Coleman Brenda L 200811 PHD Thesis PDF

THE ROLE OF DRINKING WATER
AS A SOURCE OF TRANSMISSION
OF ANTIMICROBIAL RESISTANT ESCHERICHIA COLI
by
Brenda Lee Coleman
A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy
Department of Public Health Sciences
University of Toronto
Copyright by Brenda Lee Coleman, 2008
ii
Drinking water as a source of antimicrobial resistance
The Role of Drinking Water as a Source of Transmission of Antimicrobial Resistant

Escherichia Coli
Doctor of Philosophy, 2008
Brenda Lee Coleman
Department of Public Health Sciences
University of Toronto
Abstract
Antimicrobial resistance is a serious threat to the treatment of infectious diseases and a leading public
health concern of the 21st century. Antimicrobial resistant E. coli has been detected in many places
including domestic livestock, humans, food items, surface water, and drinking water. Although the use
of antibiotics is a major contributor to the emergence of resistance, the ingestion of water
contaminated with antimicrobial resistant bacteria may contribute to the prevalence of antimicrobial
resistance in humans. Purpose: The objectives of the research were to determine the prevalence of
human faecal carriage of antimicrobial resistant E. coli in people residing in southern Ontario who used
private water sources and to determine whether the use of water contaminated with antimicrobial
resistant E. coli was associated with human carriage of same. Method: The study population consisted
of people living in Ontario households that submitted water samples from private water sources for
bacteriological testing between May 2005 and September 2006. Respondents completed a questionnaire
and submitted a self-collected rectal swab. Results: Antimicrobial resistant E. coli were detected in
the swabs of 41% of the 699 respondents, with 28% resistant to ampicillin, 25% to tetracycline, and 24%
to sulfisoxazole, and 29% that were multi-drug resistant. Subjects from households using untreated
water contaminated with antimicrobial resistant E. coli were 40% more likely to carry antimicrobial
resistant E. coli in their gastrointestinal system than people from households using uncontaminated
water, even after adjusting for the effect of other variables. Implications: The association between
the consumption of water contaminated with antimicrobial resistant E. coli and human carriage of
resistant E. coli highlights the ongoing risks associated with water contamination and antimicrobial
resistance in Ontario. The high rates of resistant E. coli in healthy non-institutional persons provides
further rationale for public health programs to reduce antibiotic use in medicine and agriculture.
iii
Acknowledgements
A sincere thank you to my thesis supervisor, Dr. Allison McGeer. Allison has been a terrific
advisor as well as a true inspiration to me. She is a brilliant researcher who effectively manages, much
to my continued admiration, her day job as the Director of Infection Control at Mount Sinai Hospital,
numerous research projects, publications, teaching commitments, speaking engagements, and
committee work - along with a family! She has the ability to see both the big picture and the little
details. Allison, I can only gawk in amazement and reiterate my thanks for mentoring me through my
PhD and introducing me to the world of research in infectious diseases. I have learned so much, but
have so much more to learn.
I cant adequately thank the other members of my committee, Dr. Susan Bondy, Dr. Iris
Gutmanis, and Dr. Ian Johnson. The fact that you took the time from your already overloaded
schedules to help me through the seemingly never-ending process of completing a PhD thesis is very
much appreciated. I cant imagine how you managed to read and re-read the never-ending litany of
drafts of the thesis. You have been supportive and effective supervisors every step of the wayfrom
hypothesis generation and questionnaire development through the protocol defence and on to the
analysis and thesis-writing.
Ian, right from our first meeting, you asked the so what questions and so often directed me
back to the public health and policy pieces of the research. Thank you for picking up on things I was
taking for granted and for keeping the focus on public health practice. Sue, you were invaluable for
your insight into the methods, statistics, and policy as well as the requirements of the public health
department. Thank you for nudging me in the right direction of continued learning. Iris, thank you for
your tireless support, your expertise in both public health and methods, and your ability to see the
veins on the leaves of the trees in that forest! It was your belief in my abilities that started this
adventure. Thank you.
Edward Woods, my soul mate. I would not have had the self-confidence to apply for my
undergraduate degree without your unfailing support and belief in me. It has been a long road and you
have been there every step of the way.even, and often most importantly, for the wobbly steps!
Thank you too for being Mr. Mom and the cruise director, for being my sounding board, and for reading
so many papers and drafts of theses that Im sure youve lost count. I know that I have. Thanks, hon.
Hey kids, thank you for your indulgence and support through the oh-so-many years of school.
Jeff and Kristy, I appreciate that you read and commented on my dissertation. Kristy, you were a huge
help working in the lab, making calls and doing interviews but I think it was your unfailing belief in me
that I appreciate the most. And, just thinkif my old brain could complete a dissertation, imagine what
you can do! Jamie and Natalie, your interest and concern has been appreciated and I thank you for
your patience and understanding through the years. All four of you have grown and matured into
beautiful, intelligent young adults. I love you and Im proud of all of you.
Thanks Mom and Dad! Thanks for everything, starting withthanks for teaching me that things
half-done are never done right and for helping me through my post-divorce return-to-school by being
iv
my day care centre, the best grandparents in the world, my coffee house of choice, and the loving
support system that I always knew would be there for me. Thanksfor everything from rescuing me
when my car broke down to being interviewed numerous times when I trained new staff to
understanding that the long stretches between visits were not because I didnt want to see you but
because I was trying to finish this work before yet another birthday passed.
Research is rarely completed in isolation and this project relied on the good-will and
participation of a number of people across Canada. A sincere thank you to all of the subjects who
participated in the study. Without you, this research would never have happened. Your willingness to
answer the survey questions as well as provide a sample for laboratory analysis was very much
appreciated and I want you to know that the information we learn from this study is already being
disseminated and will be used to promote health.
A big thank you to the staff at the public health laboratories in Ontario and Alberta that
provided thousands of E. coli slants for susceptibility testing - with a special thanks to Nancy Latimer
for also answering all of my questions about water testing in Ontario. I would also like to thank the
staff at the Safe Water Unit for working with us to contact the people who were selected for interview,
with a special thanks to Dr. Zsuzsanna Rajda for being the go-between for this study. Thanks too, to
the staff in the Ontario public health units and Alberta regional health authorities for making this
research successful.
And last, but not least, thank you to Dr. Marie Louie, Dr. Marina Salvadori, Dr. Scott McEwen,
and all of the researchers involved in the studies that made this part of the project possible. I would
also like to thank Caroline Gunette, Bryanne Crago, Kristen McLeod, Danielle Daignault, the staff at
the provincial laboratory in Calgary and the laboratory for Foodborne Zoonosis, the students who
worked on associated projects, and the staff of the Ontario Well Water Study. Your participation and
hard work are appreciated and without you, my piece of the puzzle would not exist.
Table of Contents
Abstract................................................................................................................... ii
Acknowledgements .................................................................................................... iii
Table of Contents ....................................................................................................... v
List of Tables ............................................................................................................vi
List of Figures............................................................................................................vi
List of Appendices.......................................................................................................vi
An Introduction to Antimicrobial Resistance........................................................................ 1
1.1 Background ...................................................................................................... 1
1.2 Antimicrobial Resistance in E. coli .......................................................................... 3
1.2.1 The Epidemiology of E. coli ............................................................................. 3
1.2.2 Epidemiology of Antimicrobial Resistance in E. coli ................................................ 6
Mechanisms of antimicrobial resistance in E. coli ....................................................... 6
Prevalence of antimicrobial resistant E. coli in Canadian studies..................................... 8
Prevalence across time ....................................................................................... 9
Prevalence across place ...................................................................................... 9
Prevalence across persons .................................................................................. 10
Factors affecting prevalence ............................................................................... 11
1.3 Objectives and Hypotheses .................................................................................. 21
Research Methods ...................................................................................................... 23
2.1 Study Population .............................................................................................. 23
2.1.1 Inclusion and Exclusion Criteria .................................................................... 25
2.1.2 Subject Recruitment .................................................................................... 26
Approvals and funding ....................................................................................... 27
2.2 Data Collection ................................................................................................ 27
2.2.1 London and Hamilton Regions ......................................................................... 27
2.2.2 Ottawa, Kingston, Peterborough, Orillia, and Toronto Regions.................................. 28
2.2.3 Questionnaires ........................................................................................... 29
2.2.4 Laboratory Testing of Water and Rectal Swab Samples ........................................... 30
2.3 Data Analyses .................................................................................................. 31
2.3.1 Objective 1: Prevalence of Antimicrobial Resistant E. coli ....................................... 31
2.3.2 Objective 2: Association Between Water Consumption and Human Carriage of Antimicrobial
Resistant E. coli.................................................................................................. 33
Model-building strategies. .................................................................................. 35
Focal relationship analysis. ................................................................................. 36
2.3.3 Students Role............................................................................................ 37
Results ................................................................................................................... 39
3.1 Characteristics of Water Samples, Households, and Respondents..................................... 39
3.1.1 Water samples ........................................................................................... 39
3.1.2 Participation.............................................................................................. 39
3.1.3 Households................................................................................................ 45
3.1.4 Respondents .............................................................................................. 48
3.2 Prevalence of Carriage of Antimicrobial Resistant E. coli .............................................. 51
3.2.1 Ampicillin Resistant E. coli Prevalence .............................................................. 54
3.3 Association of Human Carriage and Consumption of Contaminated Water .......................... 56
Discussion................................................................................................................ 62
4.1 Prevalence of Resistant E. coli .............................................................................. 62
4.1.1 Ampicillin Resistant E. coli............................................................................. 62
4.1.2 Antimicrobial Resistance to Other Agents ........................................................... 64
4.2 Association of Human Carriage and Consumption of Contaminated Water .......................... 66
4.2.1 Strengths and Limitations of the Study .............................................................. 67
4.3 Conclusions ..................................................................................................... 72
4.3.1 Contaminated water .................................................................................... 72
4.3.2 Prevalence of Carriage of Resistant E. coli.......................................................... 73
4.3.2 Next steps................................................................................................. 75
References .............................................................................................................. 77
Appendices .............................................................................................................. 97
vi
List of Tables
Table 1.1
Table 2.1
Table 2.2
Table 3.1
Table 3.2
Table 3.3
Table 3.4
Table 3.5
Table 3.6
Table 3.7
Table 3.8
Table 3.9
Table 3.10
Table 3.11
Table 3.12
Table 3.13
Table 4.1
Studies of E. coli contamination of private water sources, Ontario

Sampling and data collection for antimicrobial-resistance studies . . . . .
Susceptibility break-points for resistance screening and NARMS panels for
enteric bacteria, by antibiotic . . . . . . . . . . . . . . . . . . . . . . . . . . .
Details of household recruitment and participation for surveillance
project and case-control study . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Proportion of E. coli-positive water samples with antimicrobial resistant
E. coli, by antibiotic and class of antibiotic . . . . . . . . . . . . . . . . . . . .
Details of subject participation in prevalence study . . . . . . . . . . . .
Comparison of households participating in prevalence and case-control
studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Comparison of participants who completed personal questionnaires and
submitted a swab and subjects who completed questionnaires only . . . .
Descriptive statistics of participating households . . . . . . . . . . . . . . . .
Descriptive statistics of participants . . . . . . . . . . . . . . . . . . . . . . . .
Proportion of human rectal swabs with antimicrobial resistant E. Coli, by
antibiotic and class of antibiotic . . . . . . . . . . . . . . . . . . . . . . . . . . .
Intra-class resistance of antimicrobial resistant E. coli isolates from
human rectal swabs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Inter-class resistance of antimicrobial resistant E. coli isolates from
human rectal swabs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Proportion of human rectal swabs with ampicillin resistant E. coli, by
selected covariates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bivariate associations between carriage of antimicrobial resistant E. coli
and covariates. Poisson regression . . . . . . . . . . . . . . . . . . . . . . . . .
Multivariable model of association between carriage of antimicrobial
resistant E. coli, use of water contaminated with antimicrobial resistant
E. coli, and covariates. Poisson regression . . . . . . . . . . . . . . . . . . . .
Comparison of rates of antimicrobial resistance in E. coli from Canadian
studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
24
25
40
41
41
43
44
47
50
52
53
54
55
59
61
66
List of Figures
Figure 1.1
Figure 2.1
Possible routes of transmission of E. coli to humans . . . . . . . . . . . . . .

The theorized relationship between human carriage of E. coli, use of
contaminated and untreated water, and other study variables used for
multivariable model building . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13
34
List of Appendices
Appendix A
Appendix B
Appendix C
Appendix D
Appendix E
Appendix F
Appendix G
Appendix H
Appendix I
Appendix J
Appendix K
Appendix L
Studies comparing E. Coli antimicrobial resistance rates: Canadian subjects

Studies comparing E. coli antimicrobial resistance rates for human faecal
carriage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sample size calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ontario Well Water Study, information sheet . . . . . . . . . . . . . . . . . . . .
Telephone script for contact from Safe Water Unit . . . . . . . . . . . . . . . . .
Telephone script for initial study contact . . . . . . . . . . . . . . . . . . . . . . .
Ethics board approvals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Information and consent form . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Household questionnaire . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Personal questionnaire . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Rectal swab collection information sheet . . . . . . . . . . . . . . . . . . . . . . .
Variables derived from personal and household questionnaires . . . . . . . . .
97
98
104
105
106
109
111
115
117
129
137
138
Background
Chapter 1
An Introduction to Antimicrobial Resistance
1.1 Background
Resistance to penicillin was first noted in bacteria just four years after mass production of this
antibiotic began in 1943 and antimicrobial resistance has since been detected in bacteria, viruses,
fungi, and parasites (1;2). Since the 1940s the use of antibiotics has dramatically reduced death and
morbidity due to infectious diseases caused by bacteria. Yet the very use of antibiotics promotes
resistance through selective pressure. Antimicrobial resistance is considered one of the most serious
threats to the treatment of infectious diseases and is one of the leading public health concerns of the
21st century (2;3).
Antimicrobial resistance occurs when the organism is able to survive and reproduce in the
presence of the concentrations of an agent that can be achieved in target tissues/sites during therapy
(4;5). Bacteria are particularly predisposed to developing resistance due to the speed with which they
reproduce: a single bacterium can multiply within hours with several generations of bacteria being
created within days. They are also proficient at exchanging the genetic material that confers
resistance. Resistance to antibiotics has become a concern for a wide variety of bacterial species, both
pathogenic and commensal. It has been an issue for Neisseria gonorrhoea and Mycobacterium
tuberculosis for decades (6). More recently, methicillin-resistant Staphylococcus aureus (MRSA),
penicillin-resistant Streptococcus pneumoniae, and vancomycin-resistant enterococci (VRE) have been
troubling the health care systems of the world, particularly in acute and long-term care facilities (7;8).
There is a growing concern about resistance in enteric pathogens including Salmonella species, Shigella
species, Vibrio cholerae, Campylobacter species, and Escherichia coli (E. coli) (6). Antimicrobial
resistance in these enteric pathogens is of particular interest in developing countries where diarrhoeal
diseases are a leading cause of illness and death, but are also of importance in developed nations as a
reservoir for the transmission of resistance.
The health consequences of antimicrobial resistance include increased morbidity and mortality
due to delays in starting effective treatment or outright treatment failure (9). People infected with
antimicrobial resistant species of bacteria are twice as likely to be hospitalized, up to twice as likely to
die (10;11), and have as much as double the length of hospital stay as patients with susceptible strains
of bacteria (12). Other consequences include increased health care costs due to hospitalization or
prolonged stays (10;13), and additional costs for: supplementary laboratory investigations, isolation of
patients, special cleaning of health care facilities, and the use of more potent and expensive
antibacterial agents (12;14;15). It is estimated that resistant infections add $200-700 million annually
to the cost of health care in Canada (16;17).
In addition to the health and health care system consequences, no new class of antibiotic has
been introduced for several years thereby reducing the number of agents capable of combating
resistant organisms (3;18-20). Antibiotics are the foundation upon which the treatment of infectious
diseases builds and antibiotic resistance threatens not only the current management of pathogenic
bacteria, but the long-term efficacy of antimicrobial agents (21). The continuing emergence of
antimicrobial resistant Gram-negative pathogens, in particular, has not been matched by the
development of new classes of antimicrobial agents (22).
There is a general consensus that the emergence of antimicrobial resistance is largely due to
the use of antimicrobial agents (23-27). This occurs through a process called selective pressure in which
bacteria with mutations capable of surviving in the presence of the antimicrobial agent(s) persist and
pass these changes on to their offspring (28-32). The use of antimicrobial agents may promote selective
pressure in a second way: by eliminating susceptible bacteria, even of a different species, thus
permitting the resistant bacteria to multiply in the absence of competition (22;24;33;34).
The dissemination of antimicrobial resistance in bacteria is largely due to the genetic exchange
of resistance genes. Resistance genes are carried on chromosomes which enable vertical spread from
mother to daughter. However, they can also be transferred horizontally between bacteria and bacterial
species if the resistance genes are located on small segments of deoxyribonucleic acid (DNA) called
plasmids, on segments of plasmids called transposons, and on integrons which are found within
transposons and plasmids and in bacterial chromosomes (15;24;35). A resistance gene on the
chromosome of one strain of bacteria would have limited dissemination. However, resistance genes
that evolve on plasmids or transposons can be transferred to other bacteria, including other strains of
the same bacteria and other species of bacteria, making the dissemination much more efficient (24).
Resistance to one antibiotic can be conferred by one or more genes (e.g. tetA, sul1) that may
be localized on plasmids, transposons, or integrons (36). In comparison, other chromosomal mutations
Background
(e.g. mar) are capable of confering resistance to a large number of antibiotics (37;38). Resistance
genes can be spread to other bacteria by transformation, conjugation, or transduction. Transformation
is the uptake and incorporation of naked DNA and incorporation into the genome or plasmids.
Conjugation involves the donation of a copy of the plasmid (which may contain genes for resistance to
several antibiotics) to a recipient cell. Transduction is the transfer of resistance genes from one
bacterium to another through viruses (39;40). The transfer of antimicrobial resistance from one
bacterium to another and the subsequent vertical transfer to daughter bacteria is core to the
persistence and dissemination of antimicrobial resistance, including multi-drug resisitance (40).
Multiresistance integrons are important components of antimicrobial resistance in Gramnegative bacteria. Integrons are capable of acquiring, incorporating, and expressing the responses of
gene cassettes that encode for resistance against antibiotics, heavy metals, and detergents (41;42).
One integron may contain many gene cassettes with different resistance genes allowing one bacterium
to resist the effects of antimicrobial agents with varying mechanisms of action.
Although the emergence and persistence of resistance is largely due to selective pressure,
transmission of resistant bacteria and/or resistance genes contributes greatly to the overall prevalence
(26;33;43). Studies report that being hospitalized, attending a day care centre, or living with another
person who is colonized with antimicrobial resistant bacteria are risk factors for colonization or
infection with resistant bacteria suggesting transmission between humans (44-46). It is also possible
that people can become colonized after ingesting resistant strains of bacteria from food or water (4749). This thesis examines the prevalence of antimicrobial resistant E. coli in the human gastrointestinal
tract and the role of drinking water in the transmission of antimicrobial resistant E. coli to humans.
1.2 Antimicrobial Resistance in E. coli

1.2.1 The Descriptive Epidemiology of E. coli
Escherichia coli are Gram-negative bacteria of the Enterobacteriaceae genus. They are the
predominant facultative organism (capable of living in both aerobic and anaerobic environments) of the
human gastrointestinal system. There are 700 or more strains of E. coli bacteria, the majority of which
do not cause disease if they remain in the intestinal lumen. In fact, many live commensally with
humans suppressing the growth of pathogenic species of bacteria and producing vitamin K and B
complex vitamins that are necessary for health. However, some strains of E. coli are pathogenic and all
strains will cause disease if they infect areas that are normally sterile (e.g. the urinary tract) (50).
Escherichia coli are clinically significant bacteria, not only for their ability to cause
gastrointestinal disease but because they are the leading cause of Gram-negative infections.
Escherichia coli are responsible for 85-95% of urinary tract infections, 60-70% of hospital-acquired
pneumonia, and 17-37% of nosocomial bacteraemia in Europe and North America (51). Escherichia coli
also cause a high proportion of neonatal meningitis cases and abdominal, pelvic, and surgical site
infections (51). Between 2000 and 2002, E. coli comprised 13% of pathogens isolated from clinical
specimens from intensive care unit patients in Canada making it the third most common bacterial
pathogen and the most common Gram-negative pathogen (52).
Diarrhoeal infections caused by E. coli are treated with fluid and electrolyte replacement with
the role of antibiotics being uncertain. Extraintestinal infections, however, are treated with
antibiotics. The growing prevalence of antimicrobial resistance in E. coli is complicating the treatment
of all infections caused by these bacteria (53;54).
Escherichia coli are common in all warm-blooded animals meaning that humans, domestic
livestock, pets, wild animals, and birds are all reservoirs. Escherichia coli are spread by the faecal-oral
route. Transmission to humans from these reservoirs may occur in a variety ways including contact
with: other humans or animals, contaminated surfaces (e.g. door handles), manure or sewage, or
contaminated meat/poultry products, vegetables, raw milk, or untreated water (7;55-57).
Water is a known vehicle for the transmission of bacteria, including E. coli, in concentrations
large enough to cause illness in humans (58;59). In Canada and the United States, several large
outbreaks of E. coli O157:H7 have implicated drinking water as the source of infection, including a
large outbreak in Walkerton, Ontario in 2000 (60).
Escherichia coli are often used as an indicator of faecal contamination of drinking water.
Ontario microbiological drinking water standards for E. coli are set at zero colony forming units per 100
mL of water (61). In 2005-2006, E. coli were detected in only 20 of 20,000 (0.1%) routine posttreatment bacteriological tests of municipal water systems (62). In comparison, 20 of 250 tests (8%) of
small water systems i in Ontario (62) and 4% of water samples from private water supplies in southern
Small water system is one that serves a designated facility (school, nursing home, day care).
Background
Ontario (63) were determined to have E. coli contamination in the same period. Table 1.1 outlines the
proportion of private water supplies contaminated by E. coli according to published articles and
reports.
Table 1.1
Studies of E. coli Contamination of Private Water Sources, Ontario

