The Molecular Epidemiology of Antimicrobial Resistant Staphylococci in Australian Animals

198 Pages·2012·3.33 MB·English

The Molecular Epidemiology of Antimicrobial Resistant Staphylococci
in Australian Animals

© Kate Alice Worthing
BVSc (Hons I)

A thesis submitted in fulfilment of the requirements for the degree
of Doctor of Philosophy

Sydney School of Veterinary Science
Faculty of Science
The University of Sydney
November 2017
(Final version submitted March 2018)
Staphylococci in Australian Animals

TABLE OF CONTENTS

DECLARATION .....................................................................................................................V
LIST OF PEER REVIEWED PUBLICATIONS.............................................................................VI
LIST OF PEER REVIEWED CONFERENCE PROCEEDINGS ..................................................... VII
DISCLOSURE AND AUTHOR CONTRIBUTIONS ..................................................................... IX
ACKNOWLEDGMENTS ......................................................................................................... X
LIST OF TABLES .................................................................................................................. XII
LIST OF FIGURES .............................................................................................................. XIV
ABBREVIATIONS .............................................................................................................. XVI
SUMMARY .......................................................................................................................XIX

CHAPTER 1………..INTRODUCTION ....................................................................................... 1
Antimicrobial resistance and the Staphylococcus genus ...................................................... 1
Staphylococcus spp. of veterinary significance .................................................................... 2
Virulence factors of Staphylococcus spp. ............................................................................. 6
Methicillin-resistance in Staphylococcus spp. .................................................................... 11
Diagnosis and characterisation of Staphylococcus spp. ...................................................... 14
Transmission of staphylococci in veterinary medicine ....................................................... 19
Biocide tolerance in antimicrobial-resistant Staphylococcus spp........................................ 21
Scope and aims of this thesis............................................................................................. 22

CHAPTER 2……….GENERAL MATERIALS AND METHODS .................................................... 24
Staphylococcus isolate acquisition and storage ................................................................. 24
Species determination of Staphylococcus isolates ............................................................. 28
Antimicrobial susceptibility testing.................................................................................... 30
Whole genome sequencing ............................................................................................... 32
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In silico analysis ................................................................................................................. 33
CHAPTER 3………..MOLECULAR CHARACTERISATION OF METHICILLIN-RESISTANT
STAPHYLOCOCCUS AUREUS ISOLATED FROM AUSTRALIAN ANIMALS AND VETERINARIANS37
Abstract ............................................................................................................................ 37
Introduction ...................................................................................................................... 38
Materials and methods ..................................................................................................... 40
Results .............................................................................................................................. 42
Discussion ......................................................................................................................... 50
Conclusion ........................................................................................................................ 56

CHAPTER 4. .. MOLECULAR EPIDEMIOLOGY OF METHICILLIN-RESISTANT STAPHYLOCOCCUS
PSEUDINTERMEDIUS IN AUSTRALIAN ANIMALS .................................................................. 57
Abstract ............................................................................................................................ 57
Introduction ...................................................................................................................... 58
Methods ........................................................................................................................... 59
Results .............................................................................................................................. 62
Discussion ......................................................................................................................... 74
Conclusion ........................................................................................................................ 79

CHAPTER 5. ......VETS AND PETS: METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS AND
STAPHYLOCOCCUS PSEUDINTERMEDIUS IN AUSTRALIAN VETERINARY PERSONNEL, THEIR
PETS, PATIENTS AND THE VETERINARY HOSPITAL ENVIRONMENT ...................................... 80
Abstract ............................................................................................................................ 80
Introduction ...................................................................................................................... 81
Materials and methods ..................................................................................................... 83
Results .............................................................................................................................. 86
Discussion ......................................................................................................................... 92
Conclusion ........................................................................................................................ 96

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Staphylococci in Australian Animals
CHAPTER 6. ...........CHARACTERISATION OF CLINICAL STAPHYLOCOCCUS FELIS FROM CATS:
DETECTION OF COAGULASE POSITIVE ISOLATES AND GENETIC VIRULENCE DETERMINANTS97
Abstract ............................................................................................................................ 97
Introduction ...................................................................................................................... 98
Methods ........................................................................................................................... 99
Results ............................................................................................................................ 103
Discussion ....................................................................................................................... 111
Conclusion ...................................................................................................................... 115

CHAPTER 7. .. BIOCIDE TOLERANCE IN METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS
AND STAPHYLOCOCCUS PSEUDINTERMEDIUS FROM ANIMALS ......................................... 116
Abstract .......................................................................................................................... 116
Introduction .................................................................................................................... 117
Materials and methods ................................................................................................... 119
Results ............................................................................................................................ 121
Discussion ....................................................................................................................... 126
Conclusion ...................................................................................................................... 130

CHAPTER 8………..CONCLUSIONS AND FUTURE DIRECTIONS ........................................... 131

REFERENCES…………………………………………………………………………….………………………………….…….…142

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DECLARATION
This thesis is submitted to The University of Sydney in fulfilment of the requirements for the Degree of
Doctor of Philosophy.
The work presented in this thesis is, to the best of my knowledge and belief, original except as
acknowledged in the text. I hereby declare that I have not submitted this material, either in full or in part,
for a degree at this or any other institution.
Parts of this thesis have been published in the candidate’s name (see List of Peer Reviewed Publications
below).

