Examining antibiotic resistance in the feedlot cattle industry using real-time, quantitative PCR

283 Pages·2016·7.78 MB·English

Examining antibiotic resistance in the feedlot cattle industry using real-time,
quantitative PCR (qPCR) and enterococci as an indicator bacterium

Alicia Grace Beukers

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

Doctor of Philosophy
Faculty of Veterinary Science
The University of Sydney

2016
Abstract
Antibiotics are administered to livestock at subtherapeutic levels to maintain animal health.
Many of the antibiotics used are analogues or the same as those used in human medicine, raising
the possibility that genes conferring resistance arise within agricultural production systems with
implications for human health. In beef cattle, macrolides are administered for the control of
bovine respiratory disease and liver abscesses and have been identified by the World Health
Organization as critically important antibiotics for which management strategies are required to
prevent resistance development. Enterococci are present in the gastrointestinal tract of humans
and cattle and are also associated with nosocomial infections in humans. They are an indicator
bacterium that can be used to monitor macrolide resistance. This thesis examined antibiotic
resistance in the Canadian beef feedlot industry. Real-time, quantitative PCR was used to
examine differences in the relative abundance of eighteen resistance genes across five antibiotic
families including sulfonamides [sul1 and sul2], tetracyclines [tet(A), tet(B), tet(M), tet(O),
tet(Q) and tet(W)], macrolides [erm(A), erm(B), erm(C), erm(F) and mef(A)], fluoroquinolones
[qnrS and oqxB] and β-lactams [bla , bla and bla ] from feedlot cattle faeces and
SHV TEM1 CTX-M
urban environments. The effect of in-feed administration and withdrawal of tylosin phosphate on
macrolide resistance was examined using enterococci as an indicator bacterium. Resistant
enterococci (n=21) were selected for whole-genome sequencing and comparative genomics.
Results presented here show that the relative abundance of resistance genes differs between
cattle feedlots and urban environments, likely a reflection of differences in antibiotic use.
Sulfonamide, fluoroquinolone and β-lactam resistance genes predominated in urban wastewater,
whilst tetracycline resistance genes were more prevalent in cattle faeces. The inclusion of tylosin
in the diet of cattle at subtherapeutic levels increased the proportion of erythromycin- and
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tylosin-resistant enterococci. However, withdrawal of tylosin from the diet appeared to
contribute to a reduction in macrolide resistant enterococci. Comparative genomics revealed
resistance to macrolides was present on mobile genetic elements, specifically the Tn917
transposon harbouring erm(B). This transposon was identified in both Enterococcus hirae and
Enterococcus faecium, suggesting inter-species transfer of resistance genes may occur in the
bovine gastrointestinal tract. Furthermore, the integrative conjugative elements (ICEs) Tn916
and Tn5801, both conferring tetracycline resistance, were identified in E. faecium. As the cost of
genomic sequencing continues to decrease, further investigation of ICEs using whole genome
sequencing will help determine if there are linkages between enterococci isolates from bovine
environmental and human clinical sources and whether bovine enterococci represent a source of
dissemination and spread of antibiotic resistance. Although macrolide resistance in enterococci
decreased following the withdrawal of macrolides from cattle feed, this is not a reason to become
complacent with the use of macrolides in cattle production. Investigating alternatives to
macrolides for the control of bovine respiratory disease and liver abscesses, such as vaccines and
plant bioactives, is becoming increasingly important. Furthermore, implementation of
management practices by cattle producers that reduce the likelihood of disease spread is also
essential to reduce the need to use antibiotics to control infectious diseases.
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Declaration

The work described in this thesis was conducted under the supervision of Associate Professor
Alexandre V. Chaves, Faculty of Veterinary Science, The University of Sydney, Australia and
under the co-supervision of Professor Michael P. Ward, Faculty of Veterinary Science, The
University of Sydney, Australia and Dr. Tim A. McAllister, Lethbridge Research Centre,
Agriculture and Agri-Food Canada, Canada.

I declare that this thesis is the result of my own work, original and is not currently being
submitted for any other degree or qualification. Full acknowledgement has been made where the
work of others has been cited or used.