Proportion of
E. coli-positive
Year(s) of
Geographic area (reference)
sources*
testing
Ontario (64)
1950-1954
15%
Brantford (65)
1987 & 1989
2%
Ontario (64)
1991-1992
17-24%
Ontario (66)
1992
20%
Middlesex (67)
1993
2%
Halton (68)
2000
14-16%
Kingston, Frontenac, Lennox,
& Addington (69)
2000
16%
Haldimand-Norfolk (70)
2004
4-11%
* Ranges are presented for results reported separately by time period or geographic region.
Four million or more Canadians rely on private water systems that are strictly the responsibility
of the residence owner (71). While private water sources serve only about 13% of the population in
Canada, they are implicated in about 20% of waterborne outbreaks (71).
In Ontario in 2006, about 80% of the 12.1 million residents were served by municipal water
systems with another 250,000 serviced by small water systems, leaving 1.5 to 2 million residents reliant
on private water sources (62;72;73). There are a variety of systems that supply water for people using
private sources including drilled or dug wells, sand or well points, cisterns, springs, and shore or lake
wells (71;74).
Although the province of Ontario provides bacteriological testing without direct cost to the
owners of private systems, many residents do not avail themselves of the service. A postal survey of
households using private water sources in Hamilton, Ontario determined that 28% of respondents were
not aware that the testing was provided without direct cost. Further, only 8% of households tested
their water at least three times per year as recommended in Ontario (75;76). Data from the Rapid Risk
Factor Surveillance System (2001-2005) determined that 61% of surveyed Ontario households with a
private water source had their water tested for bacteriological contamination at least once in the
previous year and 17% had tested it three or more times (63).
Bacteria in drinking water may be killed by boiling, ultraviolet light, chlorine or iodine, ceramic
or glass candle filters, or ozone. Reverse osmosis, carbon filters, and softeners are not effective
against bacterial contamination (77;78). Although 56% of Hamilton, Ontario-area households using
private water sources treated their drinking water, only 26% used a device capable of killing bacteria
(75). Similar results were reported in the Households and Environment Survey conducted in 2006. Of
the households in that study that did not primarily use bottled water, 25% treated their drinking water
with the goal of removing possible bacterial contamination (73). A survey of Halton, Ontario households
using private water sources determined that only 8% of households used chlorine or ultraviolet
treatments despite the finding that 14-16% of these water supplies were contaminated with E. coli
(68). Thus, even to this day, tens of thousands of Ontario residents are regularly exposed to E. coli
from their drinking water. These residents consistute a population in which we can assess the
relationship between the carriage of antimicrobial resistant E. coli and the use of water contaminated
with same.
1.2.2 Epidemiology of Antimicrobial Resistance in E. coli

Mechanisms of antimicrobial resistance in E. coli
Escherichia coli resist the effects of antibiotics in four ways. First, by producing antibioticinactivating (or -modifying) enzymes, such as beta-lactamases, which deactivate the drug by cleaving
to the core beta-lactamase ring structure. Extended-spectrum beta-lactamases (ESBLs) mediate
resistance to penicillins and third-generation cephalosporins. Escherichia coli also resist the effects of
antibiotics by restricting the concentration of antibiotics within the cell by reducing membrane
permeability and resisting entry of a wide range of antibiotics. A third mechanism of resistance is
alteration of the antibiotic target site so the antibiotic is unable to bind properly. This type of
mechanism is used by some strains of E. coli to resist quinolones and macrolides. The fourth type of
resistance mechanism is to eliminate the antibiotic target site entirely, either through overproducing
the target enzyme or producing an alternative target enzyme (15;79). Some resistance mechanisms
confer resistance to only one class of antibiotics while other mechanisms confer resistance to many
classes of antibiotics (7). One strain of bacteria may possess several different types of resistance
mechanisms making it multi-drug resistant (79).
Background
Escherichia coli are often used in antimicrobial resistance studies because a) they are found in
high numbers in warm-blooded mammals, including humans; b) they are a common human pathogen;
c) resistance is found in both pathogenic and non-pathogenic strains; d) they have the ability to
transfer resistance between different strains of E. coli; and e) they have the ability to transfer
resistance between different strains and species of bacteria within the gastrointestinal tract (80;81).
Also, E. coli reside primarily in mammalian hosts, thus being subjected to the pressures of
antimicrobial use and other environmental pressures. This makes them an ideal agent for surveillance
and research into factors that may contribute to the selection and spread of resistant bacteria (6;8284). Further, these bacteria are abundant in the environment making them a predominant vehicle for
the transmission of resistance genes (35;55;85;86).
Antimicrobial resistance has been detected in a variety of E. coli strains including numerous
shiga toxin-producing E. coli (including O157:H7), enteropathogenic, enterohaemorrhagic,
enterotoxigenic, enteroaggregative, enteroinvasive, and others (87). Several researchers have reported
that strains of E. coli that are antimicrobial resistant are often, although not always, less virulent than
susceptible strains (88-92). This appears to be related to the particular strain of E. coli rather than
some relationship between the resistance and virulence factors per se, as non-B2 strains are much
more likely to exhibit multi-drug resistance than B2 strains. Although, Johnson et al. state that the
difference in antimicrobial resistance by virulence may be due to the tendency of non-B2 strains to
acquire and/or retain resistance genes, they hypothesize that the difference in the prevalence of
antimicrobial resistance by strain may be due to differential antibiotic selection pressure: B2 strains
are less likely to be commensal flora than non-B2 strains, thus less likely to be colonizing the
gastrointestinal system during antibiotic therapy as well as being less abundant in human
gastrointestinal systems (90).
There are no differences between pathogenic and non-pathogenic bacteria in the basic cellular
processes that affect antibacterial resistance (93) and the remaining text will not differentiate
between the two. Commensal flora have the same plasmids, transposons, and integrons as their
disease-producing brethren, thus acting as a reservoir for resistance genes for pathogenic bacteria (37).
Prevalence of antimicrobial resistant E. coli in Canadian studies

The direct comparison of studies is hampered by the wide variety of protocols, break-points,
and analytic techniques. The prevalence of antimicrobial resistance in E. coli also varies depending on
the source of the isolate. Bagger-Skjt et al. (92) and others have reported that antimicrobial
resistance rates in clinical isolates were significantly higher than those from faecal isolates from noninstitutionalized subjects from the same country and in the same year. Although the rates of resistance
are higher from clinical isolates, the patterns of resistance are similar (i.e. higher in penicillins and
tetracycline than in fluoroquinolones).
In clinical isolates from Canadian patients, the reported rates of resistance range from 34-46%
to ampicillin (94;95), 15-19% to trimethoprim-sulfamethoxazole (52;95), and 27% to tetracycline (95)
(see Appendix A). Urinary tract infections are one of the most common infections in women (96) and E.
coli is the most common pathogen; responsible for 74-84% of urinary tract infections in Canada (97;98).
Rates of resistance detected in urinary tract infections caused by E. coli range from 30-45% for
ampicillin (99;100), 12-28% for trimethoprim-sulfamethoxazole (100;101), and 0-11% for nitrofurantoin
(98;99;102). Multi-drug resistance is also common in E. coli causing urinary tract infections.
One recently published study reported that 25% of urinary tract isolates from adult Canadian
women were resistant to two or more antibiotics (97). In another study, 20% of urinary isolates from
Canadian children were resistant to both ampicillin and trimethoprim-sulfamethoxazole (100).
Similarly, the most common pattern of resistance found in urinary tract isolates collected for the ECOSENS study was ampicillin and sulfamethoxazole (101). In a separate study, 36% of ampicillin resistant
E. coli isolates and 100% of ciprofloxacin-resistant isolates from Canadian outpatients were also
resistant to trimethoprim-sulfamethoxazole (98), the antibiotic recommended for empiric treatment of
uncomplicated urinary tract infections in adults (103).
Two studies, both conducted in the 1990s, used faecal samples from healthy noninstitutionalized Canadian subjects to determine the prevalence of antimicrobial resistant E. coli. The
prevalence of resistant E. coli in 154 volunteers from St. Johns, Newfoundland was highest for
amoxicillin (22%), followed by oxytetracycline (16%), and trimethoprim (10%) (104). Similar rates of
carriage of antimicrobial resistant E. coli were reported for 115 residents of pork producing farms in
Background
Ontario and British Columbia. In these subjects the rates of resistance were 16% for ampicillin, 24% to
tetracycline, and 5% to trimethoprim-sulfamethoxazole (105).
Prevalence across time

Antimicrobial resistance in E. coli has, in general, increased over time with older antimicrobial
agents like penicillin tending to have higher rates of resistance than newer drugs like cephalosporins
(44;106-108). Houndt and Ochman compared the antimicrobial resistance patterns of two E. coli
collections: one from the pre-antibiotic era (strains collected between 1885 and 1941) and one with
strains collected between 1972 and 1982. The authors compared the minimum inhibitory
concentrations for kanamycin, ampicillin, tetracycline, and chloramphenicol and determined that there
was a higher incidence of high-level antibiotic resistance for all antibiotics tested in the more recently
collected strains of the bacteria. For example, 4 of the 32 (13%) samples collected during the preantibiotic era were resistant to kanamycin at 16 g/mL compared to 21 of 72 (29%) of those collected
between 1972 and 1982. Similarly, 1 of 32 (3%) of pre-antibiotic era samples were resistant to
ampicillin while none were resistant to tetracycline or chloramphenicol. In the samples collected after
the introduction of antibiotics, 10 of 72 (14%) strains were resistant to ampicillin, 12 of 72 (17%) to
tetracycline, and 1 of 72 (1%) were resistant to chloramphenicol at 16 g/mL (82).
The increased prevalence of antimicrobial resistant E. coli can be tracked in studies of urinary
tract infections as well (96;109). A study published in 1971 determined that only 19% of E. coli strains
from English women with urinary tract infections were resistant to one or more of the antibiotics
tested; 11% to tetracycline and 3% to ampicillin (110). In comparison, 39-45% of urinary tract isolates
collected between 2000 and 2004 from outpatients living in the United Kingdom were resistant to
amoxicillin (36-40%) or trimethoprim (7-17%) (111;112).
Prevalence across place

Rates of antimicrobial resistance in E. coli vary widely across the globe. In 2001, nonsusceptibility to ampicillin ranged from 20% to 40% in Canadian, Swedish and Japanese hospital isolates
to over 60% in isolates from Mexico, Poland, Israel, Spain, Turkey, Hong Kong, Philippines, South Africa,
and Taiwan (113). Similar results were reported for hospital isolates collected between 2004 and 2006,
10
with ampicillin resistant E. coli detected in 67-69% of isolates from Asia, the Pacific rim, and Latin
America compared with 53-59% of isolates from the United States, Canada, and Europe (114).
Appendix B outlines studies of faecal carriage of antimicrobial resistant E. coli. Similar patterns
of resistance are noted in faecal isolate studies as in those of hospital isolates: rates of ampicillin
resistance were lower in Canada, the United States, Japan, China, and Europe (12-53%) than in Mexico
(73-94%), several countries of Africa (49-89%), and Central and South America and the Phillipines (7397%). The prevalence of multi-drug resistance was also lower in developed countries than developing
ones (43;80;84;104;105;115-127).
Differences between countries may be related to the unique health care systems, regulations,
and guidelines regarding antimicrobial prescribing as well as differing standards and practice for
antimicrobial use in animal husbandry and agriculture in each country. Greater environmental exposure
to antimicrobial resistant bacteria and resistance genes through crowding, lack of sanitation, and
contamination of food and water may also be associated with higher rates of carriage of resistant
organisms.
Prevalence across persons

In studies of healthy children, a higher proportion of children under two years of age had
antibiotic resistant E. coli detected in faecal samples than children two to seven years old (45;84;115).
Similar findings were reported in studies of antimicrobial resistance in E. coli causing urinary tract
infections where higher rates of resistance were detected in isolates from younger children than older
ones (102;109;128). The frequent use of antimicrobial agents in younger children, their less than
fastidious hygienic practices, as well as high contact rates between children may account for the higher
rates in young children.
In adults, there is no clear pattern of differences in the prevalence of antimicrobial resistance
by the age of the person. Using isolates from faecal samples, no differences in colonization with
antimicrobial resistant E. coli have been reported for adults of varying ages (46;129;130). Meanwhile,
in one study using clinical isolates, higher rates of resistance were reported for isolates from older
adults for gentamicin, piperacillin, and tobramycin while isolates from younger adults were more likely
to be resistant to ciprofloxacin (113). In studies using isolates from urinary tract infections, older
Background
11
adults had higher rates of fluoroquinolone resistant E. coli (97;102;131) and trimethoprimsulphamethoxazole-resistant E. coli (97).
The prevalence of antimicrobial resistant E. coli is similar for males and females. Three studies
describing gastrointestinal carriage of antimicrobial resistant E. coli by the sex of the individual
reported no difference by sex (46;130;132) with only one reporting a higher rate in male subjects
(133). Several studies of antimicrobial resistant E. coli from urinary tract and clinical infection isolates
report higher rates of resistance for isolates from males than from females (102;109;113;131;134-136),
while others detected no difference (137), or higher rates in isolates from females (111).
Factors affecting prevalence

Factors affecting prevalence: Antibiotic use.
Antimicrobial resistance is generally agreed to be a result of the use of antimicrobial agents in
human medicine possibly contributed to by the use of antimicrobial agents in veterinary medicine,
agriculture, and aquaculture (1;25-27;35;138). At an ecological level, higher rates of resistance are
reported in countries with high consumption of antibiotics. Kenyan childrens isolates had a
significantly higher rate of resistance (68%) to trimethoprim-sulfamethoxazole than isolates from
Japanese children (2%) which was attributed to the antibiotics use in Kenya as a first-line drug for the
treatment of diarrhoea and enteric infections (139). Similar findings were reported from a study done
in Burkina Faso (west Africa), in which 80% of E. coli isolates from people with diarrhoea were resistant
to trimethoprim-sulfamethoxazole (140). In Mexico, high rates of resistance in diarrheogenic E. coli
were reported for trimethoprim-sulfamethoxazole (65%) and ampicillin (73%); antibiotics commonly
used for children with diarrhoea. In comparison, they detected no E. coli isolates resistant to
ciprofloxacin or cefotaxime, antibiotics rarely used in non-hospitalized children (141). High rates of
ampicillin resistant E. coli (90%) have also been detected in Mexican children without diarrhoea (119).
Children in these developing countries which have high rates of antibiotic use have correspondingly
high rates of antimicrobial resistant E. coli.
However, ecological comparisons between and within developed countries ascertained that
antibiotic consumption alone does not account for all of the differences in the prevalence of
antimicrobial resistant E. coli. For instance, although the overall consumption of antibiotics, defined as
12
the number of daily doses per 1,000 residents, was similar in Newfoundland and Athens, the rates of
resistance in faecal E. coli isolates from people in Athens was significantly higher (104). The authors
contend that the differences are related to the colonization pressure of living in more densely
populated regions.
Differences also exist within countries where antibiotic availability is uniform suggesting that
some mechanism beyond antibiotic use is responsible for at least part of the differences in prevalence.
Rates of antimicrobial resistant E. coli from urinary tract isolates varied significantly by region in
England (142) and by state in the United States (125). There are also studies that show high rates of
antibiotic resistance in populations with low rates of antibiotic use (117;120;143).
At an individual level, antibiotic use during hospitalization is believed to be responsible for the
higher prevalence of antimicrobial resistant E. coli reported at discharge when compared to rates on
admission for the same patients (132;144-148). In other studies, hospital patients and residents of longterm care facilities were more likely to have antimicrobial resistant E. coli infections if they had
previously been treated with antibiotics (137;149). Antibiotic use in ill community-dwelling subjects is
also associated with increased antimicrobial resistance following use of antibiotics in many studies of
adults (30;46;122;150-156). In contrast to these findings, three studies of children attending day care
centres in the United States found no association between individual consumption of antibiotics and
the carriage of antimicrobial resistant E. coli (45;157;158). These findings indicate that although
antibiotic use is likely involved in the emergence of resistance, there are other factors involved in the
persistence and dissemination.
Escherichia coli are transmitted directly from person-to-person or animal-to-person and
indirectly via contaminated surfaces, food, or water (159-163). In studies where the infectious agent
has been identified as E. coli, the most common exposures identified include beef, unpasteurized dairy
products, swimming, untreated drinking water, fresh produce, and travel to a developing country
(160;164;165). Figure 1.1 depicts some routes of transmission of E. coli to susceptible hosts. Since
there is no evidence to suggest that antimicrobial resistance confers adaptive advantages or
disadvantages to E. coli (7;166;167), the study of the spread of antimicrobial resistant E. coli must
consider the same pathways of transmission.
13
Background
Figure 1.1
Possible Routes of Transmission of E. coli to Humans

Reservoir:
Human or animal
Portal of exit: faecal
Direct contact
Environmental
o
Surfaces
o
Soil
o
Recreational water
Foods
o Meat & poultry products
o Dairy products
o Field & orchard crops
o Fish & seafood
o Drinking water*
Portal of entry: oral
Susceptible Host
* Focus of this research
Factors affecting prevalence: Person-to-person transmission.

The spread of E. coli from person-to-person is well-documented and, since most E. coli strains
are species-specific, humans serve as a main reservoir for the transmission to other humans
(33;161;162;168). As highlighted earlier, 16-22% of community-dwelling subjects in Canada were
colonized with penicillin-resistant E. coli in two recent studies (104;105). This represents a substantial
reservoir of antimicrobial resistant bacteria and resistance genes from which transmission to other
people is possible.
Infants intestinal tracts are sterile at birth but are colonized with bacteria, including E. coli,
within hours of birth (169). One of the most convincing pieces of evidence for person-to-person
transmission of commensal strains of antimicrobial resistant E. coli is the detection of resistant strains
within the faecal matter of infants that had never received antibiotics. Ampicillin resistant E. coli were
14
detected in the faecal matter of 8% of Swedish neonates and 28% of Turkish infants (8-10 weeks of age)
who had never taken an antibiotic in their lifetime (147;170).
Similarly, there was no difference in the rates of antimicrobial resistant E. coli from the stools
of young children from the United States, Venezuela, and China that had never had antibiotics
compared to those that had (127). Also, transmission from adult to child is one explanation for the
existence of fluoroquinolone and doxycycline resistant E. coli in young children and tetracycline
resistant E. coli in infants who are rarely, if ever, prescribed these antimicrobial agents (84;120;171173). Whether this transmission occurs directly from person-to-person or through less direct routes,
such as the ingestion of contaminated foods or water, was not determined.
Person-to-person transmission was likely responsible for the high rates (67%) of antimicrobial
resistance detected in faecal E. coli isolates of residents of a remote rural village in Bolivia where the
use of antimicrobials for humans was quite limited; only 7% reported antibiotic use in the previous 12
months compared to 35-40% of residents of the United States (46;158). Further, the use of antibiotics
for animal medicine was absent in this rural village (117). In a follow-up article, molecular
characterization of the isolates revealed a notable variety of resistance genes so the authors concluded
that the relatively high prevalence of resistance was likely due to the introduction of resistant strains
into the community by travellers and/or animals with subsequent horizontal gene transfer to and
between the village residents (174). Whether the transmission occurred directly between people or
indirectly through contaminated surfaces, foods, or water was not determined.
Some researchers believe that the use of antibiotics, in conjunction with high colonization
pressure (i.e. underlying rates of colonization with the organism) increases the probability of
colonization (116;175). Hospitals and long-term care facilities are sites that would have high
colonization pressure. Several studies have reported hospitalization as a significant predictor of
colonization with antimicrobial resistant E. coli, even after adjusting for antibiotic use
(44;122;155;176;177). The rates of antimicrobial resistance in faecal E. coli isolates are also higher in
residents of long-term care facilities than in community-dwelling subjects (80;149).
Day care centres are another site of enhanced transmission of enteropathogens, including E.
coli (53). Trimethoprim and ampicillin resistant E. coli have been reported to be more prevalent in
children within specific day care centres (157) and more prevalent in children attending day care
Background
15
centres compared to those not attending a centre (45). Further, person-to-person transmission was
implicated in an outbreak of antimicrobial resistant E. coli O26 in a day care centre in Japan (178).
Transmission of antimicrobial resistant E. coli likely occurs at the household level as well (179).
Having one household member with resistant E. coli was determined to be a risk factor for colonization
for other household members in several studies completed in developed countries (46;158;180-182).
Although people living within one household are more likely to share antimicrobial resistance patterns,
whether this is due to sharing a common source of resistant bacteria or resistance genes (i.e. food,
water, animals/pets) or is due to person-to-person transmission has not been determined.
Travel to areas with a high prevalence of human carriage, typically developing countries, has
been linked to colonization with antimicrobial resistant bacteria (15). Three studies of healthy college
students from the United States describe an increased prevalence of resistant E. coli after the students
spent several weeks in Mexico, a country in which a high proportion of residents are colonized
(152;183;184). Similarly, people who had travelled to a developing country in the previous year were
significantly more likely to carry antimicrobial resistant E. coli than other subjects (32% vs. 9%) in a
cross-sectional study completed in the United States (185). Exposure to the large reservoir of
antimicrobial resistant bacteria that exists in developing nations likely increases the probability of
carriage of antimicrobial resistant bacteria for visitors and residents alike. Whether higher prevalences
in people who have travelled is due to person-to-person or other form(s) of transmission is unknown.
Factors affecting prevalence: Animal-to-human transmission.

Although not all strains of E. coli are capable of colonizing the gastrointestinal tracts of both
humans and other animals, many are. Humans do become ill with pathogenic strains of E. coli whose
normal reservoir is animals after direct contact with the animals or their manure (161;163;186-188).
There are numerous studies highlighting the occurrence of antimicrobial resistant E. coli in
cattle, horses, pigs, sheep, goats, and companion animals such as dogs. Microbiological studies show
that several types of integrons (DNA element involved in antimicrobial resistance) are shared among E.
coli isolated from humans, dogs, and domestic livestock like cattle, pigs, and poultry, making the
transmission between species likely (189-191). There are also several reports of antimicrobial
16
resistance in E. coli O157:H7, an animal strain of E. coli, detected in human isolates (192-194). Thus, it
is possible that resistant bacteria are transmitted to humans from animals (26).
The Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) reported
that 33% of beef, 88% of swine, and 84% of chicken caecal samples from Canadian abattoirs were
positive for antimicrobial resistant E. coli in 2003 (195). Dogs, cats, and horses are also a potential
source of resistant bacteria and/or resistance genes (189;196-198). In one study, 25% of dogs in
breeding kennels and 12% of individually-housed dogs carried antimicrobial resistant E. coli (199).
People involved in livestock farming have been shown, in some studies, to have higher rates of
carriage of antimicrobial resistant bacteria than non-farming controls (130;200-203). However, other
studies have shown no association between farming and higher rates of carriage of antimicrobial
resistant E. coli (46;204).
A study of Canadian pig farmers determined that farmers who used in-feed antimicrobial
agent(s) were more likely to carry antimicrobial resistant E. coli than those who did not. The personal
use of antibiotics by the farmer and the number of hours he/she spent in the pig barn were positively
associated with human carriage, but, interestingly, having animals that carried antimicrobial resistant
E. coli was not (105). Similarly, pig farmers in the Netherlands had higher rates of faecal carriage of
antimicrobial resistant E. coli than urban-dwelling subjects (200). However, only 4% of E. coli from
faecal samples of farmers matched the resistance patterns for pigs from the same farm and laboratory
studies showed they had distinctly different plasmid DNA (205;206). Further, a study done in the United
States found no difference in human carriage of antimicrobial resistant E. coli for people working for
swine production facilities and people who did not (204).
A community-based study of healthy Dutch subjects six years of age and older determined that
people living on cattle farms had higher rates of carriage of tetracycline resistant E. coli than subjects
not involved in cattle farming. Of note, no differences were found for rates of carriage of ampicillin or
sulphamethoxazole resistant E. coli between the two groups (130).
Poultry workers in the United States were significantly more likely to carry E. coli resistant to
gentamicin, an antibiotic of limited human use, than community controls who were not involved with
poultry production (207). Similarly in Saudi Arabia, poultry farmers had a higher prevalence of
gentamicin resistant E. coli than hospitalized patients (38% versus 22%, respectively) (208). In Holland,
Background
17
poultry farmers had a higher prevalence of carriage of ciprofloxacin resistant E. coli (17%) than
subjects participating in other studies (<1% to 3%) in the 1990s (104;209;210).
The direct and prolonged exposure to animals, as experienced by farmers and farm-workers, is
linked with higher rates of carriage of antimicrobial resistant E. coli in some, but not all, studies. It
may be that the association is specific to the animal species, whether the animals are given antibiotics
as growth promoters, the antibiotic(s) under study, or other unmeasured variables such as the
consumption of water contaminated with bacteria originating from the animals.
Factors affecting prevalence: Environmental and foodborne transmission.

