SALMONELLA AND ANTIMICROBIAL RESISTANCE IN HUMANS, LIVESTOCK AND WILD BIRDS ...

160 Pages·2015·1.41 MB·English

by  Afema, Josephine

SALMONELLA AND ANTIMICROBIAL RESISTANCE IN HUMANS, LIVESTOCK AND
WILD BIRDS

By
JOSEPHINE AZIKURU AFEMA

A dissertation submitted in partial fulfillment of
the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY
College of Veterinary Medicine

MAY 2015
To the Faculty of Washington State University:
The members of the committee appointed to examine the dissertation of
JOSEPHINE AZIKURU AFEMA find it satisfactory and recommend that it be accepted.

___________________________________
William M. Sischo, Ph.D., Chair

___________________________________
Thomas E. Besser, Ph.D.

___________________________________
Richard Gomulkiewicz, Ph.D.

___________________________________
Devendra H. Shah, Ph.D.

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ACKNOWLEDGEMENTS
I would like to thank my main advisor Dr. William Sischo for his guidance and support
during this long journey. I especially thank him for allowing me carryout some of the work in
Uganda. I am grateful to my committee members Dr. Richard Gomulkiewicz, Dr. Devendra and
Dr. Thomas Besser for their commitment, time and critical questions that helped improve my
way of thinking and carrying out scientific research.
I thank Lindsay Parrish, Stephanie Wright, Russ McClanaghan, Christy Crudo, and
Emily Hudson of Sischo lab and Sara Packebush for help with lab work. I am also grateful to
several people in Veterinary Clinical Sciences department.
I thank Dr. Denis Byarugaba for providing lab space, field equipment and logistical
support while in Uganda. I am also grateful to the staff of Microbiology Laboratory, COVAB,
Makerere University. I appreciate the time I spent working in the field and lab with
undergraduate students Esther Atukwase, Maria Nambi and Joan Rokani. I am grateful to Jeradi
Kyeyune of Lutembe Bay and John Mulondo of Nakiwogo for providing boat rides during
fieldwork. Chief Warden QECA Nelson Guma provided boat for fieldwork and rangers. Thanks
also to James Kalyewa for assisting with fieldwork at QECA.
Part of this study was carried out in Uganda and approved by Uganda National Council
for Science and Technology under research permit NS414. Permission to sample wild birds was
provided by Uganda Wildlife Authority. I thank the staff of Uganda National Water and Sewage
Corporation for permission and help with sample collection at the wastewater treatment plant
and waste stabilization ponds. Thanks also go out to the proprietors of abattoirs and poultry
vendors for permission to collect samples.
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I appreciate funding support from Washington Safe Food Initiative for my assistantship.
Funding for fieldwork in Uganda was provided by WSU-United States Department of
Agriculture formula fund and USAID Higher Education for Development fund to North Dakota
State University.
I sincerely thank fellow graduate students, Letizia Tomassini and Diana Short for the joys
and challenges we shared along the long Ph.D. path. I also thank other graduate students that
helped me out in one way or another. Special thanks also to my old friends Karen Kearns and
Simon Hollamby for their encouragement and support over the years. I also thank my friend
Meagan Kay and her husband Mike Brands for providing me a home in Seattle and visiting me at
Pullman. I thank Dale Moore for hosting us on several occasions, listening to my presentations
and providing a bounty of vegetables from her garden during short summer seasons in Pullman.
Special thanks to my family especially Ludwig Siefert, my dad, party of sisters (Agnes,
Emily, Juliet and Eva), nieces, nephews and brothers for their moral support even though we
were thousands of miles apart.
I believe in a Creator and thank the almighty God for watching over me and guiding me
up mountains and down the valleys of the PhD path and bringing me safely to the end.
It would not have been possible without the help of many other people I have not mentioned.
Awadifo! Ashante! Thank you!

