., Casewell, M., Cox, T. (2004) Does the use of antibiotics in food animals pose a risk to human health? A critical review of published data. J Antimicrob Chemoth. 53 (1): 28-52. 11. EFSA - European Food Safety Authority. (2010) The Community Summary Report on antimicrobial resistance in zoonotic and indicator bacteria from animals and food in the European Union in 2004-2007. EFSA J. 8 (4): 1309. 12. Erskine, R.J., Wagner, S., DeGraves, F.J. (2003) Mastitis therapy and pharmacology. Vet Clin N Am-Food A. 19 (1): 109-38, vi. 13. EUCAST - European Committee for Antimicrobial Susceptibility Testing. (2000) Definitive Document E. Def 1.2, August 2000: Determination of antimicrobial susceptibility test breakpoints. Clin Microbiol Infec. 6 (10): 570-2. 14. Tenover, F.C. (2006) Mechanisms of antimicrobial resistance in bacteria. Am J Infect Control. 34 (5 Suppl 1): S3-10; discussion S64-73. 15. EMA - European Medicines Agency. (2011) Trends in the sales of veterinary antimicrobial agents in nine European countries (2005-2009). 77 16. Ostensson, K., Hageltorn, M., Astrom, G. (1988) Differential cell counting in fraction-collected milk from dairy cows. Acta Vet Scand. 29 (3-4): 493-500. 17. Kehrli, M.E., Jr. Shuster, D.E. (1994) Factors affecting milk somatic cells and their role in health of the bovine mammary gland. J Dairy Sci. 77 (2): 619-27. 18. Harmon, R.J. Physiology of mastitis and factors affecting somatic cell counts. J Dairy Sci. 77 (7): 2103-12. 19.IDF - International Dairy Federation (2005). Diagnostic potential of California Mastitis Test to detect subclinical mastitis. 26: 15-19. 20. WHO - World Health Organization. (2010) The FAO-OIE-WHO Collaboration: Tripartite Concept Note: FAO-OIE-WHO. Geneva, Switzerland. 6 21. Oliver, S.P., Murinda, S.E., Jayarao, B.M. (2011) Impact of antibiotic use in adult dairy cows on antimicrobial resistance of veterinary and human pathogens: a comprehensive review. Foodb Path Dis. 8 (3): 337-55. 22. White, D.G., McDermott, P.F. (2001) Emergence and Transfer of Antibacterial Resistance. J Dairy Sci. 84 (E. Suppl): E151-E5. 23. Barbosa, T.M., Levy, S.B. (2000) The impact of antibiotic use on resistance development and persistence. Drug Resist Update. 3 (5): 303-11. 24. Broens, E.M., Espinosa-Gongora, C., Graat, E.A. (2012) Longitudinal study on transmission of MRSA CC398 within pig herds. BMC Vet Res. 8 (1): 58. 25. McDermott, P.F., Walker, R.D., White, D.G. (2003) Antimicrobials: modes of action and mechanisms of resistance. Int J Toxicol. 22 (2): 135-43. 26. Harbottle, H., Thakur, S., Zhao, S., White, D.G. (2006) Genetics of antimicrobial resistance. Anim Biotechnol. 17 (2): 111-24. 27. Collignon, P. (2012) The Importance of a One Health Approach to Preventing the Development and Spread of Antibiotic Resistance. Curr Top Microbiol. Epub ahead of print 28. WHO - World Health Organization. (2012) The evolving threat of antimicrobial resistance: options for action. Geneva, Switzerland. 29. van den Bogaard, A.E., Stobberingh, E.E. (2000) Epidemiology of resistance to antibiotics. Links between animals and humans. Int J Antimicrob Ag. 14 (4): 327-35. 30. EFSA - European Food Safety Authority & ECDC - European Center for Disease Control. (2012) The European Union Summary Report on antimicrobial resistance in zoonotic and indicator bacteria from humans, animals and food in 2010. EFSA J. 10 (3): 233. 31. Oliver, S.P., Murinda, S.E. (2012) Antimicrobial resistance of mastitis pathogens. Vet Clin N Am-Food A. 28 (2): 165-85. 32. USDA. (2008) Dairy 2007: Antibiotic use on U.S. dairy operations, 2002 and 2007 - Info Sheet. In http://www.aphis.usda.gov/animal_health/nahms/dairy/downloads/dairy07 (accessed July 20th, 2012). pp. 1-5. 33. Owens, W.E., Ray, C.H., Watts, J.L., Yancey, R.J. (1997) Comparison of success of antibiotic therapy during lactation and results of antimicrobial susceptibility tests for bovine mastitis. J Dairy Sci. 80 (2): 313-7. 34. Sol, J., Sampimon, O.C., Barkema, H.W., Schukken, Y.H. (2000) Factors associated with cure after therapy of Clin mastitis caused by Staphylococcus aureus. J Dairy Sci. 83 (2): 278-84. 35. Ungemach, F.R., Klee, W., Heinritzi, K. (1999) [Therapeutics available in Vet Med for animals used in food production]. Tierarztl Prax A G. 27 (3): 131-43. 36. Adesiyun, A.A., Kaminjolo, J.S. (1992) Susceptibility to antibiotics of Escherichia coli strains isolated from diarrhoeic and non-diarrhoeic livestock in Trinidad. Rev Elev Med Vet Pay. 45 (3-4): 260-2. 37. Griggs, D.J., Hall, M.C., Jin, Y.F., Piddock, L.J. (1994) Quinolone resistance in veterinary isolates of Salmonella. J Antimicrob Chemoth. 33 (6): 1173-89. 38. Salyers, A.A., Amabile-Cuevas, C.F. (1997) Why are antibiotic resistance genes so resistant to elimination? Antimicrob Ag Chemoth. 41 (11): 2321-5. 39. Levy, S.B. (1998) Multidrug resistance: A sign of the times. New Engl J Med. 338 (19): 1376-8. 40. Johnston, A.M. (1998) Use of antimicrobial drugs in veterinary practice. BMJ. 317 (7159): 665-7. 41. Livermore, D.M. Bacterial resistance: origins, epidemiology and impact. (2003) Clin Infect Dis. 36 (Suppl 1): S11-23. 42. Furuya, E.Y., Lowy, F.D. (2006) Antimicrobial-resistant bacteria in the community setting. Microbiol. 4 (1): 36-45. 43. European Centre for Disease Prevention and Control (ECDC) & European Medicines Agency (EMA). (2009) The bacterial challenge: time to react. In ECDC/EMA Joint Technical Report. Stockholm, Sweden. p. 42 44. Cid, D., Blanco, M., Blanco, J.E., Ruiz Santa Quiteira, J.A., de la Fuente, R., Blanco, J. (1996) Serogroups, toxins and antibiotic resistance of Escherichia coli strains isolated form diarrhoeic goat kids in Spain. Vet Microbiol. 53 (3-4): 349-54. 45. Blanco, J.E., Blanco, M., Mora, A., Blanco, J. (1997) Prevalence of bacterial resistance to quinolones and other antimicrobials among avian Escherichia coli strains isolated from septicemic and healthy chickens in Spain. J Clinic Microbiol. 35 (8): 2184-5. 46. Davies, J.E. (1997) Origins, acquisition and dissemination of antibiotic resistance determinants. In Antibiotic Resistance: Origins, Evolution, Selection and Spread. Chadwick, D.J., Goode, J. (eds.) John Wiley & Sons Ltd. Chichester, UK. pp. 5-35. 47. Everett, M.J., Jin, Y.F., Ricci, V., Piddock, L.J. (1996) Contributions of individual mechanisms to fluoroquinolone resistance in 36 Escherichia coli strains isolated from humans and animals. Antimicrob Ag chemoth. 40 (10): 2380-6. 48. Gold, H.S., Moellering, R.C., Jr. (1996) Antimicrobial-drug resistance. New Engl J Med. 335 (19): 1445-53. 49. Qazi, S., Weber, M., Boschi-Pinto, C., Cherian, T. (2008) Global Action Plan for the Prevention and Control of Pneumonia (GAPP): Report of an informal consultation. World Health Organization. Geneva, Switzerland.

______

Balbino M. Rocha, 2013 140 Refe re nces

50. de Kraker, M.E., Davey, P.G., Grundmann, H. (2011) Mortality and hospital stay associated with resistant Staphylococcus aureus and Escherichia coli bacteremia: estimating the burden of antibiotic resistance in Europe. PLoS Med. 8 (10): e1001104. 51. Palumbi, S.R. (2001) Humans as the world's greatest evolutionary force. Science. 293 (5536): 1786-90. 52. Smith, D.H. (1967) R factor infection of Escherichia coli lyophilized in 1946. J Bacteriol. 94 (6): 2071-2. 53. Dancer, S.J., Shears, P., Platt, D.J. (1997) Isolation & characterization of coliforms from glacial ice and water in Canada's High Arctic. J Appl Microbiol. 82 (5): 597-609. 54. Song, J.S., Jeon, J.H., Lee, J.H. (2005) Molecular characterization of TEM-type beta-lactamases identified in cold-seep sediments of Edison Seamount (south of Lihir Island, Papua New Guinea). J Microbiol. 43 (2): 172-8. 55. Barlow, M., Hall, B.G. (2002) Origin and evolution of the AmpC beta-lactamases of Citrobacter freundii. Antimicrob Ag Chemoth. 46 (5): 1190-8. 56. Levy, S.B. (1992) The antibiotic paradox. Plenum Press. New York, U.S.A. 57. Levy S. (2002) The Antibiotic Paradox: How the Misuse of Antibiotics Destroys Their Curative Powers. 1st ed. Perseus Publishing. Cambridge (MA), USA. 58. D'Costa, V.M., McGrann, K.M., Hughes, D.W., Wright, G.D. (2006) Sampling the antibiotic resistome. Science. 311 (5759): 374-7. 59. Davies, J.E. (1997) Origins, acquisition and dissemination of antibiotic resistance determinants. Ciba Foundation Symposium. 207: 15-27; discussion -35. 60. Hong, H.J., Hutchings, M.I., Neu, J.M., Wright, G.D., Paget, M.S., Buttner, M.J. (2004) Characterization of an inducible vancomycin resistance system in Streptomyces coelicolor reveals a novel gene (vanK) required for drug resistance. Mol Microbiol. 52 (4): 1107-21. 61. Webb, V., Davies, J. (1993) Antibiotic preparations contain DNA: a source of drug resistance genes? Antimicrob Ag Chemoth. 37 (11): 2379-84. 62. Davies, J. (1994) Inactivation of antibiotics and the dissemination of resistance genes. Science. 264 (5157): 375-82. 63. McManus, M.C. (1997) Mechanisms of bacterial resistance to antimicrobial agents. Am J Health-Syst PH. 54 (12): 1420-33; quiz 44-6. 64. Phillips, I. (1979) Antibiotic policies. In Recent Advances in Infection. Reeves, D., Geddes, A. (eds.) Churchill Livingstone. Edinburgh, UK. pp. 151-63. 65. Storm, D.R., Rosenthal, K.S., Swanson, P.E. (1977) Polymyxin and related peptide antibiotics. Ann Rev Biochem 1977; 46: 723-63. 66. Neu, H.C. (1992) The crisis in antibiotic resistance. Science. 257 (5073): 1064-73. 67. Carpenter, CF., Chambers, HF. Daptomycin: another novel agent for treating infections due to drug-resistant gram-positive pathogens. Clin Infect Dis. 38 (7): 994-1000. 68. Tenover, F.C. (2006) Mechanisms of antimicrobial resistance in bacteria. Am J Med. 119 (6 Suppl 1): S3-10; discussion S62-70. 69. Bradford, P.A. (2001) Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology and detection of this important resistance threat. Clin Microbiol Rev. 14 (4): 933-51. 70. Poole, K. (2001) Multidrug resistance in Gram-negative bacteria. Curr Op Microbiol. 4 (5): 500-8. 71. Bugg TD, Walsh CT. (1992) Intracellular steps of bacterial cell wall peptidoglycan biosynthesis: enzymology, antibiotics, and antibiotic resistance. Nat Prod Rep. 9 (3): 199-215. 72. Wright, G.D. (2005) Bacterial resistance to antibiotics: enzymatic degradation and modification. Adv Drug Deliver Rev. 57 (10): 1451-70. 73. Gootz, T.D. (2004) Global dissemination of beta-lactamases mediating resistance to cephalosporins and carbapenems. Expert Rev Anti-infect Ther. 2 (2): 317-27. 74. Gootz, T.D. The forgotten Gram-negative bacilli: what genetic determinants are telling us about the spread of antibiotic resistance. Biochem Ph. 71 (7): 1073-84. 75. Klare, I., Heier, H., Claus, H., Reissbrodt, R., Witte, W. (1995) vanA-mediated high-level glycopeptide resistance in Enterococcus faecium from animal husbandry. FEMS Microbiol Lett. 125 (2-3): 165-71. 76. Arthur, M., Reynolds, P., Courvalin, P. (1996) Glycopeptide resistance in enterococci. Trends Microbiol. 4 (10): 401-7. 77. Casadewall, B., Courvalin, P. (1999) Characterization of the vanD glycopeptide resistance gene cluster from Enterococcus faecium BM4339. J of Bacteriol. 181 (12): 3644-8. 78. Schouten, M.A., Willems, R.J., Kraak, W.A., Top, J., Hoogkamp-Korstanje, J.A., Voss, A. (2001) Molecular analysis of Tn1546-like elements in vancomycin-resistant enterococci isolated from patients in Europe shows geographic transposon type clustering. Antimicrob Ag Chemoth. 45 (3): 986-9. 79. Abadia Patino, L., Courvalin, P., Perichon, B. (2002) vanE gene cluster of vancomycin-resistant Enterococcus faecalis BM4405. J of Bacteriol. 184 (23): 6457-64. 80. Gonzalez-Zorn, B., Courvalin, P. (2003) VanA-mediated high level glycopeptide resistance in MRSA. Lancet Infect Dis. 3 (2): 67-8. 81. Depardieu, F., Bonora, M.G., Reynolds, P.E., Courvalin, P. (2003) The vanG glycopeptide resistance operon from E. faecalis revisited. Mol Microbiol. 50 (3): 931-48. 82. Ramos, S., Igrejas, G., Rodrigues, J., Capelo-Martinez, J.L., Poeta, P. (2012) Genetic characterisation of antibiotic resistance and virulence factors in vanA-containing enterococci from cattle, sheep and pigs subsequent to the discontinuation of the use of avoparcin. Vet J. 193 (1): 301-3. 83. Chang, S., Sievert, D.M., Hageman, J.C. (2003) Infection with vancomycin-resistant Staphylococcus aureus containing the vanA resistance gene. New Engl J Med. 348 (14): 1342-7. 84. de Niederhausern, S., Bondi, M., Messi, P. (2011) Vancomycin-resistance transferability from VanA enterococci to S. aureus. Curr Microbiol. 62 (5): 1363-7. 85. CDC - Center for Disease Control and Prevention. (2002) S. aureus resistant to vancomycin - United States. MMWR Morbid Mortal Week Rep. 51 (26): 565-7. 86. Neu, H.C., Duma, R.J., Jones, R.N. (1992) Antibiotic resistance. Epidemiology and therapeutics. Diag Microbiol Infect Dis. 15 (2 Suppl): 53S-60S. 87. Wright, G.D. (1999) Aminoglycoside-modifying enzymes. Curr Op Microbiol. 2 (5): 499-503. 88. Trieu-Cuot, P., de Cespedes, G., Bentorcha, F., Delbos, F., Gaspar, E., Horaud, T. (1993) Study of heterogeneity of chloramphenicol acetyltransferase (CAT) genes in streptococci and enterococci by polymerase chain reaction: characterization of a new CAT determinant. Antimicrob Ag Chemoth. 37 (12): 2593-8. 89. Vassort-Bruneau, C., Lesage-Descauses, M.C., Martel, J.L., Lafont, J.P., Chaslus-Dancla, E. (1996) CAT III chloramphenicol resistance in Pasteurella haemolytica and Pasteurella multocida isolated from calves. J Antimicrob Chemoth. 38 (2): 205-13. 90. Bissonnette, L., Champetier, S., Buisson, J.P., Roy, P.H. (1991) Characterization of the nonenzymatic chloramphenicol resistance (cmlA) gene of the In4 integron of Tn1696: similarity of the product to transmembrane transport proteins. J Bacteriol. 173 (14): 4493-502. 91. White, D.G., Hudson, C., Maurer, J.J. (2000) Characterization of chloramphenicol and florfenicol resistance in Escherichia coli associated with bovine diarrhea. J Clin Microbiol. 38 (12): 4593-8. 92. Chopra, I., Roberts, M. (2001) Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev. 65 (2): 232-60. 93. Testa, R.T., Petersen, P.J., Jacobus, N.V., Sum, P.E., Lee, V.J., Tally, F.P. (1993) In vitro and in vivo antibacterial activities of the glycylcyclines, a new class of semisynthetic tetracyclines. Antimicrob Ag Chemoth. 37 (11): 2270-7. 94. Simjee, S., McDermott, P.F., Wagner, D.D., White, D.G. (2001) Variation within the vat(E) allele of Enterococcus faecium isolates from retail poultry samples. Antimicrob Ag Chemoth. 45 (10): 2931-2. 95. Lin, A.H., Murray, R.W., Vidmar, T.J., Marotti, K.R. (1997) The oxazolidinone eperezolid binds to the 50S ribosomal subunit and competes with binding of chloramphenicol and lincomycin. Antimicrob Ag Chemoth. 41 (10): 2127-31. 96. Prystowsky, J., Siddiqui, F., Chosay, J. Resistance to linezolid: characterization of mutations in rRNA and comparison of their occurrences in vancomycin-resistant enterococci. Antimicrob Ag Chemoth. 45 (7): 2154-6. 97. Yao, J., Moellering, R.J. (2003) Antibacterial agents. In: Manual of Clin Microbiology. Murray, P.R., Baron, E.J., Jorgensen, J.H., Pfaller, M.A., Yolken, R.H. (eds.) ASM Press. 8th ed. Washington, D.C., U.S.A. pp. 1039-73. 98. Petri, W.J. (2006) Antimicrobial agents: sulfonamides, trimethoprim-sulfamethoxazole, quinolones, and agents for urinary tract infections. In: The Pharmacological Basis of Therapeutics. Brunton, L.L., Lazo, J.S., Parker, K.L. (eds.) Goodman & Gilman’s, McGraw-Hill. 11th ed. New York, U.S.A. pp. 1111-26. 99. White, D.G., Zhao, S., Sudler, R. (2001) The isolation of antibiotic-resistant salmonella from retail ground meats. New Engl J Med. 345 (16): 1147-54.