Food crops fertilized or otherwise contaminated by animal manure or human excrement are
another possible source of transmission of resistant bacteria and/or resistance genes to humans
(47;211-214). Antimicrobial resistant E. coli have been detected on a variety of foods in a number of
countries including Finland (214), Spain (198), and the United States (215;216). Escherichia coli were
detected on 9% of vegetables, fruit, and other foods purchased in stores in the United States and 27%
of these isolates were found to be antimicrobial resistant (211;217). Antimicrobial resistant E. coli has
also been detected in several different brands of ready-to-eat shrimp that was purchased in the United
States but caught and packaged in a variety of countries (218). The Canadian Integrated Program for
Antimicrobial Resistance Surveillance reported that 25% of raw beef, 60% of pork, and 70% of chicken
sampled from Canadian retail sources in 2003 were contaminated with antimicrobial resistant E. coli
(195). And, an Ontario-based study detected ampicillin resistance in 25% of E. coli isolates recovered
from the milk of cattle with mastitis (219).
Although antimicrobial resistant E. coli has been detected in numerous foods, only one study
has shown an association between consumption of meat and colonization with resistant E. coli. The
transfer of antimicrobial resistant E. coli from a raw chicken carcass to a human who handled, cooked,
and ate it was demonstrated in 1 of 14 trials within the same study, showing that transmission is
possible but infrequent (220). Several other studies from developed countries have found no
differences in the prevalence of carriage of antimicrobial resistant E. coli between people who
consume meat or poultry infrequently or not at all compared with those who eat it more often
(46;155;180;185). The lack of differentiation in the frequency of meat consumption may be a factor in
18
the lack of findings. Other reasons for the lack of findings include the fact that meat is one of myriad
possible sources of antimicrobial resistant bacteria and resistance genes. Additionally, foods are often
prepared (i.e. washed or cooked) which may dislodge and/or destroy the bacteria before consumption.
Factors affecting prevalence: Waterborne transmission.

Ground and surface water contaminated by animal or human waste is a reservoir of
antimicrobial resistant bacteria (7;221). Antimicrobial resistant E. coli have been detected in surface
waters in a number of countries and various water sources. Antimicrobial resistant E. coli, often multidrug resistant, has been detected in surface water (222-226), rivers and streams (227-234), farm
retention ponds (235), and ground well water (234).
Studies have detected antimicrobial resistant E. coli in the effluent of sewage treatment plants
as well as in the river water into which the effluent is discharged and in the sludge that is either
spread on agricultural land or dumped into landfill sites (212;230;236). A Canadian study completed in
1979 detected transferable drug resistance factors in 6-13% of faecal coliforms from the final effluent
of sewage treatment facilities in the Northwest Territories, Alberta, Manitoba, and Saskatchewan
(237). A study done in the late 1970s in Manitoba detected antibiotic resistant faecal coliforms in 47%
and 59% of sewage and river water samples, respectively (238). Antimicrobial resistant E. coli carrying
known resistance genes was detected in 11% of isolates from water sampled from the St. Clair and
Detroit rivers in Southern Ontario (239).
In Ontario, 95% of E. coli isolates from surface water sampled within a ten kilometre radius of
the Hamilton harbour in 2002 were resistant to one or more antibiotics (236) as were 81% of bacterial
isolates (not limited to E. coli) from sewage treatment effluent in Ottawa, Ontario (240). Escherichia
coli-positive beach water isolates submitted to the public health regional laboratories as part of the
requirements for surveillance of recreational water safety were tested for antimicrobial resistance as
part of the surveillance project connected with this research. Antimicrobial resistance was detected in
10% of isolates submitted from Alberta, 15% from Quebec, and 27% from Ontario public beaches for the
summer of 2004. Surface water is of importance not only for its recreational use but also because it
provides water for most large municipal drinking water systems in Ontario.
Background
19
Untreated water used for drinking could be a source of transmission for antimicrobial resistant
strains and genes (48;49;241). A study of drinking water from both surface and ground water sources in
the United States determined that 34% of the bacteria (not just E. coli) were multi-drug resistant in
1980 (242). In 1984, 13% of E. coli isolates from a public water supply in Connecticut (detected during a
period of elevated total coliform count) were antimicrobial resistant (243). In the same era, 10 of 18
wells tested in the United States contained antimicrobial resistant bacteria, with 16% of faecal
coliforms being multi-drug resistant (241). A study of 44 private groundwater supplies in West Virginia
determined that 46% of E. coli isolates (n=28) were resistant to one or more antibiotics (241).
Antimicrobial resistant E. coli was also isolated from drinking water in Montana with 70% and 55% of
isolates resistant to carbenicillin and tetracycline, respectively (244). In a pilot study for this project,
14% and 16% of E. coli-positive water samples submitted from Ontario and Alberta private water
sources were antimicrobial resistant. The highest rates of resistance were to tetracycline (11%),
sulfamethoxazole (6%), streptomycin (6%), and ampicillin (5%) (245). These findings emphasize the
potential magnitude of the problem of antimicrobial resistant E. coli in drinking water sources and
further support the role of water as a source of antimicrobial resistant bacteria.
One positive link between the consumption of antimicrobial resistant E. coli-contaminated
water and carriage of resistant E. coli comes from an animal study. One flock of grazing sheep in
Oregon had a significantly higher prevalence of multi-drug resistant E. coli than the other nine flocks
studied. Upon investigation, it was determined that their water source was contaminated with multidrug resistant E. coli likely originating from a nearby human septic system. No further study of the
issue was completed as this finding was coincidental to the original research (246). However, it raises
the possibility of the transmission of antimicrobial resistant E. coli to mammals through the ingestion
of contaminated drinking water.
A connection between drinking contaminated water and human carriage of antimicrobial
resistant E. coli was reported as a result of the investigation of three outbreaks that occurred on cruise
ships in 1997-1998. In all three outbreaks, the ingestion of the ships tap water or ice made from tap
water was significantly associated with diarrhoeal illness and implicated as the likely cause of the
outbreaks. Faecal samples collected from ill passengers were cultured for a variety of pathogenic
organisms. The investigators determined that 38% of the enterotoxigenic E. coli-positive isolates from
20
these ill passengers were resistant to three or more antibiotics (247). The authors hypothesized that
the ships potable water was contaminated with antimicrobial resistant E. coli thus infecting the
passengers. Unfortunately, by the time the investigations were conducted, it was not possible to
isolate E. coli from the water supplies of the cruise ships. Thus, no direct association could be made
between ingestion of water contaminated with antimicrobial resistant E. coli and infection with
resistant strain(s).
Shanahan et al. reported that there was no association between consuming water
contaminated with bacteria and human carriage of resistant bacteria in a 1992 study conducted with
healthy South Africans (248). The rural and urban dwelling subjects had similar rates of carriage of
resistant enterobacteria for three antibiotics (ampicillin, trimethoprim, and nalidixic acid) but rural
dwelling subjects had a higher prevalence of gentamicin (11% versus 4%) and chloramphenicol (60%
versus 46%) resistant enterobacteria. Although the authors reported no association between the use of
water contaminated with bacteria and the carriage of resistant enterobacteria, water was tested for
bacterial contamination at only one of two urban locations and two of four rural locations (a school and
a clinic). No tests were completed on the household water supplies, either rural or urban, and no tests
of antimicrobial resistance were completed on the bacteria that were isolated. Thus, it was not
possible to determine whether there was exposure to water contaminated with bacteria let alone
exposure to water contaminated with antimicrobial resistant bacteria. Therefore, we cannot support
nor refute the conclusion of no association between consumption of water contaminated with bacteria
and human carriage of resistant bacteria.
The prevalence of carriage of resistant aerobic faecal bacteria, not E. coli specifically, was
studied by Amyes et al. in 1989 in four villages in India (249). In this study, urban and rural subjects
were equally likely to carry bacteria resistant to ampicillin (98%), trimethoprim (98%), and
chloramphenicol, (97%), but urban dwelling subjects were more likely to be colonized with bacteria
resistant to nalidixic acid (35%) than the rural dwelling subjects (13%). The authors reported that the
rural village wells and the municipal drinking water both carried antimicrobial resistant bacteria
although no further details were given. The authors concluded that the high rates of human carriage of
resistant bacteria were due to high use of antibacterial drugs combined with the ingestion of faecal
bacteria from contaminated water supplies. However, without further details and with no statistical
21
Background
tests of association reported, the authors conclusion that the ingestion of contaminated water was
associated with human carriage of resistant bacteria cannot be substantiated.
Despite the lack of more direct evidence, it is biologically feasible that humans will become
infected with antimicrobial resistant E. coli after consuming water contaminated with antimicrobial
resistant bacteria. In support of this idea is the fact that several outbreaks of antimicrobial resistant E.
coli have been reported indicating that the transmission of antimicrobial resistant bacteria is
occurring. These include an outbreak of antimicrobial resistant E. coli O26 in a day care nursery (178),
outbreaks of extended beta-lactamase producing E. coli in long-term care facilities (250) and acute
care hospitals (251), a foodborne outbreak caused by a resistant strain of E. coli O153:H45 (87).
Antimicrobial resistant E. coli have been found in numerous water sources (241;242;244) and E. coli is
known to be transmitted in large enough quantities in drinking water to cause disease (58;252-255).
Since there are no differences in the basic cellular processes that affect antimicrobial resistance in
pathogenic and non-pathogenic bacteria (93) and the acquisition of antimicrobial resistance does not
appear to confer adaptive advantage or disadvantages to E. coli (7;166), it is reasonable to assume that
antimicrobial resistant E. coli may be transmitted through water.
Whether the ingestion of water contaminated with antimicrobial resistant E. coli is associated
with human colonization with resistant strains of E. coli has not been definitively determined and was
the focus of this thesis.
1.3 Objectives and Hypotheses

The objectives of the research were to determine the prevalence of human faecal carriage of
antimicrobial resistant E. coli in people residing in southern Ontario who used private water sources
and whether the use of water contaminated with antimicrobial resistant E. coli was associated with
human carriage. We proposed to:
1. measure the prevalence of faecal carriage of antimicrobial resistant strains of E. coli in people
using private water sources in southern Ontario, by antimicrobial agent, with a focus on
ampicillin; and
2. determine whether the consumption of water contaminated with antimicrobial resistant E. coli
was associated with faecal carriage of antimicrobial resistant strains of E. coli.
22
We were most interested in examining the prevalence of resistance specific to ampicillin based
on the fact that penicillins are the most frequently prescribed antibacterial agents in Canada and
ampicillin is the first line choice for treatment of urinary tract infections due to E. coli when resistance
is below 20% (256). Ampicillin was also the only non-combination penicillin used in the 2002 or 2004
National Antimicrobial Resistance Monitoring System (NARMS) panels for enteric bacteria (the antibiotic
panels used to assess for antimicrobial resistance in this study) (257). Ampicillin is used for both
children and adults to treat urinary tract infections, bacterial respiratory tract infections,
gastrointestinal infections, bacterial meningitis, septicaemia, and endocarditis caused by E. coli,
enterococci, S. pneumoniae, H. influenza, P. mirabilis, and S. epidermis (258).
Research hypotheses were:

1. The prevalence of human carriage of ampicillin resistant E. coli in southern Ontario residents
using private water sources will be greater than or equal to the prevalence rate of 22% ii as
described in previous studies (104;105); and
2. The use of water contaminated with antimicrobial resistant E. coli will be associated with
faecal carriage of antimicrobial resistant E. coli in this population.
ii
The hypothesized prevalence of ampicillin resistant E. coli was determined using an estimate from the paper by
Bruinsma et al. (104). We hypothesized that the prevalence in Ontario, a province that is more densely populated
and which is more intensively cultivated, 6-7 years after the collection of data in Newfoundland would be at least,
if not greater than, the 22% prevalence of amoxicillin resistant E. coli (breakpoint concentration 25g/mL)
reported in that study. Another reason for using the 22% hypothesized prevalence was based on the Infectious
Diseases Society of America recommendation that empirical treatment of urinary tract infections change when
resistance to the recommended antibiotic reaches 20% (103).
Methods
23
Chapter 2
Research Methods
This study was one component of a three-part project starting with of a multi-province
surveillance project investigating the prevalence and geospatial distribution of antimicrobial resistant
E. coli in beach water samples submitted to provincial laboratories in Ontario, Alberta, and Quebec in
2004 and 2005, and private water samples submitted from participating public health laboratories in
Ontario and Alberta between April 2004 and September 2006.
In the second year of the surveillance project, April 2005 to September 2006, a case-control
study was conducted to determine the risk factors for contamination of private water sources with
antimicrobial resistant E. coli. The case-control study used water sample results from the surveillance
study to identify case and control water sources (households).
The research for this thesis was comprised of a cross-sectional human prevalence study of
antimicrobial resistant E. coli colonizing the gastrointestinal tract of healthy human adults and
adolescents. It used a convenience sample of people living in households who agreed to participate in
the case-control study (see Table 2.1). All consenting household members who were 12 years of age
and older and capable of completing the questionnaire in English were eligible for inclusion. This
sample was then used to determine whether consumption of water contaminated with antimicrobial
resistant E. coli was associated with faecal carriage of antimicrobial resistant E. coli. Sample sizes
calculated for the protocol are available in Appendix C.
2.1 Study Population

The sampling frame consisted of all suitable iii water samples submitted iv for bacteriological
testing at the participating public health laboratories in Ontario between May 1, 2005 and September
30, 2006. All E. coli-positive water samples tested at the London and Hamilton laboratories and a
monthly quota of E. coli-positive samples, selected at random, from the Ottawa, Kingston,
Peterborough, Orillia, and Toronto laboratories were screened for antimicrobial resistance.
iii
Water submitted from a private water source, tested within 48 hours of collection, containing adequate contact
information, and sent in an approved bottle that is not broken, leaking, or frozen
iv
Submission of water samples from private water sources was voluntary in Ontario
24
Table 2.1
Sampling and Data Collection for Antimicrobial-Resistance Studies; Survey of
Households using Private Water Sources, Southern Ontario, 2005-2006
Water samples tested for bacteriological contamination
by participating public health laboratories
E. coli positive water samples

(All from London & Hamilton; Random selection from
Surveillance project
Kingston, Orillia, Ottawa, Peterborough, & Toronto*)
(Water tests)
Antimicrobial resistant
Antimicrobial susceptible
E. coli
E. coli
All non-repeated
Random sample from
Random sample of
samples from
surveillance project
bacteria-free water
surveillance project
tests from lab data base
Case-control study
(Water source of
(Case)
(A control)
(B control)
households)
Eligible & agreed to share contact information
Site visit
Telephone interview
Interviewer called to explain study
Survey company explained study &
& arrange site visit
completed household interview by
telephone
Household interview at site visit
Personal interview(s) & rectal swab(s)

Personal interview(s) by telephone
Prevalence study
from eligible individuals in household
Swab kits mailed & follow-up call
(Human subjects)
Interviewer mailed swab(s)
Subject mailed swab & consent form
* A random selection of E. coli-positive water samples were submitted from these laboratories due to
workload issues
Cases for the case-control study included households with E. coli-positive water samples that
were resistant to one or more of the antibiotic agents included in the National Antimicrobial Resistance
Monitoring System (NARMS) panel for enteric bacteria (Table 2.2). A controls were households
randomly selected from the E. coli-positive samples submitted to the study that were susceptible to all
antibiotics on the screening panel. B controls were households that were randomly selected from the
provincial database of all water submissions that tested negative for contamination with E. coli or nonE. coli coliform. Both A and B controls were frequency matched by laboratory region from samples
submitted within one month of the date of the case submission, with an average of 1.3 controls per
case to ensure that an adequate number of controls were available.
25
Methods
Table 2.2
Susceptibility Breakpoints for Screening and NARMS Panels for Enteric Bacteria, by
Antibiotic; Survey of Households using Private Water Sources, Southern Ontario, 2005-2006
NARMS Panels
Screening1
2002
2004
breakpoints
breakpoints
Antibiotic
Concentration
(260)
(259)
Aminoglycoside
Amikacin
64 g/mL
64 g/mL
Gentamicin
16 g/mL
16 g/mL
8 g/mL
Kanamycin
64 g/mL
64 g/mL
Streptomycin
64 g/mL
64 g/mL
32 g/mL
Beta-lactam
Amoxicillin/clavulanic acid
32/16g/mL
32/16g/mL
Ampicillin
32 g/mL
32 g/mL
8 g/mL
Cefoxitin
32 g/mL
32 g/mL
Ceftiofur
8 g/mL
8 g/mL
Ceftriaxone
64 g/mL
64 g/mL
Fluoroquinolone
Ciprofloxacin
4 g/mL
4 g/mL
Nalidixic acid
32 g/mL
32 g/mL
4 g/mL
Sulphonamide
Trimethoprim/sulphamethoxazole
4/76 g/mL
4/76 g/mL
--Sulphamethoxazole
512 g/mL
128 g/mL
Sulfisoxazole
--256g/mL2
--Cephalothin
32 g/mL
32 g/mL
Chloramphenicol
32 g/mL
32 g/mL
Tetracycline
16 g/mL
16 g/mL
4 g/mL
1
Screening of E. coli-positive samples was done using antibiotic concentrations lower than or equal to
the NARMS breakpoints to reduce the number of samples requiring full NARMS panel analysis (samples
susceptible on screening were not tested using NARMS)
2
Breakpoint concentration was 512 g/mL in NARMS, but 256 g/mL was the highest concentration
available on the test plate
The case-control and human prevalence studies were initially conducted only in the London and
Hamilton public health laboratory regions of Ontario. It was expanded in January 2006 to include five
other laboratory regions: Ottawa, Kingston, Peterborough, Orillia, and Toronto. Escherichia colipositive water samples were already being submitted from these laboratory regions as part of the
surveillance project so that the same time period of water sample submission (May 2005 to September
2006) was used for all regions.
2.1.1 Inclusion and Exclusion Criteria

Inclusion in the case-control study was limited to households that a) submitted a suitable water
sample to a participating Ontario public health laboratory for bacteriological testing during the study
26
period, b) consented to have their contact information disclosed to the study group, c) resided on the
property from which the water was submitted, d) had at least one household member who was 18 years
of age or older, e) spoke English, and f) could be contacted by telephone.
Water samples were not eligible if they were from a) households that had already been
contacted by the study and submitted a subsequent water sample, b) sites outside the study area,
c) real estate brokers, d) commercial properties, or e) households from which the submitter had
moved. Also ineligible were households selected as B controls that had a water sample that tested
positive for bacterial (E. coli or other coliform) contamination within the previous twelve months.
All people living in case and control households that were 12 years and older were eligible for
inclusion in this human prevalence study. Only people who agreed to submit a rectal swab were
interviewed for the prevalence study.
2.1.2 Subject Recruitment

The case-control and prevalence studies were first introduced to potential participants with an
information sheet (Appendix D) that was mailed, along with the bacteriological test results, to all
households that submitted a water sample to a participating laboratory during the study period. In the
Hamilton and London areas, information sheets were also attached to water sample kits that were
distributed between May 2005 and September 2006.
Following public health laboratory testing, screening, and final resistance testing, case and A
control records were forwarded to the study assistant who was located at the Safe Water Unit of the
Ministry of Health and Long-Term Care. The study assistant matched the cases and A controls
records (accession number, laboratory, and date of sample submission) with the provincial database
and then randomly selected A controls from within the matches. B controls were randomly
selected from results without bacterial contamination from within the provincial database. Households
selected for inclusion in the case-control study were telephoned by the study assistant.
Using an approved script (Appendix E), the study assistant solicited the consent of the submitter
of the water sample to share their contact information with the study investigators. When submitters
agreed to be contacted, the study assistant screened them for eligibility and sent their contact
27
Methods
information to the study coordinator. The study coordinator stripped the case-control designation from
the contact information and sent it to the appropriate interviewer.
The interviewer called consenting households and, using an approved script (Appendix F),
described the research in more detail. The person submitting the water sample, or his/her designate,
was asked to participate in both the case-control and prevalence sections of the study. They were
informed that they would be requested to provide a rectal swab as part of the prevalence study and, if
they refused, were given the option of completing the case-control study (household questionnaire)
only. For those households that participated in the case-control section of the study, the interviewer
asked if other eligible household members would be willing to participate in the prevalence section of
the study.
The study assistant, interviewers, and the telephone survey company made a minimum of ten
attempts to contact each submitter. These calls were made at various times of day, between 9:00 a.m.
and 8:00 p.m., throughout the week and on weekends. Response files were kept by each caller
detailing the date and time of attempted calls, eligibility to participate, reason for no contact, and
response.
Approvals and funding

Ethics approvals were granted by the Universities of Western Ontario and Toronto (Appendix G).
Approval was also granted by the Ontario Ministry of Health and Long-Term Care. The research was
funded by the Canadian Institute of Health Research-Health Canada and the Physicians Services
Incorporated Foundation (Ontario).
2.2 Data Collection

2.2.1 London and Hamilton Regions
Visits were made to many of the study participants homes to collect data in the London and
Hamilton regions. Trained interviewers called the person who submitted the water sample to explain
the study, arranged a site visit with those who consented to participate, and solicited assent to speak
with other household members during the visit. Parental or custodial assent was obtained before
approaching youths 12 to 15 years of age. The submitter completed a written consent (Appendix H) and
28
was interviewed using the household questionnaire (Appendix I). All household members who agreed to
submit a rectal swab provided written consent and were interviewed using the personal questionnaire
(Appendix J). Responses of participants were recorded on paper copies of the questionnaires with
responses entered into a database by one data entry professional.
At site visits, the participant was given the labelled rectal swab, a swab collection information
sheet (Appendix K), a bio-hazard specimen bag, and asked to collect the swab. The interviewer mailed
the swabs to the study laboratory within 24 hours of collection. Rectal swabs were chosen over stool
samples as the preferred method of collection for several reasons. At the site visits, interviewers could
request the sample be collected immediately, the interviewers could demonstrate how to collect the
swab, and we expected, given results of other research, that requesting the subject collect the swab
at the time of the interview would increase the proportion of swabs submitted versus having the
subject collect and mail the swab to the study laboratory at a later date (261-264). For subjects
interviewed by telephone, swab kits were more easily mailed to participants than stool collection kits.
Previous research has shown that rectal swabs are a good tool for the detection of antimicrobial
resistant E. coli. Lautenbach studied the sensitivity and specificity of rectal and peri-rectal swabs for
the detection of fluoroquinolone resistant E. coli compared to stool samples. In their sample, 90% of
the patients with positive stool samples also had positive rectal swabs and 90% had positive peri-rectal
swabs. The authors noted that the two samples in which the fluoroquinolone resistant E. coli were not
detected using the rectal or peri-rectal swabs had low concentrations of E. coli (168).
Site visit interviewers were allowed to follow the protocol for telephone surveys (see Section
2.2.2) for subjects living in remote areas and for individuals who refused a site visit.
2.2.2 Ottawa, Kingston, Peterborough, Orillia, and Toronto Regions

