Antibacterial Resistance Patterns and Over Time Trends of Major Mastitis Pathogens from Dairy Cattle in Portugal

. 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 ____________________________________________________________________________________ Balbino M. Rocha, 2013 V 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 VI 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 shortto 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 VII 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 mastitis worldwide ............................................................................................................................................................................................. iv 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 cows with mastitis worldwide ............................................................................................................................................................................ vi 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 worldwide .......................................................................................................................................................................................................... vii Table 45: Summary of some of the pertinent literature on the ABR of S. agalactiae, esculin-positive Streptococcus sp. and Enterococcus spp. isolated in milk from cows with mastitis worldwide .................................................................................................................................... viii 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 VIII 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 Resumo ____________________________________________________________________________________ Balbino M. Rocha, 2013 XVI 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. Abstract ____________________________________________________________________________________ Balbino M. Rocha, 2013 XV 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. STRUCTURE OF DISSERTATION Structure of the Dissertation ____________________________________________________________________________________ Balbino M. Rocha, 2013 XVIII 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. INTRODUCTION Introduction ____________________________________________________________________________________ Balbino M. Rocha, 2013 2 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 Introduction ____________________________________________________________________________________ Balbino M. Rocha, 2013 3 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 Introduction ____________________________________________________________________________________ Balbino M. Rocha, 2013 4 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 OBJECTIVES Objectives ____________________________________________________________________________________ Balbino M. Rocha, 2013 6 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. Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 13 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 14 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 Nacetylmuramic 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 Land 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 15 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 16 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 17 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 effluxand 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 18 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 19 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 20 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 21 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 plasmidencoded 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 22 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 29 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 30 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 standardsetting 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 31 Table 5: World organizations with published breakpoints (Adapted from Turnidge, 2007). 151 Organization or test [method reference(s)] Method(s) and main media used Breakpoint-setting parameters [reference(s)] Arbeidsgruppen for antibiotikaspørsmål (Norwegian Working Group on Antibiotics [APA]) 158 Disk diffusion — Mueller-Hinton or IsoSensitest Resistance markers, MIC distributions, PK/PD, clinical and bacteriological outcomes159, 160 British Society for Antibacterial Chemotherapy (BSAC) 161,162 Agar dilution, broth dilution, broth microdilution, disk diffusion — Iso-Sensitest agar and broth PK and protein binding (formula), MIC distributions 163 Calibrated dichotomous sensitivity test CDS; promulgated by a single laboratory in Sydney, Australia) 164 Disk diffusion — Sensitest agar Mainly zone diameter distributions 164 Clinical and Laboratory Standards Institute (CLSI) 155 and the U.S. Food and Drug Administration For aerobic and facultative bacteria, broth dilution, broth microdilution, disk diffusion — Mueller-Hinton agar and broth; for anaerobic bacteria, agar dilution, broth microdilution — supplemented Brucella agar and broth MIC distributions, PK/PD, clinical/bacteriological outcome correlations167 Commissie Richtlijnen Gevoeligheidsbepalingen (CRG) 165,166 Disk diffusion — Iso-Sensitest MIC distributions, PK/PD, clinical and bacteriological outcome correlates 167 Comité de l’Antibiogramme de la Société Française de Microbiologie (CA-SFM) 168 Agar dilution, broth