______

Balbino M. Rocha, 2013 141 Refe re nces

100. Zhao, S., White, D.G., Ge, B. Identification and characterization of integron-mediated antibiotic resistance among Shiga toxin-producing Escherichia coli isolates. Appl Environ Microbiol. 67 (4): 1558-64. 101. Spratt, B.G. (1994) Resistance to antibiotics mediated by target alterations. Science. 264 (5157): 388-93. 102. Piddock, L.J., Williams, K.J., Ricci, V. (2000) Accumulation of rifampicin by Mycobacterium aurum, Mycobacterium smegmatis and Mycobacterium tuberculosis. J Antimicrob Chemoth. 45 (2): 159-65. 103. Drlica, K., Zhao, X. (1997) DNA gyrase, topoisomerase IV and the 4-quinolones. Microbiol Mol Biol Rev. 61 (3): 377-92. 104. Hooper, D.C. (1999) Mechanisms of fluoroquinolone resistance. Drug Resist Update. 2 (1): 38-55. 105. Achouak, W., Heulin, T., Pages, J.M. (2001) Multiple facets of bacterial porins. FEMS Microbiol Lett.199 (1): 1-7. 106. Poole, K. (2002) Outer membranes and efflux: the path to multidrug resistance in Gram-negative bacteria. Curr Ph Biotech. 3 (2): 77-98. 107. White, D.G., Goldman, J.D., Demple, B., Levy, S.B. (1997) Role of the acrAB locus in organic solvent tolerance mediated by expression of marA, soxS, or robA in Escherichia coli. J Bacteriol. 179 (19): 6122-6. 108. McDermott, P.F., White, D.G., Podglajen, I., Alekshun, M.N., Levy, S.B. (1998) Multidrug resistance following expression of the Escherichia coli marA gene in Mycobacterium smegmatis. J Bacteriol. 180 (11): 2995-8. 109. Alekshun, M.N., Levy, S.B. (1999) The mar regulon: multiple resistance to antibiotics and other toxic chemicals. Trends Microbiol. 7 (10): 410-3. 110. Poole, K. (2004) Efflux-mediated multiresistance in Gram-negative bacteria. Clin Microbiol Infect. 10 (1): 12-26. 111. Piddock, L.J. (2006) Clinly relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. Clin Microbiol Rev. 19 (2): 382-402. 112. Oethinger, M., Kern, W.V., Jellen-Ritter, A.S., McMurry, L.M., Levy, S.B. (2000) Ineffectiveness of topoisomerase mutations in mediating Clinly significant fluoroquinolone resistance in Escherichia coli in the absence of the AcrAB efflux pump. Antimicrob Ag Chemoth. 44 (1): 10-3. 113. Bass, L., Liebert, C.A., Lee, M.D. (1999) Incidence and characterization of integrons, genetic elements mediating multiple-drug resistance, in avian Escherichia coli. Antimicrob Ag Chemoth. 43 (12): 2925-9. 114. Frost, L.S., Leplae, R., Summers, A.O., Toussaint, A. (2005) Mobile genetic elements: the agents of open source evolution. Nature Rev Microbiol. 3 (9): 722-32. 115. Watanabe, T., Fukasawa, T. (1961) Episome-mediated transfer of drug resistance in Enterobacteriaceae. I. Transfer of resistance factors by conjugation. J Bacteriol. 81: 669-78. 116. Li, X.Z. (2005) Quinolone resistance in bacteria: emphasis on plasmid-mediated mechanisms. Int J Antimicrob Ag. 25 (6): 453-63. 117. Gay, K., Robicsek, A., Strahilevitz, J. (2006) Plasmid-mediated quinolone resistance in non-Typhi serotypes of Salmonella enterica. Clin Infect Dis. 43 (3): 297-304. 118. Jacoby, G.A., Walsh, K.E., Mills, D.M. (2006) qnrB, another plasmid-mediated gene for quinolone resistance. Antimicrob Ag Chemoth. 50 (4): 1178-82. 119. Sherley, M., Gordon, D.M., Collignon, P.J. (2004) Evolution of multi-resistance plasmids in Australian Clin isolates of Escherichia coli. Microbiology. 150 (Pt 5): 1539- 46. 120. Gebreyes, W.A., Thakur, S. (2005) Multidrug-resistant Salmonella enterica serovar Muenchen from pigs and humans and potential interserovar transfer of antimicrobial resistance. Antimicrob Ag Chemoth. 49 (2): 503-11. 121. Michael ,G.B., Butaye, P., Cloeckaert, A., Schwarz, S. (2006) Genes and mutations conferring antimicrobial resistance in Salmonella: an update. Microbes and infection/Institut Pasteur. 8 (7): 1898-914. 122. Martinez-Martinez, L., Pascual, A., Jacoby, G.A. (1998) Quinolone resistance from a transferable plasmid. Lancet. 351 (9105): 797-9. 123. Tran, J.H., Jacoby, G.A. (2002) Mechanism of plasmid-mediated quinolone resistance. In Proceedings of the National Academy of Sciences of the United States of America. 99 (8): 5638-42. 124. Poirel, L., Cabanne, L., Vahaboglu, H., Nordmann, P. (2005) Genetic environment and expression of the extended-spectrum beta-lactamase blaPER-1 gene in gram- negative bacteria. Antimicrob Ag Chemoth. 49 (5): 1708-13. 125. Olsen, J.E. (1999) Antibiotic resistance: genetic mechanisms and mobility. Acta Vet Scand Suppl. 92:15-22. 126. Hall, R.M., Collis, C.M., Kim, M.J., Partridge, S.R., Recchia, G.D., Stokes, H.W. (1999) Mobile gene cassettes and integrons in evolution. Annals NY Acad Sci. 870: 68-80. 127. Hall, R.M. (1997) Mobile gene cassettes and integrons: moving antibiotic resistance genes in gram-negative bacteria. Ciba Foundation Symposium. 207: 192-202; discussion - 5. 128. Recchia, G.D., Hall, R.M. (1997) Origins of the mobile gene cassettes found in integrons. Trends Microbiol. 5 (10): 389-94. 129. Goldstein, C., Lee, M.D., Sanchez, S. (2001) Incidence of class 1 and 2 integrases in Clin and commensal bacteria from livestock, companion animals and exotics. Antimicrob Ag Chemoth. 45 (3): 723-6. 130. Zhao, S., Qaiyumi, S., Friedman, S. (2003) Characterization of Salmonella enterica serotype newport isolated from humans and food animals. J Clin Microbiol. 41 (12): 5366-71. 131. White, D.G., Zhao, S., McDermott, P.F. (2003) Characterization of integron mediated antimicrobial resistance in Salmonella isolated from diseased swine. Can J Vet Res. 67 (1): 39-47. 132. Threlfall, E.J., Frost, J.A., Ward, L.R., Rowe, B. (1994) Epidemic in cattle and humans of Salmonella typhimurium DT 104 with chromosomally integrated multiple drug resistance. Vet Rec. 134 (22): 577. 133. Besser, T.E., Gay, C.C., Gay, J.M. (1997) Salmonellosis associated with S. typhimurium DT104 in the USA. Vet Rec. 140 (3): 75. 134. Glynn, M.K., Bopp, C., Dewitt, W., Dabney, P., Mokhtar, M., Angulo, F.J. (1998) Emergence of multidrug-resistant Salmonella enterica serotype typhimurium DT104 infections in the United States. New Engl J Med. 338 (19): 1333-8. 135. Ng, L.K., Mulvey, M.R., Martin, I., Peters, G.A., Johnson, W. (1999) Genetic characterization of antimicrobial resistance in Canadian isolates of Salmonella serovar Typhimurium DT104. Antimicrob Ag Chemoth. 43 (12): 3018-21. 136. Mulvey, M.R., Boyd, D.A., Olson, A.B., Doublet, B., Cloeckaert, A. (2006) The genetics of Salmonella genomic island 1. Microbes & infection/Institut Pasteur. 8 (7): 1915-22. 137. Butaye, P., Michael, G.B., Schwarz, S., Barrett, T.J., Brisabois, A., White, D.G. (2006) The clonal spread of multidrug-resistant non-typhi Salmonella serotypes. Microbes & infection/Institut Pasteur. 8 (7): 1891-7. 138. Schmieger, H., Schicklmaier, P. (1999) Transduction of multiple drug resistance of Salmonella enterica serovar typhimurium DT104. FEMS Microbiol Lett. 170 (1): 251-6. 139. Arcangioli, M.A., Leroy-Setrin, S., Martel, J.L., Chaslus-Dancla, E. (1999) A new chloramphenicol and florfenicol resistance gene flanked by two integron structures in Salmonella typhimurium DT104. FEMS Microbiol Lett. 174 (2): 327-32. 140. Toleman, M.A., Bennett, P.M., Walsh, T.R. (2006) Common regions e.g. orf513 and antibiotic resistance: IS91-like elements evolving complex class 1 integrons. J Antimicrob Chemoth. 58 (1): 1-6. 141. Beaber, J.W., Hochhut, B., Waldor, M.K. (2002) Genomic and functional analyses of SXT, an integrating antibiotic resistance gene transfer element derived from Vibrio cholerae. J Bacteriol. 184 (15): 4259-69. 142. Sandvang, D., Aarestrup, F.M., Jensen, L.B. (1997) Characterisation of integrons and antibiotic resistance genes in Danish multiresistant Salmonella enterica Typhimurium DT104. FEMS Microbiol Lett. 157 (1): 177-81. 143. Blahova, J., Kralikova, K., Krcmery, V., Jezek, P. (2000) Low-Frequency transduction of imipenem resistance and high-frequency transduction of ceftazidime and aztreonam resistance by the bacteriophage AP-151 isolated from a Pseudomonas aeruginosa strain. J Chemoth. 12 (6): 482-6. 144. Eagle, H., Fleischman, R., Musselman, A.D. (1950) Effect of schedule of administration on the therapeutic efficacy of penicillin; importance of the aggregate time penicillin remains at effectively bactericidal levels. Am J Med. 9 (3): 280-99. ______