Data for respondents living in the Ottawa, Kingston, Peterborough, Orillia, and Toronto public
health laboratory regions were collected by telephone interview. Trained professional interviewers
from a telephone survey company called the submitters. After explaining the study, they completed
household interviews with consenting submitters. Personal questionnaires were completed for
submitters and other eligible household members who verbally consented and agreed to submit a rectal
Methods
29
swab. Telephone surveys were entered directly into a database using a computer assisted telephone
interview (CATI) system.
Rectal swab sampling kits v were mailed to all participants who completed personal
questionnaires by telephone. Swabs and consent forms were mailed to the laboratory and study
coordinator, respectively, by the participant. Participants were contacted by telephone if the rectal
swab had not arrived at the laboratory within four weeks of mailing it to the participant.
2.2.3 Questionnaires
The household questionnaire was completed by an adult, either the person who submitted the
water sample for bacteriological testing or their designate (e.g. spouse/partner). The household
questionnaire included information about the water source as well as several questions about the
household including the number and age of household members, occupations of household members,
county of residence, pets, livestock, water treatment(s), and household income. It took about 20
minutes to complete. When data were collected by site visit, physical measurements were made by the
interviewer to determine distances between the water source and possible sources of contamination.
People completing questionnaires by telephone were asked to estimate these distances.
Individual (personal) questionnaires were completed by household members, 12 years of age
and older, who consented to participate and to provide a rectal swab for analysis. The questionnaire
collected information about the participants age, sex, length of residence at the site, underlying
disease processes, hospitalization, antibiotic use, and contact with or consumption of possible sources
of antimicrobial resistant E. coli including animals, meat, water, and human or animal waste.
Questionnaires took about 10 minutes to complete and were not necessarily collected in isolation from
other household members. No proxy interviews were allowed.
Household and personal questionnaire items were developing following a review of the literature
to determine the topic areas that needed to be covered to illicit information on risk factors for
bacterial contamination of well water and the development and transmission of antimicrobial resistant
enteric bacteria as well as variables that might confound the associations under study. Items were
fashioned after items from a variety of questionnaires including the Canadian Community Health Survey
v
rectal swab sampling kits contained a letter from the study coordinator, swab collection instruction sheet,
stamped self-addressed envelopes, swabs, labels, and consent forms
30
(265), the Canadian Census (72), the draft Households and Environment Survey (73), the Rapid Risk
Factor Surveillance System (266), and an enteric outbreak case management questionnaire from a local
public health unit (267). The questionnaires were reviewed by several content experts including M.
Louie, S. McEwen, A. McGeer, I. Johnson, I. Gutmanis, and S. Bondy. They were pilot tested with 12
volunteers, including 8 adults and 4 adolescents of both sexes who lived on farm and non-farm rural
properties.
2.2.4 Laboratory Testing of Water and Rectal Swab Samples

Water samples were processed and E. coli-positive water samples identified by public health
laboratory personnel according to standard methods (268). All E. coli colonies on each selected plate
were picked onto a swab creating a single pool of colonies to enhance the likelihood of identifying
antimicrobial resistant isolates from an individual water sample. Swabs were shipped to the study
laboratory (Provincial Laboratory, Calgary) on trypicase soy agar slants. Screening for antibiotic
resistance was performed by the agar screen plate method using antibiotic concentrations listed in
Table 2.1. Isolates growing on agar screen plates were confirmed as E. coli using standard biochemical
tests vi . Escherichia coli isolates that screened positive for resistance to any antibiotic were sent to the
Laboratory for Foodborne Zoonoses, St. Hyacinthe, Quebec to have broth microdilution susceptibility
testing using the 2002 and 2004 NARMS antimicrobial susceptibility panel for enteric bacteria (259;260).
Rectal swabs collected by study participants were mailed to the study laboratory in Carey Blair
transport media. Upon receipt, swabs were inoculated into tryptic soy broth and incubated overnight.
A 1 mL aliquot of the overnight broth was archived and frozen at -70C for future susceptibility testing.
A swab of the frozen sample was inoculated onto MacConkey agar with crystal violet. Lactosefermenting colonies growing on the antibiotic screen plates were screened to identify E. coli isolates.
Antimicrobial resistant E. coli were sent to the Laboratory for Foodborne Zoonoses to be tested using
the 2004 NARMS panel.
Susceptibility testing was limited to water and faecal samples that were not susceptible to
antibiotics used in the screening panel and also confirmed as being E. coli using API-E20 (BioMerieux
Canada Inc.) assay. Antimicrobial susceptibility results were interpreted using resistance breakpoints
vi
Citrate, indole, malonate metabolism
31
Methods
relevant to human health as outlined by the National Committee for Clinical Laboratory Standards
(269).
2.3 Data Analyses

These analyses were based on the prevalence study conducted with a convenience sample of
people living in households that participated in the case-control study (Table 2.1). Questionnaires of
people who did not submit a swab or submitted a swab that was not screened for antimicrobial
resistance were excluded from analyses. Because of the change from the 2002 to the 2004 NARMS
panels part way through the study, with the substitution of sulphamethoxazole with sulfisoxazole and
the elimination of cephalothin, we excluded households that had water samples that were resistant
only to sulphamethoxazole or cephalothin and individuals that had rectal swabs that were resistant
only to sulfisoxazole.
Data from six different sources were ultimately joined to create one data file. These sources
included the Calgary laboratory files for rectal swab and water screening results, the Laboratory for
Foodborne Zoonoses results files for rectal swabs and water tests, the Compustat Consultants file of
telephone interviews, and the file of site interviews.
A comparison of households participating in the case-control study only (household questionnaire
only) and the prevalence/cross-sectional study (household questionnaire and one or more personal
questionnaires with rectal swabs) was completed using Pearson chi square tests of independence.
Similarly, a comparison of participants who completed a questionnaire without submitting a swab (or
who submitted a swab not eligible for inclusion) and subjects who completed a questionnaire and
submitted a swab that was used in the analyses was completed using Pearson chi square without
adjustment for household clustering.
2.3.1 Objective 1: Prevalence of Antimicrobial Resistant E. coli

Prevalence of carriage of antimicrobial resistant E. coli in this study sample was determined
using the total number of isolates (1 per rectal swab) in which the E. coli was resistant to at least one
antibiotic included in the 2004 NARMS for enteric bacteria panel (see Table 2.2 for breakpoints) divided
by the total number of isolates that grew E. coli. Antibiotic specific prevalence was calculated as the
32
proportion of all E. coli-positive isolates that were resistant to the individual antibiotic. Multi-drug
resistance was defined as resistance to two or more classes of antibiotics included in the panel.
Estimate intervals were presented with 95% probability confidence limits. A Z-approximation was used
to test the first hypothesis. Chi-square tests were used to determine if there were differences in the
proportion of subjects carrying ampicillin resistant E. coli, by risk factor. The variances for these
estimates were adjusted to account for the non-independence of observations within households using
the household identifier as the primary sampling unit in the Stata survey command which restricts the
degrees of freedom to the number of clusters rather than the number of observations and adjusts the
variance for non-independence of observations.
Inter-class resistance is a measure of bacterial resistance between two classes of antibiotics
while intra-class antimicrobial resistance is resistance to two or more drugs within the same class of
antibiotics. Both were calculated by taking the sum of the observed resistances to both antibiotics (or
classes) divided by the observed resistances in the antibiotic (or class) of interest (e.g. inter-class
resistance of tetracycline with -lactam: number of samples resistant to -lactam and tetracycline
divided by number resistant to tetracycline).
Resistance scores (the total number of observed resistances divided by the total number of
possible resistances) were reported to reflect the burden of multi-drug resistance (184;197). Resistance
scores are presented to enable readers to compare the extent of multi-drug resistance when the
studies use a different number of antibiotics in their panels.
The observations were re-weighted to provide a prevalence estimate removing the impact of
the sampling strategy. Since case and A control households were over-represented in the sample,
the households were given weights that represented their probability of selection from the sampling
frame. The weights were based on the known results of water tests completed during the study period
but could not be adjusted for repeat submissions from the same household. The variances for these
estimates were also adjusted to account for non-independence of observations within households
(clustering).
The direct age-standardized prevalence was also calculated. Since the vast majority of
residences supplied with private water sources are located in rural areas, this was done using the 2006
estimated population for rural Ontario residents as the standard population (270).
33
Methods
2.3.2 Objective 2: Association Between Water Consumption and Human Carriage of Antimicrobial
Resistant E. coli
The dependent variable was the subjects laboratory results regarding whether the E. coli
detected in their rectal swab was either (0) susceptible to all or (1) resistant to one or more of the
antibiotics included in the 2004 NARMS susceptibility panel for enteric bacteria.
The independent variable of primary interest was the use of water that was either (1)
contaminated with E. coli that was resistant to one or more antimicrobial agents included in both the
2002 and 2004 NARMS panels and which was not treated for bacterial contamination or (0) water that
was not contaminated with E. coli, contaminated with E. coli that was susceptible to all antibiotics in
the panel, or contaminated with antimicrobial resistant E. coli but treated for bacterial contamination
for one year or longer from the date of the interview. Treatment for bacterial contamination was
defined as drinking water that was boiled or treated with chlorine, ultraviolet light, or ozone (77;78).
We determined that one year of treatment was required to take into account the possible lag time
between beginning the water treatment and potential elimination of antimicrobial resistant E. coli
from the users gastrointestinal system (carriage in humans was detected for up to 10 months in one
longitudinal study (149)).
The covariates of interest as possible predictors of human carriage of antimicrobial resistant E.
coli or confounders of the association of interest are presented in Figure 2.1. The covariates, including
how variables were derived, are outlined in Appendix L.
34
Figure 2.1
The Theorized Relationship between Human Carriage of E. coli, Use of Contaminated
and Untreated Water, and Other Study Variables Used for Multivariable Model Building; Survey of
Households using Private Water Sources, Southern Ontario, 2005-2006
Potential effect modifier:

- Used bottled water
Primary predictor:
- Water used
Outcome:
- AR E. coli carriage
Potential confounders:
- Age
- Antibiotic use
- Sex
- Hospitalization
- Household education
- Travel
- Household income
- Child in day care
- Laboratory region
- Household size
- Mode of data collection
- Contact with livestock
- Days between water
- Farming property
sample & interview
- Contact with dog/cat
- Contact with raw meat
AR: antimicrobial resistant (to any antibiotic in the both the 2002 and 2004 panels)
Primary predictor: Use of untreated, antimicrobial resistant E. coli-contaminated water versus
uncontaminated water; contaminated, treated water; or water with susceptible E. coli (water sample)
Outcome: Faecal carriage of antimicrobial resistant E. coli versus susceptible E. coli (rectal swab)
Potential effect modifier: Use of tap water versus use of both tap and bottled water versus use of
bottled water
Potential confounders:
- Demographic variables: age, sex, education, income
- Based on study design: laboratory region, mode of data collection, days between water sample
and personal interview (rectal swab collection)
- As identified by literature review: antibiotic use, hospitalization, travel, child in day care,
household size, contact with livestock, farming property, contact with pets, contact with raw
meat/poultry
Methods
35
Although the dependent variable was binary, logisitic regression was not ideal for the analyses
since data collection was cross-sectional. The preferred measure was the relative risk or prevalence
ratio rather than the odds ratio. Poisson regression has been identified as an alternative that can
provide correct estimates of relative risk while adequately representing the association between the
dependent and independent variables (271-273) and hence was chosen for these analyses.
The observations in this data set were not independent as several individuals from one
household were eligible to participate in the prevalence study. To account for the statistical nonindependence of the observations, we analyzed the data using a generalized linear model with the
cluster estimation option in Stata, version 9.2 (274). This estimation option specifies that
observations are independent across groups but not necessarily independent within groups/clusters.
The equation uses a robust (or Huber-White) estimate of variance that produces correct standard errors
even if observations are correlated (275) and adjusts the Poisson regression error variance to account
for its conservative results when the dependent variable is binary (273).
Multivariable Poisson regression was used to determine the relationship between the
dependent and independent variables while taking into account other covariates; the second objective.
How a regression equation is constructed drives the process and depends on the goal of the analysis
(276).
Model-building strategies.
There are generally three distinct forms of research objectives that can be met through the
development of regression models and each argues for a different approach to selection of the best
model. The first is exploratory (277-280). This approach is used to determine multiple important
predictors of an outcome. The emphasis of model-building is on what covariates to include in the
model.
In contrast to the exploratory approach is the predictive approach. This second reason for
developing regression models is to predict the outcome of future observations (278-283). This approach
is usually more concerned with the accuracy of the prediction of the overall model rather than the
value of the individual coefficients. The goal is to include as many variables as necessary to accurately
36
predict the outcome of current and/or future observations. Cross validation and/or data splitting is
often used to determine the predictive ability of the covariates (281).
Kleinbaum, Klein, Austin, Aneschensel, Stahel, Sauerbrei, Vittinghoff, and others describe an
explanatory model-building process. Explanatory model-building assesses the association between an
exposure and an outcome of primary interest with the goal of producing an accurate, unbiased
estimate of the relationship between them (277-283). This is a hypothesis-driven process that takes
into account apriori-identified confounders, moderators, and effect modifiers. The goal is to develop a
model that rules out confounding of the focal relationship by other variables.
Focal relationship analysis.

The goal of our analysis was explanatory: to determine whether the focal relationship
(between human carriage and use of water contaminated with resistant E. coli) remained after
adjusting for confounding and effect modification. We used the strategy outlined by Vittinghoff et al.
(280) who suggest including all potential confounders including those established through previous
studies, those hypothesized to matter on substantive grounds, and any variables that act like
confounders during statistical analysis. This is done to rule out confounding as much as possible.
Confounding occurs when the crude association fails to reflect the size and/or direction of the
exposure effect because of different distributions of a third variable among exposed and unexposed
subjects. A variable is confounding to the association if it is a) predictive of the outcome, b) associated
with exposure, and c) not affected by exposure or outcome (284). Meaningfully different
interpretations of the focal relationship occur when a confounding variable is included or excluded
from the analysis. Confounding can be controlled for through stratification, matching, or regression
analyses. We used regression analysis as it provides an estimate of the focal relationship while holding
the other variables constant, which is the main goal of explanatory model building (280).
Variables were entered into the original (full) model if the association with the dependent
variable had a p-value of 0.25 on bivariate analysis (276). The product term of the primary predictor
and the theorized effect modifier were also included in the full model, along with the product term of
the primary predictor and biologically-plausible confounding variables. Interaction, or effect
modification, exists when the association between the outcome and exposure is different at different
37
Methods
levels of a third variable (effect modifier). The inclusion of biologically-plausible combinations of

variables are included in multivariable regression analyses to determine whether effect modification
exists (276;280).
The model was reduced by removing variables with the largest p-value, one at a time, until
only variables with a p-value of 0.20 (276;283;285-287) and those that changed the estimate of the
primary predictor by more than 10% (280;282;288) remained. To reduce the potential bias of missing a
confounding variable, all variables were added back into the model, one at a time, to identify variables
that were important to the model in the presence of other variables (276). Continuous variables were
assessed for linearity and transformed or categorized, if necessary. Analyses were run again after
variables were transformed.
Regression diagnostics were also performed. Tables of residual values were examined and
residual values were graphed to determine if there were covariate patterns that were outliers or had
high leverage influence values (276;280). The statistical impact of outlying or high leverage
observations was examined by analyzing the data after removing the observations from the data set.
The final model was assessed to determine if the Poisson distribution adequately described the
association between the dependent and independent variables. The response variance was not
expected to be meaningfully different than the mean as using Poisson regression with binary data
(denominator of 1) can not produce over-dispersed errors (280;289). The specification test, a summary
measure of the models goodness of fit, and the Akaike and Bayesian information criteria were
reviewed for the final model.
The data were also analyzed using generalized estimating equations with exchangeable
correlation within households (clusters), a Poisson distribution, robust standard error estimates, and
population-averaged equations. The parameter estimates, standard errors, and model-building results
were compared with those resulting from the generalized linear model equation.
2.3.3 Students Role