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SALMONELLA AND ANTIMICROBIAL RESISTANCE IN HUMANS, LIVESTOCK AND
WILD BIRDS
Abstract
by Josephine Azikuru Afema, Ph.D.
Washington State University
May 2015
Chair: William M. Sischo
Salmonella are Gram negative bacteria that are classified into over 2600 serovars. The
serovars that cause typhoid fever are restricted to humans, whereas non-typhoidal Salmonella
serovars (NTS) infect multiple hosts worldwide. However, fundamental differences exist
between developed countries and sub-Saharan Africa with regards to sources, transmission, host
and environmental risk factors, and disease outcome. In United States, although NTS are mainly
foodborne, control measures implemented within slaughter and processing facilities have failed
to reduce disease incidence implying transmission is not fully understood. In Africa, limited
research has been done; hence NTS transmission is poorly understood. This study was conducted
to better understand sources and transmission of NTS and associated antimicrobial resistance
(AMR).
We analyzed long-term human and cattle AMR data from Pacific Northwest using
diversity measures and bootstrapping techniques. We show that some AMR profiles in the
predominant serovars were common between host populations, but more profiles were unique to
source populations than were shared. Also, the expected and observed number of AMR profiles
in humans and cattle were different. These results suggest Salmonella and AMR from humans
and cattle may not be wholly derived from a common population.
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We also determined phenotypic antimicrobial structure and genotypic structure of
common serovars from various sources (storm-water, human wastewater, abattoir flush-water
and floor swab, poultry and swine feces) in Kampala, Uganda. We found similar strains among
different sources suggesting NTS disseminates amongst humans, livestock and the environment.
Also, more resistance was detected in poultry suggesting poultry are potential reservoirs.
Next, we assessed the risk of Salmonella shedding in water-birds utilizing impacted and
conserved areas, but found no difference in prevalence by location. Some strains were distinct to
birds, while other strains in birds reflected strains in the urban environment. Of concern, an
important bird area close to the city attracts high bird diversity, and could favor reemergence of
fecal zoonotic pathogens.
Finally, we used a mathematical modeling approach to gain insights into NTS
transmission. We find the proportion of humans infected via animal contact depends on
assumptions of NTS prevalence in animals, and proportion of humans that contact animals.

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TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS……………………………………………………………………... iii
ABSTRACT………………………………………………………………………………… v–vi
LIST OF TABLES…………………………………………………………………………… viii
LIST OF FIGURES…………………………………………………………………………… xii
GENERAL INTRODUCTION…………………………………………………………………. 1
CHAPTER ONE
1. ANTIMICROBIAL RESISTANCE PROFILES AND DIVERSITY IN SALMONELLA
FROM HUMANS AND CATTLE, 2004-2011…………………………………………3
2. POTENTIAL SOURCES AND TRANSMISSION OF SALMONELLA AND
ANTIMICROBIAL RESISTANCE IN KAMPALA, UGANDA…………………….. 41
3. SALMONELLA IN WILD BIRDS UTILIZING CONSERVED AND HUMAN
IMPACTED HABITATS IN UGANDA……………………………………………… 87
4. INSIGHTS INTO TRANSMISSION OF NON-TYPHOIDAL SALMONELLA BY A
MATHEMATICAL MODELING APPROACH……………………………………. 119
GENERAL CONCLUSIONS………………………………………………………………... 142

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LIST OF TABLES
CHAPTER ONE
1. Table 1. Antimicrobials and interpretation of zone of inhibition (mm) to evaluate
antimicrobial resistance from human and cattle sources in northwestern United States, 2004-
2011……………………………………………………………………………………….. 27
2. Table 2. Yearly distribution of NTS isolates from human and cattle sources in northwestern
Unites States used for this study…………………………………………………………... 27
3. Table 3. The 20 most frequently isolated NTS serovars from human and cattle sources in
northwestern United States, 2004–2011…………………………………………………... 28
4. Table 4. Percentage of important resistance phenotypes in Salmonella and common serovars
from human and cattle sources in northwestern United States, 2004–2011………………. 29
5. Table 5. Diversity of antimicrobial resistance profiles in common NTS serovars in human
and cattle sources…………………………………………………………………………. 29
6. Table 6. Multinomial logistic regression indicating the probability of resistance to one or
more classes of antimicrobial over pan–susceptible (reference class) in cattle isolates
compared to human isolates………………………………………………………………. 30
7. Table S1. Antimicrobial resistance profiles observed in Salmonella Typhimurium from
human and cattle sources in northwestern United States, 2004–2011……………………. 31
8. Table S2. Antimicrobial resistance profiles in Salmonella Newport from human and cattle
sources in northwestern United States, 2004–2011……………………………………….. 33
9. Table S3. Antimicrobial resistance profiles in Salmonella Montevideo from human and cattle
sources in northwestern United States, 2004–2011……………………………………….. 34
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10. Table S4. Diversity of antimicrobial resistance profiles in the five most common Salmonella
serovars in humans………………………………………………………………………… 35
11. Table S5. Diversity of antimicrobial resistance profiles in the five most common Salmonella
serovars in cattle…………………………………………………………………………... 35