Kate Worthing, BVSc (Hons I)
5th November, 2017

Signed:_ _______

Date:__________5/11/17_______________________________________________________

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LIST OF PEER REVIEWED PUBLICATIONS
1. Worthing KA, Coombs GW, Pang S, Abraham S, Saputra S, Trott DJ, Jordan D, Wong HS, Abraham RJ,
Norris JM. (2016) Isolation of mecC MRSA in Australia. Journal of Antimicrobial Chemotherapy 71,
2348-2349
2. Worthing KA, Abraham S, Pang S, Coombs GW, Saputra S, Jordan D, Wong HS, Abraham RJ, Trott DJ,
Norris JM. (2017) Molecular characterisation of methicillin-resistant Staphylococcus aureus isolated
from Australian animals and veterinarians. Microbial drug resistance. 24: 203-212
3. Saputra S, Jordan D, Worthing KA, Norris JM, Wong HS, Abraham R, Trott DJ, Abraham S. (2017)
Antimicrobial resistance in coagulase-positive staphylococci isolated from companion animals in
Australia: a one year study. PLOS ONE: 12(4): e0176379
4. Worthing KA, Abraham S, Pang S, Coombs GW, Saputra S, Jordan D, Trott DJ, Norris JM. (2017) Clonal
diversity and geographic distribution of methicillin-resistant Staphylococcus pseudintermedius from
Australian animals: discovery of novel sequence types. Veterinary Microbiology. 213, 58-65
5. Worthing KA, Marcus, A, Abraham S, Trott DJ, Norris JM. (2018) Qac genes and biocide tolerance in
clinical veterinary methicillin-resistant and methicillin-susceptible Staphylococcus aureus and
Staphylococcus pseudintermedius. Veterinary Microbiology. 216: 153-158

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Veterinary drugs and superbugs – Kate Worthing – 2017
LIST OF PEER REVIEWED CONFERENCE PROCEEDINGS
1. Worthing KA, Norris JM. Rumble in the microbial jungle: feline-derived Staphylococcus
pseudintermedius and Staphylococcus felis inhibit growth of human-derived MRSA in vitro. Exploring
human host-microbiome interactions in health and disease 2017 Conference, Wellcome Genome
Campus, Cambridge, United Kingdom. 13th-15th September 2017. (Awarded travel bursary for
abstract presentation).
2. Worthing KA. Methicillin-resistant Staphylococcus pseudintermedius in Australia. Invited speaker:
Dermatology Chapter of the Australian College of Veterinary Science Conference, 6th-8th July, 2017.
Gold Coast, Australia.
3. Worthing KA, Abraham S, Pang S, Coombs G, Saputra S, Jordan D, Wong H, Abraham R, Trott D, Norris
JM. Characterisation of novel SCCmec elements from methicillin-resistant Staphylococcus
pseudintermedius from Australian animals. StaphPath/Antimicrobials 2017. 23rd – 25th February
2017. Adelaide, Australia. (Awarded Australian Society for Antimicrobials Travel Award).
4. Worthing KA, Abraham S, Coombs G, Pang S, Saputra S, Trott D, Jordan D, Norris JM. Molecular
epidemiology of methicillin-resistant Staphylococcus aureus from animals and veterinarians in
Australia. Marie Bashir Institute Colloquium. 3rd November, 2016. Sydney, Australia
5. Worthing KA, Abraham S, Pang. S, Marcus, A, Saputra S, Trott D, Wong, H, Jordan D, Norris JM. What
makes a superbug? Antimicrobial and biocide tolerance in methicillin-resistant Staphylococcus
pseudintermedius. Australian College of Veterinary Science Conference, July 7th-9th, 2016. Gold Coast,
Queensland, Australia.
6. Worthing KA, Coombs G, Pang. S, Abraham S, Saputra S, Trott D, Wong, H, Abraham R, Jordan D,
Norris JM. First report of mecC MRSA in Australia. Antimicrobials 2016. 25-27th February, 2016.
Melbourne, Australia
7. Worthing KA, Abraham S, Saputra S, Trott D, Pang, S, Coombs G, Wong, H, Abraham R, Jordan D,
Norris JM. Characterisation of methicillin-susceptible and resistant staphylococcal infections in
Australian companion animals. 4th ASM-ESCMID Conference on MRSA in Animals: Veterinary and
Public Health Implications, 3rd-5th November, 2015. Chicago, USA.