The candidate was involved in the CIHR-Health Canada-funded surveillance study since
September 2004. She was instrumental in determining the design of the case-control and prevalence
studies. Her original proposal was pared down, due to budget constraints, by excluding a three-month
38
follow-up of wells and participants. She worked closely with other members of the research group in
writing the proposal for additional funding, which was granted by the Physicians Services Incorporated
Foundation, Ontario. The proposal was mirrored for funding received from the Alberta Heritage
Foundation for an expansion to the Ontario study completed in Alberta.
The candidate was instrumental in the implementation of the Ontario case-control and
prevalence studies. She worked with the laboratories, public health units, and Ministry of Health and
Long-Term Care to ensure their cooperation in the study. Further, she drafted and pre-tested the
questionnaires, scripts, consent forms, and information sheets including necessary changes for the
telephone surveying component of the study. The candidate determined the need to implement
changes to the protocol and implemented the resulting expansion and extension of the study. She
wrote the applications for ethics review and amendments to the approvals received from the University
of Western Ontario and University of Toronto research ethics boards.
The candidate was the coordinator of the Ontario site of the well water study, which included
hiring, training (including the development of a training manual), and supervising interviewers, callers
at the Ministry of Health and Long-Term Care, and the telephone survey company. She managed the
flow of information between the laboratories, callers, interviewers, and data entry clerk and managed
the databases. The candidate was also the contact person listed on the information sheet and consent
forms making her the contact for the public.
The candidate was responsible for the cleaning, editing, and analysis of the case-control and
prevalence studies and will also be responsible for providing data to other students and researchers
involved in the CIHR-Health Canada surveillance study. She has been and will be involved in the
preparation and submission of the case-control findings and will be lead author on publications
submitted from the prevalence study.
Results
39
Chapter 3
Results
3.1 Characteristics of Water Samples, Households, and Respondents
3.1.1 Water samples
In total, 342,009 water samples submitted to the seven participating public health laboratories
during the study period (May 2005 to September 2006) were tested for bacterial contamination.
Recalling that multiple water samples may be submitted from one household, 15,238 (4.5%) water
samples were contaminated with E. coli, 60,540 (18%) were contaminated with non-E. coli coliform
bacteria, 12,139 (3.5%) were overgrown with non-E. coli bacteria, and 254,092 (74%) had no bacterial
contamination (see Table 3.1). Of the E. coli-positive samples, 6,492 were sent to the study laboratory
for susceptibility testing and 645 (10%) were antimicrobial resistant. Assuming the samples sent to the
study laboratory were representative of all samples, about 0.4% of all water samples tested by the
participating public health laboratories were contaminated with antimicrobial resistant E. coli.
Due to selection of households for participation in the case-control study, 22% of households
participating in the study had water contaminated with antimicrobial resistant E. coli (Table 3.2).
3.1.2 Participation
Dwelling questionnaires were completed by 880 of the 1,717 (51%) eligible households in
southern Ontario (Tables 3.1 & 3.3). Personal questionnaires were completed by 1,007 individuals from
671 households (average 1.5 interviews per household). Three-quarters of individual respondents (752
or 75%) submitted a rectal swab and the laboratory was able to detect E. coli on 703 (94%) of the 746
swabs that were screened. Four swabs were not eligible for the final analysis because the E. coli were
resistant only to sulfisoxazole (which was not tested for in all submitted water samples). The final
analyses were conducted using the resulting 699 swabs and personal survey responses with the
associated 488 household questionnaires.
40
Table 3.1
Details of Household Recruitment and Participation for Surveillance Project and CaseControl Study, Antimicrobial Resistant E. coli in Private Water Sources, Southern Ontario, 2005-2006
342,009 water samples2
Water samples: total tested1
Water samples : results of bacteriological
15,238 (4.5%)
254,092
E. coli-positive
No bacteria
testing
Water samples: screened for resistance
6,492 water samples
Water samples: results of antimicrobial
645 (9.9%)
5,856
susceptible
resistance testing
resistant
645
840
846
(Case)
(A control)
(B control)
Water samples: selected for study
TOTAL
3
Households: not eligible for study
202
180
232
614
eligible for study
443
660
614
1,717
143
151
164
458
Households: refused
unable to contact
29
29
27
85
agreed to be called by study
272
480
423
1,175
Households: refused survey
88
107
92
287
completed survey
184
369
327
880
Households: eligible for analysis
108
196
184
488
1
Includes all water samples tested for bacteriological contamination, including multiple samples from
the same household
2
Includes 72,679 not eligible for the study (contaminated with non-E. coli coliform bacteria [n=60,540]
or overgrown with no E. coli detected [n=12,139])
3
Unique and eligible households (see section 2.1.1 for criteria)
Results
41
Table 3.2
Proportion of E. coli-positive Water Samples with Antimicrobial Resistant
E. coli, by Antibiotic and Class of Antibiotic, Survey of Households using Private Water Sources,
Southern Ontario, 2005-2006
All water samples
Participating households only
Number2
%
Number
%
positive
positive
CI95%
CI95%
ResisResis(N=6,492)
(N=488)
Antibiotic1
tant
tant
Resistant to one or more antibiotics
645
10%
9, 11
108
22%
18, 26
Multi-drug resistance (2+ classes)
394
6%
5, 7
64
13%
10, 16
236
4%
3, 4
38
Aminoglycoside
8%
5, 10
Amikacin
0
----0
----Gentamicin
25
0.4%
0, 1
6
1%
0, 2
Kanamycin
69
1%
1, 1
11
2%
1, 4
Streptomycin
209
3%
3, 4
33
7%
5, 9
Beta-lactam
268
4%
3, 5
49
10%
7, 14
58
1%
1, 1
10
2%
1, 3
Ampicillin
268
4%
4, 5
49
10%
7, 13
Cefoxitin
15
0.2%
--9
2%
1, 3
Ceftiofur
38
0.6%
0, 1
7
1%
0, 3
Ceftriaxone
3
0
--1
0.2%
0, 1
1%
0, 2
Fluoroquinolone
0, 1
5
30
0.5%
0.2%
0, 1
--1
Ciprofloxacin
5
0.1%
0, 2
5
1%
0.5%
0, 1
Nalidixic acid
30
295
Sulphonamide
5%
4, 5
53
11%
8, 14
133
Trimethoprim-sulfamethoxazole
2%
2, 2
22
5%
3, 6
Sulfisoxazole (n=3602)
1022
3%
2, 3
20
10%
6, 14
Sulphamethoxazole (n=2890)
1792
6%
5, 7
29
10%
7, 14
Tetracycline
498
8%
7, 8
86
18%
14, 21
Chloramphenicol
50
1%
1, 1
13
3%
1, 4
Cephalothin (n=2890)
562
2%
1, 2
14
5%
2, 7
CI: Confidence interval (95%)
1
Using the 2004 NARMS panel for enteric bacteria
2
Number of E. coli-positive water samples tested is 6,492 unless otherwise stated (to account for
change from 2002 to 2004 NARMS panel)
Table 3.3
Details of Subject Participation in Prevalence Study, Survey of Households using Private
Water Sources, Southern Ontario, 2005-2006
Households
Individuals
Eligible for case-control study
1,717
Completed household survey
880
Household survey only
209
Completed household & personal surveys
671
1,007
Submitted rectal swab
752
Lost or damaged in transit
6
Swabs screened for E. coli
746
No E. coli
43
Swabs screened for resistance
703
Not eligible (only sulfisoxizole resistant)
4
Swabs used in analysis
488
699
42
As shown in Table 3.4, there were few differences between the households that participated in
the prevalence study (completed a household questionnaire and one or more individuals completed a
personal questionnaire and submitted a swab) and those that participated only in the case-control
study (completed only a household survey). Households that participated in the prevalence study had
fewer household residents, with an average of 2.6 household members compared to 3.0 members in
households that took part only in the case-control study (p=0.001). Households that participated in the
prevalence study were also more likely to state their household income than households than only took
part in the case-control section. Households in the London and Hamilton regions were more likely to
participate in the prevalence study, which is associated with the mode of interview: people
interviewed face-to-face were significantly more likely to participate in both the case-control and
prevalence studies than people interviewed by telephone and asked to post the swabs at a later date.
On crude analysis (not adjusted for household clustering), individuals who participated in the
prevalence study by completing a personal questionnaire and submitting a rectal swab were older
(mean: 58 years) than individuals who completed a personal questionnaire without submitting a swab
or for whom the swab was not usable (mean: 52 years; p<0.001). As shown in Table 3.5, individuals 49
years of age and younger were less likely to submit a swab after completing a personal questionnaire
than older individuals (p<0.01). Similarly, individuals in recent contact with horses were less likely to
submit a swab than those without recent contact but who completed a personal questionnaire
(p=0.03). In comparison, individuals who used tap water exclusively were more likely to submit a swab
than people who used bottled water (p=0.01). Subjects who travelled outside Canada in the previous
year were more likely to submit a swab than those who did not travel (p=0.05), and participants who
were in contact with poultry in the previous three months were more likely to submit a swab than
those without contact but who completed a questionnaire (p=0.02).
Results
43
Table 3.4
Comparison of Households Participating in Prevalence and Case-control Studies; Survey
of Households using Private Water Sources, Southern Ontario, 2005-2006
Prevalence with
Case-control only
swab
p-value
% of
% of
Household-level item
2
Number
Number
(N=488)*
households
(N=392)*
households
Water source
Contaminated with resistant E. coli
108
22%
76
19%
Contaminated with susceptible E. coli
196
40%
173
44%
No bacterial contamination past year
184
38%
143
36%
NS
Water treated1 (n=487; 390)
243*
50%
177*
45%
NS
Household size (n=488; 390)
53
36
1
11%
9%
2
273
56%
163
42%
3 or 4
24%
115
127
33%
5 to 10
10%
47
64*
<0.01
16%
Children in household (1 or more)
20%
77
13%
64
12-19 years
19%
76
11%
56
4-11
12%
48
7%
32
<4
11%
45
8%
40
In diapers
NS
4%
17
2%
9
Attend day care
Farming property
134
27%
92
23%
NS
Highest education in household
26
7%
38
8%
Less than high school
67
17%
74
15%
Graduated high school
31%
122
30%
148
College or trade school
39%
44%
153
University
214
NS
6%
3%
24
Not stated
14
Household income2
10%
41
16%
80
<$40,000 annually
27%
104
32%
156
$40,000-79,999
31%
121
29%
140
$80,000 or more
<0.01
32%
126
23%
112
Not stated
Laboratory region3
38%
150
46%
223
London
15%
59
27%
131
Hamilton
13%
50
8%
37
Kingston
10%
39
7%
34
Orillia
11%
44
7%
34
Peterborough
6%
25
4%
18
Ottawa
<0.01
6%
25
2%
11
Toronto
Mode of interview
356
91%
63%
308
Telephone interview
37%
36
9%
<0.01
180
Site visit (face-to-face interview)
*
N=488 and 392 households unless otherwise stated (i.e. incomplete data for item)
1
Water treated by boiling, chlorination, ultraviolet light, or ozonation
2
Annual household income, before taxes, for all members in household (including net farm income)
3
Laboratory to which the water sample was submitted (not necessarily household location)
Chi square test of independence between case and control households
44
Table 3.5
Comparison of Participants who Completed Personal Questionnaires and Submitted a
Rectal Swab and Subjects Who Completed Questionnaires Only or Submitted an Ineligible Swab, Survey
of Households using Private Water Sources, Southern Ontario, 2005-2006
Questionnaire &
Questionnaire
p-value
only or ineligible
swab
swab
Item
2
Respondents age at time of interview
12-29 years
30-39
40-49
50-59
60-69
70-79
80 & older (n=699; 305)
Respondents sex
Female
Male
Hospitalized (over night) in past year
Antibiotic used, past 3 months
Currently using antibiotic
Chronic condition
Diabetes mellitus
Crohns, celiac, IBS, etc.
Heart disease/high blood pressure
Arthritis/rheumatism
Tap or bottled water1
Tap water only
Tap and bottled
Bottled only
Raw milk consumed, regularly (n=698)
Travelled outside Canada, past year
Contact in past 3 months
Raw red meat (n=697; 306)
Raw poultry products (n=696; 305)
Dogs (n=698; 308)
Cats (n=699; 307)
Horses (n=699; 306)
Poultry (chicken,turkey)(n=699; 307)
Cattle (dairy or beef) (n=698; 306)
Sheep or goats (n=699; 306)
Pigs (n=697; 305)
*
1
Number
(N=699)*
% of
subjects
Number
(N=308)*
% of
subjects
31
49
97
175
207
122
18*
4%
7%
14%
25%
30%
17%
3%
30
32
74
73
51
35
10*
9%
10%
24%
24%
17%
11%
3%
<0.01
346
353
55
85
6
49%
51%
8%
12%
1%
143
165
34
33
2
46%
54%
11%
11%
1%
NS
NS
NS
NS
41
51
206
233
6%
7%
29%
33%
16
25
79
85
5%
8%
26%
28%
NS
NS
NS
NS
433
133
133
48*
376
62%
19%
19%
7%
54%
161
79
68
18
145
52%
26%
22%
6%
47%
0.01
NS
0.05
562*
543*
528*
392
94
80
79*
39
27*
81%
78%
76%
56%
13%
11%
11%
6%
4%
250*
241*
243
171*
57*
20*
36*
20*
9*
82%
79%
79%
56%
19%
7%
12%
7%
3%
NS
NS
NS
NS
0.03
0.02
NS
NS
NS
N=699 and 308 subjects unless otherwise stated (i.e. incomplete data for item)
Tap water only: do not used bottled water at home on regular basis; Tap and bottled: glasses
of water [total] > glasses bottled; Bottled water only: glasses of water [total] = glasses bottled
Unadjusted Chi square test of association between respondents submitting swabs and those
who only completed the personal questionnaire or submitted an ineligible swab
Results
45
3.1.3 Households
Table 3.6 highlights the demographic information about the 488 households in which the survey
respondents lived. One hundred eight households (22%) had water that was contaminated with
antimicrobial resistant E. coli, 196 (40%) had water that was contaminated with E. coli that was
susceptible to the tested antibiotics, and 184 (38%) had water than had not been contaminated with E.
coli or other coliform bacteria for one year or longer. This finding mirrors the selection of households
for the case-control section of the project, not the probability of contamination of private water
sources (see Section 3.1.1).
One-half of the households in this study (243 of 488) used a treatment to kill water-borne
bacteria (boiling, chlorine, ozone, or ultraviolet light). Households that had E. coli-contaminated water
were significantly more likely to treat their water than households with no recent history of
contamination (54% versus 43%; p=0.02).
Of the 108 households that had water contaminated with antimicrobial resistant E. coli, 54
households (50%) met the criteria to be classified, for this study, as treating their water for bacterial
contamination (chlorine, ultraviolet light, ozone, or boiled). However, 15 of these households had been
treating their water for less than one year. Thus, 69 households, representing 94 individuals, were
classified as exposed to untreated, contaminated water for the analyses. The only difference between
case and control households was the proportion of households that treated their water for bacterial
contamination.
Household size ranged from 1 to 10 people, with the median size of 2 people per household
(mean 2.7). Sixty-four households (13%) had youths 12 to 18 years of age living in them, 11% had
children 4 to 11 years of age, and 7% had children under 4 years old. Forty households (8%) had one or
more children that used diapers while only nine (2%) of these largely rural households had a child that
attended day care.
Three hundred sixty-two households (74%) had one or more household members with a college or
university education. Two hundred ninety-six households (61%) earned $40,000 or more per year
although 112 (23%) did not answer the questions regarding household income. One hundred thirty-four
households (27%) were located on farming properties.
46
As expected, given the sampling strategy used for the case-control study, most households (73%)
were located in the London (n=223) and Hamilton (n=131) public health laboratory regions. Three
hundred eight households (63%) were interviewed by telephone and the remaining 180 were
interviewed face-to-face in their home (site visit). Households in the London and Hamilton regions
were less likely to be interviewed by telephone than households in the Ottawa, Kingston,
Peterborough, Orillia, and Toronto regions (49% versus 99%; p<0.01).
Results
47
Table 3.6
Descriptive Statistics of Participating Households; Survey of Households using Private
Water Sources, Southern Ontario, 2005-2006
Resistant E. coli
Susceptible
All households
Household-level item
Number
(N=488)*
% of
households
Number
(N=69)*
% of
households
Number
(N=419)*
% of
households
Water source
69
100%
39
22%
9%
Contaminated with resistant E. coli
108
40%
196
196
47%
Contaminated with susceptible E. coli
38%
184
184
No bacterial contamination past year
44%
Water treated1 (n=487)
243*
50%
15
22%
228
54%
Household size2
1
53
11%
11
16%
10%
42
2
273
56%
31
45%
242
58%
3 or 4
115
24%
19
27%
96
23%
5 to 10
47
10%
8
39
12%
9%
Children in household (1 or more)
13%
54
10
14%
13%
12-19 years
64
11%
47
9
13%
11%
4-11
56
6%
26
9%
7%
6
32
<4
6%
24
7%
8%
5
40
In diapers
2%
9
0
2%
0
9
Attend day care
3
Farming property
134
27%
24
35%
110
26%
Highest education in household4
33
8%
7%
5
38
8%
15%
16%
63
11
15%
74
31%
29%
128
20
30%
148
43%
46%
182
32
44%
University
214
3%
1%
13
1
3%
Not stated
14
Household income5
16%
69
16%
11
16%
80
<$40,000 annually
32%
133
33%
23
32%
156
$40,000-79,999
28%
119
30%
21
29%
140
$80,000 or more
23%
98
20%
14
23%
112
Not stated
Laboratory region6
46%
194
42%
29
46%
223
London
26%
108
33%
23
27%
131
Hamilton
8%
32
7%
5
8%
37
Kingston
7%
30
6%
4
7%
34
Orillia
4%
16
3%
2
7%
34
Peterborough
7%
30
6%
4
4%
18
Ottawa
2%
9
3%
2
2%
11
Toronto
Mode of interview
64%
270
55%
38
63%
308
Telephone interview
36%
149
45%
31
37%
180
Site visit (face-to-face interview)
*
N=488, 69, or 619 households unless otherwise stated (i.e. incomplete data for item)
1
Water treated by boiling, chlorination, ultraviolet light, or ozonation
2
Household size (all): mean: 2.6; median: 2; range: 1-10 people
3
As described by person completing household questionnaire
4
Highest level of education attained by any member in household
5
6
Chi square test of independence of households with/without contaminated water: p-value of 0.05
48
3.1.4 Respondents
As detailed in Table 3.7, the age of the 699 respondents ranged from 12 to 87 years (median 59;
mean 58). Three hundred eighty-two respondents (55%) were 50 to 69 years of age with only 31
respondents (4%) 12 to 19 years old. Three hundred fifty-three (51%) respondents were male and 346
(49%) were female. The ages and sex of respondents were similar for those from households that had
water that was or was not contaminated with antimicrobial resistant E. coli.
The number of days between the submission of the water sample to the regional public health
laboratory for bacteriological testing and the date of the interview (proxy for date of rectal swab
collection) ranged from 3 to 439 days. The number of days between water submission and interview
had a skewed distribution with the median lower than the mean (125 and 142, respectively). The
respective median (122 and 125 days) and mean (155 and 140 days) lag times were similar for
respondents from households that had water that was and was not contaminated with E. coli that was
antimicrobial resistant (p=0.12).
Fifty-five respondents (8%) reported being hospitalized in the previous year and 85 (12%) reported
using an antibiotic in the previous three months (6 were using antibiotics at the time of the interview).
Forty-one respondents (6%) reported having been diagnosed with diabetes mellitus, 51 (7%) with an
underlying gastrointestinal condition (e.g. Crohns disease, celiac disease, colitis, ileitis, or irritable
bowel syndrome), 206 (29%) with heart disease, and 233 (33%) with arthritis or rheumatism.
Four hundred thirty-three respondents (62%) did not use bottled water regularly (most days of
the week) at home. Of the 266 respondents (38%) who reported using bottled water at home on a
regular basis, half (n=133) used bottled water almost exclusively vii while the other half (n=133) used
both bottled and tap water. Respondents from households with E. coli-contaminated water (n=429),
not antimicrobial resistant E. coli specifically, were significantly more likely than respondents from
households with no recent history of bacterial contamination (n=270) to use bottled water exclusively
(24% and 15%, respectively) or to use both bottled and tap water (21% and 12%, respectively) (p<0.001).
Similarly, respondents in households using water contaminated with antimicrobial resistant E. coli were
significantly more likely than subjects in other households to use bottled water (p=0.02).
vii
Glasses of water consumed (total) = glasses of bottled water consumed
Results
49
Although only 185 respondents (27%) had direct contact with farm livestock or their manure, 528
(76%) had direct contact with dogs and 392 (56%) had contact with cats in the previous three months.
Of interest, although males were more likely than females (14% versus 8%) to have had direct contact
with cattle (or cattle manure) in the previous three months (p=0.02), there were no significant
differences between the sexes regarding contact with any other animals.
Forty-eight subjects (7%) drank raw milk or ate dairy products made with unpasteurized raw milk
in the previous three months. Raw dairy product consumption was more common among people living
on a farming property (n=30 or 15%) than subjects not living on a farm (n=18 or 4%; p<0.001).
Five hundred sixty-two respondents (81%) had handled raw red meat (beef, pork, or lamb) or raw
poultry (78%). Females were significantly more likely than males (95% vs. 75%) to have handled raw
meat or poultry in the previous three months (p<0.001).
Over one-half (n=376 or 54%) of respondents had travelled outside Canada in the previous year.
One hundred seventy-one (25%) subjects travelled outside both Canada and the United States with 73
respondents visiting countries in the Caribbean and Central or South America, 34 visiting Mexico, and
the remainder visiting Europe, Australia, New Zealand, Asian or Indonesian countries, or Russia.
50
Table 3.7
Descriptive Statistics of Participants, Survey of Households using Private Water Sources,
All subjects
Resistant E. coli
Susceptibile
Number
% of
Number
% of
Number
% of
(N=699)*
subjects (N=94)* subjects (N=605) subjects
Individual-level item
Respondents age at time of interview
12-29 years
31
4%
5
5%
26
4%
30-39
49
7%
12
13%
37
6%
40-49
97
14%
15
16%
82
14%
50-59
175
25%
22
23%
153
25%
60-69
207
30%
29
31%
178
29%
70-79
122
17%
11
12%
111
18%
80 & older
18
3%
0
0
18
3%
Respondents sex
49%
298
51%
48
49%
Female
346
51%
307
49%
46
51%
353
Male
Lag: water submission to interview
130
3-59 days
21%
135
5%
5
19%
90
60-99
15%
29%
117
27
17%
100-139
114
19%
19%
18
132
19%
140-199
130
17%
21%
16
146
21%
200-439
141
30%
28
23%
169
24%
Hospitalized (over night) in past year
55
8%
9
10%
46
8%
Antibiotic used, past 3 months
85
12%
10
11%
75
12%
Currently using antibiotic
6
1%
0
0
6
1%
Chronic condition
6%
37
4%
4
6%
Diabetes mellitus
41
8%
48
3%
3
7%
Crohns, celiac, IBS, etc.
51
31%
186
21%
20
29%
206
Heart disease, high blood pressure
34%
206
29%
27
33%
233
Arthritis/rheumatism
63%
384
52%
49
62%
433
Tap water only
19%
116
18%
17
19%
133
Tap and bottled
17%
105
30%
28
19%
133
Bottled only
Raw milk consumed, regularly (n=698)
48*
7%
7
7%
41
7%
Travelled outside Canada, past year
376
54%
47
50%
329
54%
Travelled outside Canada & USA
171
25%
23
24%
148
24%
80%
481
86%
81
81%
562*
Raw red meat (n=697)
77%
467
82%
76
78%
543*
Raw poultry products (n=696)
74%
449
84%
79
76%
528*
Dogs (n=698)
55%
333
63%
59
56%
392
Cats
12%
75
20%
19
13%
94
Horses
9%
52
30%
28
11%
80
Poultry (chickens, turkeys, etc)
10%
62
18%
17
11%
79*
Cattle (dairy or beef) (n=698)
5%
32
7%
7
6%
39
Sheep or goats
3%
20*
7%
7*
4%
27*
Pigs (n=697,93,604)
* N=699, 94, or 605 subjects unless otherwise stated (i.e. incomplete data for item)
2
Age (all): mean: 58 years, median: 59 years, range: 12-87 years
3
Lag (all): days from water submission to interview: mean:142; median:124; range:3-439
4
Tap water only: do not used bottled water at home on regular basis; Tap and bottled: glasses
of water [total] > glasses bottled; Bottled water only: glasses of water [total] = glasses bottled
Chi square test of independence of subjects using/not contaminated water: p-value of <0.05
Results
51
3.2 Prevalence of Carriage of Antimicrobial Resistant E. coli

The prevalence of antimicrobial resistant E. coli detected on the rectal swabs, defined as
resistance to one or more of the antibiotics in the 2004 NARMS panel, was 41% (95% confidence
interval, CI95%, 37, 45). Since the weighting strategy was rudimentary, and because the prevalence
estimates were comparable, the un-weighted estimates of prevalence, adjusted for non-independence
of observations within households, are presented in the text.
The highest rates of resistance were to ampicillin (n=194 or 28%), tetracycline (n=176 or 25%),
and sulfisoxazole (n=164 or 24%). The prevalence of streptomycin resistant E. coli in this population
was 17% and trimethoprim-sulfamethoxazole resistance was 14%. The most common patterns of
resistance were between ampicillin and sulfisoxazole with 120 (17%) isolates resistant to both of these
antibiotics. Resistance to both tetracycline and sulfisoxazole (n=114 or 16%) and ampicillin and
tetracycline (n=110 or 16%) was also common.
As shown in Table 3.8, the classes of drugs with the highest rates of resistance were the betalactam (n=194 or 28%), tetracycline (n=176 or 25%), sulphonamide (n=166 or 24%), and aminoglycoside
(n=127 or 18%) agents. Escherichia coli from the faecal swabs were less likely to be resistant to the
fluoroquinolone and chloramphenicol classes of antibiotics (6% (n=42) and 5% (n=32) respectively). The
resistance score (observed resistances divided by total possible resistances), a summary measure of the
prevalence of multi-drug resistance, was 8.5% in this sample.
Table 3.9 highlights the intra-class resistance detected in the E. coli from human faecal samples.
Within the beta-lactam class of antibiotics, all E. coli isolates that were resistant to amoxicillinclavulanic acid (n=10), cefoxitin (n=8), and ceftiofur (n=7) were also resistant to ampicillin. All
ciprofloxacin resistant E. coli (n=18) were resistant to nalidixic acid. Most (98 of 99 or 98%) of the
trimethoprim-sulphamethoxazole resistant isolates were resistant to sulfisoxazole. There was slightly
lower intra-class resistance in the aminoglycoside agents with 9 of 14 (64%) of gentamicin resistant
isolates and 9 of 12 (75%) kanamycin resistant E. coli isolates being resistant to streptomycin as well.
For this study, multi-drug (or inter-class) resistance was defined as resistance to two or more
classes of antibiotics included in the NARMS panel. Multi-drug resistance was detected in 204 of the 699
swabs (29%; CI95% 26, 33). As shown in Table 3.10, greater than 70% of aminoglycoside resistant E. coli
isolates were simultaneously resistant to a sulphonamide (108 of 127 or 85%), a beta-lactam (97 of 127
52
or 76%), or tetracycline (91 of 127 of 72%). Similarly, greater than 70% of fluoroquinolone (31 of 42 or
74%) or sulphonamide (121 of 166 or 73%) resistant isolates were resistant to one of the beta-lactam
agents included in the 2004 NARMS enteric bacteria panel. Further, more than 80% of chloramphenicol
resistant E. coli isolates were also resistant to one of the sulphonamide (29 of 32 or 91%) drugs or
tetracycline (26 of 32 or 81%). In fact, 18 of 32 (56%) chloramphenicol resistant isolates were resistant
to tetracycline, a sulphonamide, and a beta-lactam agent.
Table 3.8
Proportion of Human Rectal Swabs with Antimicrobial Resistant E. coli, by Antibiotic
and Class of Antibiotic, Survey of Households using Private Water Sources, Southern Ontario, 2005-2006
Number
Weighted
Design-adjusted
resistant
Estimate2,3
estimate2
(N=699)
Antibiotic1
Resistant
CI95%
Resistant
CI95%
Resistance to one or more antibiotics
285
41%
37, 45
39%
33, 45
Multi-drug resistance (2+ classes)
204
29%
26, 33
28%
23, 33
14, 23
18%
15, 21
18%
127
Aminoglycoside
--0
--0
0
Amikacin
0, 3
2%
1, 3
2%
14
Gentamicin
1, 5
3%
1, 3
2%
12
Kanamycin
13, 22
17%
14, 20
17%
119
Streptomycin
194
21, 32
28%
24, 31
27%
Beta-lactam
10
0, 1
1%
1, 2
0.4%
194
21, 32
28%
24, 31
27%
Ampicillin
8
0, 1
Cefoxitin
1%
0, 2
0.4%
Ceftiofur
7
1%
0, 2
1%
0, 2
Ceftriaxone
0
--0
0
--3,
8
5%
4,
8
6%
42
Fluoroquinolone
1, 5
3%
1, 4
3%
18
Ciprofloxacin
3, 8
5%
4, 8
6%
42
Nalidixic acid
16, 25
21%
20, 27
24%
166
Sulphonamide
10, 17
13%
12, 17
14%
99
Trimethoprim-sulfamethoxazole
16, 25
20%
20, 27
23%
164
Sulfisoxazole
Tetracycline
176
25%
22, 29
25%
20, 30
Chloramphenicol
32
5%
3, 6
5%
3, 8
1
Using the 2004 NARMS panel for enteric bacteria
2
Variances adjusted to account for non-independence of observations within households
3
Weighted by households probability of selection from Safe Water Unit data base
Results
53
Table 3.9
Intra-class Resistance of Antimicrobial Resistant E. coli Isolates from Human Rectal
Swabs; Survey of Households using Private Water Sources, Southern Ontario, 2005-2006
(a) Beta-lactam
Resistant isolates (n)

(%)
AmoxicillinClavulanic acid
Ampicillin
Cefoxitin
Ceftiofur
Amoxicillinclavulanic
acid
n=10
(1%)
Ampicillin
n=194
(28%)
Cefoxitin
n=8
(1%)
Ceftiofur
n=7
(1%)
--100%
80%
60%
5%
--4%
4%
100%
100%
--75%
86%
100%
86%
---