Balbino M. Rocha, 2013 142 Refe re nces

145. McKinnon, P.S., Davis, S.L. (2004) Pharmacokinetic & pharmacodynamic issues in the treatment of bacterial Infect Dis. Eur J Clin Microbiol. 23 (4): 271-88. 146. Forrest, A., Nix, D.E., Ballow, C.H., Goss, T.F., Birmingham, M.C., Schentag, J.J. (1993) Pharmacodynamics of intravenous ciprofloxacin in seriously ill patients. Antimicrob Ag Chemoth. 37 (5): 1073-81. 147. Nightingale, C.H. (2001) Antimicrobial Pharmacodynamics in Theory and Clinical Practice. Murakawa, T. Ambrose, P.G. (eds.) Marcel Dekker Inc. New York, USA. 148. Craig, W. (1993) Pharmacodynamics of antimicrobial agents as a basis for determining dosage regimens. Eur J Clin Microbiol. 12 Suppl 1: S6-8. 149. Quintiliani, R. (2012) Pharmacodynamics of Antimicrobial Agents: Time-Dependent vs. Concentration-Dependent Killing. In Antimicrobe.org (accessed September 14th, 2012). 150. Tenover, F.C. (2001) Development and spread of bacterial resistance to antimicrobial agents: an overview. Clin Infect Dis. 33 Suppl 3: S108-15. 151. Turnidge, J., Paterson, D.L. (2007) Setting and revising antibacterial susceptibility breakpoints. Clin Microbiol Rev. 20 (3): 391-408. 152. Wikler, M., Ambrose, P. (2005) The breakpoint. In Antibiotics in laboratory medicine. Lorian, V. (ed.) 5th ed. Philadelphia, USA. Lippincott Williams & Wilkins: 1-7. 153. Kahlmeter, G., Brown, D.F., Goldstein, F.W. (2003) European harmonization of MIC breakpoints for antimicrobial susceptibility testing of bacteria. J Antimicrob Chemoth. 52 (2): 145-8. 154. International Organization for Standards. (2006) ISO 20776-1: Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility testing devices. 1. Reference method for testing the in vitro activity of antimicrobial agents against rapidly growing anaerobic bacteria involved in Infectious Disease. Geneva, Switzerland. 155. Watts, J.L., Shryock, T.R., Apley, M. (2008) Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated From Animals. 3rd ed. Pfizer Animal Health. Pennsylvania, USA. 28: 112. 156. EUCAST - European Committee on Antimicrobial Susceptibility Testing. (2012) EUCAST procedure for harmonising and defining breakpoints. In http://www.eucast.org (accessed August 28th, 2012). 157. Watts, J., Lindeman, C. Chapter 3: Antimicrobial Susceptibility Testing of Bacteria of Veterinary Origin. In Antimicrobial Resistance in Bacteria of Animal Origin. Aarestrup, F. (ed.) ASM Press. Washington DC, USA. 158. Bergan, T., Bruun, J.N., Digranes, A., Lingaas, E., Melby, K.K., Sander, J. (1997) Susceptibility testing of bacteria and fungi. Report from "the Norwegian Working Group on Antibiotics". Scand J Infect Dis Suppl. 103: 1-36. 159. Arbeidsgruppen for Antibiotikaspørsmål. (2005) Nødvendig bakgrunnsmateriale og prosedyrer for etablering og justering av mm-brytningssoner. Arbeidsgruppen for Antibiotikaspørsmål. Tromso, Norway. 160. Arbeidsgruppen for Antibiotikaspørsmål. (2006) AFAs brytningspunkter for bakteriers antibiotikafølsomhet, versjon 1.9. Arbeidsgruppen for Antibiotikaspørsmål. Tromso, Norway. 161. Ambrose, P.G., Quintiliani, R. (2000) Limitations of single point pharmacodynamic analysis. Pediatr Infect Dis J. 19 (8): 769. 162. Andrews, J. (2005) BSAC disc diffusion method for antimicrobial susceptibility testing, version 4. In www.bsac.org.uk. British Society for Antimicrobial Chemotherapy. Birmingham, UK. 163. MacGowan, A.P., Wise, R. (2001) Establishing MIC breakpoints and the interpretation of in vitro susceptibility tests. J Antimicrob Chemoth. 48 Suppl 1: 17-28. 164. Bell, S.M. (1975) The CDS disc method of antibiotic sensitivity testing (calibrated dichotomous sensitivity test). Pathology. 7 (4 Suppl): Suppl 1-48. 165. Werkgroep Richtlijnen Gevoelighheidsbepalingen. (1991) Standaadisatie van gevoelighheidsbepalingen. Bilthoven, The Netherlands: RIVM. 166. Commissie Richtlijnen Gevoelighheidsbepalingen. (1996) Standaadisatie van gevoelighheidsbepalingen. Ned Tijschr Med Microbiol. 4: 104-6. 167. Commissie Richtlijnen Gevoelighheidsbepalingen. (2000) Interpretie van gevoelighheidsonderzoek en gevoelighheidscriteria voor antibacterie¨le middelen in Nederland. Ned Tijdschr Med Microbiol. 8: 79-81. 168. Comite de l'Antibiogramme de la Societe Francaise de Microbiologie. (2003) Report 2003. Int J Antimicrob Ag. 21 (4): 364-91. 169. Deutches Institut für Normung. (2002) Medical Microbiology - susceptibility testing of pathogens to antimicrobial agents. 10. Criteria for the evaluation of the in vitro efficacy and the admission of new antimicrobial agents. Beuth Verlag GmbH. 170. EUCAST. (2000) Definitive Document E. DEF 3.1, June 2000. Determination of minimum inhibitory concentrations (MICs) of antibacterial agents by agar dilution. Clin Microbiol Infect. 6 (9): 509-15. 171. Paterson, D.L. (2006) Resistance in gram-negative bacteria: enterobacteriaceae. Am J Med. 119 (6 Suppl 1): S20-8; discussion S62-70. 172. Erskine, R., Cullor, J., Schaelibaum, M. (2004) National Mastitis Council Research Committee Report: Bovine mastitis pathogens and trends in resistance to antibacterial drugs. Verona (WI), USA. pp. 400-14. 173. Japanese Society for Chemotherapy. (1990) Method for the determination of minimum inhibitory concentration (MIC) of aerobic bacteria by microdilution method. Chemotherapy - Tokyo. 38: 102-5. 174. Japanese Society for Chemotherapy. (1993) Method for the determination of minimum inhibitory concentration (MIC) of fastidious bacteria and anaerobic bacteria by microdilution method. Chemotherapy - Tokyo. 41: 183–9. 175. Saito, A., Inamatsu, T., Okada, J. (1999) Clinical breakpoints in pulmonary infections and sepsis: new antimicrobial agents and supplemental information for some agents already released. J Infect Chemoth. 5 (4): 223-6. 176. Rosco Diagnostica. (2012) Neo-Sensitabs susceptibility testing: User’s guide (2005-2006). 17th ed. Rosco Diagnostica A/C. Taastrup, Denmark. 177. Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. (2000) Procedimientos en microbiología clínica: métados básicos para el estudio de la sensibilidad a los antimicrobianos. Madrid, Spain. 178. Baquero, F., Martínez-Beltrán, J., Cánton, R. (1997) Criterios del grupo MENSURA para la definición de los puntos críticos de sensibilidad a los antibióticos. Rev Esp Quimioter. 10: 103-313. 179. Swedish Reference Group on Antibiotics. (2004) The principles behind SRGA breakpoints. Vaxjo, Sweden. 180. Turnidge, J., Bordash, G. (2007) Statistical methods for establishing quality control ranges for antibacterial agents in Clinical and Laboratory Standards Institute susceptibility testing. Antimicrob Ag Chemoth. 51 (7): 2483-8. 181. Fleming, A. (2001) On the antibacterial action of cultures of a penicillium, with special reference to their use in the isolation of B. influenzae - 1929 re-issue. B World Health Organ. 79 (8): 780-90. 182. Moore, R.D., Lietman, P.S., Smith, C.R. (1987) Clinical response to aminoglycoside therapy: importance of the ratio of peak concentration to minimal inhibitory concentration. J Infect Dis. 155 (1): 93-9. 183. Koeth, L.M., King, A., Knight, H. (2000) Comparison of cation-adjusted Mueller-Hinton broth with Iso-Sensitest broth for the NCCLS broth microdilution method. J Antimicrob Chemoth. 46 (3): 369-76. 184. Jorgensen, J.H., Ferraro, M.J. (2009) Antimicrobial susceptibility testing: a review of general principles and contemporary practices. Clinic Infect Dis. 49(11): 1749-55. 185. Michigan State University. (2011) In Antimicrobial Resistance Learning Site for Veterinary Students: http://amrls.cvm.msu.edu/microbiology (accessed September 17th, 2012) 186. Chitwood, L.A. (1969) Tube dilution antimicrobial susceptibility testing: efficacy of a microtechnique applicable to diagnostic laboratories. Appl Microbiol. 17(5): 707-9. 187. EMEA - European Agency for the Evaluation of Medicinal Products. (1999) Antibiotic Resistance in the European Union Associated with Therapeutic Use of Vet Meds. London, UK. Committee for Veterinary Medicinal Products. 188. Jorgensen, J.H., Turnidge, J.D. (2007) Antibacterial susceptibility tests: dilution and disk diffusion methods. In Manual of Clinical Microbiology. Murray, P.R., Baron, E.J., Jorgensen, J.H., Landry, M.L., Pfaller, M.A. (eds.) 9th ed. American Society for Microbiology. Washington, DC, USA. 1152–72. 189. Bauer, A.W., Kirby, W.M., Sherris, J.C., Turck, M. (1966) Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Path. 45 (4): 493-6.

______

Balbino M. Rocha, 2013 143 Refe re nces

190. CLSI - The Clinical and Laboratory Standards Institute. (2008) Performance standards for antimicrobial disk and difussion susceptibility tests for bacteria isolated from animals - Approved standard M31-A3. Wayne (PA), USA. 28 (8). 191. Korgenski, E.K., Daly, J.A. (1998) Evaluation of the BIOMIC video reader system for determining interpretive categories of isolates on the basis of disk diffusion susceptibility results. J Clin Microbiol. 36 (1): 302-4. 192. Nijs, A., Cartuyvels, R., Mewis, A., Peeters, V., Rummens, J.L., Magerman, K. (2003) Comparison and evaluation of Osiris and Sirscan 2000 antimicrobial susceptibility systems in the Clinical Microbiology laboratory. J Clin Microbiol. 41 (8): 3627-30. 193. LeCorn, D.W., Vertucci, F.J., Rojas, M.F., Progulske-Fox, A., Belanger, M. (2007) In vitro activity of amoxicillin, clindamycin, doxycycline, metronidazole and moxifloxacin against oral Actinomyces. J Endodont. 33 (5): 557-60.

194. Citron, D.M., Ostovari, M.I., Karlsson, A., Goldstein, E. (1991) Evaluation of the E-test for susceptibility testing of anaerobic bacteria. J Clin Microbiol. 29(10):2197-203. 195. Huang, M.B., Baker, C.N., Banerjee, S., Tenover, F.C. (1992) Accuracy of the E-test for determining antimicrobial susceptibilities of staphylococci, enterococci, Campylobacter jejuni, and gram-negative bacteria resistant to antimicrobial agents. J Clin Microbiol. 30 (12): 3243-8. 196. Jorgensen, J.H., Ferraro, M.J., McElmeel, M.L., Spargo, J., Swenson, J.M., Tenover, F.C. (1994) Detection of penicillin and extended-spectrum cephalosporin resistance among Streptococcus pneumoniae Clinical isolates by use of the E-test. J Clin Microbiol. 32 (1): 159-63. 197. Baker, C.N., Stocker, S.A., Culver, D.H., Thornsberry, C. (1991) Comparison of the E-Test to agar dilution, broth microdilution and agar diffusion susceptibility testing techniques by using a special challenge set of bacteria. J Clin Microbiol. 29 (3): 533-8. 198. Rennie, R.P., Turnbull, L., Brosnikoff, C., Cloke, J. (2012) First comprehensive evaluation of the M.I.C. evaluator device compared to E-test and CLSI broth microdilution for MIC testing of aerobic Gram-positive and Gram-negative bacterial species. J Clin Microbiol. 50 (4): 1147-52. 199. Prakash, V., Lewis, J.S., Jorgensen, J.H. (2008) Vancomycin MICs for methicillin-resistant Staphylococcus aureus isolates differ based upon the susceptibility test method used. Antimicrob Ag Chemoth. 52 (12): 4528. 200. Boudewijns, M., Vandeven, J., Verhaegen, J., Ben-Ami, R., Carmeli, Y. (2005) Vitek 2 automated identification system and Kocuria kristinae. J Clin Microbiol. 43 (11): 5832. 201. Cruinn Diagnostics Limited. (2012) Siemens MicroScan® WalkAway®-96 Plus System. In http://www.cruinn.ie/home/Default.aspx?id=11706 (accessed September 18th, 2012). 202. TREK Diagnostic Systems. (2012) Thermo Scientific. In http://www.trekds.com (accessed September 18th, 2012). 203. Abbott Laboratories. (2012) In http://www.abbott.com (accessed September 18th, 2012). 204. MERLIN Diagnostika GmbH. (2012) In http://www.merlin-diagnostika.de (accessed September 18th, 2012). 205. Liu, Z.K., Ling, T.K., Cheng, A.F. (2005) Evaluation of the BD Phoenix Automated Microbiology System for identification and antimicrobial susceptibility testing of common Clinical isolates. Int J Kuwait Univ. 14 (4): 250-4. 206. Felmingham, D., Brown, D.F. (2001) Instrumentation in antimicrobial susceptibility testing. J Antimicrob Chemoth. 48 Suppl 1: 81-5. 207. Tenover, F.C. (1989) DNA probes for antimicrobial susceptibility testing. Clin Laboratory Med. 9 (2): 341-7. 208. Rice, L.B., Bonomo, R.A. (2007) Mechanisms of resistance to antibacterial agents. In: Manual of Clinical Microbiology. Murray, P.R., Baron, E.J., Jorgensen, J.H., Landry, M.L., Pfaller, M.A. (eds.) 9th ed. American Society for Microbiology. Washington, DC, USA. pp. 1114–45. 209. Zadoks, R.N., Middleton, J.R., McDougall, S., Katholm, J., Schukken, Y.H. (2011) Molecular epidemiology of mastitis pathogens of dairy cattle and comparative relevance to humans. J Mammary Gland Biol. 16 (4): 357-72. 210. Zadoks, R.N., Schukken, Y.H. (2006) Use of molecular epidemiology in veterinary practice. Vet Clin N Am-Food A. 22 (1): 229-61. 211. McCaig, L.F., Hughes, J.M. (1995) Trends in antimicrobial drug prescribing among office-based physicians in the United States. Jama - J Am Med Assoc. 273 (3): 214-9. 212. Davey, P.G., Bax, R.P., Newey, J. (1996) Growth in the use of antibiotics in the community in England and Scotland in 1980-93. BMJ. 312 (7031): 613. 213. Guillemot, D., Maison, P., Carbon, C. Trends in antimicrobial drug use in the community - France, 1981-1992. J Infect Dis. 177 (2): 492-7. 214. van Bijnen, E.M., den Heijer, C.D., Paget, W.J. (2011) The appropriateness of prescribing antibiotics in the community in Europe: study design. BMC Infect Dis. 11:293. 215. Guillemot, D., Carbon, C., Vauzelle-Kervroedan, F. (1998) Inappropriateness and variability of antibiotic prescription among French office-based physicians. J Clin Epidemiol. 51 (1): 61-8. 216. Okeke, I.N., Lamikanra, A., Edelman, R. (1999) Socioeconomic and behavioral factors leading to acquired bacterial resistance to antibiotics in developing countries. Emerg Infect Dis. 5 (1): 18-27. 217. Bergus, G.R., Levy, B.T., Levy, S.M., Slager, S.L., Kiritsy, M.C. (1996) Antibiotic use during the first 200 days of life. Arch Fam Med. 5 (9): 523-6. 218. Nyquist, A.C., Gonzales, R., Steiner, J.F., Sande, M.A. (1998) Antibiotic prescribing for children with colds, upper respiratory tract infections, and bronchitis. Jama - J Am Med Assoc. 279 (11): 875-7. 219. Gonzales, R., Steiner, J.F., Sande, M.A. (1997) Antibiotic prescribing for adults with colds, upper respiratory tract infections, and bronchitis by ambulatory care physicians. Jama - J Am Med Assoc. 278 (11): 901-4. 220. Baquero, F. (1995) Pneumococcal resistance to beta-lactam antibiotics: a global geographic overview. Microb Drug Resist. 1 (2): 115-20. 221. Ena, J., Lopez-Perezagua, M.M., Martinez-Peinado, C., Cia-Barrio, M.A., Ruiz-Lopez, I. (1998) Emergence of ciprofloxacin resistance in Escherichia coli isolates after widespread use of fluoroquinolones. Diag Microbiol Infect Dis. 30 (2): 103-7. 222. Guillemot, D. (1999) Antibiotic use in humans and bacterial resistance. Curr Op Microbiol. 2 (5): 494-8. 223. Dowell, S.F., Schwartz, B. (1997) Resistant pneumococci: protecting patients through judicious use of antibiotics. Am Fam Physician. 55 (5): 1647-54, 57-8. 224. Eady, E.A. (1998) Bacterial resistance in acne. Dermatology. 196 (1): 59-66. 225. Jones, R.N. (1998) Important and emerging beta-lactamase-mediated resistances in hospital-based pathogens: the Amp C enzymes. Diag Microbiol Infectious Dis. 31 (3): 461-6. 226. Rahal, J.J., Urban, C., Horn, D. (1998) Class restriction of cephalosporin use to control total cephalosporin resistance in nosocomial Klebsiella. Jama - J Am Med Assoc. 280 (14): 1233-7. 227. Fish, D.N., Piscitelli, S.C., Danziger, L.H. (1995) Development of resistance during antimicrobial therapy: a review of antibiotic classes and patient characteristics in 173 studies. Pharmacotherapy. 15 (3): 279-91. 228. Brennen, C., Wagener, M.M., Muder, R.R. (1998) Vancomycin-resistant Enterococcus faecium in a long-term care facility. J Am Geriatr Society. 46(2): 157-60. 229. Espersen, F. (1998) Resistance to antibiotics used in dermatological practice. Brit J Dermatology. 139 Suppl 53: 4-8. 230. Raad, I., Alrahwan, A., Rolston, K. (1998) Staphylococcus epidermidis: emerging resistance and need for alternative agents. Clin Infect Dis. 26 (5): 1182-7. 231. Chen, D.K., McGeer, A., de Azavedo, J.C., Low, D.E. (1999) Decreased susceptibility of Streptococcus pneumoniae to fluoroquinolones in Canada - Canadian Bacterial Surveillance Network. New Engl J Med. 341 (4): 233-9. 232. Cohen, R., Bingen, E., Varon, E. Change in nasopharyngeal carriage of Streptococcus pneumoniae resulting from antibiotic therapy for acute otitis media in children. Pediatr Infect Dis J. 16 (6): 555-60. 233. Guillemot, D., Carbon, C., Balkau, B. (1998) Low dosage and long treatment duration of beta-lactam: risk factors for carriage of penicillin-resistant Streptococcus pneumoniae. Jama - J Am Med Assoc. 279 (5): 365-70. 234. Baquero, F. (1999) Evolving resistance patterns of Streptococcus pneumoniae: a link with long-acting macrolide consumption? J Chemother. 11 Suppl 1: 35-43. 235. AGISAR - WHO Advisory Group on Integrated Surveillance of Antimicrobial Resistance. (2009) Critically Important Antimicrobials for Human Medicine. In Zoonoses WHO (ed.) 2nd ed. WHO. Copenhagen, Denmark. ______