Alicia Grace Beukers
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Acknowledgements
First of all I would like to thank my supervisors Associate Professor Alex V. Chaves and
Professor Michael Ward from the University of Sydney and Dr. Tim A. McAllister from
Agriculture and Agri-Food Canada for your continued support, encouragement and guidance. I
especially would like to thank Associate Professor Alex V. Chaves and Dr. Tim A. McAllister
for giving me the opportunity to conduct my research at Agriculture and Agri-Food Canada. It
was a truly invaluable experience and one that I will never forget.
Thank you to Dr. Rahat Zaheer for your unwavering support and assistance. I cannot
express how grateful I am for your patience and the knowledge you have shared with me. Thank
you to Shaun Cook for showing me the ‘ropes’, your technical assistance and always being there
to discuss ideas. To the staff at Agriculture and Agri-Food Canada, thank you for making me feel
so welcome when I was so far from home.
A special thanks to Krysty Munns for being my first Canadian friend. Thank you for
organising the many social events and introducing me to so many wonderful people. I have made
so many lifelong friends and I have you to thank for that. To Cassidy and Pam, I cannot thank
you enough for all your support and friendship. I’m not sure what I would have done without
‘Soup Sundays’. To Steph, my fellow Aussie, thank you for always being there, for our many
Costco visits and cooking endeavours. To Brie, thank you for your encouragement and support,
for being my ‘Pen Pal’, escorting me back to Australia and being an overall great friend. To the
many other beautiful people (my Canadian family) I met along the way, my sincere thanks for
your friendship and sharing so many wonderful experiences with me.
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Lastly I would like to thank my family for their love, support and encouragement to
follow my dreams, even if those dreams took me halfway across the world. To my parents,
Leanne and Tony, none of this would have been possible if it wasn’t for you. Thank you for
always being there through the good and the bad times, putting up with my mood swings and
providing me with the love and the support I needed to complete my PhD. To my sisters, Maddy
and Shonai, I cannot thank you enough for being there when I needed you. You are truly my best
friends and I am so blessed to have you in my life.
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Table of Contents
Abstract .......................................................................................................................................... ii
Declaration ................................................................................................................................... iv
Acknowledgements ...................................................................................................................... v
Table of Contents ....................................................................................................................... vii
List of Figures .............................................................................................................................. ix
List of Tables ............................................................................................................................. xiii
List of Abbreviations ................................................................................................................ xiv
List of Publications and Presentations .................................................................................... xix
Chapter 1 – General Introduction ................................................................................................. 1
Chapter 2 – Literature Review ..................................................................................................... 5
Chapter 3 – Objectives ............................................................................................................... 88
Chapter 4 – Antimicrobial resistance genes within feedlots and urban wastewater ................... 89
Chapter 5 – Effect of in-feed administration and withdrawal of tylosin phosphate
on antibiotic resistance in enterococci isolated from feedlot steers ..................... 116
Chapter 6 – Draft genome sequence of an Enterococcus thailandicus strain
isolated from bovine faeces .................................................................................. 156
Chapter 7 – Comparative genomic analysis of Enterococcus spp. isolated from
bovine faeces ........................................................................................................ 163
Chapter 8 – General Discussion ............................................................................................... 217
Appendices ................................................................................................................................ 241
Appendix 1 ..................................................................................................................... 242
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Appendix 2 ..................................................................................................................... 248
Appendix 3 ..................................................................................................................... 253
Appendix 4 ..................................................................................................................... 261
Appendix 5 ...................................................................................................................... 262