(%)
Gentamicin
Kanamycin
Streptomycin
Ceftriaxone
n=0
---
(b) Aminoglycoside
Amikacin
Gentamicin
n=0
n=14
(2%)
------0
--64%
(c) Fluoroquinolone
Ciprofloxacin
n=18
(%)
(3%)
Ciprofloxacin
--Nalidixic acid
100%
(d) Sulphonamide
Trimethoprimsulphamethoxazole
n=99
(%)
(14%)
Trimethoprim--sulphamethoxazole
Sulfisoxazole
98%
Kanamycin
n=12
(2%)
0
--75%
-------
Streptomycin
n=119
(17%)
8%
8%
---
Nalidixic acid
n=42
(6%)
43%
---
Sulfisoxazole
n=164
(24%)
59%
---
Column identifies antibiotic used in denominator: it specifies the percentage of isolates resistant to
that antibiotic that are also resistant to the antibiotic in the corresponding row. E.g. Gentamicin: of
the 14 isolates resistant to gentamicin, none were resistant to kanamycin but 64% (n=9) were resistant
to streptomycin.
54
Table 3.10
Inter-class Resistance of Antimicrobial Resistant E. coli Isolates from Human Rectal
Swabs; Survey of Households using Private Water Sources, Southern Ontario, 2005-2006
AminoBetaFluoroSulphonTetraChloramglycoside
lactam
quinolone
amide
cycline
phenicol
n=127
n=194
n=42
n=166
n=176
n=32
(%)
18%
28%
6%
24%
25%
4%
Aminoglycoside
-50%
48%
65%
52%
44%
eta-lactam
76%
-74%
73%
63%
63%
Fluoroquinolone
16%
16%
-17%
14%
9%
Sulphonamide
85%
62%
67%
-65%
91%
Tetracycline
72%
57%
57%
69%
-81%
Chloramphenicol
11%
10%
7%
18%
15%
-Column identifies antibiotic used in denominator: it specifies the percentage of isolates resistant to
that class of antibiotic that are also resistant to the class of antibiotic in the corresponding row
(e.g. aminoglycoside: of the 127 isolates resistant to an aminoglycoside, 76% (n=97) were resistant to a
beta-lactam).
3.2.1 Ampicillin Resistant E. coli Prevalence

The prevalence (variance adjusted for non-independence of observations) of ampicillin resistant
E. coli from rectal swabs of non-institutionalized people living in southern Ontario and using private
water sources was 28% (CI95% 24, 31). This corresponds to the hypothesized prevalence of greater than
or equal to 22% (p<0.001). The age-standardized prevalence (30%; CI95% 26, 33) and weighted
prevalence (27%; CI95% 21, 32) of ampicillin resistant E. coli were not significantly different than the
original estimate of 28% (Table 3.8).
As shown in Table 3.11, several variables were associated with the prevalence of carriage of
ampicillin resistant E. coli in these subjects. The prevalence in the population of respondents who used
water contaminated with antimicrobial resistant E. coli was significantly higher than for those who did
not use water that was contaminated with antimicrobial resistant E. coli; 38% versus 26% (p=0.01).
Males were significantly more likely than females to carry ampicillin resistant E. coli (32% versus 23%;
p=0.007) and people who had travelled outside of Canada in the previous year were more likely to
carry resistant E. coli than people who had either not travelled or had travelled only within Canada
(32% versus 23%; p=0.008).
Results
55
Table 3.11
Proportion of Human Rectal Swabs with Ampicillin Resistant E. coli by Selected
Covariates, Variances Adjusted for Household Clustering; Survey of Households using Private Water
Sources, Southern Ontario, 2005-2006
Number
p-value1
(N=699)*
(2)
Covariate
Percent
CI95%
Water
Contaminated with resistant E. coli, untreated
94
38%
29, 48
No resistant E. coli or treated 12+ months
605
26%
23, 30
0.01
Respondents sex
Female
346
23%
19, 28
Male
353
32%
27, 37
0.007
Travel outside Canada past year
376
32%
27, 37
No travel or only within Canada
323
23%
18, 27
0.008
Household size
1
53
17%
7, 27
2
410
28%
24, 32
3 or 4
160
27%
20, 34
5 to 10
76
36%
25, 46
0.13
Hospitalized past year
55
20%
11, 32
Not hospitalized
644
28%
25, 32
0.18
Child in day care
14
43%
21, 68
No child or no child in day care
685
27%
24, 31
0.20
Contact with sheep or goats past 3 months
39
18%
8, 36
No contact
660
28%
25, 32
0.22
Interviewed face-to-face (site visit)
266
30%
25, 37
Telephone interview
433
26%
22, 31
0.23
2
16, 30
23%
135
3-59 days
24, 41
32%
117
60-99
25, 41
33%
132
100-139
19, 34
27%
146
140-199
0.26
18, 31
25%
169
200-439
Household income in previous year
105
< $40,000
25%
17, 34
$40,000-79,999
229
24%
19, 30
$80,000 or more
209
32%
26, 39
Not stated
156
30%
23, 37
0.27
Respondents age at interview
25, 39
32%
177
12-49 years
23, 37
30%
175
50-59
19, 31
25%
207
60-69
0.30
17, 31
24%
140
70 & older
Contact with poultry past 3 months (n=696)
80
33%
23, 44
No contact
619
27%
24, 31
0.34
85
24%
15, 34
Antibiotic used past 3 months
No antibiotic
614
28%
25, 32
0.37
23, 32
27%
Tap only
433
19, 34
26%
133
Tap and bottled
0.42
25, 41
32%
133
Bottled only
Contact with cattle past 3 months (n=698)
79
32%
22, 43
No contact
619
27%
24, 31
0.42
Table continued on next page
56
Table 3.11, continued
p-value1
(2)
Covariate
Number
Percent
CI95%
Property
Farming
194
30%
24, 37
Not farming
505
27%
23, 31
0.45
Contact with pigs past 3 months (n=697)
27
22%
11, 39
No contact
670
28%
24, 31
0.48
Laboratory region
London
318
30%
25, 35
Hamilton
192
30%
23, 37
Kingston
55
25%
16, 38
Orillia
50
22%
12, 36
Ottawa
27
26%
11, 49
Peterborough
45
16%
8, 28
Toronto
12
33%
14, 60
0.51
Contact with horses past 3 months
94
26%
17, 36
No contact
605
28%
25, 32
0.62
Contact with cats past 3 months
392
27%
23, 32
No contact
307
29%
24, 34
0.65
528
28%
24, 32
Contact with dogs past 3 months (n=698)
No contact
170
26%
20, 34
0.67
Highest education in household4
51
25%
15, 40
High school graduate
99
30%
21, 41
213
27%
22, 34
University
319
28%
23, 33
Not stated
17
24%
9, 48
0.96
*N=699 unless otherwise stated
1
Variances adjusted for non-independence of observations within households
2
Lag: days between water sample collection for submission to public health laboratory for
bacteriological testing and date of interview (proxy for the date of rectal swab collection)
3
Tap water only: do not used bottled water at home on regular basis (most days)
Tap and bottled: glasses of water [total] > glasses of bottled water
Bottled water only: glasses of water [total] = glasses of bottled water
4
3.3 Association of Human Carriage and Consumption of Contaminated Water

The prevalence of carriage of antimicrobial resistant E. coli, adjusted for non-independence of
observations within households, was higher in subjects living in households with untreated,
antimicrobial resistant E. coli-contaminated water (50 of 94 or 53%) than in other subjects (235 of 605
or 39%) (p=0.01). Of note, there was no difference between subjects living in households with no
history of E. coli or coliform bacterial contamination of their water for one year or longer (i.e. people
living in B control households), those with water that was contaminated with E. coli that was
Results
57
sensitive to the antibiotics in the NARMS panel (i.e. people living in A control households), or people
living in households with antimicrobial resistant E. coli-contaminated water that was treated for
bacterial contamination for one year or longer (39%, 40%, and 32%, respectively; p=0.54).
Poisson regression, adjusted for non-independence of observations of people living within the
same household, was used to determine that people living in households that used water contaminated
with antimicrobial resistant E. coli were 1.4 times (CI95% 1.1, 1.7) more likely to be colonized with
antimicrobial resistant E. coli than people living in households without antimicrobial resistant E. colicontaminated water sources (p=0.007; n=699 respondents from 488 households; AIC:1081; BIC:1091).
Variables that were associated with the dependent variable (carriage of resistant E. coli) at a
p-value of 0.25 or less on bivariate analyses were used to construct the multivariable models (see Table
3.12). The initial (full) model included the following variables: the focal independent variable: water
source (contaminated/not); the potential confounders: contact with cattle, respondents sex, travel,
contact with poultry, property type (farming/non-farming), contact with horses, household income,
antibiotic use, hospitalization history, contact with raw red meat; the hypothesized effect modifier
(tap or bottled water use) and its product term (tap or bottled water use by water source); and the
product terms of the focal independent variable by property type and by household income.
As discussed in section 2.3.2, the final model included the focal independent variable (water
source) and the following potentially confounding variables: travel outside of Canada in the previous
year, respondents sex, and contact with dairy or beef cattle in the previous three months (see Table
3.13). Although the potential interaction term and product term (tap or bottled water use by water
source) were also entered into the final model, they were removed as they did not improve the fit of
the model (p=0.72 for partial F-test; AIC:1081; BIC:1117). No other variables were detected that
affected the focal relationship or improved the fit of the model when the variables were added back
into the model after it was reduced.
The final model had no high leverage or outlying residual patterns and used 698 observations
from 487 clusters/households. Only one observation was not included in the final model due to itemspecific missing data. The goodness of fit test for applicability of the Poisson distribution was not
significant showing that the Poisson distribution was not over-dispersed and was appropriate for this
analysis (289). Also, the information criteria (Akaike and Bayesian) confirmed that the full model fit
58
the data as well as or better than the bivariate association (AIC:1076; BIC:1099 for n=698 respondents
and n=487 households).
The data were also analyzed using generalized estimating equations with exchangeable
correlation within households (clusters), a Poisson distribution, robust standard error estimates, and
population-averaged equations. The parameter estimates, standard errors, and model-building results
were virtually identical (< 0.001% difference) to those achieved using the generalized linear model
equation as described above. We chose to present the generalized Poisson regression results since the
statistical package provided a wider array of model-testing tools and because the results did not differ.
After adjusting for the effect of other variables, it was determined that people living in
households that used water contaminated with antimicrobial resistant E. coli were 1.4 times (CI95% 1.1,
1.7) more likely to be colonized with antimicrobial resistant E. coli than people living in households
without antimicrobial resistant E. coli-contaminated water sources (p<0.0001).
The risk difference or attributable risk between the subjects who were exposed and subjects
who were not exposed to water contaminated with antimicrobial resistant drinking water was 14%. The
attributable fraction, both crude (based on the crude prevalence rates) and adjusted (based on the
multivariable model), for subjects exposed to water contaminated with antimicrobial resistant E. coli
was 26%.
Based on samples tested in the participating public health laboratories during the study period,
4.5% of water samples were contaminated with E. coli, 8-50% of water sources have treatment systems
for eradicating bacteria, and 10% of the E. coli-positive samples were resistant to one or more of the
antimicrobial agents in the NARMS panel. Thus, about 8-14 cases of antimicrobial resistant E. coli
carriage per 10,000 Ontario residents using private water sources (1-2 cases per 10,000 Ontario
residents) would be attributable to drinking untreated water contaminated with antimicrobial resistant
E. coli.
Results
Table 3.12
Bivariate Associations between Carriage of Antimicrobial Resistant E. coli and
Covariates. Poisson Regression, Variances Adjusted for Household Clustering; Survey of Households
using Private Water Sources, Southern Ontario, 2005-2006
Prevalence
Standard
Covariates
Ratio
p-value
CI95%
error1
Focal independent variable
Water source
Referent
Not contaminated or treated2
Contaminated & not treated
1.37
0.16
0.007
1.09, 1.72
Potential confounders
No contact with cattle
Referent
1.39
0.16
0.003
1.11, 1.74
Respondents sex
Female
Referent
Male
1.25
0.11
0.009
1.06, 1.48
No travel, or only within Canada
Referent
Travel outside Canada, past year
1.29
0.13
0.011
1.06, 1.56
No contact with poultry
Referent
1.23
0.16
0.127
0.94, 1.59
Non-farming property
Referent
Farming property
1.16
0.12
0.135
0.95, 1.42
No contact with horses
Referent
1.17
0.15
0.199
0.92, 1.50
Household income, past year3
<$40,000
Referent
$40,000-79,999
0.97
0.15
0.878
0.71, 1.33
$80,000 or more
1.24
0.18
0.154
0.92, 1.66
Not stated
1.16
0.18
0.356
0.85, 1.57
overall test (3 df)
0.200
No antibiotic used
Referent
Antibiotic in past 3 months
0.82
0.13
0.211
0.60, 1.12
Referent
No hospitalization4
Hospitalized in past 12 months
0.79
0.15
0.228
0.54, 1.16
No contact with raw red meats
Referent
1.16
0.14
0.243
0.90, 1.48
Household education, highest5
Less than grade 9
Referent
High school
1.24
0.33
0.429
0.73, 2.09
College or trade
1.39
0.34
0.175
0.86, 2.23
University
1.47
0.35
0.103
0.92, 2.34
Not stated
1.80
0.60
0.076
0.94, 3.45
overall test (4 df)
0.332
Respondents age at interview
12-49 years
Referent
50-59
1.02
0.12
0.842
0.81, 1.30
60-69
0.87
0.11
0.259
0.68, 1.11
70 & older
0.85
0.12
0.268
0.64, 1.13
overall test (3 df)
0.379
1
Robust variance estimate, adjusted for household clustering
2
Water source not contaminated with antimicrobial resistant E. coli OR- contaminated but treated
with ultraviolet light, chlorine, boiling, or ozone for 12 months or longer OR- contaminated with
susceptible E. coli
3
4
Hospitalized for one night or longer in past 12 months
Table continued on next page
59
60
Table 3.12, Continued

Covariates
3-59 days
60-99
100-139
140-199
200-439
Prevalence
Ratio
Standard
error1
Referent
1.15
1.28
1.10
0.20
overall test (4 df)
Referent
1.32
1.17
1.39
overall test (3 df)
p-value
CI95%
0.19
0.19
0.18
0.18
0.391
0.104
0.561
0.240
0.379
0.83, 1.60
0.95, 1.72
0.80, 1.50
0.89, 1.62
0.28
0.26
0.33
0.181
0.485
0.166
0.385
0.88, 1.99
0.75, 1.81
0.87, 2.23
0.134
0.551
0.84, 1.37
0.13
0.18
0.17
0.29
0.16
0.32
0.218
0.993
0.410
0.687
0.348
0.634
0.583
0.92, 1.43
0.70, 1.42
0.58, 1.25
0.66, 1.87
0.57, 1.22
0.40, 1.76
0.10
0.653
0.77, 1.17
0.23
0.670
0.72, 1.66
0.12
0.811
0.82, 1.28
0.32
0.869
0.57, 1.93
0.22
0.976
0.65, 1.55
0.09
0.979
0.83, 1.20
0.11
0.13
0.244
0.416
0.278
0.67, 1.11
0.88, 1.37
Household size
1 person
2
3 or 4
5 to 10
Interview mode6
Site visit
Telephone interview
Laboratory region7
London
Hamilton
Kingston
Orillia
Ottawa
Peterborough
Toronto
No contact with raw poultry product
No contact with pigs
No contact with dogs
No child or not in day care
Child in day care centre
No contact with sheep/goats
No contact with cats
Potential effect modifier
Tap only
Tap and bottled
Bottled only
Referent
1.07
Referent
1.14
1.00
0.85
1.11
0.83
0.83
overall test (6 df)
Referent
0.95
Referent
1.09
Referent
1.03
Referent
1.05
Referent
1.01
Referent
1.00
Referent
0.86
1.10
overall test (2 df)

1
5
6
Mode of interview: site visit to home versus telephone interview
7
8
Lag: days between water sample collection for submission to public health laboratory for
bacteriological testing and date of interview (proxy for the date of rectal swab collection)
9
Tap water only: Do not used bottled water at home on regular basis (most days)
Tap and bottled: Glasses of water [total] > glasses of bottled water
Bottled water only: Glasses of water [total] = glasses of bottled water
Results
61
Table 3.13
Multivariable Model of Association between Carriage of Antimicrobial Resistant E. coli,
Use of Water Contaminated with Antimicrobial Resistant E. coli, and Covariates. Poisson Regression,
Variances Adjusted for Household Clustering; Survey of Households using Private Water Sources,
Prevalence
Standard
p-value
CI95%
Variable
ratio
error1
Water source
Referent
Not contaminated or treated2
Contaminated & not treated
1.35
0.15
0.009
1.08, 1.69
Travel in past year
No travel or only within Canada
Referent
Travelled outside Canada
1.30
0.13
0.007
1.08, 1.58
Contact with cattle
No contact past 3 months
Referent
Contact
1.33
0.15
0.011
1.03, 1.45
Respondents sex
Female
Referent
Male
1.22
0.11
0.019
1.03, 1.45
1
2
Water source not contaminated with antimicrobial resistant E. coli OR- contaminated but
treated with ultraviolet light, chlorine, boiling, or ozone for 12 months or longer
62
Chapter 4
Discussion
4.1 Prevalence of Resistant E. coli
4.1.1 Ampicillin Resistant E. coli
The prevalence of faecal carriage of ampicillin resistant E. coli in 699 non-institutionalized
subjects who lived in southern Ontario, Canada and used private water sources was 28% (CI95% 24, 31),
which was greater than the hypothesized prevalence of 22%. This estimate was quite stable, with little
change noted after weighting the observations to account for the over-sampling of households with
contaminated water sources or after age-standardizing the estimate to reflect the age of the rural
Ontario population.
The prevalence estimates of this study were not statistically different than two smaller Canadian
studies, both completed in the 1990s, that also estimated the prevalence of antimicrobial resistant E.
coli by using faecal samples. Akwar et al. determined that 16% of E. coli isolates from 115 subjects who
lived on swine farms located in Ontario and British Columbia were resistant to ampicillin (105).
Bruinsma et al. reported that 22% of 154 non-institutionalized subjects living in St. Johns,
Newfoundland carried amoxicillin resistant E. coli (104). Ampicillin and amoxicillin are comparable in
that they are both bactericidal aminopenicillin agents that are used to treat systemic and urinary tract
infections caused by E. coli and other Gram-negative and Gram-positive organisms (290).
The prevalence of carriage of ampicillin resistant E. coli in our study was lower than rates from
studies of clinical and urinary tract infections. Reported rates of ampicillin resistance originating from
clinical isolates (including urinary tract isolates) ranged from 30-46% (94;95;97-99;102;291). The lower
rate of resistance in our study likely reflects the fact that the samples were drawn from a noninstitutionalized population. It is expected that higher rates of resistance would be reported from
clinical isolates because many clinical isolates originate from hospitalized individuals who are more
likely to be exposed to antimicrobial agents and to the nosocomial transmission of antimicrobial
resistant strains of bacteria (92). Also, since most uncomplicated urinary tract infections are treated
empirically, without submission of urine for laboratory analysis (97;103), laboratory-based studies
likely reflect isolates of individuals who failed initial treatment or had complicated infections, thus
biasing reported rates of antibiotic resistance (292). In addition, Laupland et al. determined that rates
Discussion
63
of antimicrobial resistance from urine samples were about 30% higher when samples from the same
patient were not excluded for at least one year of the two-year study period (293). For these reasons,
the estimated prevalence of the carriage of ampicillin resistant E. coli from our study is likely a more
accurate reflection of the prevalence of resistance in the community than that from studies based on
clinical isolates.
Although not part of the hypothesis, a preliminary examination of the factors associated with
ampicillin resistance was made. Three subgroups had different prevalences of ampicillin resistant E.
coli in this sample. First, a higher proportion of people from households with untreated water supplies
that were contaminated with antibiotic resistant E. coli carried ampicillin resistant E. coli than people
from households with water that was either not contaminated or was contaminated but treated with
chlorine, ultraviolet light, ozone, or by boiling (38% versus 26%; p=0.01). This association is explored
further in the following discussion (see Section 4.2).
Second, subjects who travelled beyond the borders of Canada within the previous year had a
higher prevalence of carriage of ampicillin resistant E. coli than subjects who did not travel or
travelled only within Canada (32% and 23%, respectively; p=0.008). In several other studies, travel to
areas with a high prevalence of human carriage of antimicrobial resistant E. coli was linked to higher
rates of colonization with antimicrobial resistant bacteria upon return from these areas (15;152;183185). Similary, travel has been identified as a risk factor for infection with a resistant strain of E. coli
in women with urinary tract infections (109;294). The prevalence of faecal carriage of ampicillin
resistant E. coli ranges from 73-94% in Mexico, a common destination for many of our subjects who
travelled in the previous year (106;141). It is likely that people travelling to foreign countries become
colonized with the predominant strains of that country while visiting.
Also, a higher proportion of males than females carried ampicillin resistant E. coli (32% and
23%, respectively; p=0.007). Although some other studies of ampicillin or amoxicillin resistance in E.
coli have detected a higher prevalence in males than females (102;136), the opposite has also been
reported (111), and others report no difference by sex (132;134). In our study, males and females were
equally likely to be exposed to water contaminated with antimicrobial resistant E. coli and equally
likely to have travelled outside of Canada. Although it is possible that this association was found purely
64
by chance, it may be that there are unmeasured factors driving the difference in the prevalence of
ampicillin resistance by sex in these subjects.
Of note, neither antibiotic use in the previous three months nor hospitalization in the previous
year were associated with a higher prevalence of carriage of ampicillin resistant E. coli in our study.
These factors have been identified as risk factors for carriage or infection with resistant E. coli in
several other studies (129;146;295-298). However, our sample size was not large enough to refine the
items further to detect an association; only 55 respondent had been hospitalized and 85 respondents
had taken an antibiotic (28 of whom had taken penicillin). The power to detect a two-fold difference in
the association between prior antibiotic use and the prevalence of ampicillin resistance was only 11%
( = 0.05) in this study.
The results of our study, that 28% of healthy non-institutionalized people living in southern
Ontario carried E. coli that was resistant to ampicillin, is probably a less biased estimate than that
available from clinical laboratory based studies. Further, the estimate is quite stable, showing little
change when standardized to the age of the underlying population or weighted to reflect the
underlying risk of exposure to antimicrobial resistant E. coli from consumption of household water.
Although the current estimate of ampicillin resistance was not statistically significantly different than
the estimates from the earlier Canadian studies using faecal samples, the difference is clinically
relevant and likely reflects an increased prevalence of carriage of ampicillin resistant E. coli over time.
Guidelines written for the Infectious Diseases Society of America recommend changing the agents
used for the empiric treatment of urinary tract infections when the rates of resistance to an agent
reach 20% in the geographic region (103). Following these guidelines, ampicillin should not be used for
the empiric treatment of infections suspected to be caused by E. coli among residents of southern
Ontario.
4.1.2 Antimicrobial Resistance to Other Agents