Balbino M. Rocha, 2013 144 Refe re nces

236.ESAC - European Surveillance of Antimicrobial Consumption.(2010) In ESAC Yearbook 2009. p. 141. 237. Commonwealth Department of Health and Aged Care & Commonwealth Department of Agriculture. (1999) The Use of antibiotics in food-producing animals: Antibiotic-resistant bacteria in animals and humans. Report of the Joint Expert Advisory Committee on Antibiotic Resistance (JETACAR). Canberra, Australia. p. 249. 238. McClary, D., Vogel, G. (1999) Effect of timing of tilmicosin metaphylaxis on control of bovine respiratory disease and performance in feeder cattle. Bov Practitioner. 33: 155–61. 239. Guthrie, C.A., Vogel, G.J., Laudert, S.B. (1997) Effects of tilmicosin on the incidence of bovine respiratory disease and animal performance when used in temperature-based therapy and complete metaphylaxis treatment programs. 30th Annual Conference of the American Association of Bovine Practitioners With the Society for Theriogenology. Montreal, Quebec, Canada. p. 134. 240. Stokstad, E.L., Jukes, T.H. (1949) The multiple nature of the animal protein factor. J Biolog Chem. 180 (2): 647-54. 241. Nagaraja, T.G., Chengappa, M.M. (1998) Liver abscesses in feedlot cattle: a review. J Anim Sci. 76 (1): 287-98. 242. Gaskins, H.R., Collier, C.T., Anderson, D.B. (2002) Antibiotics as growth promotants: mode of action. Anim Biotechnol. 13 (1): 29-42. 243. Dennis, S.M., Nagaraja, T.G., Bartley, E.E. (1981) Effects of lasalocid or monensin on lactate-producing or - using rumen bacteria. J Anim Sci. 52 (2): 418-26. 244. Nagaraja, T.G., Taylor, M.B., Harmon, D.L., Boyer, J.E. (1987) In vitro lactic acid inhibition and alterations in volatile fatty acid production by antimicrobial feed additives. J Anim Sci. 65 (4): 1064-76. 245. Anderson, D.B., McCracken, V.J., Aminov, R.I. Gut Microbiology and growth-promoting antibiotics in swine. Pig News and Information. 20(4): 115N-22N. 246. Pieterman, R., Hanekamp, J. (2001) Resistance against antibiotics. In The Cautious Society? An Essay on the Rise of the Precautionary Culture The Precautionary Principle or Striving for Ignorance. Heidelberg Appeal Nederland Foundation. Amsterdam, The Netherlands. p. 76. 247. Preston, R. (1987) The role of animal drugs in food animal production. In Symposium on Animal Drug Use - Dollars and Sense. Center for Veterinary Medicine & Food and Drug Administration (eds.) Food and Drug Administration. Washington, DC, USA. pp.127-34. 248. Lees, P., Shojaee Aliabadi, F. (2002) Rational dosing of antimicrobial drugs: animals versus humans. Int J Antimicrob Ag. 19 (4): 269-84. 249. European Medicines Agency Website. (2012) In http://www.ema.europa.eu (accessed September 11th, 2012). 250. European Centre for Disease Control. (2012) In http://ecdc.europa.eu (accessed September 11th, 2012). 251. European Food Safety Authority. (2012) In http://www.efsa.europa.eu/en/topics/topic/amr.htm (accessed September 11th, 2012). 252. European Union. (2009) Comission Directive 2009/9/EC of 10 February 2009 amending Directive 2001/82/EC of the European Parliament and of the Council on the Community code relating to medicinal products for veterinary use. Official J European Union. p. 52. 253. Portuguese Government. (2009) Decreto-Lei n.º 314-2009 - Código comunitário relativo aos medicamentos veterinários. In Diário da República. Portuguese Government (ed.) 2nd ed. Lisbon, Portugal. p. 110. 254. Direcção-Geral de Alimentação e Veterinária, DRAV – Direção Regional de Alimentação e Veterinária, DGAMPV – Divisão de Gestão e Autorização de Medicamentos e Produtos Veterinários. (2012) Relatório Nacional de Monitorização do Consumo de Antimicrobianos - Portugal: Ano 2011. Ministério da Agricultura, Mar, Ambiente e Ordenamento do Território. p. 19. 255. Prescott, J.F., Baggot, J.D., Walker, R.D. (2000) Antimicrobial Therapy in Veterinary Medicine. 3rd ed. Iowa State University Press. Ames (IA), USA. 256. USDA. (2008) Dairy 2007: Part III - Reference of Dairy Cattle Health and Management Practices in the United States - Info sheet. United States Department of Agriculture, Animal Plant Health Inspection Service & National Animal Health Monitoring System (eds.). p. 154. 257. Witte, W. (1998) Medical consequences of antibiotic use in agriculture. Science. 279 (5353): 996-7. 258. O'Brien, T.F. (2002) Emergence, spread, and environmental effect of antimicrobial resistance: how use of an antimicrobial anywhere can increase resistance to any antimicrobial anywhere else. Clin Infect Dis. 34 Suppl 3: S78-84. 259. Mateu, E., Martin, M. (2001) Why is anti-microbial resistance a veterinary problem as well? J Vet Med. 48 (8): 569-81. 260. Summers, A.O. (2002) Generally overlooked fundamentals of bacterial genetics and ecology. Clin Infect Dis. 34 Suppl 3: S85-92. 261. Anderson, A.D., Nelson, J.M., Rossiter, S., Angulo, F.J. (2003) Public health consequences of use of antimicrobial agents in food animals in the United States. Microb Drug Resist. 9 (4): 373-9. 262. Holmberg, S.D., Wells, J.G., Cohen, M.L. (1984) Animal-to-man transmission of antimicrobial-resistant Salmonella: investigations of U.S. outbreaks, 1971-1983. Science. 225 (4664): 833-5. 263. Harwood, V.J., Brownell, M., Perusek, W., Whitlock, J.E. (2001) Vancomycin-resistant Enterococcus spp. isolated from wastewater and chicken feces in the United States. Appl Environ Microbiol. 67 (10): 4930-3. 264. Vidaver, A.K. (2002) Uses of antimicrobials in plant agriculture. Clin Infect Dis. 34 Suppl 3: S107-10. 265. World Health Organization. (2009) Report of the 1st meeting of the WHO advisory group on integrated surveillance of antimicrobial resistance (AGISAR), Copenhagen. In http://www.who.int/foodsafety/foodborne_disease/AGISAR_2009_report_final.pdf (accessed September 26th, 2012). 266. Wall, P.G., Threllfall, E.J., Ward, L.R., Rowe, B. (1996) Multiresistant Salmonella typhimurium DT104 in cats: a public health risk. Lancet 348 (9025): 471. 267. Centers for Disease Control and Prevention. (1999) Reptile associated salmonellosis - selected States, 1996-1998. In CDC Morbidity and Mortality Weekly Report. pp. 1009-13. 268. Centers for Disease Control and Prevention. Outbreaks of multidrug-resistant Salmonella Typhimurium associated with veterinary facilities - Idaho, Minnesota, and Washington. In Morbidity and Mortality Weekly Report. 50: 701-4. 269. Olsen, S.J., DeBess, E.E., McGivern, T.E. (2001) A nosocomial outbreak of fluoroquinolone-resistant Salmonella infection. New Engl J Med. 344 (21): 1572-9. 270. Barker, J., Bloomfield, S.F. (2000) Survival of Salmonella in bathrooms and toilets in domestic homes following salmonellosis. J Appl Microbiol. 89 (1): 137-44. 271. Schutze, G.E., Sikes, J.D., Stefanova, R., Cave, M.D. (1999) The home environment and salmonellosis in children. Pediatrics. 103 (1): E1. 272. ECDC - European Centre for Disease Prevention and Control. (2011). Reporting on 2009 surveillance data and 2010 epidemic intelligence data. In Annual epidemiological report. ECDC (ed.) Stockholm, Sweden. p. 239. 273. Gibreel, A., Taylor, D.E. (2006) Macrolide resistance in Campylobacter jejuni and Campylobacter coli. J Antimicrob Chemoth. 58 (2): 243-55. 274. EARS-Net - European Antimicrobial Resistance Surveillance Network. (2012) Antimicrobial resistance surveillance in Europe 2011. Annual Report of the European Antimicrobial Resistance Surveillance Network (EARS-Net). European Centre for Disease Prevention and Control (ed.). p. 209. 275. Werner, G., Coque, T.M., Hammerum, A.M. (2008) Emergence and spread of vancomycin resistance among enterococci in Europe. In Euro surveillance: European communicable disease bulletin. 13 (47). 276. van Den Bogaard, A.E., London, N., Stobberingh, E.E. (2000) Antimicrobial resistance in pig faecal samples from the Netherlands (five abattoirs) and Sweden. J Antimicrob Chemoth. 45 (5): 663-71. 277. Cauwerts, K., Decostere, A., De Graef, E.M., Haesebrouck, F., Pasmans, F. (2007) High prevalence of tetracycline resistance in Enterococcus isolates from broilers carrying the erm(B) gene. Avian Pathol. 36 (5): 395-9. 278. Collignon, P., Dreimanis, D., Ferguson, J. (2008) Reducing harm to patients from health care associated infection: the role of surveillance. In Bloodstream infections. Cruickshank, M., Ferguson, J. (eds.) Australian Commission on Safety and Quality in Health Care. Sydney, Australia 53–89. 279. Hujer, K.M., Hujer, A.M., Hulten, E.A. (2006) Analysis of antibiotic resistance genes in multidrug-resistant Acinetobacter sp. isolates from military and civilian patients treated at the Walter Reed Army Medical Center. Antimicrob Ag Chemoth. 50 (12): 4114-23. 280. Fernando, G.A., Collignon, P.J., Bell, J.M. (2010) A risk for returned travellers: the "post-antibiotic era". Med J Australia. 193 (1): 59. 281. Li, J., Nation, RL., Turnidge, JD. (2006) Colistin: the re-emerging antibiotic for multidrug-resistant Gram-negative bacterial infections. Lancet Infect Dis 6 (9): 589-601. 282. Collignon, P., Nimmo, GR., Gottlieb, T., Gosbell, IB., (2005) Australian Group on Antimicrobial R. Staphylococcus aureus bacteremia. Emerg Infect Dis.11(4): 554-61. ______