microdilution, disk diffusion — Mueller-Hinton MIC distributions, PK, correlation with clinical and bacteriological outcomes 163 Deutches Institut für Normung (DIN) 169 Agar dilution, broth microdilution, disk diffusion — Mueller-Hinton MIC distributions, PK, correlation with clinical and bacteriological outcomes 169 EUCAST 7,170 Agar dilution, broth dilution, broth microdilution — Mueller-Hinton In vitro drug characteristics, MIC distributions, PK/PD, clinical outcome correlations 13 Japanese Society for Chemotherapy (JSC)173,174 Broth microdilution — Mueller-Hinton MIC - clinical outcome correlations 175 Rosco Diagnostica (a commercial company based in Denmark) 179 Disk (pressed tablet) diffusion — MuellerHinton, Iso-Sensitest, PDM, and Danish blood agar Zone diameters are calibrated against a range of different national and international MIC breakpoints as well as unique breakpoints for tests performed on Danish blood agar 176 Mesa Española de Normalización de la Sensibilidad y Resistencia a los Antimicrobianos (MENSURA [Spain]) 177 Disk diffusion, broth dilution, agar dilution — Mueller-Hinton MIC distributions, PK/PD, clinical and bacteriological outcomes 178 Swedish Reference Group for Antibiotics (SRGA) 179 Agar dilution, disk diffusion, gradient diffusion — Iso-Sensitest “Pharmacological breakpoints” with species 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 32 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 twofold 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 33 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 34 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 35 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 36 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 37 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 38 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 45 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 Country Penicillins Cephalosporins and other β-lactams Tetracyclines MLS Quinolones Sulfonamides and Trimethoprim Other Classes Total (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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 46 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 47 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 48 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 49 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 50 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 foodproducing 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 51 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 52 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 53 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 54 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 2ndgeneration 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 61 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 62 Table 15: Examples of diseases on the different food-producing animal species including organ, pathogen and type of treatment (Adapted from EMA, 2009). 1 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 63 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 foodproducing 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., macrolideor fluoroquinolone-resistant Campylobacter spp., multidrugresistant E. coli, glycopeptideor streptogramin-resistant enterococci, S. pneumoniae and S. aureus – particularly MRSA. 10 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 64 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 65 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 crosscontamination 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 66 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 EUMS 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 67 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 68 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 GuillainBarré 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 69 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 Grampositive 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 70 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 aminoglycosideresistant E. faecalis and vancomycin-resistant E. faecium (%R), including 95% CI, reported per country in 2011 (Adapted from EARS-Net, 2012). 274 Country High-level Aminoglycoside-resistant E. faecalis Vancomycin-resistant E. faecium 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%. Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 77 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-careassociated MRSA infections add to the number of infections caused by methicillinsusceptible 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 Country Methicillin Rifampin 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 78 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 79 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 LAMRSA, 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 80 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 81 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 82 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 Region Program Name Years of Activity Participants Microorganisms under Surveillance AFR Integrated Disease Surveillance and Response (IDSR) 2002–present 43 countries 8 epidemic-prone pathogens ABR Red Latinoamericana de Vigilancia a las Resistencias Antimicrobianas (Re-LAVRA) 1996–present 21 countries 519 laboratories 16 pathogens All sample types EMR Antibacterial Resistance in the Mediterranean (ARMed) 2001–2005 9 countries 27 laboratories 7 pathogens Blood and CSF Regional Program for Surveillance of ABR Proposed 28 species All sample types EUR European Antibacterial Resistance Surveillance (EARSS) 1999–2009 33 countries 917 laboratories European Antibacterial Resistance Surveillance Network (EARS-Net) 2010–present 28 countries 886 laboratories 7 pathogens Blood and CSF SEAR National and regional surveillance system Proposed in 2010 WPR Regional Program for Surveillance of ABR 1990–2000 13 countries 22 species 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). Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 83 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 84 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, underprescription, 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 85 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 multidrugresistant 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 Chapter I: The Big Picture on Antibacterial Resistance ____________________________________________________________________________________ Balbino M. Rocha, 2013 86 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 casemanagement 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 Chapter II: Antibacterial Resistance of Mastitis Pathogens ____________________________________________________________________________________ Balbino M. Rocha, 2013 93 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 bulktank 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 Chapter II: Antibacterial Resistance of Mastitis Pathogens ____________________________________________________________________________________ Balbino M. Rocha, 2013 94 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 Chapter II: Antibacterial Resistance of Mastitis Pathogens ____________________________________________________________________________________ Balbino M. Rocha, 2013 95 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 Coagulasenegative staphylococci (CNS) are generally the cause of moderate infections, frequently associated with SCM. 18,361 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 ECO: E coli; SAG: S. agalactiae; SAU: S. aureus; SDY: S. dysgalactiae; SUB: S. uberis Chapter II: Antibacterial Resistance of Mastitis Pathogens ____________________________________________________________________________________ Balbino M. Rocha, 2013 96 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 Isolate Number of Isolates % of Results % of Positive Culture 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 Chapter II: Antibacterial Resistance of Mastitis Pathogens ____________________________________________________________________________________ Balbino M. Rocha, 2013 97 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 Chapter II: Antibacterial Resistance of Mastitis Pathogens ____________________________________________________________________________________ Balbino M. Rocha, 2013 98 causative microorganisms. There have also been significant developments in nucleic-acidbased 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) This assay indirectly measures the SCC in milk samples. A bromocresol-purple-containing detergent is used to break down the cell membrane of somatic cells, and the subsequent release and aggregation of nucleic acid forms a gel-like matrix with a viscosity that is proportional to the leukocyte number.  Advantages: cost effective (~ €10 for 350 tests), rapid, user friendly and can be used ‘on-site’ or in the laboratory.  Disadvantages: can be difficult to interpret and has low sensitivity.  Portacheck PortaSCC® milk test This assay uses an esterase-catalyzed enzymatic reaction to determine the SCC in milk.  Advantages: cost effective (~€2 per test), rapid and user friendly.  Disadvantage: low sensitivity at low SCCs.  FossomaticTM SCC This counter operates on the principle of optical fluorescence. Ethidium bromide penetrates and intercalates with nuclear DNA, and the fluorescent signal generated is used to estimate the SCC in milk.  Advantages: rapid and automated.  Disadvantages: expensive deice (~€5500) and complex to use.  DeLaval Cell Counter DCC This counter operates on the principle of optical fluorescence, whereby propidium iodide is used to stain nuclear DNA to estimate the SCC in milk.  Advantages: rapid and the device is easily transportable.  Disadvantage: relatively expensive.  Electrical conductivity (EC) test This test measures the increase in conductance in milk caused by the elevation in levels of ions such as sodium, potassium, calcium, magnesium and chloride during inflammation.  Advantage: can be used ‘on-site’.  Disadvantage: non-mastitis-related variations in EC can present problems in diagnosis.  Culture tests Laboratory-based tests use selective culture to identify different microorganisms involved in causing mastitis.  Advantage: identifies specific pathogens causing mastitis.  Disadvantages: cannot be used ‘on-site’ and long waiting times (days).  pH tests The rise in milk pH, due to mastitis, is detected using bromothymol blue.  Advantage: user friendly, cost effective and rapid.  Disadvantages: not as sensitive as other tests.  Enzymes Assays are used to detect enzymes, such as NAGase and LDH.  