The prevalence of faecal carriage of antimicrobial resistant E. coli in our study was not
statistically significantly different, when examined by individual antibiotic, than that reported in two
other studies of non-institutionalized Canadian subjects that estimated prevalence of colonization
(104;105). As shown in Table 4.1, however, the prevalence of resistance to all antibiotics except one
Discussion
65
(kanamycin) is higher in the current study. This trend of increased prevalence makes the findings
clinically relevant.
Tetracycline is not recommended for empiric use against infections caused by E. coli due to the
high rates of resistance (258). However, it is one of the three most frequently dispensed classes of
antibiotics for human use in Canada (256) and is commonly prescribed for the treatment of acne. The
finding, that 25% of subjects carried E. coli resistant to tetracycline, confirms the recommendation not
to use tetracycline against infections caused by E. coli since there is a high probability that the strain
causing the infection will be resistant to this antibiotic.
Trimethoprim-sulfamethoxazole is recommended for empiric treatment of uncomplicated
urinary tract infections in adult women as long as the prevalence of resistance to the antibiotic agent
remains below 20% (103). The prevalence in our study was below that threshold at 14% (CI95% 11, 16)
but, comparing results from this and other studies (97;299), appears to be increasing over time. Since
the use of this antibiotic is associated with the development of antimicrobial resistance (136;298), and
because urinary tract infections are quite common in women, recommendations regarding its use as
the first-line empiric treatment of uncomplicated urinary tract infections in women should be reviewed
before it reaches this threshold.
Patients with multi-drug resistant infections are at higher risk of morbidity and mortality
because of delayed or incorrect treatment with effective antimicrobial therapy (14) and there are
fewer treatment options for their treatment. Multi-drug resistance (resistance to two or more
antibiotic classes) was detected in 29% (CI95% 26, 33) of E. coli isolates in this study of residents of
households using private water sources. The most common pattern of inter-class/multi-drug resistance
in our study was between sulphonamide and beta-lactam agents, with over 70% of the sulphonamide
resistant E. coli isolates being resistant to one of the beta-lactam agents included in the study. This
pattern of resistance was also noted in other Canadian studies where 72-80% of the trimethoprimsulphamethoxazole resistant E. coli isolates from outpatient urine samples were also resistant to
ampicillin (98;291). Howard reported that co-resistance to ampicillin and trimethoprimsulphamethoxazole was associated with the use of either antibiotic (136). If this relationship is borne
out in other studies, it will need to be considered in future recommendations of treatment of
infections caused by E. coli. Although much can be learned about resistance when researching one
66
antimicrobial agent in isolation, the high rates of multi-drug resistance demand epidemiological and
microbiological research into this perplexing problem. Ultimately, the prevalence of multi-drug
resistance and the associated treatment issues argue for strong action to slow the rise in the
prevalence of antimicrobial resistance for all antibiotics, not just those that have high rates of
resistance at present.
Table 4.1
Comparison of Rates of Antimicrobial Resistance in E. coli from Canadian Studies
Antibiotic
Current study
Akwar (105)
Bruinsma (104)
UTI Studies1
Ampicillin
28%
16%
22% (AMX)
30-42%
Tetracycline
25%
24%
16% (OXT)
19%
Sulfisoxazole
24%
17% (SMX)
NA
NA
Streptomycin
17%
10%
NA
NA
Trimethoprim14%
5%
10% (TMP)
12-19%
sulphamethoxazole
Nalidixic acid
6%
<1%
1%
1%
Chloramphenicol
5%
3%
1%
NA
Ciprofloxacin
3%
0
1%
0-7%
Gentamicin
2%
1%
1%
1-3%
Kanamycin
2%
4%
NA
NA
Cefoxitin
1%
NA
NA
4%
Ceftiofur
1%
0
NA
NA
Ceftriaxone
0
0
NA
NA
Amikacin
0
0
NA
1%
UTI: urinary tract infection; AMX: amoxicillin; OXT: oxytetracycline; SMX: sulfamethoxazole;
TMP: trimethoprim
1
(97-99;102;291;300)
4.2 Association of Human Carriage and Consumption of Contaminated Water

The prevalence of carriage of antimicrobial resistant E. coli, adjusted for non-independence of
observations, was 40 percent higher in subjects living in households that used water contaminated with
antimicrobial resistant E. coli than for people living in households that used uncontaminated or treated
water (53% vs 39%, respectively). This relationship persisted when associated covariates (travel outside
Canada in the previous year, direct contact with cattle in the previous three months, and respondents
sex) were included in the multivariable regression model. This finding shows that, for the people
involved in this study, the use of water contaminated with antimicrobial resistant E. coli increased the
likelihood of carriage of resistant E. coli even after accounting for other associated factors.
This research is the first to show a link between the consumption of water contaminated with
antimicrobial resistant E. coli and human carriage of resistant E. coli. Two other studies attempted to
Discussion
67
determine if an association existed between the consumption of water contaminated with, and human
carriage of, antimicrobial resistant bacteria. However, neither of the studies looked at E. coli
specifically and the findings of the studies were inconclusive.
As detailed in the literature review, Amyes et al. compared the faecal carriage of antimicrobial
resistant aerobic faecal bacteria in subjects from four villages in India in 1989 (249). The authors
concluded that the consumption of water contaminated with antimicrobial resistant bacteria coupled
with the high use of antibiotics was likely responsible for the high rates of carriage of resistant bacteria
in the inhabitants of the four villages. Although the authors presented details about the differences in
human carriage of resistant bacteria by village, no details were given about the resistance profiles of
the water supplies. Thus, no direct association between water consumption and human carriage was
made and the authors conclusion could not be substantiated.
Shanahan et al. studied the faecal carriage of antimicrobial resistant enterobacteria of
residents of South Africa in 1992 (248). The authors concluded that there was no association between
consuming water contaminated with bacteria and the carriage of resistant enterobacteria. However,
the water was not tested in all study locations and the bacteria present in the water that was collected
were not tested for antimicrobial resistance. Thus, it was not possible to determine if there was an
association between consuming water contaminated with antimicrobial resistant bacteria and human
carriage of resistant bacteria.
In our study, the prevalence of carriage of antimicrobial resistant E. coli was 40% higher for
individuals using water contaminated with antimicrobial resistant E. coli than for subjects consuming
uncontaminated water or water contaminated with E. coli sensitive to antibiotics. Although this
association was not unexpected, the fact that the association was still significantly associated even
after accounting for the effect of other associated variables highlights the importance of contaminated
water as a risk factor for colonization with antimicrobial resistant E. coli.
4.2.1 Strengths and Limitations of the Study

As with all studies, this one had a number of strengths and limitations. One of the strengths of
this study was that it provided a sample of people living across a wide geographic area in southern
Ontario. The results included responses from people living in Elgin County (southwestern Ontario),
68
Peterborough (central Ontario), Ottawa (eastern Ontario) and all counties in between. The survey
included people living in a variety of settings including farms, non-farm rural residences, villages, and
small towns. In this population, we were able to estimate the prevalence of antimicrobial resistant E.
coli in non-institutionalized individuals. This group of subjects were more representative of the general
population than studies based on clinical isolates which largely rely on isolates from individuals
screened for infections.
Another strength of this study was that the E. coli isolates tested for resistance were not
necessarily pathogenic. It is important to know the rates of resistance in all strains of E. coli since they
are all able to cause urinary tract and bloodstream infections and are able to transfer resistance to
other strains of E. coli as well as other types of bacteria. Of similar importance, commensal bacteria
are the largest reservoir for the transmission of antimicrobial resistant bacteria to other humans,
animals, and the environment (33;301).
The design of the study allowed for the systematic collection of household and personal level
variables hypothesized to be confounding or modifying to the relationship between human carriage of
resistant bacteria and the consumption of water contaminated with antimicrobial resistant E. coli. The
questionnaires were designed to collection information on variables a) identified in the literature as
being associated with antimicrobial resistance, b) influenced by the design of the study, and c) that
were common epidemiological confounders. The use of regression analyses allowed for the control of
these variables.
The sample size for this study was large enough to estimate the prevalence of antimicrobial
resistant carriage with reasonable accuracy; enough to detect clinically meaningful and statistically
significant differences in the rates of carriage. It also provided enough data for more complex
statistical analysis with the power to detect factors associated with the carriage of antimicrobial
resistant E. coli. Because our study was nested within the case-control project, we were able to
accurately locate a large enough sample of people using water contaminated with antimicrobial
resistant E. coli to allow the study of the effects of consuming contaminated water. Since the
identification of water sources contaminated with antimicrobial resistant E. coli is not routine, and
because of the low prevalence of contamination (4 per 1,000 water tests), the cost of identifying these
households would have been prohibitive for a traditional cohort or cross-sectional study.
Discussion
69
Also, our choice of control groups for the case-control project allowed us to compare subjects
who used water contaminated with antimicrobial resistant E. coli with those using water contaminated
with E. coli susceptible to antibiotics and people who used water not contaminated by coliform
bacteria for one year or longer. By determining the use of water treatment(s) and the date the
treatment was started, we were able to further refine the exposure of participants. This allowed us to
conclude that it was not the consumption of E. coli in general that was associated with carriage, but
the consumption of antimicrobial resistant E. coli, specifically.
Another strength of the study was that all subjects and interviewers in the study were blinded
to the specific exposure status of the subjects. Although subjects knew whether their household water
source was contaminated with E. coli, no information was given to them or the interviewer, about the
results of the laboratory analyses for antimicrobial susceptibility. This prevented biased responses
specific to the antimicrobial resistant status of the E. coli in the water source. Also, since subjects did
not know the antimicrobial resistance status of the E. coli, we could reasonably assume that the
differences between the two groups with E. coli contaminated water were not related to some other
unmeasured factor (e.g. disinterest in the health-related concerns of using E. coli contaminated
water).
One of the limitations of the study was that it was based on a convenience sample of people
living in households participating in the case-control project. The case-control project itself was
limited by having access only to households that submitted water samples to a participating regional
public health laboratory for bacteriological testing during the study period. Since not all households
with private water sources submit their water for bacteriological testing, the sampling frame itself may
not accurately reflect the target population: that of all Ontario households using private water sources
(302;303). However, although our response rate was only 49%, it was similar for case and control
households and for individuals within case and control households. Furthermore, we had questionnaires
and rectal swabs from people between the ages of 12 and 87 years, from a wide geographic area of
southern Ontario including both farming and non-farming households located in rural areas, villages,
and small towns without public water systems, and from households with E. coli-contaminated and
uncontaminated water.
70
In the same vein, we found that the mean age of the respondents in this study (58 years) was
significantly older than the 45 year average age reported for residents in rural Ontario (270). Since
there was no sampling frame of households using private water sources, it was not possible to
determine whether our sample was representative of the underlying population. However, we did
determine that younger subjects were less likely to submit a rectal swab than older participants. In our
study, there was a non-significant trend of a lower prevalence of carriage of antimicrobial resistant E.
coli in the older age groups. However, the direct age-standardized (to the 2006 estimate for rural
Ontario residents-our best estimate of the underlying population) prevalence was not significantly
different than the original estimate. Further, the prevalence of antimicrobial resistant E. coli was not
statistically different by age group in other studies estimating the prevalence of faecal carriage in
adults (46;129;130).
Another limitation of the study was that the questionnaire items, although pilot-tested,
reviewed by content experts, and based on items from other surveys, were not necessarily validated.
Although it would be ideal to validate the items, it was not an objective of this thesis to do so.
The time lag between the collection of the water sample for bacteriological testing and the
date of interview (proxy for the date of collection of the rectal swab) was another limitation of the
study. This time lag represented a potential separation between the subjects exposure to water
contaminated with antimicrobial resistant E. coli and measurement of the outcome (rectal swab to
assess carriage of resistant E. coli). Earlier studies have reported varying lengths of colonization with
antimicrobial resistant E. coli; from days (181;213) to months (34;120;304;305). In one follow-up study,
the median time for clearance of fluoroquinolone-resistant E. coli was five months (range 2 to 10)
(149). Thus, if some of the subjects were no longer exposed to water contaminated with antimicrobial
resistant E. coli they may have cleared resistant strains of E. coli from their system before the swab
was collected. However, we know that 8.4% of the samples of antimicrobial resistant water submissions
from Hamilton and London households had already been contacted by the study signifying that the
contamination of the water sources with antimicrobial resistant E. coli was not a one-time occurrence.
Thus, although we had no measure of the length of exposure, exposure did not likely stop at the time
of the water submission. Also, statistical analysis showed that the number of days between water
Discussion
71
sampling and interview was not associated with the prevalence of human carriage. Nevertheless, we
recommend that future studies cut the time lag to reduce this potential bias.
Another limitation with our study was the inability to determine the exposure dose (i.e. the
concentration of antimicrobial resistant E. coli per quantity of water and/or the length of exposure).
Similarly, we were unable to determine the efficacy of the water treatment: were they correctly
installed and maintained? Thus, some subjects living in households with water contaminated with
antimicrobial resistant E. coli may have been incorrectly classified as using treated water (i.e.
classified as not exposed when they were exposed). We recommend that future studies collect samples
of the water from the point of consumption, as well as the source, and to do so over a period of weeks
or months to determine whether antimicrobial resistant E. coli is detected in repeated measures. It
would also be informative to determine the concentration of E. coli in the water. We note however,
given the results of this study, it would not be ethical to knowingly allow continued consumption of
water contaminated with antimicrobial resistant E. coli.
Finally, although we analysed both the water and human faecal swabs for the specific
resistance patterns to determine their relationship, it was not possible to determine causality. It is
possible that the contaminated water did not transmit resistance to humans, but rather, that both
were contaminated from a separate source (or sources) such as other humans or animals. However, we
have included a number of variables in the regression models to help rule out confounding, including
variables hypothesized to be associated with carriage of resistant bacteria in other research (antibiotic
use, hospitalization, travel, child in day care, household size, contact with livestock, farming, contact
with pets, contact with raw meat), demographic variables (age, sex, education, income), and study
design variables (laboratory region, mode of data collection, days between water sample and
interview).
The probability of reverse causation; that the people colonized with antimicrobial resistant E.
coli contaminated their water supply rather than the opposite (that the people carrying antimicrobial
resistant E. coli were infected through contaminated water), is low. First, the association between
human carriage of antimicrobial resistant E. coli and the use of water contaminated with resistant E.
coli was not significant (relative risk: 1.1; p=0.19); the association was dependent on the use of
untreated water (see section 3.3). Also, for human sewage to contaminate the water source, the
72
bacteria would have to be transported from the septic system to the water source. Analysis of factors
associated with water source contamination (not presented in this thesis) determined that there was
no association between the distance between the water source and the septic system and the presence
of antimicrobial resistant E. coli in the water sample (p=0.61). Thus, it is more likely that the
contaminated water was the source of colonization for the subjects in this study than visa versa.
4.3 Conclusions
4.3.1 Contaminated water
In this study, the consumption of water contaminated with antimicrobial resistant E. coli was
associated with carriage of antimicrobial resistant E. coli. Although this association may seem obvious,
our findings confirm the association. In Canada, the microbiological quality of drinking water is
determined through testing for the presence of E. coli since it indicates faecal contamination of the
water supply. The Canadian drinking water guidelines state that all systems should be tested and that
the maximum acceptable concentration is zero colony forming units of E. coli per 100 mL for public,
semi-public, and private drinking water systems (306;307). In our study, 4.5% of water samples
submitted for bacteriological testing were contaminated with E. coli and an additional 18% were
contaminated with coliform bacteria above the accepted limit. Yet, according to earlier studies, only
8-26% of the households with private water supplies treat the water to destroy bacteria (68;73;75).
Since only 50-60% of households in recent surveys tested their water in the year before the interview
(73;266), it is likely that many household residents dont even know they are consuming contaminated
water, which may also be contaminated with other pathogenic bacterial species such as Salmonella,
Shigella, Campylobacter, or Legionella (308;309). This would put them at increased risk of contracting
a gastrointestinal illness and for acquiring antimicrobial resistant bacteria, including E. coli. However,
since there is no registry of private water sources, and thus no denominator data, it makes it very
difficult to quantify the size of the problem with any accuracy.
There are several steps required to reach the goal of treating all contaminated water sources.
First, is a registry of all public and private water systems. Second, is the routine monitoring of these
systems through bacteriological testing of the water. Third, is the follow-up of systems that have
significant levels of bacterial contamination. Not only would this reduce the number of cases of
Discussion
73
gastrointestinal illness caused by waterborne bacteria, but would reduce the transmission of
antimicrobial resistant E. coli and, perhaps, the transmission of other resistant waterborne bacteria.
Preventing people from becoming colonized with antimicrobial resistant E. coli is important for
several reasons. First, people carrying antimicrobial resistant E. coli have the potential to develop
antimicrobial resistant infections if the bacteria infect the carriers urinary tract, a wound, or blood
system. Second, the carriage of resistant E. coli increases the probability of transferring resistance
genes to other types of bacteria within the individuals gastrointestinal system. In addition, carriage of
resistant bacteria creates a reservoir for the transmission of resistant bacteria and resistance genes to
other humans, other mammals, and the environment.
The impact of the transmission of antimicrobial resistance through the ingestion of
contaminated drinking water is not inconsequential. To put this issue in perspective, 1.5 to 2 million
Ontario residents and over 4 million Canadians rely on private drinking water sources (62;71;73). In
Ontario only about 1 case of antimicrobial resistant E. coli carriage per 1,000 residents who use private
water sources can be attributed to drinking untreated water contaminated with antimicrobial resistant
E. coli. However, there are hundreds of millions of people across the globe, many of them in less
developed countries without the resources to treat drinking water, who are exposed to water
contaminated with E. coli and other bacteria that are resistant to frequently-prescribed antibiotics
(234;241-244;249). If the attributable fraction was similar in a developing country as it was in Ontario,
the population attributable risk fraction would be 60 to 260 per 1,000 resident of India, for example,
where even the treated water is often contaminated with antimicrobial-resistant bacteria (310).
Since people who ingest water contaminated with antimicrobial resistant E. coli are more likely to
carry resistant E. coli than people who consume uncontaminated water, the list of strategies to reduce
the prevalence and transmission of antimicrobial resistance needs to include the adequate treatment
of contaminated water.
4.3.2 Prevalence of Carriage of Resistant E. coli

The prevalence of antimicrobial resistant E. coli appears to be increasing in Canada, a nation
with relatively strict regulations on the distribution of antimicrobial agents (see Table 4.1). The
findings, that 28% of rural Ontario residents were colonized with ampicillin resistant E. coli and over
74
40% were colonized with E. coli resistant to one or more antibiotics, is a clear indication that antibiotic
resistance has made its way from the clinical setting to the rural population.
Although the prevalence of carriage of antimicrobial resistant E. coli in this study was
estimated among subjects that used private drinking water sources, and thus from rural areas, the
findings may be generalizable to the Ontario population in general. Although rural and urban residents
have somewhat varied colonization pressures due to different human and animal population densities,
the subjects included in our sample were not from remote rural areas. Thus, they likely had similar
access to medical treatment, antimicrobial agents, travel, and food sources as residents of urban
Ontario. Study subjects also included people living in urban areas who used private water sources at
their cottage and people living in rural areas and urban fringes but who worked in larger urban centres.
The colonization pressures for these subjects would not differ significantly from residents of urban
centres. Also, the majority of subjects were not exposed to water contaminated with antimicrobial
resistant E. coli (605 of 699 were not exposed). As noted above, the attributable risk fraction is low, at
1 per 1,000 people using private water sources. Thus, the drinking water exposure of most subjects was
similar to that of residents of urban centres who use municipally treated water. It is probable that the
prevalence of carriage of antimicrobial resistant E. coli is comparable for urban and rural residents of
southern Ontario.
It is troubling that the prevalence has reached this height in Ontario and that it is increasing
despite the plethora of literature about the issue. Antibiotic resistance threatens the management of
infectious diseases through increased morbidity and mortality. People infected with resistant strains of
bacteria are more likely to be hospitalized, have longer hospital stays, and are more likely to die than
people with susceptible strains of the same bacteria (9-13).
We strongly encourage a reduction in the use of antibiotics for animal, agricultural, and human
use to reduce the effects of selective pressure on the development of antimicrobial resistance. This,
along with infection control techniques to reduce the transmission of resistant bacteria will help slow
the rising prevalence of resistant bacteria (16;311;312). Physicians, nurse practitioners, dentists, and
veterinarians must prescribe antibiotics only for bacterial infections, use laboratory tests to confirm
the cause of the infection, and change the prescription to a more effective (often more narrow
spectrum) antibiotic if necessary (22). The development of rapid diagnostic tests may aid in the early
Discussion
75
diagnosis of disease thereby reducing unnecessary and/or ineffective antibiotic use (8;55). Also,
consulting infectious disease specialists would improve the rational use of antimicrobial agents, help
prevent infections, and reduce the transmission of infectious agents (23).
Health care providers must also communicate effectively with patients to ensure that
antimicrobial agents are used properly (i.e. completing the full course of antibiotics and not using
other peoples prescriptions)(15). Guidelines for the use of antibiotics should also be written,
distributed, and updated to help clinicians treat their patients effectively in the ever-changing world of
infectious disease medicine (27;313).
Research needs to continue into the development of new antimicrobial agents. It is also
essential that we learn the factors associated with the emergence, persistence, and transmission of
antimicrobial resistance. Ongoing surveillance of antibiotic use and antimicrobial resistance in humans,
animals, and the environment could provide timely information on the state of antimicrobial resistance
in Canada (25;83;314). Research into vaccines and other preventive mechanisms, including public
health measures to reduce the transmission of infectious organisms, would also help reduce the need
for antimicrobial agents, thereby reducing our dependence on an ever-dwindling collection of effective
antibiotics (8;55).
4.3.2 Next steps