CHAPTER TWO
1. Table 1. Minimum inhibitory concentration of antimicrobials used for the agar dilution assay
to evaluate antimicrobial resistance in Salmonella from Uganda, 2012–2013……………. 72
2. Table 2. Salmonella detection from sites within Kampala, Uganda, 2012–2013…………. 73
3. Table 3. Salmonella serovars recovered from different sources in Kampala, Uganda……. 74
4. Table 4. Antimicrobial resistance in Salmonella from various sources in Kampala……… 75
5. Table 5. Criteria for Latent Class Analysis model selection based on resistance to 10
antimicrobials in Salmonella from various sources in Kampala, 2012–2013…………….. 75
6. Table 6. Latent classes of antimicrobial resistance in Salmonella from various sources in
Kampala…………………………………………………………………………………… 76
7. Table 7. Latent classes of antimicrobial resistance in Salmonella from bovine, small
ruminant, and swine sources………………………………………………………………. 76
8. Table 8. LCA of antimicrobial resistance in Salmonella from Nakivubo Channel……….. 77
9. Table 9. LCA of antimicrobial resistance in Salmonella from human sources…………… 77
10. Table 10. LCA of antimicrobial resistance in Salmonella from poultry…………………...78
11. Table 11. Historical Salmonella from Uganda, 2003–2010………………………………. 78
12. Table 12. Pulsed field gel electrophoresis and antimicrobial resistance patterns in common
Salmonella serovars……………………………………………………………………….. 79
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13. Table S1. Antimicrobial resistance profiles in Salmonella Kentucky according to source.. 81
14. Table S2. Comparison of AMR profiles in serovars found in poultry……………………. 81
15. Table S2. Antimicrobial resistance profiles in S. Newport according to source………….. 82
16. Table S3. Statistics for test of measurement invariance across sources for latent class model
of antimicrobial resistance patterns……………………………………………………….. 82
17. Table S5. Antimicrobial resistance profiles in Salmonella collected from 2003-2010…… 83
18. Table S6. Antimicrobial resistance profiles in S. Typhimurium according to source……. 83

CHAPTER THREE
1. Table 1. Antimicrobial resistance panel and interpretation used to evaluate antimicrobial
resistance in Salmonella from wild birds and storm/wastewater in Kampala, Uganda…. 111
2. Table 2. List of birds observed during sample collection at Lake Victoria bird areas and
Queen Elisabeth Conservation Area……………………………………………………... 112
3. Table 3. Prevalence of Salmonella in wild birds and storm/wastewater………………… 113
4. Table 4. Salmonella serovars isolated from wild bird feces from Lake Victoria Bird Areas,
Queen Elisabeth Conservation Area, and Nakivubo Channel, Uganda………………….. 114
5. Table 5. PFGE types in Salmonella serovars common to birds and storm/wastewater….. 115
6. Table 6. Antimicrobial resistance profiles of Salmonella from wild bird feces and
storm/wastewater, Kampala, Uganda……………………………………………………. 116

CHAPTER FOUR
1. Table 1. Model parameters………………………………………………………………. 138
2. Table 2. Proportion of people with occupation related contact with animals……………. 138
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