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 Chapter II: Antibacterial Resistance of Mastitis Pathogens ____________________________________________________________________________________ Balbino M. Rocha, 2013 99 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 Chapter II: Antibacterial Resistance of Mastitis Pathogens ____________________________________________________________________________________ Balbino M. Rocha, 2013 100 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 Chapter II: Antibacterial Resistance of Mastitis Pathogens ____________________________________________________________________________________ Balbino M. Rocha, 2013 101 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). Table 25: Summary of the 3-compartment model to target mastitis pathogens (Adapted from Erskine et al., 2003).12 Mastitis Pathogens Pharmacologic Compartment 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 Chapter II: Antibacterial Resistance of Mastitis Pathogens ____________________________________________________________________________________ Balbino M. Rocha, 2013 102 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 nonionized 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 consequently increasing the need to develop longer withholding periods for the treated cows.12 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 Chapter II: Antibacterial Resistance of Mastitis Pathogens ____________________________________________________________________________________ Balbino M. Rocha, 2013 109 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 usually lower and the t1/2 longer for IM and SC administrations. 12 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 Chapter II: Antibacterial Resistance of Mastitis Pathogens ____________________________________________________________________________________ Balbino M. Rocha, 2013 110 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. Chapter II: Antibacterial Resistance of Mastitis Pathogens ____________________________________________________________________________________ Balbino M. Rocha, 2013 111  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 E. coli 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 Tetracycline 2 > 16 > 16 0.25 - >16 Ceftiofur 0.25 0.5 0.5 0.12-1 Klebsiella spp. Ampicillin >16 >16 >16 4 - >16 Cephapirin 2 2 4 1-16 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 Ceftiofur 0.5 0.5 0.5 0.12-1 S. aureus 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 Penicillin 0.12 0.25 2 0.12-4 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 S. uberis 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 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 S. dysgalactiae 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 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. Chapter II: Antibacterial Resistance of Mastitis Pathogens ____________________________________________________________________________________ Balbino M. Rocha, 2013 112 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, RajalaSchultz 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 - Chapter II: Antibacterial Resistance of Mastitis Pathogens ____________________________________________________________________________________ Balbino M. Rocha, 2013 113 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 shortto 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. Erskine et al. 432, USA, 2002 Analysis for linear trends indicated increased susceptibility by some pathogens to some 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. Nam et al. 443, PRK, 2009 Wide differences in the prevalence of resistance were apparent among individual Streptococcus spp. Some were 100% susceptible, but others showed varying rates of resistance. Nam et al. 433, PRK, 2009 There was no significant change in the prevalence of bacterial and the proportion of antimicrobial resistance among gram-negative bacteria isolates during a 6-y period. A relatively high resistance to tetracycline was observed. Persson 437, Sweden, 2011 S. aureus and CNS were the most frequently isolated pathogens. Whereas, 45% of S. aureus isolates and 35% of the CNS isolates were resistant to penicillin G. Resistance to other antimicrobials was uncommon. Pol & Ruegg 438, USA, 2007 Most isolates of S aureus, CNS, and Streptococcus spp. were inhibited at the lowest dilution of most antimicrobial drugs tested. Exposure to most antimicrobial drugs commonly used for prevention and treatment of mastitis was not associated with resistance. Rajala-Schultz et al. 439, USA, 2004 Differences in the proportions of resistant isolates of CNS between first lactation and older 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. Rossitto et al. 396, USA, 2002 Enterococcus spp. were the most resistant organisms tested. Environmental streptococci 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. Sahebekhtiari et al. 444, Iran, 2011 All S. aureus isolates were susceptible to ciprofloxacin, gentamicin, imipenem. minocycline, oxacillin, and vancomycin and demonstrated highest resistance to ampicillin (64%) and penicillin (56%), and median resistance to other antimicrobials. San Martin et al. 441, Chile, 2012 E. coli was sensitive to most antimicrobials. CNS demonstrated greatest resistance (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%). Suriyasathaporn 445, Thailand, 2010 Percentages of antimicrobial-resistant bacteria (CNS, environmental streptococci, A pyogenes, C. bovis) at a former organic farm decreased after 6 months operating as an organic farm system. Chapter II: Antibacterial Resistance of Mastitis Pathogens ____________________________________________________________________________________ Balbino M. Rocha, 2013 114 Table 27 summarizes the conclusions from fifteen shortto 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 MATERIALS & METHODS Materials & Methods ____________________________________________________________________________________ Balbino M. Rocha, 2013 116 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. Materials & Methods ____________________________________________________________________________________ Balbino M. Rocha, 2013 117 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 Materials & Methods ____________________________________________________________________________________ Balbino M. Rocha, 2013 118 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.). Results ____________________________________________________________________________________ Balbino M. Rocha, 2013 125 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). Antibacterial Agent Year 2004 2005 2006 2007 2008 2009 2010 Total AUG 0/31 (0%) 0/27 (0%) 0/18 (0%) 0/26 (0%) 0/76 (0%) 0/40 (0%) 0/89 (0%) 0/307 (0%) OB/CX 1/31 (3.2%) 0/27 (0%) 1/19 (5.3%) 1/27 (3.7%) 1/78 (1.3%) 3/40 (7.5%) 10/85 (11.8%) 17/307 (5.5%) P 0/31 (0%) 1/27 (3.7%) 0/20 (0%) 0/19 (0%) 0/33 (0%) NT NT 1/130 (0.8%) KZ 0/31 (0%) 0/27 (0%) 1/20 (5.0%) 0/23 (0%) 1/78 (1.3%) 0/40 (0%) 0/88 (0%) 2/307 (0.7%) SXT 1/13 (7.7%) 0/24 (0%) 1/10 (10.0%) 0/29 (0%) 4/58 (6.9%) 0/35 (0%) 1/11 (9.1%) 7/180 (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). Antibacterial Agent Year 2004 2005 2006 2007 2008 2009 2010 Total AUG 4/58 (6.9%) 1/52 (1.9%) 1/20 (5.0%) 2/50 (4.0%) 0/19 (0%) NT 2/58 (3.4%) 10/257 (3.9%) OB/CX 57/58 (98.3%) 49/53 (92.5%) 20/21 (95.2%) 47/49 (95.9%) 32/32 (100%) NT 48/50 (96.0%) 253/263 (96.2%) P 4/6 (66.7%) 5/8 (62.5%) 8/9 (88.9%) 9/16 (56.3%) 9/11 (81.8%) NT NT 35/50 (70.0%) KZ 44/59 (74.6%) 39/49 (79.6%) 18/22 (81.8%) 24/35 (68.6%) 31/32 (96.9%) NT 47/57 (82.5%) 203/254 (79.9%) SXT 0/18 (0%) 11/43 (25.6%) 4/12 (33.3%) 11/49 (22.4%) 8/25 (32.0%) NT 3/8 (37.5%) 37/155 (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 Results ____________________________________________________________________________________ Balbino M. Rocha, 2013 126 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 Agent Year 2004 2005 2006 2007 2008 2009 2010 2011 2012 Total AUG NT 7/31 (22.6%) 13/86 (15.1%) 22/181 (12.2%) 35/206 (17.0%) 49/206 (23.8%) 83/239 * (34.7%) 110/212 * (51.9%) 72/131 (55.0%) 391/1292 (30.3%) OB/CX 43/43 (100%) 31/31 (100%) 94/94 (100%) 45/46 (97.8%) NT NT NT NT NT 213/214 (99.5%) P NT 31/31 (100%) 99/99 (100%) 124/125 (99.2%) 77/78 (98.7%) NT NT NT NT 331/333 (99.4%) KZ 2/41 (4.9%) 4/29 (13.8%) 10/85 (11.8%) 8/124 (6.5%) 14/213 (6.6%) 17/211 (8.1%) 11/215 (5.1%) 20/175 * (11.4%) 32/131 * (24.4%) 118/1224 (9.6%) CEQ NT NT NT 0/28 (0%) 0/94 (0%) 43/155 * (27.7%) 6/240 * (2.5%) 4/85 (4.7%) 5/105 (4.8%) 58/707 (8.2%) CN 0/43 (0%) 2/28 (7.1%) NT 3/114 (2.6%) 14/211 (6.6%) 3/205* (1.5%) 9/243 (3.7%) 5/203 (2.5%) 6/132 (4.5%) 42/1179 (3.6%) SXT NT NT NT 18/187 (9.6%) 16/133 (12.0%) 27/201 (13.4%) 13/149 (8.7%) 35/171 * (20.5%) 18/126 (14.3%) 127/967 (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 Results ____________________________________________________________________________________ Balbino M. Rocha, 2013 127 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). Antibacterial Agent Year 2004 2005 2006 2007 2008 2009 2010 2011 2012 Total AUG 8/14 (57.1%) NT 1/29 * (3.4%) 4/32 (12.5%) 5/19 (26.3%) 22/34 * (64.7%) 12/41 * (29.3%) 17/22 * (77.3%) 5/7 (71.4%) 74/198 (37.4%) OB/CX 14/14 (100%) 5/5 (100%) 31/31 (100%) 8/8 (100%) NT NT NT NT NT 58/58 (100%) P NT 5/5 (100%) 34/34 (100%) 27/27 (100%) 7/7 (100%) NT NT NT NT 73/73 (100%) KZ 1/14 (7.1%) 0/5 (0%) 2/26 (7.7%) 3/20 (15.0%) 1/19 (5.3%) 1/36 (2.8%) 1/40 (2.5%) 6/22 * (27.3%) 1/7 (14.3%) 16/189 (8.5%) CN 0/14 (0%) NT NT 1/19 (5.3%) 0/19 (0%) 1/34 (2.9%) 0/43 (0%) 0/21 (0%) 0/7 (0%) 2/157 (1.3%) SXT NT NT 0/11 (0%) 2/31 (6.5%) 2/12 (16.7%) 1/34 (2.9%) 1/10 (10.0%) NT NT 6/98 (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. DISCUSSION Discussion ____________________________________________________________________________________ Balbino M. Rocha, 2013 129 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 Discussion ____________________________________________________________________________________ Balbino M. Rocha, 2013 130 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 βLactam is among the most frequently used IMM antibacterials in lactatingand 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 g enerally extremely high for t he 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 βlactam 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 penicillinasesusceptible 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 Discussion ____________________________________________________________________________________ Balbino M. Rocha, 2013 131 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 Lforms, 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 (4thgeneration cephalosporin), are broad spectrum agents with greater activity against Gramnegative 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 Discussion ____________________________________________________________________________________ Balbino M. Rocha, 2013 132 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). Discussion ____________________________________________________________________________________ Balbino M. Rocha, 2013 133 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 Discussion ____________________________________________________________________________________ Balbino M. Rocha, 2013 134 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