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Antibiotic Resistance in Enteric Escherichia Coli and Enterococcus Spp. Isolated from Ungulates at Marwell Zoo, England

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Antibiotic Resistance in Enteric Escherichia Coli and Enterococcus Spp. Isolated from Ungulates at Marwell Zoo, England Antibiotic resistance in enteric Escherichia coli and Enterococcus spp. isolated from ungulates at Marwell Zoo, England By Mary-Anne Frank A thesis submitted in partial fulfilment of the requirements for the degree of Magister Scientiae (Animal/Human/Ecosystem Health) In the Department of Veterinary Tropical Diseases Faculty of Veterinary Science University of Pretoria, South Africa Supervisor: PROF MORITZ VAN VUUREN 2016 1 © University of Pretoria DECLARATION I declare that this dissertation hereby submitted to the University of Pretoria for the degree of Master of Science (Animal/Human/Ecosystem Health) has not been previously submitted by me for the degree at this or any other University, that it is my own work in design and in execution, and that all material contained therein has been duly acknowledged. Signed: Date: 07/11/2016 2 © University of Pretoria ACKNOWLEDGEMENTS As a veterinary surgeon with little microbiology experience, much help and advice was needed to complete this project. Agriseta supplied funding for both my tuition fees and for the research project. Professor Moritz van Vuuren supervisor at University of Pretoria helped develop the project design, provided support and guidance during the project. The University of Surrey allowed me to use their microbiology laboratory. Professor Mark Chambers supervised my work at their laboratory and helped with the design of the project. Professor Roberto la Ragione assisted with many aspects including support and advice during the planning and execution of the project. Will Justice and Phil Robbins at Marwell Zoo who facilitated sample collection and supplied medical records for the animals in this project. Laboratory technicians: Jon Ridgeon, Tim Baker and Ian Wright helped order supplies for project, showed me how to use equipment, gave general advice on microbiology methods and taught me how to perform PCR. Other researchers at the laboratory: Mary Howlett helped with the LAMP and antibiotic sensitivity testing, Simon Dickson assisted with advice for microbiology methods and Jai Mehat helped with problem solving and technical advice for the MLST work. My husband, Richard Tozer, assisted with spreadsheet formulae and extra parent duties while I was working in the lab and writing this dissertation. My mother-in-law, Fiona Tozer, provided childcare while she was visiting so I could work on my dissertation. My brother, Stephen Frank, added some professional touches to the map of the zoo (Figure 4). I have learnt a huge amount from doing this project and hope to use this experience for future research in veterinary microbiology. I am very grateful to everyone for their support, patience and guidance. 3 © University of Pretoria Table of Contents DECLARATION ................................................................................................................................... 2 ACKNOWLEDGEMENTS ..................................................................................................................... 3 LIST OF ABBREVIATIONS ................................................................................................................... 6 LIST OF FIGURES ................................................................................................................................ 7 LIST OF TABLES .................................................................................................................................. 7 SUMMARY......................................................................................................................................... 8 INTRODUCTION ................................................................................................................................. 9 AIMS AND OBJECTIVES .................................................................................................................... 10 LITERATURE REVIEW ....................................................................................................................... 11 METHODS AND MATERIALS ............................................................................................................ 15 Study Location ............................................................................................................................ 15 Target Animals ............................................................................................................................ 15 Sample Collection ....................................................................................................................... 16 Bacterial isolation and identification .......................................................................................... 16 Antibiotic sensitivity testing ........................................................................................................ 17 Multilocus sequence typing (MLST) ............................................................................................ 19 Investigation of Medical Records at Marwell Zoo ....................................................................... 20 RESULTS .......................................................................................................................................... 21 Bacterial isolation and identification .......................................................................................... 21 Antibiotic sensitivity testing ........................................................................................................ 21 Medical history report ................................................................................................................ 22 Multilocus sequence typing ........................................................................................................ 25 DISCUSSION ................................ .................................................................................................... 27 CONCLUSIONS ................................................................................................................................. 31 APPENDICES .................................................................................................................................... 33 Appendix A .................................................................................................................................. 33 Appendix B .................................................................................................................................. 34 Appendix C .................................................................................................................................. 35 Appendix D .................................................................................................................................. 36 Appendix E .................................................................................................................................. 37 Appendix F .................................................................................................................................. 38 Certificate of Approval by the Animal Ethics Committee, University of Pretoria ........................ 45 REFERENCES .................................................................................................................................... 46 4 © University of Pretoria LIST OF ABBREVIATIONS AMR Antimicrobial Resistance APHA Animal and Plant Health Agency (United Kingdom) BSAC British Society for Antimicrobial Chemotherapy ⁰C Degrees Celsius CBP Clinical break point CLSI Clinical and Laboratory Standards Institute DNA Deoxyribonucleic acid ECDC European Centre for Disease Prevention and Control ECOFF Epidemiological cut off EDTA Ethylenediaminetetraacetic acid EFSA European Food Safety Authority e.g. For example ESBL Extended-spectrum beta-lactamase et al. And others etc. Et cetera EU European Union EUCAST European Committee on Antimicrobial Susceptibility Testing FAO Food and Agriculture Organisation of the United Nations g Gram(s) xg Times gravity (relative centrifugal forces) i.e. That is KOH Potassium hydroxide LAMP Loop-Mediated Isothermal Amplification assay MDR Multi-drug resistance MIC Minimum inhibitory concentration ml Millilitre(s) MLST Multi-locus sequence typing mm Millimetre(s) NaCl Sodium chloride NCTC National Collection of Type Cultures No. Number OIE World Organisation for Animal Health PCR Polymerase Chain Reaction rpm Revolutions per minute spp. Species µg Microgram(s) UK United Kingdom µl Microlitre(s) V Volts WHO World Health Organisation w/v Weight per volume % 5 © University of Pretoria LIST OF FIGURES Figure 1 View of one of the enclosures at Marwell Zoo ...................................................................... 16 Figure 2 Photograph of an antibiotic sensitivity test showing how the antibiotic disks have inhibited growth of a susceptible isolate ........................................................................................................... 18 Figure 3 Photograph of an antibiotic sensitivity test on a resistant strain. Note the absence of zones of inhibition around some of the antibiotic disks ............................................................................... 18 Figure 4 Map of zoo showing geographical locations and resistance phenotypes of E. coli ............... 26 Figure 5 Comparison of rotors to spin individual tubes (A) vs strips of tubes (B). .............................. 35 Figure 6 Drawing of an agar plate following incubation showing inhibition
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