Balbino M. Rocha, 2013 145 Refe re nces

283. Cosgrove, S.E., Sakoulas, G., Perencevich, E.N., Schwaber, M.J., Karchmer, A.W., Carmeli, Y. (2003) Comparison of mortality associated with methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteremia: a meta-analysis. Clin Infect Dis. 36 (1): 53-9. 284. De Oliveira, A.P., Watts, J.L., Salmon, S.A., Aarestrup, F.M. (2000) Antimicrobial susceptibility of Staphylococcus aureus isolated from bovine mastitis in Europe and the United States. J Dairy Sci. 83 (4): 855-62. 285. Moon, J.S., Lee, A.R., Kang, H.M. (2007) Phenotypic and genetic antibiogram of methicillin-resistant staphylococci isolated from bovine mastitis in Korea. J Dairy Sci. 90 (3): 1176-85. 286. Aarestrup, F.M., Wegener, H.C., Collignon, P. (2008) Resistance in bacteria of the food chain: epidemiology and control strategies. Exp Rev Anti-infective Therap. 6 (5): 733-50. 287. Khanna, T., Friendship, R., Dewey, C., Weese, J.S. (2008) Methicillin resistant Staphylococcus aureus colonization in pigs and pig farmers. Vet Microbiol. 128(3-4): 298-303. 288. Lewis, H.C., Molbak, K., Reese, C. (2008) Pigs as source of methicillin-resistant Staphylococcus aureus CC398 infections in humans, Denmark. Emerg Infect Dis. 14 (9): 1383-9. 289. Bos, M.E., Graveland, H., Portengen, L., Wagenaar, J.A., Heederik, D.J. (2012) Livestock-associated MRSA prevalence in veal calf production is associated with farm hygiene, use of antimicrobials and age of the calves. Prev Vet Med. 105(1-2): 155-9. 290. Nemati, M., Hermans, K., Lipinska, U. (2008) Antimicrobial resistance of old and recent Staphylococcus aureus isolates from poultry: first detection of livestock- associated methicillin-resistant strain ST398. Antimicrob Ag Chemoth. 52 (10): 3817-9. 291. Devriese, L.A., Hommez, J. (1975) Epidemiology of methicillin-resistant Staphylococcus aureus in dairy herds. Res Vet Sci. 19 (1): 23-7. 292. van Loo, I.H., Diederen, B.M, Savelkoul, P.H. (2007) Methicillin-resistant Staphylococcus aureus in meat products, the Netherlands. Emerg Infect Dis. 13(11): 1753-5. 293. Weese, J.S., Avery, B.P., Reid-Smith, R.J. (2010) Detection and quantification of methicillin-resistant Staphylococcus aureus (MRSA) clones in retail meat products. Letters Appl Microbiol. 51 (3): 338-42. 294. van Loo, I., Huijsdens, X., Tiemersma, E. (2007) Emergence of methicillin-resistant Staphylococcus aureus of animal origin in humans. Emerg Infect Dis. 13 (12): 1834-9. 295. Hallin, M., De Mendonça, R., Denis, O. (2011) Diversity of accessory genome of human and livestock-associated ST398 methicillin resistant Staphylococcus aureus strains. J Mol Epidemiol Evol Gen Infect Dis. 11 (2): 290-9. 296. van Cleef, B.A., Monnet, D.L., Voss, A. (2011) Livestock-associated methicillin-resistant Staphylococcus aureus in humans, Europe. Emerg Infect Dis. 17 (3): 502-5. 297. Wassenberg, M.W., Bootsma, M.C., Troelstra, A., Kluytmans, J.A., Bonten, M.J. (2011) Transmissibility of livestock-associated methicillin-resistant Staphylococcus aureus (ST398) in Dutch hospitals. Clin Microbiol Infect. 17 (2): 316-9. 298. Bootsma, M.C., Wassenberg, M.W., Trapman, P., Bonten, M.J. (2011) The nosocomial transmission rate of animal-associated ST398 meticillin-resistant Staphylococcus aureus. J Royal Society. 8 (57): 578-84. 299. Health Council of the Netherlands. (2011) Antibiotics in food animal production and resistant bacteria in humans. The Hague, The Netherlands. p. 45. 300. Hsueh, P.R., Teng, L.J., Lee, L.N., Yang, P.C., Ho, S.W., Luh, K.T. (1999) Dissemination of high-level penicillin-, extended-spectrum cephalosporin-, and erythromycin-resistant Streptococcus pneumoniae clones in Taiwan. J Clin Microbiol. 37 (1): 221-4. 301. Pallares, R., Linares, J., Vadillo, M. Resistance to penicillin and cephalosporin and mortality from severe pneumococcal pneumonia in Barcelona, Spain. New Engl J Med. 333 (8): 474-80. 302. Collignon, P.J., Turnidge, J.D. (2000) Antibiotic resistance in Streptococcus pneumoniae. Med J Australia. 173 Suppl: S58-64. 303. Kahn, L., Kaplan, B., Monath, T.P. (2012) One Health Initiative. In http://www.onehealthinitiative.com (accessed October 29th, 2012). 304. Huovinen, P., Cars, O. (1998) Control of antimicrobial resistance: time for action. The essentials of control are already well known. BMJ. 317 (7159): 613-4. 305. Holloway, K., van Dijk, L. (2011) The World Medicines Situation 2011 - Rational Use of Medicines. World Health Organization. (ed.) 3rd ed. Geneva, Switzerland. 306. Duse, A.G. (2011) The Global Antibiotic Resistance Partnership (GARP). South African Med J. 101 (8 Pt 2): 551. 307. Grundmann, H., Klugman, K.P., Walsh, T. (2011) A framework for global surveillance of antibiotic resistance. Drug Resist Update. 14 (2): 79-87. 308. ANSORP - Asian Network of Surveillance of Resistant Pathogens. (2012) In http://www.ansorp.org (accessed September 28th, 2012). 309. JMI Laboratories. (2012) SENTRY Antimicrobial Surveillance Program 1997–2009. In http://www.jmilabs.com (accessed September 28th, 2012). 310. Turner, P.J. (2000) MYSTIC (Meropenem Yearly Susceptibility Test Information Collection): a global overview. J Antimicrob Chemoth. 46 Suppl T2: 9-23. 311. APUA - Alliance for the Prudent Use of Antibiotics. (2012) International Surveillance of Reservoirs of Antibiotic Resistance (ISRAR). In http://www.tufts.edu/med/apua/research/israr.shtml (accessed September 28th, 2012). 312. AGISAR - WHO Advisory Group on Integrated Surveillance of Antimicrobial Resistance. (2012) In http://www.who.int/foodborne_disease/resistance/agisar/en/index.html (accessed September 28th, 2012). 313. Dancer, S.J. (2008) Importance of the environment in meticillin-resistant Staphylococcus aureus acquisition: the case for hospital cleaning. Lancet Infect Dis. 8 (2): 101-13. 314. Singh, M.P., Greenstein, M. (2000) Antibacterial leads from microbial natural products discovery. Curr Opin Drug Disc Develop. 3 (2): 167-76. 315. Lim, Y.W., Steinhoff, M., Girosi, F. (2006) Reducing the global burden of acute lower respiratory infections in children: the contribution of new diagnostics. Nature. 444 Suppl 1: 9-18. 316. Collignon, P., Powers, J.H., Chiller, T.M., Aidara-Kane, A., Aarestrup, F.M. (2009) World Health Organization ranking of antimicrobials according to their importance in human medicine: A critical step for developing risk management strategies for the use of antimicrobials in food production animals. Clin Infect Dis. 49 (1): 132-41. 317. Food and Drug Administration. (2005) Animal Drugs, Feeds and Related Products; Enrofloxacin for Poultry; Withdrawal of Approval of New Animal Drug Application. In http://edocket.access.gpo.gov/2005/05-15223.htm - United States Department of Health and Human Services (ed.), Federal Register Online. Rockville, USA. p.44048-9. 318. European Parliament. (2003) Regulation (EC) No. 1831/2003 of the European Parliament and of the Council of 22 September 2003 on additives for use in animal nutrition. Council of the European Parliament (ed.). L 268-29. 319. European Union. (2005) Ban on antibiotics as growth promoters in animal feed enters into effect. Brussels, Belgium. IP/05/1687. 320. SCAN - Scientific Committee for Animal Nutrition. (1996) Report of the Scientific Committee for Animal Nutrition (SCAN) on the possible risk for humans of the use of avoparcin as a feed additive. Opinion expressed 21 May 1996. Luxembourg: Office for EC Publications. 321. SCAN - Scientific Committee for Animal Nutrition.(1998) Opinion of the Scientific Committee for Animal Nutrition (SCAN) on the immediate and longer-term risk to the value of streptogramins in human medicine posed by the use of virginiamycin as an animal growth promoter, 10 July 1998. Luxembourg: Office for EC Publications. 322. Aarestrup, F.M., Seyfarth, A.M., Emborg, H.D., Pedersen, K., Hendriksen, R.S., Bager, F. (2001) Effect of abolishment of the use of antimicrobial agents for growth promotion on occurrence of antimicrobial resistance in fecal enterococci from food animals in Denmark. Antimicrob Ag Chemoth. 45 (7): 2054-9. 323. Bager, F., Emborg, H.D., Heuer, O.E. (2002) DANMAP 2001- Use of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, food and humans in Denmark. 324. Schrag, S.J., Perrot, V. (1996) Reducing antibiotic resistance. Nature. 381 (6578): 120-1. 325. Morell, V. (1997) Antibiotic resistance: road of no return. Science. 278 (5338): 575-6. 326. Langlois, B.E., Dawson, K.A., Cromwell, G.L., Stahly, T.S. (1986) Antibiotic resistance in pigs following a 13 year ban. J Anim Sci. 62 (Supplement 3): 18-31. 327. Blix, H.S., Grave, K., Lund, A. (2011) Usage of Antimicrobial Agents and Occurrence of Antimicrobial Resistance in Norway. In NORM/NORM-VET 2010. Lund, A., Simonsen, G.S. (eds.) Tromsø, Oslo. 328. Hasman, H., Aarestrup, F.M. (2002) tcrB, a gene conferring transferable copper resistance in Enterococcus faecium: occurrence, transferability, and linkage to macrolide and glycopeptide resistance. Antimicrob Ag Chemoth. 46 (5): 1410-6. ______

Balbino M. Rocha, 2013 146 Refe re nces

329. Wegener, H.C. (2003) Antibiotics in animal feed and their role in resistance development. Curr Opin Microbiol. 6 (5): 439-45. 330. Johnsen, P.J., Townsend, J.P., Bohn, T., Simonsen, G.S., Sundsfjord, A., Nielsen, K.M. (2009) Factors affecting the reversal of antimicrobial-drug resistance. Lancet Infect Dis. 9 (6): 357-64. 331. Johnsen, P.J., Townsend, J.P., Bohn, T., Simonsen, G.S., Sundsfjord, A., Nielsen, K.M. (2011) Retrospective evidence for a biological cost of vancomycin resistance determinants in the absence of glycopeptide selective pressures. J Antimicrob Chemoth. 66 (3): 608-10. 332. Dutil, L., Irwin, R., Finley, R. (2010) Ceftiofur resistance in Salmonella enterica serovar Heidelberg from chicken meat and humans, Canada. Emerg Infect Dis. 16 (1): 48-54. 333. Aarestrup, F.M., Jensen, V.F., Emborg, H.D., Jacobsen, E., Wegener, H.C. (2010) Changes in the use of antimicrobials and the effects on productivity of swine farms in Denmark. Am J Vet Res. 71 (7): 726-33. 334. Food and Agriculture Organization of the United Nations, World Organization for Animal Health. (2006) Report of a joint FAO/OIE/WHO Expert Consultation on Antimicrobial Use in Aquaculture and Antimicrobial Resistance. In http://www.who.int/topics/foodborne_diseases/aquaculture_rep_13_16june2006%20.pdf (accessed September 26th, 2012). 335. Food and Agriculture Organization of the United Nations, World Health Organization. Codex Alimentarius. (2012) In http://www.codexalimentarius.org (accessed September 26th, 2012). 336. International Dairy Federation. (1987) Bovine Mastitis: Definition and Guidelines for Diagnosis. B Int Dairy Fed. (211): 24. 337. Fetrow, J. (2000) Mastitis: An economic consideration. In Proceedings of the 2000 National Mastitis Council Meeting. Madison, WI. p. 3-47. 338. Odegard, J., Klemetsdal, G., Heringstad, B. (2003) Genetic improvement of mastitis resistance: validation of somatic cell score and clinical mastitis as selection criteria. J Dairy Sci. 86 (12): 4129-36. 339. Osteras, O., Edge, V.L. (2000) Factors prior to dry period associated with high & low levels of cow milk SCC in next lactation. Acta Vet Scand. 41 (1): 63-77. 340. Pyorala, S. (2003) Indicators of inflammation in the diagnosis of mastitis. Vet Res. 34 (5): 565-78. 341. Merck. (2005) In The Merck Veterinary Manual. 9th - 50th Anniversary Edition. Gary Zelko (ed.) Philadelphia, USA. 342. Schukken, Y.H., Wilson, D.J., Welcome, F., Garrison-Tikofsky, L., Gonzalez, R.N. (2003) Monitoring udder health and milk quality using somatic cell counts. Vet Res. 34 (5): 579-96. 343. Akers, R.M. (2002) In Lactation and the Mammary Gland. John Wiley & Sons. Iowa, USA. p. 278. 344. Suriyasathaporn, W., Schukken. Y.H., Nielen, M., Brand, A. (2000) Low somatic cell count: a risk factor for subsequent clinical mastitis in a dairy herd. J Dairy Sci. 83 (6): 1248-55. 345. Oviedo-Boyso, J., Valdez-Alarcon, J.J., Cajero-Juarez, M. (2007) Innate immune response of bovine mammary gland to pathogenic bacteria responsible for mastitis. J Infect. 54 (4): 399-409. 346. Emanuelson, U., Oltenacu, P.A., Grohn, Y.T. (1993) Nonlinear mixed model analyses of five production disorders of dairy cattle. J Dairy Sci. 76 (9): 2765-72. 347. Rajala-Schultz, P.J., Grohn, Y.T., McCulloch, C.E., Guard, C.L. Effects of clinical mastitis on milk yield in dairy cows. J Dairy Sci. 82 (6): 1213-20. 348. Barkema, H.W., Schukken, Y.H., Lam, T.J. Incidence of clinical mastitis in dairy herds grouped in three categories by bulk milk SCC. J Dairy Sci. 81 (2): 411-9. 349. Steeneveld, W., Hogeveen, H., Barkema, H.W., van den Broek, J., Huirne, R.B. (2008) The influence of cow factors on the incidence of clinical mastitis in dairy cows. J Dairy Sci. 91 (4): 1391-402. 350. Persson Waller, K., Bengtsson, B., Lindberg, A., Nyman, A., Ericsson Unnerstad, H. (2009) Incidence of mastitis and bacterial findings at clinical mastitis in Swedish primiparous cows - influence of breed and stage of lactation. Vet Microbiol. 134 (1-2): 89-94. 351. Busato, A., Trachsel, P., Schallibaum, M., Blum, J.W. (2000) Udder health and risk factors for subclinical mastitis in organic dairy farms in Switzerland. Prev Vet Med. 44 (3-4): 205-20. 352. Wilson, D.J., Gonzalez, R.N, Hertl, J. (2004) Effect of clinical mastitis on the lactation curve: a mixed model estimation using daily milk weights. J Dairy Sci. 87 (7): 2073-84. 353. Grohn, Y.T., Wilson, D.J., Gonzalez, R.N. (2004) Effect of pathogen-specific clinical mastitis on milk yield in dairy cows. J Dairy Sci. 87 (10): 3358-74. 354. Svensson, C., Nyman, A.K., Persson Waller, K., Emanuelson, U. (2006) Effects of housing, management, and health of dairy heifers on first-lactation udder health in southwest Sweden. J Dairy Sci. 89 (6): 1990-9. 355. Schreiner, D.A., Ruegg, P.L. (2003) Relationship between udder and leg hygiene scores and subclinical mastitis. J Dairy Sci. 86 (11): 3460-5. 356. Elbers, A.R., Miltenburg, J.D., De Lange, D., Crauwels, A.P., Barkema, H.W., Schukken, Y.H. (1998) Risk factors for clinical mastitis in a random sample of dairy herds from the southern part of The Netherlands. J Dairy Sci. 81 (2): 420-6. 357. Nyman, A.K., Ekman, T., Emanuelson, U. Risk factors associated with the incidence of veterinary-treated clinical mastitis in Swedish dairy herds with a high milk yield and a low prevalence of subclinical mastitis. Prev Vet Med. 78 (2): 142-60. 358. Barkema, H.W., Schukken, Y.H., Lam, T.J., Beiboer, M.L., Benedictus, G., Brand, A. (1999) Management practices associated with the incidence rate of clinical mastitis. J Dairy Sci. 82 (8): 1643-54. 359. Olde Riekerink, R.G., Barkema, H.W., Stryhn, H. (2007) The effect of season on somatic cell count and the incidence of clinical mastitis. J Dairy Sci. 90 (4): 1704-15. 360. Bradley, A. (2002) Bovine mastitis: an evolving disease. Vet J. 164 (2): 116-28. 361. Djabri, B., Bareille, N., Beaudeau, F., Seegers, H. (2002) Quarter milk somatic cell count in infected dairy cows: a meta-analysis. Vet Res. 33 (4): 335-57. 362. Kapur, V., Sischo, W.M., Greer, R.S., Whittam, T.S., Musser, J.M. (1995) Molecular population genetic analysis of Staphylococcus aureus recovered from cows. J Clin Microbiol. 33 (2): 376-80. 363. Rivas, A.L., Gonzalez, R.N., Wiedmann, M. (1997) Diversity of Streptococcus agalactiae and Staphylococcus aureus ribotypes recovered from New York dairy herds. Am J Vet Res. 58 (5): 482-7. 364. Zadoks, R., van Leeuwen, W., Barkema, H. Application of pulsed-field gel electrophoresis and binary typing as tools in veterinary clinical microbiology and molecular epidemiologic analysis of bovine and human Staphylococcus aureus isolates. J of Clin Microbiol. 38 (5): 1931-9. 365. Joo, Y.S., Fox, L.K., Davis, W.C., Bohach, G.A., Park, Y.H. (2001) Staphylococcus aureus associated with mammary glands of cows: genotyping to distinguish different strains among herds. Vet Microbiol. 80 (2): 131-8. 366. Zadoks, R. (2007) Sources and epidemiology of Streptococcus uberis, with special emphasis on mastitis in dairy cattle. CAB Rev: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources. Oxfordshire, UK. 2 (30): 15. 367. Hogeveen, H. (2005) Gram-positive mastitis - Streptococcus species. In Mastitis in Dairy Production: Current knowledge and future solutions. Hogeveen, H. (ed.) Wageningen Academic. p. 111. 368. Bradley, A.J., Green, M.J. (2001) Adaptation of Escherichia coli to the bovine mammary gland. J Clin Microbiol. 39 (5): 1845-9. 369. Pinho, L.A., Ferreira, J., Cabral, C. (2008) Prevalence of mastitis pathogens in milk samples from Portuguese dairy cattle. In Mastitis control: from science to practice. Lam, T. (ed.) The Hague, Netherlands. pp. 47-53. 370. European Parliament. Corrigendum to Regulation (EC) No 853/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific hygiene rules for food of animal origin. In: European Parliament C, editor. 835/2004; 2004. p. L 226/22. 371. Viguier, C., Arora, S., Gilmartin, N., Welbeck, K., O'Kennedy, R. (2009) Mastitis detection: current trends and future perspectives. Trends Biotech. 27 (8): 486-93. 372. Schalm, O.W., Noorlander, D.O. (1957) Experiments and observations leading to development of the California mastitis test. J Am Vet Med Assoc. 130 (5): 199-204. 373. Sears, P.M., Fettinger, M., Marsh-Salin, J. Isolation of L-form variants after antibiotic treatment in Staphylococcus aureus bovine mastitis. J Am Vet Med Assoc. 191 (6): 681-4.