Appendix 6 ...................................................................................................................... 263
Appendix 7 ...................................................................................................................... 264
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List of Figures
Figure 1.1. Potential routes of transmission of antibiotic resistant bacteria to
humans. Figure adapted from Centers for Disease Control and
Prevention (2013) ........................................................................................................ 2
Figure 2.1. Timeline of antibiotic discovery and development of antibiotic
resistance. Figure adapted from Centers for Disease Control and
Prevention (2013); Zaffiri et al. (2012) and Zaffiri et al. (2013) ................................ 8
Figure 2.2. Selective pressure and antibiotic resistance development. (A)
Bacterial population consists of a mixture of susceptible and resistant
bacteria; (B) Antibiotics provide selective pressure, eliminating
susceptible bacteria whilst resistant bacteria survive; (C) Resistant
bacteria predominant the population. Figure adapted from Centers for
Disease Control and Prevention (2013) ...................................................................... 9
Figure 2.3. Schematic demonstrating how different concentrations of antibiotics
influence the characteristics of resistant mutants in terms of their
fitness and level of resistance. At high (lethal) antibiotic
concentrations, highly resistant mutants are selected, with either a
high or low fitness cost. At low (sub-lethal) antibiotic
concentrations, mutants with a low fitness cost are selected that are
either highly resistant or low level resistant. At both high or low
antibiotic concentrations, highly resistant mutants with a low fitness
cost can be selected, indicated by the blue shaded box. Figure adapted
from Andersson and Hughes (2012) ......................................................................... 16
Figure 4.1. Relative abundance (copies of ARGs/copies of 16S-rRNA) of
sulfonamide resistance genes. (a) sul1 and (b) sul2. Error bars
represent standard deviation of the means. A = feedlot A, B = feedlot
B, C = feedlot C, DC = feedlot D (conventional production), DN =
feedlot D (natural production), CB = catch basin, Influent = sewage
influent, Effluent = sewage effluent, and Creek = Ephemeral creek.
Means with different letters significantly differ (P ≤ 0.05) ...................................... 98
Figure 4.2. Relative abundance (copies of ARGs/copies of 16S-rRNA) of
tetracycline resistance genes. (a) tet(A), (b) tet(B), (c) tet(M), (d)
tet(O), (e) tet(Q) and (f) tet(W). Error bars represent standard
deviation of the means. A = feedlot A, B = feedlot B, C = feedlot C,
DC = feedlot D (conventional production), DN = feedlot D (natural
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production), CB = catch basin, Influent = sewage influent, Effluent =
sewage effluent, and Creek = Ephemeral creek. Means with different
letters significantly differ (P ≤ 0.05). w - unable to be detected/outside
standard curve range ................................................................................................. 99
Figure 4.3. Relative abundance (copies of ARGs/copies of 16S-rRNA) of
macrolide resistance genes. (a) erm(A), (b) erm(B), (c) erm(C), (d)
erm(F) and (e) mef(A). Error bars represent standard deviation of the
means. A = feedlot A, B = feedlot B, C = feedlot C, DC = feedlot D
(conventional production), DN = feedlot D (natural production), CB =
catch basin, Influent = sewage influent, Effluent = sewage effluent,
and Creek = Ephemeral creek. Means with different letters
significantly differ (P ≤ 0.05). w - unable to be detected/outside
standard curve range ............................................................................................... 100
Figure 4.4. Relative abundance (copies of ARGs/copies of 16S-rRNA) of
fluoroquinolone resistance genes. (a) qnrS and (b) oqxB. Error bars
represent standard deviation of the means. A = feedlot A, B = feedlot
B, C = feedlot C, DC = feedlot D (conventional production), DN =
feedlot D (natural production), CB = catch basin, Influent = sewage
influent, Effluent = sewage effluent, and Creek = Ephemeral creek.
Means with different letters significantly differ (P ≤ 0.05). w - unable
to be detected/outside standard curve range ........................................................... 101
Figure 4.5. Relative abundance (copies of ARGs/copies of 16S-rRNA) of β-
lactam resistance genes. (a) bla , (b) bla and (c) bla . Error
SHV CTX-M TEM1
bars represent standard deviation of the means. A = feedlot A, B =
feedlot B, C = feedlot C, DC = feedlot D (conventional production),
DN = feedlot D (natural production), CB = catch basin, Influent =
sewage influent, Effluent = sewage effluent, and Creek = Ephemeral
creek. Means with different letters significantly differ (P ≤ 0.05). w -
unable to be detected/outside standard curve range ................................................ 103
Figure 5.1. Schematic representation of experiment timeline (Figure reproduced
from Sharma et al., 2008). Numbers indicate day of feeding period.
Periodic orange rectangles indicate points where faecal samples were
collected from steers. A, B, D, E and I represent points where isolates
were selected for assessing antibiotic susceptibility, PFGE profiles
and identifying resistance determinants. Grey shaded area represents
the period that tylosin was administered in the diet ................................................ 121
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