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8. Worthing KA, Abraham S, Saputra S, Trott D, Wong, H, Abraham R, Jordan D, Norris JM.
Characterisation of antimicrobial susceptible and resistant staphylococcal infections in Australian
cats and dogs. Australian College of Veterinary Science Conference, July 9th-11th, 2015. Gold Coast,
Queensland, Australia. (Won first prize: young speaker section)

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DISCLOSURE AND AUTHOR CONTRIBUTIONS
Aspects of Chapters 3, 4 and 7 are published in four peer-reviewed journal articles as follows:
• Isolation of mecC MRSA in Australia. Journal of Antimicrobial Chemotherapy (2016) 71,
2348-2349. Doi: https://doi.org/10.1093/jac/dkw138 (Chapter 3)
• Molecular characterisation of methicillin-resistant Staphylococcus aureus isolated from
Australian animals and veterinarians. Microbial drug resistance (2017) Available ahead of
print 9th June, 2017: Doi: https://doi.org/10.1089/mdr.2017.0032 (Chapter 3)
• Clonal diversity and geographic distribution of methicillin-resistant Staphylococcus
pseudintermedius from Australian animals: discovery of novel sequence types. Veterinary
Microbiology (2018) 213, 58-65 (Chapter 4)
• Qac genes and biocide tolerance in clinical veterinary methicillin-resistant and
methicillin-susceptible Staphylococcus aureus and Staphylococcus pseudintermedius.
Veterinary Microbiology (2018) 216, 153-158 (Chapter 7)

I, Kate Worthing, was primarily responsible for study design, conducting the laboratory work,
statistical analysis of the data, interpretation of the findings and writing the manuscripts. I am very
grateful to: Associate Professor Jacqui Norris, Professor Darren Trott, Professor Geoff Coombs, Dr
Sam Abraham, Dr David Jordan, Mr Sugiyono Saputra, Dr Stanley Pang, Dr Hui San Wong, Mrs
Rebecca Abraham, Associate Professor Tom Gottlieb, Dr James Brown and Ms Laura Gerber for
their assistance with study designs, laboratory work, data analyses and editing of the manuscripts.

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ACKNOWLEDGEMENTS
I am lucky to have received help and support from an amazing group of people during my PhD
candidature. I have been supported financially by several scholarships for which I am grateful: the
Australian Postgraduate Award, the University of Sydney Merit Award, Eric Horatio Maclean
Scholarship, Royston George Booker Grant and the Postgraduate Research Support Scheme. My
work has been generously funded by the Australian Research Council linkage scheme with Zoetis
Pty Ltd and three grants from the Australian Companion Animal Health Foundation.
I am indebted to Jacqui Norris (University of Sydney), Sam Abraham (Murdoch University), David
Jordan (NSW DPI) and Darren Trott (University of Adelaide) for conceiving and laying the ground
work for the surveillance study. Without their work, this thesis would not have been possible. Thank
you to my hard working fellow PhD student from the University of Adelaide, Sugiyono Saputra, for
all the work done on MIC testing of surveillance isolates. To the following people from Murdoch
University for their collaboration: Geoff Coombs for graciously sharing his world class expertise on
MRSA; Stanley Pang for teaching me how to do whole genome sequencing and starting me on the
right track with bioinformatics and Tanya Laird for processing the subsequent WGS samples. Thank
you to Tom Gottlieb and his fabulous team at Concord Hospital for allowing me to conduct MALDI-
TOF in their lab and for their passion in moving the One Health paradigm forward.
To the veterinarians that have assisted with sample collection: Teresa Fair and Gwilym Hunt at
Annandale Animal Hospital whose flexibility allowed me continue as a practicing veterinarian while
conducting clinical research. To the Animal Referral Hospital and its staff, particularly James Brown,
for allowing sample collection at their hospital. Thanks to animal owners for their interest and
consent in participating in the study, and for the approximately 1200 animals who tolerated sample
collection across the various studies. Thank you to Denise Wigney for her endless support in
fulfilling my lab supply orders and generally organising my lab work, and Veronica Ventura for her
amazing patience and diligence in preparing all laboratory supplies. To the undergraduate students
who have helped in sample processing, particularly Emily Hudson but also Laura Gerber, Suzanne
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