______

Balbino M. Rocha, 2013 147 Refe re nces

374. Erskine, R.J., Unflat, J.G., Eberhart, R.J., Hutchinson, L.J., Hicks, C.R., Spencer, S.B. (1987) Pseudomonas mastitis: difficulties in detection and elimination from contaminated wash-water systems. J Am Vet Med Assoc. 191 (7): 811-5. 375. Newbould, F.H. (1974) Antibiotic treatment of experimental Staphylococcus aureus infections of the bovine mammary gland. Can J Compar Med. 38 (4): 411-6. 376. Sears, P.M., Smith, B.S., English, P.B., Herer, P.S., Gonzalez, R.N. (1990) Shedding pattern of Staphylococcus aureus from bovine intramammary infections. J Dairy Sci. 73 (10): 2785-9. 377. Ruegg, P.L., Reinemann, D.J. (2002) Milk Quality and Mastitis Tests. In http://milkquality.wisc.edu/wp-content/uploads/2011/09/milk-quality-and-mastitis-diagnostic- tests.pdf. University of Madison (WI), USA. pp. 33 378. Dohoo, I.R., Leslie, K.E. (1991) Evaluation of changes in somatic cell counts as indicators of new intramammary infections. Prev Vet Med. 10: 225. 379. McDermott, M.P., Erb, H.N., Natzke, R.P. (1982) Predictability by SCC related to prevalence of intrammary infection within herds. J Dairy Sci. 65 (8): 1535-9. 380. Timms, L.L., Schultz, L.H. (1987) Dynamics and significance of coagulase-negative staphylococcal intramammary infections. J Dairy Sci. 70 (12): 2648-57. 381. Sargeant, J.M., Leslie, K.E., Shirley, J.E., Pulkrabek, B.J., Lim, G.H. (2001) Sensitivity and specificity of somatic cell count and California Mastitis Test for identifying intramammary infection in early lactation. J Dairy Sci. 84 (9): 2018-24. 382. Ruegg, P.L., Tabone, T.J. (2000) The relationship between antibiotic residue violations and somatic cell counts in Wisconsin dairy herds. J Dairy Sci. 83 (12): 2805-9. 383. Wallace, R.L. (2000) Detecting herd mastitis problems by computer. 39th Annual Meeting of the National Mastitis Council. Atlanta (GA), USA: National Mastitis Council, Inc. p. 68-78. 384. Ziv, G. (1980) Practical pharmacokinetic aspects of mastitis therapy - 2: practical & therapeutic applications. Vet Med, SAC 75 (3): 469-74. 385. Owens, W.E., Watts, J.L., Boddie, R.L., Nickerson, S.C. (1988) Antibiotic treatment of mastitis: comparison of intramammary and intramammary plus intramuscular therapies. J Dairy Sci. 71 (11): 3143-7. 386. Sol, J., Harink, J., van Uum, A. (1990) Factors affecting the result of dry cow treatment. In International Symposium on Bovine Mastitis. National Mastitis Council, Inc. Madison (WI), USA. p. 118-23. 387. Cebra, C.K., Garry, F.B., Dinsmore, R.P. (1996) Naturally occurring acute coliform mastitis in Holstein cattle. J Vet Inter Med. 10 (4): 252-7. 388. Wenz, J.R., Barrington, G.M., Garry, F.B. Bacteremia associated with naturally occuring acute coliform mastitis in dairy cows. J Am Vet Med Assoc. 219 (7): 976-81. 389. Erskine, R.J., Bartlett, P.C., VanLente, J.L., Phipps, C.R. (2002) Efficacy of systemic ceftiofur as a therapy for severe clinical mastitis in dairy cattle. J Dairy Sci. 85 (10): 2571-5. 390. Morin, D.E., Shanks, R.D., McCoy, G.C. (1998) Comparison of antibiotic administration in conjunction with supportive measures versus supportive measures alone for treatment of dairy cows with clinical mastitis. J Am Vet Med Assoc. 213 (5): 676-84. 391. Groothuis, D.G., Frik, J.F. (1982) [Comparison of the sensitivity patterns of Staphylococci isolated from bovine udders in 1974 and 1980]. Tijdschr Diergeneesk. 107 (23): 883-8. 392. Watts, J.L., Salmon, S.A. (1997) Activity of selected antimicrobial agents against strains of Staphylococcus aureus isolated from bovine intramammary infections that produce beta-lactamase. J Dairy Sci. 80 (4): 788-91. 393. Schlegelova, J., Rysanek, D., Sediva, I., Babak, V. (2001) Comparison of methods for the determination of antimicrobial resistance in Staphylococcus aureus from bovine mastitis. J Vet Med B. 48 (1): 21-9. 394. Brown, M.B., Scasserra, A.E. (1990) Antimicrobial resistance in streptococcal species isolated from bovine mammary glands. Am J Vet Res. 51 (12): 2015-8. 395. Owens, W.E., Watts, J.L., Greene, B.B., Ray, C.H. (1990) Minimum inhibitory concentrations and disk diffusion zone diameter for selected antibiotics against streptococci isolated from bovine intramammary infections. J Dairy Sci. 73 (5): 1225-31. 396. Rossitto, P.V., Ruiz, L., Kikuchi, Y. (2002) Antibiotic susceptibility patterns for environmental streptococci isolated from bovine mastitis in central California dairies. J Dairy Sci. 85 (1): 132-8. 397. Sogaard, H. (1982) In vitro antibiotic susceptibility of E. coli isolated from acute and chronic bovine mastitis with reference to clinical efficacy. Nord Veterinaermed. 34 (7-9): 248-54. 398. Walser, K., Gandorfer, B., Steinberger, A. (1993) Studies of the antibacterial activity and pharmacokinetics of enrofloxacin (Baytril) in lactating cows. Tierarztl Umsch. 48: 414-9. 399. Gedek, W. (1981) Determination of the susceptibility of mastitis pathogens to penicillins. 1. Streptococci. Tierarztl Umsch. 36: 688-91. 400. Thornsberry, C., Marler, J.K., Watts, J.L., Yancey, R.J., Jr. (1993) Activity of pirlimycin against pathogens from cows with mastitis and recommendations for disk diffusion tests. Antimicrob Ag Chemoth. 37 (5): 1122-6. 401. Thornsberry, C., Burton, P.J., Yee, Y.C., Watts, J.L., Yancey, R.J., Jr. (1997) The activity of a combination of penicillin and novobiocin against bovine mastitis pathogens: development of a disk diffusion test. J Dairy Sci. 80 (2): 413-21. 402. Sandholm, M., Kaartinen, L., Pyorala, S. (1990) Bovine mastitis: why does antibiotic therapy not always work? An overview. J Vet Ph Therap. 13 (3): 248-60. 403. Pyorala, S.H., Pyorala, E.O. (1998) Efficacy of parenteral administration of three antimicrobial agents in treatment of clinical mastitis in lactating cows: 487 cases (1989-1995). J Am Vet Med Assoc. 212 (3): 407-12. 404. Gallian, M.J., Heather, C.D., Luther, H.G., Price, K.E. (1955) The influence of milk and other media on antibiotic sensitivity of mastitis organisms. Antibiotics annual. 3: 753-62. 405. Owens, W.E., Ray, C.H., Washburn, P.J. (1993) Effect of selected antibiotics on Staphylococcus aureus present in milk from infected mammary glands. B J Vet Med. 40 (7): 508-14. 406. Mercer, H.D., Geleta, J.N., Baldwin, R.A, Shimoda, W. (1976) Viewpoint and current concepts regarding accepted and tried products for control of bovine mastitis. J Am Vet Med Assoc. 169(10): 1104-14. 407. Ziv, G. (1980) Drug selection and use in mastitis: systemic vs local therapy. J Am Vet Med Assoc. 176(10 Spec No): 1109-15. 408. Ziv, G. (1980) Practical pharmacokinetic aspects of mastitis therapy - 1: parenteral treatment. Vet Med, SAC. 75 (2): 277-90. 409. Deubler, M.J., Cole, E.J. (1955) Preliminary studies on Pseudomonas mastitis. Am J Vet Res. 16 (61 Part 1): 511-4. 410. Owens, W.E., Watts, J.L. (1987) Effects of milk on activity of antimicrobials against Staphylococcus aureus isolated from bovine udders. J Dairy Sci. 70 (9): 1946-51. 411. Myllys, V., Louhi, M., Ali-Vehmas, T. (1992) Comparison of penicillin-G susceptibility testing methods of staphylococci isolated from bovine mastitis. B J Vet Med. 39 (10): 723-31. 412. Constable, P.D., Morin, D.E. (2002) Use of antimicrobial susceptibility testing of bacterial pathogens isolated from the milk of dairy cows with clinical mastitis to predict response to treatment with cephapirin and oxytetracycline. J Am Vet Med Assoc. 221 (1): 103-8. 413. Cattell, M.B., Dinsmore, R.P., Belschner, A.P., Carmen, J., Goodell, G. (2001) Environmental gram-positive mastitis treatment: in vitro sensitivity and bacteriologic cure. J Dairy Sci. 84 (9): 2036-43. 414. Pyorala, S., Syvajarvi, J. (1987) Bovine acute mastitis. Part II. Effect of mastitis pathogen, initial inflammatory reaction and therapy on the outcome of the disease. B J Vet Med. 34 (9): 629-39. 415. Shpigel, N.Y., Winkler, M., Ziv, G., Saran, A. (1998) Relationship between in vitro sensitivity of coliform pathogens in the udder and the outcome of treatment for clinical mastitis. Vet Rec. 142 (6): 135-7. 416. Sol, J., Sampimon, O.C., Snoep, J.J., Schukken, Y.H. (1997) Factors associated with bacteriological cure during lactation after therapy for sub clinical mastitis caused by Staphylococcus aureus. J Dairy Sci. 80 (11): 2803-8. 417. Ziv, G., Storper, M. (1985) Intramuscular treatment of subclinical staphylococcal mastitis in lactating cows with penicillin G, methicillin and their esters. J Vet Ph Therap. 8 (3): 276-83.

______

Balbino M. Rocha, 2013 148 Refe re nces

418. Louhi, M., Inkinen, K., Myllys, V., Sandholm, M. (1992) Relevance of sensitivity testing (MIC) of S. aureus to predict the antibacterial action in milk. J Vet Med. 39 (4): 253-62. 419. Louhi-Lehtio, M., Sandholm, M., Myllys, V., Honkanen-Buzalski, T. (1994) Antibacterial susceptibility of bovine-mastitis pathogens tested directly in milk from infected quarters. J Vet Med. 41 (2): 101-12. 420. Fang, W., Pyorala, S. (1996) Mastitis-causing Escherichia coli: serum sensitivity and susceptibility to selected antibacterials in milk. J Dairy Sci. 79 (1): 76-82. 421. Hoblet, K.H., Queen, W.G., Howard, R.R. (1993) Antibiotic susceptibility testing in mastitis: a comparison of a commercial test kit with Kirby Bauer methods. Agri- Practice. 14: 45-51. 422. Hoe, F. (2002) Comparison of antimicrobial susceptibility testing using a commercial milk microdilution test (Mastik®) with broth microdilution using Sensititre®. In 41st Annual Meeting of the National Mastitis Council. National Mastitis Council, Inc. Arlington (VA), USA. p. 163-4. 423. Hinckley, L.S, Benson, R.H, Post, J.E., DeCloux JC. (1985) Antibiotic susceptibility profiles for mastitis treatment. J Am Vet Med Assoc. 187 (7): 709-11. 424. Owens, W.E., Watts, J.L. (1988) Antimicrobial susceptibility and beta-lactamase testing of staphylococci isolated from dairy herds. J Dairy Sci. 71 (7): 1934-9. 425. Vuye, A., Pijck, J., Soep, H. (1976) In vitro activity of BL-s640 against gram-negative bacilli and Staphylococcus aureus compared with activity of four other semisynthetic cephalosporins. Antimicrob Ag Chemoth. 9 (3): 422-32. 426. Ziv, G. (1971) The in vitro effects of pH and type of milk media on the growth of mastitis organisms as related to antibiotic sensitivity tests. J Vet Med. 18(1): 72-9. 427. Sanchez, M.S., Watts, J.L. (1999) Enhancement of the activity of novobiocin against Escherichia coli by lactoferrin. J Dairy Sci. 82 (3): 494-9. 428. Lintner, T.J., Eberhart, R.J. Effects of antibiotics on phagocyte recruitment, function, and morphology in the bovine mammary gland during the early nonlactating period. Am J Vet Res. 51 (4): 533-42. 429. Nickerson, S.C., Paape, M.J., Dulin, A.M. (1985) Effect of antibiotics and vehicles on bovine mammary polymorphonuclear leukocyte morphologic features, viability, and phagocytic activity in vitro. Am J Vet Res. 46 (11): 2259-65. 430. Myers, M.J., Farrell, D.E., Henderson, M. (1995) In vitro modulation of bovine blood neutrophils and mononuclear cells by oxytetracycline. Am J Vet Res. 56 (8): 1007-11. 431. Owens, W.E., Nickerson, S.C. (1990) Treatment of Staphylococcus aureus mastitis with penicillin and novobiocin: antibiotic concentrations and bacteriologic status in milk and mammary tissue. J Dairy Sci. 73 (1): 115-24. 432. Erskine, R.J., Walker, R.D., Bolin, C.A., Bartlett, P.C., White, D.G. (2002) Trends in antibacterial susceptibility of mastitis pathogens during a seven-year period. J Dairy Sci. 85 (5): 1111-8. 433. Nam, HM., Lim, SK., Kang, HM. (2009) Antimicrobial resistance of streptococci isolated from mastitic bovine milk samples in Korea. J Vet Diag Invest.21(5): 698-701. 434. Bengtsson, B., Unnerstad, H.E., Ekman, T., Artursson, K., Nilsson-Ost, M., Waller, K.P. (2009) Antimicrobial susceptibility of udder pathogens from cases of acute clinical mastitis in dairy cows. Vet Microbiol. 136 (1-2): 142-9. 435. Botrel, M.A., Haenni, M., Morignat, E., Sulpice, P., Madec, J.Y., Calavas, D. (2010) Distribution and antimicrobial resistance of clinical and sub clinical mastitis pathogens in dairy cows in Rhone-Alpes, France. Foodb Path Dis. 7 (5): 479-87. 436. Kalmus, P., Aasmae, B., Karssin, A., Orro, T., Kask, K. (2011) Udder pathogens and their resistance to antimicrobial agents in dairy cows in Estonia. Acta Vet Scand. 53: 4. 437. Persson, Y., Nyman, A.K., Gronlund-Andersson, U. (2011) Etiology and antimicrobial susceptibility of udder pathogens from cases of sub clinical mastitis in dairy cows in Sweden. Acta Vet Scand. 53: 36. 438. Pol, M., Ruegg, P.L. (2007) Relationship between antimicrobial drug usage and antimicrobial susceptibility of gram-positive mastitis pathogens. J Dairy Sci. 90 (1): 262-73. 439. Rajala-Schultz, P.J., Smith, K.L., Hogan, J.S., Love, B.C. (2004) Antimicrobial susceptibility of mastitis pathogens from first lactation and older cows. Vet Microbiol. 102 (1-2): 33-42. 440. Roesch, M., Perreten, V., Doherr, M.G., Schaeren, W., Schallibaum, M., Blum, J.W. (2006) Comparison of antibiotic resistance of udder pathogens in dairy cows kept on organic and on conventional farms. J Dairy Sci. 89 (3): 989-97. 441. San Martín, B., Kruze, J., Morales, M.A. (2003) Antimicrobial Resistance in Bacteria Isolated From Dairy Herds in Chile. Int J Appl Res Vet Med. 442. Ebrahimi, A., Kheirabadi, K.H., Nikookhah, F. (2007) Antimicrobial susceptibility of environmental bovine mastitis pathogens in west central Iran. Pakistan J Biolog Sci. 10 (17): 3014-6. 443. Nam, H.M., Lim, S.K., Kang, H.M. (2009) Prevalence and antimicrobial susceptibility of gram-negative bacteria isolated from bovine mastitis between 2003 and 2008 in Korea. J Dairy Sci. 92 (5): 2020-6. 444. Sahebekhtiari, N., Nochi, Z., Eslampour, M.A. (2011) Characterization of Staphylococcus aureus strains isolated from raw milk of bovine subclinical mastitis in Tehran and Mashhad. Acta Microb Immunolog Hungarica. 58 (2): 113-21. 445. Suriyasathaporn, W. (2010) Milk Quality and Antimicrobial Resistance against Mastitis Pathogens after Changing from a Conventional to an Experimentally Organic Dairy Farm. Asian-Aust J Anim Sci. 23(5): 659-64. 446. Rollins, L.D., Pocurull, D.W., Mercer, H.D., Natzke, R.P., Postle, D.S. (1974) Use of antibiotics in a dairy herd and their effect on resistance determinants in enteric and environmental Escherichia coli. J Dairy Sci. 57 (8): 944-50. 447. Call, D.R., Davis, M.A., Sawant, A.A. (2008) Antimicrobial resistance in beef and dairy cattle production. Anim Heal Res Rev. 9 (2): 159-67. 448. Sato, K., Bennedsgaard, T.W., Bartlett, P.C., Erskine, R.J., Kaneene, J.B. (2004) Comparison of antimicrobial susceptibility of Staphylococcus aureus isolated from bulk tank milk in organic and conventional dairy herds in the midwestern United States and Denmark. J Food Protect. 67 (6): 1104-10. 449. Apifarma - Associação Portuguesa da Indústria Farmacêutica. (2011) Simposium Veterinário Apifarma Online. In http://www.apifarma.pt/simposiumvet/Paginas/Pesquisaavancada.aspx (accessed January 22nd, 2013). 450. Bronzo, V., Piepers, S., Shukken, Y. (2012) Hipra Symposium - Results of Mastitis Vaccination. World Buiatrics Conference. Lisbon, Portugal. p. 16. 451. Nunes, S.F., Cavaco, L.M., Vilela, C.L., Bexiga, R. (2007) Antimicrobial susceptibility traits of subclinical bovine mastitis pathogens in Portugal. Rev Port Ciên Vet. 102: 275-80. 452. Rato, M.G., Bexiga, R., Florindo, C., Cavaco, L.M., Vilela, C.L., Santos-Sanches, I. (2012) Antimicrobial resistance and molecular epidemiology of streptococci from bovine mastitis. Vet Microbiol. 453. Nunes, S.F., Bexiga, R., Cavaco, L.M., Vilela, C.L. (2007) Technical note: Antimicrobial susceptibility of Portuguese isolates of Staphylococcus aureus and Staphylococcus epidermidis in subclinical bovine mastitis. J Dairy Sci. 90(7): 3242-6. 454. Gentilini, E., Denamiel, G., Llorente, P., Godaly, S., Rebuelto, M., DeGregorio, O. (2000) Antimicrobial susceptibility of Staphylococcus aureus isolated from bovine mastitis in Argentina. J Dairy Sci. 83 (6): 1224-7. 455. Tenhagen, B.A., Koster, G., Wallmann, J., Heuwieser, W. (2006) Prevalence of mastitis pathogens and their resistance against antimicrobial agents in dairy cows in Brandenburg, Germany. J Dairy Sci. 89 (7): 2542-51. 456. Murray, B.E. (2000) Vancomycin-resistant enterococcal infections. New Engl J Med. 342 (10): 710-21. 457. Wells, V.D., Wong, E.S., Murray, B.E., Coudron, P.E., Williams, D.S., Markowitz, S.M. (1992) Infections due to beta-lactamase-producing, high-level gentamicin- resistant Enterococcus faecalis. Annals Intern Med. 116 (4): 285-92. 458. Patterson, J.E., Masecar, B.L., Zervos, M.J. (1988) Characterization and comparison of two penicillinase-producing strains of Streptococcus (Enterococcus) faecalis. Antimicrob Ag Chemoth. 32 (1): 122-4. 459. Rhinehart, E., Smith, N.E., Wennersten, C. (1990) Rapid dissemination of beta-lactamase-producing, aminoglycoside-resistant Enterococcus faecalis among patients and staff on an infant-toddler surgical ward. New Engl J Med. 323 (26): 1814-8.

______

Balbino M. Rocha, 2013 149 Refe re nces

460. Rice, L.B., Eliopoulos, G.M., Wennersten, C., Goldmann, D., Jacoby, G.A., Moellering, R.C., Jr. (1991) Chromosomally mediated beta-lactamase production and gentamicin resistance in Enterococcus faecalis. Antimicrob Ag Chemoth. 35 (2): 272-6. 461. Morris, J.G., Jr., Shay, D.K., Hebden, J.N. (1995) Enterococci resistant to multiple antimicrobial agents, including vancomycin. Establishment of endemicity in a university medical center. Annals Intern Med. 123 (4): 250-9. 462. Lin, R.V., Tan, A.L. (1991) Enterococcus faecium with high-level resistance to gentamicin. Lancet. 338 (8761): 260-1. 463. Mullarky, I.K., Su, C., Frieze, N., Park, Y.H., Sordillo, L.M. (2001) Staphylococcus aureus agr genotypes with enterotoxin production capabilities can resist neutrophil bactericidal activity. Infect Immun. 69 (1): 45-51. 464. Yancey, R.J., Sanchez, M.S., Ford, C.W. (1991) Activity of antibiotics against Staphylococcus aureus within polymorphonuclear neutrophils. Eur J Clin Microbiol Infect Dis. 10 (2): 107-13. 465. Brouillette, E., Grondin, G., Lefebvre, C., Talbot, B.G., Malouin, F. (2004) Mouse mastitis model of infection for antimicrobial compound efficacy studies against intracellular and extracellular forms of Staphylococcus aureus. Vet Microbiol. 101 (4): 253-62. 466. Sordillo, L.M., Nickerson, S.C., Akers, R.M. (1989) Pathology of Staphylococcus aureus mastitis during lactogenesis: relationships with bovine mammary structure and function. J Dairy Sci. 72 (1): 228-40. 467. Kerro Dego, O., van Dijk, J.E., Nederbragt, H. (2002) Factors involved in the early pathogenesis of bovine Staphylococcus aureus mastitis with emphasis on bacterial adhesion and invasion. A review. Vet Quart. 24 (4): 181-98. 468. Lammers, A., Kruijt, E., van de Kuijt, C., Nuijten, P.J., Smith, H.E. (2000) Identification of Staphylococcus aureus genes expressed during growth in milk: a useful model for selection of genes important in bovine mastitis? Microbiology.146 (Pt 4): 981-7. 469. Makovec, J.A., Ruegg, P.L. (2003) Antimicrobial resistance of bacteria isolated from dairy cow milk samples submitted for bacterial culture: 8,905 samples (1994- 2001). J Am Vet Med Assoc. 222 (11): 1582-9.

______

Balbino M. Rocha, 2013 150 APPENDICES Appen dices

Appendix 1:

et al. al. et

0 0 2

– – – – – – – –

444

64 22 56 22

Iran

tiari tiari

6 6 months

Sahebekh

† †

et et

†

† † † † †

† †

440

– – – – –

0/0 0/0 0/0

al.

2.2/0 2.2/0 4.3/0 8.7/0 6.5/0 2.2/0

1 year

13.0/3 13.0/3

19.6/6.1 19.6/6.1 10.9/6.1

Roesch Roesch Switzerland

434

0 0

– – – – – – – – – –

0.5 7.1

1.9

1 1 year

et al. al. et Sweden

Bengtsson

0 0 4 3

0

– – – – – – – – – –

437

1 1 year

Sweden

Persson

•

• • •

et et

•

435

– – – – – – – – – –

0/0 0/0

Isolates (%) Isolates

al.

2 2 years

3.1/1.0 3.1/1.0 0.8/2.4 1.6/1.4

14.1/17 14.1/17 Botrel Botrel

Germany

et et

S. S. aureus

436

– – – – – – –

3.8 4.8 6.8 3.4 4.1

59.5 18.1 61.4

al.

2 2 years Estonia

isolated in milk from cows with mastitis worldwide (Adapted from Oliver &

Kalmus Kalmus

Resistant

et et

441

– – – – – – –

26

6.1 3.3 6.8 8.3 6.9

S. S. aureus

San San

38.1 28.8 al.

Chile

2 2 years

Martin Martin

†

†

† †

† †

438

† †

– – – – – – –

USA

Pol & Pol &

0/1.2 0/1.2 1.9/0

1 1 year

6.5/2.4 6.5/2.4 5.8/3.6

11.8/2.4 11.8/2.4 23.1/5.9

Ruegg Ruegg 28.9//25.9

43.1/44.7

et et

-

439

– – – – – – – –

–

2.4

months

USA

al.

2.4/0 2.4/0 *

Rajala

3.6/1.8 * 3.6/1.8 * 3.6/7.1

Schultz Schultz

16 16 26.5/39.3 * 26.5/39.3

* 10.8/14.3

et et

432

– – – – –

0.2 0.2 6.9 1.1 0.6 2.1 8.5 0.6

49.6 49.6

USA

al.

7 7 years

Erskine

31

Clav. Acid Clav.

-

Summary Summary of some of the pertinent literature on the ABR of

SMX -

Vs. Lactation OlderCows

Antibacterial Agent

st

Nottested

Vs. Organic Conventional dairying

ClinicalVs. Subclinical mastitis cows

Amoxicillin Ampicillin Ceftiofur Cephalothin Chloramphenicol Clindamycin Erythromycin Gentamicin Oxacillin Oxytetracycline Penicillin Pirlimycin Tetracycline TMP Vancomycin Country Durationof Study Reference – *1 † † • Murinda, 2012). Table 39:

______

Balbino M. Rocha, 2013 ii Appen dices

▲

442

0

– – – – – – – – – – –

Iran

42.86 14.29 42.86 42.86

et al. al. et

Ebrahimi Ebrahimi

2 seasons 2

om om Oliver &

445

– – – – – – – – – – – –

Thailand

33.33/100 8 months

Suriyasat

16.67/57.14 16.67/28.57 16.67/57.14

haporn haporn

438

– – – – –

USA

0.6/3 0.6/3 1.3/0

Pol & Pol &

18.1/7

1 1 year

1.3/6.4

16.7/12 16.7/12 17/14.3

19.6/9.7 33.1/3.7

52.5/61.5

Ruegg Ruegg

et et

-

439

– – – – – – –

USA

2.4/0 2.4/0

al.

1 1 year

3.6/1.8 3.6/7.1

Rajala

12/12.5

4.8/12.5 13.2/8.9

10.8/14.3 26.5/39.3

Schultz Schultz

† †

et et

†

†

† †

† † † †

440

– – – – – –

0/0 0/0 0/0 1/0 0/0

al.

0/5.3 0/5.3 5.3/0

1 1 year

10.5/0 10.5/0

5.3/5.3 5.3/5.3

Roesch Roesch

26.3/42.1 26.3/42.1 31.6/47.4

Switzerland

434

0 0

– – – – – – – –

ResistantIsolates CNS (%)

3.6 1.8 5.4 7.2 0/0

12.5

1 1 year

et al. al. et

Sweden

Bengtsson

0 0 0 0 0 4 3

– – – – – – – – –

437

1 1 year

Sweden

Persson Persson

•

•

•

et et

•

435

– – – – – – – – – – – –

0/0 0/0

al.

2 2 years

1.2/1.5 1.2/1.5

40/30.7 40/30.7

Botrel Botrel

13.8/7.3 13.8/7.3

Germany

et et

436

– – – – – – – –

3.6 1.4 2.6

38.5 17.6 14.5 34.5 11.6

al.

2 2 years

Estonia

Kalmus Kalmus

31

Clav. Acid Clav.

-

Summaryof someof the pertinent literature on ABR the of CNS isolated inmilk from cows withmastitis worldwide (Adapted fr

SMX

-

Lactation Vs. Lactation OlderCows

Antibacterial Agent

st

Assumed Assumed to translateto ~8 months

Nottested

Vs. Organic Conventional dairying

ClinicalVs. Subclinical mastitis cows

Amoxicillin Ampicillin Ceftiofur Cephalothin Chloramphenicol Clindamycin Erythromycin Gentamicin Oxacillin Oxytetracycline Penicillin Pirlimycin Tetracycline TMP Sulfadimethoxine Vancomycin Country Durationof Study Reference – *1 † † • ▲

Table 40: Murinda, 2012).

______

Balbino M. Rocha, 2013 iii Appen dices

Table 41: Summary of some of the pertinent literature on the ABR of environmental Streptococcus sp. isolated in milk from cows with mastitis worldwide (Adapted from Oliver & Murinda, 2012). 31

Antibacterial Agent Resistant Environmental Streptoccus sp. Isolates (%)

Ampicillin 5.88/43.48 * – – Cephalothin – – 11.3 Chloramphenicol – 6.67 – Cloxacillin 5.88/4.35 * – –

Colistin – 46.67 – Enrofloxacin – 0 – Erythromycin – 40 0-30.7

Furazolidone – 33.34 – Gentamicin 29.41/26.09 * 13.34 28.9 Kanamycin – 20 –

Lincomycin – – 52.8 Lincospectine – 0 – Oxacillin – – 39.28

Oxytetracycline – 80 – Penicillin 17.65/8.70 * 73.34 0-8.7 Streptomycin 35.29/30.43 * 20 –

Tetracycline – – 62.2 TMP-SMX 35.29/30.43 * – – Country Thailand Iran PRK Duration of Study 8 months 2 seasons † 4/5 months Reference Suriyasathaporn 445 Ebrahimi et al. 442 ▲Nam et al. 433

– Not tested * Organic Vs. Conventional dairying † Assumed to translate to ~8 months ▲ Includes Streptococcus spp., S. bovis, S. oralis, S. salivarius, and S. intermedius; all except S. oralis are esculin positive.

______

Balbino M. Rocha, 2013 iv Appen dices

Table 42: Summary of some of the pertinent literature on the ABR of E. coli isolated in milk from cows with mastitis worldwide (Adapted from Oliver & Murinda, 2012). 31

Antibacterial Agent Resistant E. coli Isolates (%)

Ampicillin 7.4 5.9 15.7 – – 31.3 – Cefoperazone – – – – 3.6 0 0.8

Ceftiofur 0 0 4.6 – 11.2 – 0.8 Cephalothin – – 25.5 – – – 0.7 Chloramphenicol 1.8 0 – 0 – – – Erythromycin – – – 88.24 – – – Gentamicin 0 0 2 70.59 15.7 5.7 0.7

Oxytetracycline – – – 88.24 20.6 – 0.8 Penicillin – – – 88.24 – – – Streptomycin 11 5.9 – 88.24 – 21.4 13.4 TMP-SMX – – 2.8 – 0 15.7 – Tetracycline 4.9 5.9 33.2 – – 22.2 10.4 Country Sweden Sweden USA Iran Chile Estonia Germany Duration of Study 1 year 2 years 7 years 2 seasons † 2 years 2 years 2 years

Bengtsson 437 Erskine Ebrahimi et San Martin Kalmus et Botrel et al. Reference 434 Persson 432 442 441 436 435 et al. et al. al. et al. al. – Not tested † Assumed to translate to ~8 months

______

Balbino M. Rocha, 2013 v Appen dices

Table 43: Summary of some of the pertinent literature on the ABR of other Gram-negative bacteria (besides E. coli) isolated in milk from cows with mastitis worldwide (Adapted from Oliver & Murinda, 2012). 31

Antibacterial Agent Other Gram-negative Resistant Isolates (%)

Amikacin – – 4 – – Amoxicillin-Clav. Acid – – 19 – – Ampicillin 44.4 15.7/81.1/100 * 32.2 0/50 • 0/100 ▲

Ceftiofur 16.7 14.1/2.6/100 * – – – Cephalothin 50 14.1/2.6/100 * 15 – – Chloramphenicol – 14.1/2.6/100 * 18.4 – –

• ▲ Cloxacillin – – – 0/0 0/0 Erythromycin 100 – – – –

• ▲ Gentamicin – 0/0/0 * 10.3 0/100 75/100 Kanamycin – – 30 – – Oxacillin 100 – – – –

• ▲ Penicillin 100 – – 0/0 25/0 Pirlimycin 100 – – – –

• ▲ Streptomycin – – 52.8 0/0 0/0 Tetracycline 50 33/97.5/96.2 * 47.3 – –

• ▲ TMP-SMX – 3.7/5/98.1 * – 0/0 0/0 Trimethoprim – – 14.6 – – Country USA USA PRK Thailand Thailand Duration of Study 16 months 7 years 6 years 8 months 8 months

Rajala-Schultz et 432 Nam et Suriyasathaporn Suriyasathaporn 439 Erskine et al. 433 445 445 Reference al. al.

– Not tested * K. pneumonia, S. marcescens and P. aeruginosa, respectively † The most commonly observed Gram-negative bacteria were E. coli, P. fluorescens, K. pneumonia, E. cloacae, A. lwaoffiljunii, P. aeruginosa and S. marcescens. • A. pyogenes isolates; Organic Vs. Conventional ▲C. bovis isolates; Organic Vs. Conventional

______

Balbino M. Rocha, 2013 vi Appen dices

et et

435

0

– – – – –

0/0

al.

4.1/5.5 4.1/4.3 1.4/1.0 5.5/2.2

2 2 years

Botrel Botrel

42.5/35.2

Germany

et et

436

0 0 0 1

– – – – –

11.7 11.4 51.1

al.

2 2 years

Estonia

Kalmus Kalmus

Isolates(%)

396

0 0 2

– – – –

0.7 6.6 1.1 7.9

71.7

USA

3 3 years

et al. et

Rossitto Rossitto

S. dysgalactiae S. dysgalactiae

et et

440

– – – – – – –

0/0

0/50 0/25

al.

1 year 1

22.2/25

55.6/100

Roesch Roesch

Switzerland

Resistant

et et

432

0

– –

16 11

0.8 0.3 3.2 1.9 5.5 3.5

60.2

USA

al.

7 7 years

Erskine Erskine

et et

435

– – – – – –

isolated in milkfrom cows withmastitis worldwide (Adapted from Oliver &

0.5/0

al.

2 2 years

7.2/12.9

Botrel Botrel

16.5/12.9 15.4/14.1 12.6/12.9 17.1/18.8

Germany

et et

436

– – – – – –

0.4 0.4 4.1

10.4 28.1 20.1

al.

2 2 years

Estonia

S. S. dysgalactiae

Kalmus Kalmus

(%)

and and

396

– – –

6.8 6.8 2.8 3.8

48.1 49.6 49.6 39.1 72.9

Isolates USA

3 3 years

et al. et

Rossitto Rossitto

S. S. uberis

S. uberis S. uberis

et al. et

1

– – – – –

433

8.1

34.3 42.4 41.4 33.3 57.6

PRK

4/5 months 4/5

Nam Nam

et et

Resistant

440

– – – – – – –

0/0

0/0.9

al.

1 year 1

10.5/0

5.3/11.1

52.6/33.3

Roesch Roesch

Switzerland

et et

432

0 0

– –

0.2 5.5 4.4

31.9 34.2 41.7 20.1 45.2

USA

al.

7 7 years

Erskine Erskine

31

Subclinicalmastitis cows

Summary Summary of some of the pertinent on literature ABR the of

SMX

-

Nottested

Antibacterial Agent

Vs. Organic Conventional dairying

Ampicillin Ceftiofur Cephalothin Erythromycin Gentamicin Lincomycin Oxacillin Penicillin Pirlimycin Streptomycin Tetracycline TMP Country Durationof Study Reference – *Clinical Vs. † †

Table 44: Murinda, 2012).

______

Balbino M. Rocha, 2013 vii Appen dices

438

(%)

– – –

USA

4.8/0 4.8/0

sp. and

Pol Pol &

1 1 year

7.1/9.4 4.8/1.9 9.5/9.4

35.7/7.0

spp. spp.

85.7/90.6 42.9/60.4 48.8/16.7

Ruegg Ruegg

et et

441

– – – – – – –

San San

34.9 14.2 36.7 10.1 17.4

Chile al.

2 2 years

Enterococcus

Martin Martin

Streptococcus

31

sp. and and sp.

396

positive

0

– – – – –

-

60 60

2.5

77.5 97.5 22.5

USA

3 3 years

et al. et

Rossitto Rossitto

, , esculin

& Murinda, & 2012).

396

Streptococcus

– – –

73

21.6 13.5 10.8 18.9 24.3 43.2 21.6 37.8

USA

3 3 years

et al. et

Oliver Oliver

Rossitto Rossitto

S. S. agalactiae

et et

-

439

– – –

50

77.3 63.6 63.6 59.1 36.4 90.9 86.4 68.2

USA

al.

Rajala

16 months 16

Resistant Esculin+ Esculin+ Resistant

Schultz Schultz

(Adapted from (Adapted

et et

436

0 0 0 0

– – – –

3.9 6.4

(%)

36.4 21.9

al.

2 2 years

Estonia

Kalmus Kalmus

et al. et

0 0

– – – – –

433

20 60 40 20 60

PRK

S. agalactiae S. agalactiae

4/5 months 4/5

Nam Nam

et et

432

0 0

– –

2.6 3.8 3.9 7.1

Resistant

15.4 76.9 46.2 50.5

USA

al.

7 7 years

Erskine Erskine

isolated in milk from cows with mastitis worldwide mastitismilkworldwide cows from with in isolated

spp.

Summary of some of the pertinent literature on the ABR of

:

SMX

-

Antibacterial Agent

Nottested

Ampicillin Ceftiofur Cephalothin Erythromycin Gentamicin Lincomycin Oxacillin Penicillin Pirlimycin Sulfadimethoxine Tetracycline TMP Country Durationof Study Reference – *Organic Conventional Vs. dairying

Table 45 Enterococcus

______

Balbino M. Rocha, 2013 viii Appen dices

Appendix 2:

2012).

-

along each year (2004 tested along

,

among each tested antibacterial agent antibacterial tested each among

isolates,

aureus

Staphylococcus

of

esistance proportions esistance

R

: Figure 36

______

Balbino M. Rocha, 2013 ix Appen dices

Figure 37: Resistance proportions of Staphylococcus aureus see Table 46). ______

Balbino M. Rocha, 2013 x Appen dices

2012).

-

along each tested year (2004 eachtested along

,

each tested antibacterial eachtested agent

among

isolates,

agalactiae agalactiae

Streptococcus

of

esistance proportions esistance

R

: Figure 38

______

Balbino M. Rocha, 2013 xi Appen dices

Figure 39: Resistance proportions of Streptococcus agalactiae isolates, among each tested antibacterial agent, along each tested year. Asterisks represent see Table 47).

______

Balbino M. Rocha, 2013 xii Appen dices

2012).

-

along each testedalongyear each (2004

,

among each tested antibacterialagent tested each among

isolates,

uberis uberis

Streptococcus

of

esistance proportions esistance

R

: Figure 40

______

Balbino M. Rocha, 2013 xiii Appen dices

Figure 41: Resistance proportions of Streptococcus uberis see Table 48).

______

Balbino M. Rocha, 2013 xiv Appen dices

2010).

-

along each tested year (2004 eachtested along

,

among each tested antibacterialagent tested amongeach

isolates,

dysgalactiae dysgalactiae

subsp. subsp.

dysgalactiae dysgalactiae

Streptococcus

of

esistance proportions esistance

R

:

Figure 42

______

Balbino M. Rocha, 2013 xv Appen dices

Figure 43: Resistance proportions of Streptococcus dysgalactiae isolates, among each tested antibacterial agent, along see Table 49).

______

Balbino M. Rocha, 2013 xvi Appen dices

2010).

-

tested year tested (2004

along each along

,

among each tested antibacterialagent tested each among

isolates,

)

E. faecalis and E. and faecium faecalis E.

. ( .

p

sp

Enterococcus

of

esistance proportions esistance

R

:

Figure 44

______

Balbino M. Rocha, 2013 xvii Appen dices

Figure 45: Resistance proportions of Enterococcus spp. (E. faecalis and E. faecium) isolates, among each tested antibacterial agent, along each tested year. Asterisks represent statistically significant changes see Table 50).

______

Balbino M. Rocha, 2013 xviii

2012).

-

along each tested year each (2004 tested along

,

among each tested antibacterial agent antibacterial each tested among

isolates,

coli

Escherichia

of

esistance proportions esistance

R

: Figure 46

Appen dices

______

Balbino M. Rocha, 2013 xix Appen dices

Figure 47: Resistance proportions of Escherichia coli isolates, see Table 51).

______

Balbino M. Rocha, 2013 xx Appen dices

2012).

-

along each year (2004 tested along

,

among each tested antibacterial agent testedantibacterial each among

isolates,

pneumoniae

Klebsiella

of

esistance proportions esistance

R

: Figure 48

______

Balbino M. Rocha, 2013 xxi Appen dices

Figure 49: Resistance proportions of Klebsiella pneumoniae see Table 52).

______

Balbino M. Rocha, 2013 xxii Appen dices

Table 46: SPSS outputs for logistic regression analysis to determine, for the Staphylococcus aureus isolates, whether the percentage of isolates resistant to the various antibacterial agents changed throughout the study period. Top table: variable "year" treated as continuous; Bottom table: variable "year" treated as ordinal.

AUG: OB/CX:

P: KZ:

CEQ: CN:

______

Balbino M. Rocha, 2013 xxiii Appen dices

SXT:

Table 47: SPSS outputs for logistic regression analysis to determine, for the Streptococcus agalactiae isolates, whether the percentage of isolates resistant to the various antibacterial agents changed throughout the study period. Top table: variable "year" treated as continuous; Bottom table: variable "year" treated as ordinal.

OB/CX: P:

KZ: CEQ:

______

Balbino M. Rocha, 2013 xxiv Appen dices

SXT:

Table 48: SPSS outputs for logistic regression analysis to determine, for the Streptococcus uberis isolates, whether the percentage of isolates resistant to the various antibacterial agents changed throughout the study period. Top table: variable "year" treated as continuous; Bottom table: variable "year" treated as ordinal.

OB/CX: P:

KZ: CEQ:

______

Balbino M. Rocha, 2013 xxv Appen dices

CN: SXT:

Table 49: SPSS outputs for logistic regression analysis to determine, for the Streptococcus dysgalactiae isolates, whether the percentage of isolates resistant to the various antibacterial agents changed throughout the study period. Top table: variable "year" treated as continuous; Bottom table: variable "year" treated as ordinal.

OB/CX: P:

KZ: SXT:

______

Balbino M. Rocha, 2013 xxvi Appen dices

Table 50: SPSS outputs for logistic regression analysis to determine, for the Enterococcus spp. (E. faecalis and E. faecium) isolates, whether the percentage of isolates resistant to the various antibacterial agents changed throughout the study period. Table above: variable "year" treated as continuous; Table below: variable "year" treated as ordinal.

AUG: OB/CX:

P: KZ:

SXT:

______

Balbino M. Rocha, 2013 xxvii Appen dices

Table 51: SPSS outputs for logistic regression analysis to determine, for the Escherichia coli isolates, whether the percentage of isolates resistant to the various antibacterial agents changed throughout the study period. Top table: variable "year" treated as continuous; Bottom table: variable "year" treated as ordinal.

AUG: OB/CX:

P: KZ:

CEQ: CN:

______

Balbino M. Rocha, 2013 xxviii Appen dices

SXT:

Table 52: SPSS outputs for logistic regression analysis to determine, for the Klebsiella pneumoniae isolates, whether the percentage of isolates resistant to the various antibacterial agents changed throughout the study period. Top table: variable "year" treated as continuous; Bottom table: variable "year" treated as ordinal.

AUG: KZ:

CN: SXT:

______

Balbino M. Rocha, 2013 xxix Appen dices

Appendix 3:

Article under submission for publication in international journal:

______

Balbino M. Rocha, 2013 xxx Appen dices

______

Balbino M. Rocha, 2013 xxxi Appen dices

______

Balbino M. Rocha, 2013 xxxii Appen dices

______

Balbino M. Rocha, 2013 xxxiii Appen dices

______

Balbino M. Rocha, 2013 xxxiv Appen dices

______

Balbino M. Rocha, 2013 xxxv Appen dices

______

Balbino M. Rocha, 2013 xxxvi Appen dices

______

Balbino M. Rocha, 2013 xxxvii Appen dices

Appendix 4:

Slide presentation of short communication presented by the author: "Evolução de Padrões de Resistência a Antibióticos em Agentes Etiológicos da Mastite Bovina em Portugal". II Conferência Anual do Conselho Português de Saúde do Úbere. Santarém, Portugal – February 23rd, 2013.

______

Balbino M. Rocha, 2013 xxxviii Appen dices

______

Balbino M. Rocha, 2013 xxxix Appen dices

Appendix 5:

Poster presentation: XV Jornadas da APB. Ílhavo, Portugal – May 24th to 26th, 2013.

______

Balbino M. Rocha, 2013 xl