We plan to publish several articles based on the results of this thesis including an alreadydrafted article on the difference in participation by mode of interview (face-to-face versus telephone).
We plan to submit articles on the prevalence of antimicrobial resistant E. coli in this sample of healthy
subjects using private water sources and one on the association between carriage of resistant bacteria
and the consumption of water contaminated with antimicrobial resistant E. coli. A submission has been
accepted to present a poster at the Interscience Conference on Antimicrobial Agents and
Chemotherapy conference in October 2008. The PhD student will also be involved in the publication of
the case-control project and other articles based on the personal and household questionnaires.
Beyond academic outputs, there are several avenues available to influence policy development
within the province. The student has already presented results to staff at the Public Health Laboratory
and will be drafting a brief to be given to Fred Ruf, the director of the Safe Water Unit in the Ministry
76
of Health and Long-Term Care and to all medical officers of health of Ontario. Since drinking water is
under the jurisdiction of more than one ministry in Ontario, we will also provide a synopsis of the
findings to the Ministry of the Environment and Health Canada. The goal is to provide information that
will help guide informed decision-making and aid in the provision of safe water to all residents in the
province of Ontario and Canada.
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96
Appendices
Appendices
Appendix A
a) Studies Comparing E. coli Antimicrobial Resistance Rates in Canadian Subjects: Clinical Isolates
Study
Zhanel
Lin (316)
Jones
Jones (52)
Wenzel
Anon.
(315)
(317)
(318)
(94)
Year
2008
2004
2004
2004
2003
2003
Age of subjects
All
Children
All
All
All
Subjects
Hospital
Inpt &
Hospital
Hospital
Hospital
Hospital
ICU
B.S.I.
ICU
outpt
Total isolates
536
6,860
87
2,013
Amikacin
0
0.4%
Amoxicillin-CA
7%
20%
14%
Ampicillin
37%
35%
34%
Cefoxitin
Ceftiofur
Ceftriaxone
4%
6%
2%
1%
Cephalothin
Chloramphenicol
Ciprofloxacin
21%
10%
4%
10%
7%
4%
Gentamicin
3%
5%
7%
5%
5%
9%
Kanamycin
Nalidixic acid
Nitrofurantoin
Piperacillin
30%
Sulphamethoxazole
Tetracycline
Trimethoprim
TMP/SMX
25%
21%
15%
17%
18%
Pfaller
(95)
1998
All
Hospital
B.S.I.
939
1%
23%
46%
7%
1%
3%
6%
40%
27%
19%
b) Studies Comparing E. coli Antimicrobial Resistance Rates in Canadian Subjects: Urinary Tract Infection Isolates
Study
Zhanel
McIsaac
Zhanel
Kahlmeter
Zhanel
Jones
Preston
(291)
(97)
(102)
(99)
(98)
(300)
(319)
Year
2006
2006
2005
2003
2000
1997
1992
Age of subjects
All
All
All
18-65 yrs
All
All
All
Subjects
Outpt
Comm
Outpt
Comm
Outpt
Hosp
Total isolates
280
2,199
496
166
1,681
182
716
Amikacin
1%
1%
Amoxicillin-CA
4%
21%
Ampicillin
33%
32%
42%
30%
41%
33%
31%
Cefoxitin
4%
Ceftiofur
Ceftriaxone
Cephalothin
13%
Chloramphenicol
Ciprofloxacin
1%
7%
7%
0
1%
1%
0
Gentamicin
1%
3%
Kanamycin
Nalidixic acid
1%
1%
Nitrofurantoin
0
1%
12%
1%
0.1%
4%
0
Sulphamethoxazole
25%
30%
Tetracycline
19%
Trimethoprim
11%
13%
TMP/SMX
17%
15%
15%
12%
19%
19%
11%
CA: clavulanic acid
TMP/SMX: trimethoprim-sulfamethoxazole
Comm: community
Hosp: hospitalized subjects
Outpt: outpatient
NOTE: empty cells reflect the fact that the antimicrobial agent was not used in susceptibility testing.
97
Appendices
Appendix B
a) Studies Comparing Escherichia Coli Antimicrobial Resistance Rates: Faecal Carriage in the United Kingdom and the
Netherlands (Holland)
Study
Cooke
Jenkins
Filius
Bruinsma
Bruinsma
Jonkers
Bruinsma
(295)
(320)
(321)
(146)
(104)
(144)
(210)
Year
1971
2006
2005
2003
2003
2002
2002
Country
UK
UK
Holland
Holland
Holland
Holland
Holland
Subject age
All ages
Adult
Adult
Adult
Adult
Subject
Comm
Outpt
Hosp
Hosp
Comm
Hosp
Comm
Isolates/subject
5
1
Total subjects
41
241
411
183
129
180
438
Amikacin
Amoxicillin
11%
28%
30%
12%
12%
Amoxicillin-CA
1%
Ampicillin
15%
13%
Cefoxitin
Ceftiofur
Ceftriaxone
Cephalothin
4%
Chloramphenicol
7%
6%
3%
Ciprofloxacin
1%
<1%
3%
0
0
<1%
Gentamicin
<1%
2%
1%
0
1%
Kanamycin
Naladixic acid
5%
2%
1%
1%
Nitrofurantoin
0
0
0
Streptomycin
37%
Sulphamethoxazole
Tetracycline
39%
Oxytetracycline
26%
33%
12%
11%
Trimethoprim
17%
23%
9%
8%
TMP/SMX
b) Studies Comparing Escherichia Coli Antimicrobial Resistance Rates: Faecal Carriage in the Netherlands (Holland),
continued
Study
Nijsten
London
London
Bonten
Bonten
Degener
Degner
(200)
(151)
(121)
(116)
(43)
(130)
(129)
Year
1994
1994
1994
1992
1990
1983
1983
Country
Holland
Holland
Holland
Holland
Holland
Holland
Holland
Subject age
Adult
1-82 yr
Adult
Adult
17-32 yr
All
All
Subject
Comm
Outpt
Comm
Comm
Comm
Comm
Hosp
Isolates/subject
1
7.4
1.5
5
Total subjects
150
168
183
310
172
680
286
Amikacin
Amoxicillin
13%
13%
Amoxicillin-CA
10%
Ampicillin
16%
84%
26%
24%
Cefoxitin
Ceftiofur
Ceftriaxone
Cephalothin
13%
10%
Chloramphenicol
3%
9%
55%
73%
Ciprofloxacin
0
Gentamicin
27%
23%
Kanamycin
4%
4%
47%
34%
Naladixic acid
0
2%
1%
16%
9%
Nitrofurantoin
0
0
1%
10%
9%
Streptomycin
24%
24%
26%
34%
Sulphamethoxazole
10%
24%
32%
44%
82%
46%
27%
Tetracycline
20%
29%
42%
18%
Oxytetracycline
8%
16%
21%
8%
Trimethoprim
3%
8%
9%
29%
10%
3%
TMP/SMX
98
Appendices
Appendix B
c) Studies Comparing Escherichia Coli Antimicrobial Resistance Rates: Faecal Carriage in Scandanavia
Study
BaggerKronvall
Leegaard Leistevuo Osterblad Osterblad Osterblad
Skjt (92)
(322)
(132)
(323)
(80)
(80)
(80)
Year
2007
2005
2002
1996
2000
2000
2000
Country
Denmark
Sweden
Norway
Finland
Finland
Finland
Finland
Subject age
Adult
Adult
All
Adult
Subjects
Comm
Comm
Outpt
Comm
Comm
Hospital
LTC
Isolates/subject
3
3
5
5
5
Total subjects
85
50
83
334
125
159
74
Amikacin
0
Amoxicillin
40%
Amoxicillin-CA
0
0
0
3%
1%
Ampicillin
26%
31%
38%
12%
14%
25%
Cefoxitin
Ceftiofur
Ceftriaxone
0
Cephalothin
1%
5%
8%
Chloramphenicol
4%
12%
0
4%
7%
3%
Ciprofloxacin
1%
2%
0
0
0
Gentamicin
0
0%
4%
0
3%
0
Kanamycin
6%
Naladixic acid
1%
5%
1%
4%
13%
Nitrofurantoin
3%
Streptomycin
24%
18%
14%
Sulphamethoxazole
27%
24%
16%
13%
28%
Tetracycline
16%
23%
21%
14%
13%
25%
Oxytetracycline
Trimethoprim
9%
14%
9%
12%
40%
TMP/SMX
45%
8%
8%
15%
d) Studies Comparing Escherichia Coli Antimicrobial Resistance Rates: Faecal Carriage in Europe and Israel
Study
Sturmer
Lietzau
Lietzau
Bruinsma
VatopoEylan
Eylan
(155)
(180)
(171)
(104)
lous (115)
(324)
(324)
Year
2004
2006
2007
2003
1998
1979
1979
Country
Germany
Germany
Germany
Greece
Greece
Israel
Israel
Subject age
Adult
Adult
<5 yr
Adult
<7 yr
Adult
Infant
Subjects
Outpt
Comm
Outpt
Comm
Comm
Outpt
Isolates/subject
3
1
6
6
Total subjects
484
412
492
179
181
100
85
Amikacin
Amoxicillin
39%
Amoxicillin-CA
2%
8%
Ampicillin
17%
17%
17%
41%
8%
40%
Cefoxitin
Ceftiofur
Ceftriaxone
Cephalothin
Chloramphenicol
13%
7%
24%
Ciprofloxacin
1%
3%
Gentamicin
2%
Kanamycin
1%
11%
Naladixic acid
2%
12%
5%
Nitrofurantoin
Streptomycin
23%
40%
Sulphamethoxazole
Tetracycline
Oxytetracycline
50%
Trimethoprim
31%
TMP/SMX
9%
10%
9%
99
Appendices
Appendix B
e) Studies Comparing Escherichia Coli Antimicrobial Resistance Rates: Faecal Carriage in Europe
Study
Dunman
Mora (325)
Domnguez
Saenz
Carratala
(170)
(118)
(198)
(148)
Year
2005
2005
2002
2001
1996
Country
Turkey
Spain
Spain
Spain
Spain
Subject age
Infant
< 2 yr
Adult
Subjects
Hosp
Clinical
Comm
Comm
Outpt
Isolates/subject
4
1
1
5
Total subjects
118
138
41
40
25
Amikacin
0
0
Amoxicillin
58%
60%
Amoxicillin-CA
11%
1%
11%
0
Ampicillin
31%
19%
31%
Cefoxitin
1%
0
0
Ceftiofur
Ceftriaxone
0
Cephalothin
6
Chloramphenicol
10
12%
6%
Ciprofloxacin
0
5%
0
12%
Gentamicin
1%
2%
0
10%
Kanamycin
8%
10%
6%
Naladixic acid
4%
22%
6%
Nitrofurantoin
Streptomycin
32%
37%
Sulphamethoxazole
Tetracycline
32%
51%
25%
Oxytetracycline
Trimethoprim
8%
TMP/SMX
7%
24%
3%
70%
f) Studies Comparing Escherichia Coli Antimicrobial Resistance Rates: Faecal Carriage in China, the Phillipines, and Japan
Study
Lester
Nys (106)
Nys (106)
Bii (139)
(127)
Year
1990
2004
2004
2005
Country
China
PhilliPhilliJapan
pines
pines
Subject age
<7 yr
18-50 yr
18-50 yr
< 5 yr
Subjects
Comm
Comm
Comm
Outpt
Isolates/subject
9.9
Total subjects
53
105
106
47
Amikacin
Amoxicillin
Amoxicillin-CA
Ampicillin
26%
87%
75%
38%
Cefoxtin
Ceftiofur
Ceftriaxone
Cephalothin
Chloramphenicol
19%
68%
60%
Ciproflox.
63%
35%
Gentamicin
19%
46%
20%
0
Kanamycin
11%
Naladixic acid
Nitrofurantoin
Streptomycin
51%
Sulphamethoxazole
58%
Tetracycline
75%
Oxytetracycline
94%
85%
Trimethoprim
38%
93%
86%
TMP/SMX
100
Appendices
Appendix B
g) Studies Comparing Escherichia Coli Antimicrobial Resistance Rates: Faecal Carriage in Mexico and Venezuela
Study
Nys (106)
Nys (106)
EstradaCalva
van de
Lester
Nys (106)
Garcia
(119)
Mortel
(127)
(141)
(123)
Year
2004
2004
2005
1996
1998
1990
2004
Country
Mexico
Mexico
Mexico
Mexico
VenezVenezVenezuela
uela
uela
Subject age
18-50 yr
18-50 yr
< 5 yr
Infant
All
<7 yr
18-50 yr
Subjects
Comm
Comm
Hosp
Comm
Comm
Comm
Comm
Isolates/subject
4.7
13
1
9.6
Total subjects
99
99
62
20
161
41
230
Amikacin
Amoxicillin
53%
Amoxicillin-CA
Ampicillin
94%
78%
73%
90%
32%
43%
Cefoxtin
Ceftiofur
Ceftriaxone
Cephalothin
Chloramphincol
75%
45%
19%
62%
15%
30%
Ciprofloxacin
51%
15%
0
1%
1%
Gentamicin
14%
10%
5%
0
3%
Kanamycin
12%
Naladixic acid
Nitrofurantoin
2%
0
Streptomycin
56%
Sulphamethoxazole
62%
63%
Tetracycline
82%
62%
66%
Oxytetracycline
97%
86%
64%
52%
Trimethoprim
96%
76%
77%
41%
12%
33%
TMP/SMX
65%
h) Studies Comparing Escherichia Coli Antimicrobial Resistance Rates: Faecal Carriage in South America
Study
Nys (106)
Bartoloni
Bartoloni
Nys (106)
Bartoloni
Bartoloni
Bartoloni
(326)
(84)
(326)
(84)
(117)
Year
2004
2008
2006
2004
2008
2006
2004
Country
Curacao
Peru
Peru
Peru
Bolivia
Bolivia
Bolivia
Subject age
18-50 yr
< 6 yr
< 6 yr
18-50 yr
< 6 yr
< 6 yr
All
Subjects
Comm
Comm
Comm
Comm
Comm
Comm
Comm
Isolates/subject
~6
~6
~6
~6
Total subjects
149
1593
1590
95
1600
1584
108
Amikacin
0
1%
0.1%
Amoxicillin
Amoxicillin-CA
Ampicillin
48%
96%
92%
95%
97%
97%
58%
Cefoxtin
Ceftiofur
Ceftriaxone
2%
0.1%
2%
0.1%
Cephalothin
Chloramphenicol
8%
72%
71%
52%
67%
70%
41%
Ciprofloxacin
1%
39%
21%
36%
26%
16%
Gentamicin
4%
24%
20%
33%
30%
23%
Kanamycin
24%
22%
34%
34%
Naladixic Acid
62%
38%
51%
36%
Nitrofurantoin
Streptomycin
91%
79%
94%
92%
Sulphamethoxazole
Tetracycline
94%
76%
92%
86%
64%
Oxytetracycline
56%
93%
Trimethoprim
32%
93%
TMP/SMX
94%
91%
94%
96%
50%
101
Appendices
Appendix B
i) Studies Comparing Escherichia Coli Antimicrobial Resistance Rates: Faecal Carriage in the United States and Canada
Study
Sannes
Price
Hannah
Putnam
Scott
Akwar
Bruinsma
(185)
(207)
(46)
(30)
(204)
(105)
(104)
Year
2008
2007
2005
2005
2005
2007
2003
Country
USA
USA
USA
USA
USA
Canada
Canada
Subject age
>13 yrs
Adult
All
Adult
Adult
Adult
Subjects
Comm
Comm
Outpt
Comm
Comm
Comm
Isolates/subject
5
5
Total subjects
667
49
517
188
308
115
154
Amikacin
0
Amoxicillin
22%
Amoxicillin-CA
2%
Ampicillin
61%
46%
16%
16%
Cefoxitin
3%
2%
Ceftiofur
0
Ceftriaxone
2%
0
0
Cephalothin
28%
2%
Chloramphenicol
16%
3%
3%
1%
Ciprofloxacin
1%
6%
13%
<1%
0
1%
Gentamicin
18%
1%
1%
1%
Kanamycin
<1%
4%
Naladixic acid
5%
3%
4%
<1
1%
Nitrofurantoin
2%
<1
0
Streptomycin
12%
10%
Sulphamethoxazole
12%
17%
Tetracycline
41%
47%
19%
23%
Oxytetra.
16%
Trimethoprim
10%
TMP/SMX
8%
11%
36%
8%
5%
j) Studies Comparing Escherichia Coli Antimicrobial Resistance Rates: Faecal Carriage in the United States
Study
Murray
Lester
Levy (122) Levy (122)
Siegel
(184)
(127)
(203)
Year
1990
1990
1988
1988
1975
Country
USA
USA
USA
USA
USA
Subject age
<7 yr
Subjects
Comm
Comm
Hosp
Comm
Isolates/subject
9.3
9.9
1.5
1.5
2.3
Total subjects
13
39
289
189
130
Amikacin
Amoxicillin
Amoxicillin-CA
Ampicillin
38%
13%
17%
27%
22%
Cefoxitin
Ceftiofur
Ceftriaxone
Cephalothin
5%
Chloramphenicol
8%
3%
2%
2%
<1%
Ciprofloxacin
Gentamicin
0
0
<1%
1%
0
Kanamycin
0
0
4%
8%
Naladixic acid
1%
1%
2%
Nitrofurantoin
Streptomycin
38%
15%
16%
21%
37%
Sulphamethoxazole
13%
Tetracycline
46%
15%
19%
23%
Oxytetracycline
39%
Trimethoprim
0
3%
TMP/SMX
0
102
Appendices
Appendix B
k) Studies Comparing Escherichia Coli Antimicrobial Resistance Rates: Faecal Carriage in Africa
Study
Okeke
Ahmed
Bii (139)
Nys
Nys
Bonfiglio
Nys
(327)
(328)
(106)
(106)
(140)
(106)
Year
Country
Subject age
Subjects
Isolates/subject
Total subjects
Amikacin
Amoxicillin
Amoxicillin-CA
Ampicillin
Cefoxtin
Ceftiofur
Ceftriaxone
Cephalothin
Chloramphenicol
Ciprofloxacin
Gentamicin
Kanamycin
Naladixic acid
Nitrofurantoin
Streptomycin
Sulphamethoxazole
Tetracycline
Oxytetracycline
Trimethoprim
TMP/SMX
2000
Nigeria
2000
Sudan
2005
Kenya
2004
Kenya
2004
Ghana
Comm
Infant
Hosp
< 5 yr
Outpt
18-50 yr
Comm
18-50 yr
Comm
1
26
82
100
2002
Burkina
Faso
All
Hosp &
outpt
2004
Zimbabwe
18-50 yr
Comm
100
1405
207
361
95%
47%
95%
49%
73%
38%*
80%
69%
60%
4%
0
3%
Shanahan
(248)
1993
S. Africa
66%
89%
89%
6%
45%
1%
2%
82%
8%
2%
19%
1%
1%
92%
88%
90%
89%
59%
64%
All
Comm
4%
55%
100%
73%
48%
83%
80%
Amoxicillin-CA: Amoxicillin clavulanic acid

Sulphamethox: Sulphamethoxazole
TMP/SMX: Trimethoprim-sulfamethoxazole
Comm: Community-based
Hosp: Hospital
LTC: Long-term care facility
Outpt: outpatient
NOTE: empty cells reflect the fact that the antimicrobial agent was not used in susceptibility testing
103
Appendices
Appendix C
Sample Size Calculations
Sample size for proportions (329)
Hypothesis 1: the prevalence of human carriage of ampicillin resistant E. coli will be greater than or equal to 22% The
hypothesized proportion is based on 22% carriage in Canadian residents in Bruinsma, et als article (104) and assuming a
type I error probability of 5%.
H0: P < 0.22
H1: P 0.22
n = (Z/m)2 * pq
p
0.22
0.22
0.22
0.22
0.05
0.05
0.05
0.05
m
0.035
0.03
0.025
0.02
n
539
733
1,055
1,648
Where:
Z = ([p p0] 1/2n) / pq/n
m = confidence width / 2
p = hypothesized proportion
q = 1-p
= probability of Type I error
Sample size for logistic regression (276)

This was used to estimate the sample size required for the case-control study from which the subjects for the prevalence
study were drawn.
The estimates for p0 and p1 were based on the probability of the contaminated water sources being located on farms that
housed livestock.
H0: 1 = 0
H1: 1 0
n = (Z1-/22pq + Z1-p0q0 + p1q1)2 / (p1-p0)2
p0
p1
0.05
0.05
0.05
0.05
0.05
0.05
0.20
0.20
0.20
0.20
0.20
0.20
.30
.30
.35
.35
.40
.40
.39
.46
.45
.52
.50
.57
Odds
Ratio
1.5
2.0
1.5
2.0
1.5
2.0
Where p1 = OR x p0 / [(1-p0)+OR*p0]
OR = odds ratio (looking for ability to detect difference)
= probability of Type I error
= probability of Type II error
p1 = probability of outcome in group 1
p0 = probability of outcome in group 2
104
n
(per group)
425
141
401
135
388
133
Appendix D
Appendices
Ontario Well Water Study

Information Sheet
Safe drinking water is important for everyone. In Ontario, about 90% of all drinking
water samples that test positive for bacteria are from private drinking water sources
such as wells. Some of the bacteria in well water, including certain kinds of bacteria
called Escherichia coli (E. coli), can be resistant to a number of the antibiotics used to
treat infections. There are many unanswered questions about how antibiotic resistance
develops; some scientists think that antibiotic resistant E. coli in water may be one
source of antibiotic resistant infections in people.
From May 2005 to December 2006, a group of doctors and scientists from the
University of Western Ontario and the University of Toronto will be studying how
drinking or using contaminated well water affects people. This study will determine
possible factors (e.g. type of farm, soil, or well) that could contaminate private wells
with antibiotic resistant bacteria. It will also determine whether antibiotic resistant
bacteria in drinking water might be a source of antibiotic resistant bacteria in humans.
Since you have submitted a water sample for testing, you may be telephoned a few
weeks after your water sample is tested to ask if you would be willing to take part in the
study. The researchers need to talk to some people with water that tested positive for E.
coli and some people whose water tested negative. Only a small fraction of people, who
will be selected at random, will be telephoned. If you do receive a call, it will be from a
member of the Safe Water Unit at the Ministry of Health and Long-Term Care. This
person will ask permission to give your phone number to the researchers. You do not
have to take part in the study if you do not want to. If you consent to participate in the
study, the Ministry of Health and Long-Term Care will disclose your contact
information to the researchers. The researchers will then contact you to arrange an
interview. If you decide not to participate in the study, the Ministry of Health and LongTerm Care will not disclose your contact information to the researchers. Whether or not
you decide to take part in the study will not affect future water testing in any way.
If you have any questions about this study, you may contact Brenda Coleman, the study
coordinator, at (519) 631-3159 ext. 265 weekdays between 8:30 a.m. and 4:30 p.m. or
e-mail her at b.coleman@utoronto.ca. If you are interested, more information about the
study is available at http://microbiology.mtsinai.on.ca/research/wellwaterstudy/
For information about the results of your water test please call your local public
health unit.
Thank you.
105
Appendices
Appendix E

Telephone Script for Contact from Safe Water Unit
Cases and A Controls (E. coli positive)
Hello, my name is <FILL: First and last name>. I am calling from the Safe Water Unit at the Ministry of
Health and Long-Term Care. May I please speak with <FILL: name of submitter>.
Submitter/designate not available:
I am calling to talk to you about the water sample that was sent from your household for testing. Is there
someone else that I could talk to?
[NO] Is there a good time to call back so I can talk with <FILL: Name of submitter>?
[YES] Continue
Submitter (or designate) speaking:
Thank you. I am calling for a group of doctors and scientists at the University of Western Ontario and the
University of Toronto who are studying private well water. They want to know why the water from some
wells is contaminated with bacteria that are resistant to antibiotics while water in other wells is not. They
are also looking at whether using contaminated water affects the health of people who drink it. To help
with this study, they need to talk to people who have had their well water tested. Because you have
submitted a sample for testing, they would like to talk with you about taking part in their study.
I am phoning to ask if you would agree to allow the Safe Water Unit to give your name and phone
number to these researchers. They would then contact you to explain the study and ask if you would be
willing to participate. Would it be okay if they called you to explain the study?
[NO]
Thank you very much for your time. Good bye.
[YES] I have a few other questions I need to ask to see if you are eligible to take part in the study. Do
you use the water that was sent for testing on <FILL: Date Collected>?
[NO real estate, moved, submitted for someone else] The researchers need to talk with someone
who can answer questions about the water source and possible sources of contamination, so you are not
eligible for the study. Thank you very much for your time. Good bye.
[NO use bottled/filtered/treated water] Is the water from a well or other water source in Southern
Ontario?
[YES] Is the water from a well or other water source in Southern Ontario? (Note: see attached)
[NO] The researchers are only interested in talking with people who are using water from Southern
Ontario, so you are not eligible for the study. Thank you very much for your time. Good bye.
[YES] Good. The Ontario Well Water Study will be calling you in the next week or two to explain the
study and ask if you will agree to participate. They will also be asking you if other members of your
household would be willing to take part in the study. Thank you for your time and good bye.
106
Appendices

Telephone Script for Contact from Safe Water Unit
B Controls (uncontaminated)
Hello, my name is <FILL: First and last name >. I am calling from the Safe Water Unit at the Ministry of
Health and Long-Term Care. May I please speak with <FILL: Name of submitter >.
Submitter/designate not available:
I am calling to talk to you about the water sample that was sent from your household for testing. Is there
someone else that I could talk to?
[NO] Is there a good time to call back so I can talk with < FILL: Name of submitter >?
[YES] Continue
Submitter (or designate) speaking:
Thank you. I am calling for a group of doctors and scientists at the University of Western Ontario and the
University of Toronto who are studying private well water. They want to know why the water from some
wells is contaminated with bacteria that are resistant to antibiotics while water in other wells is not. They
are also looking at whether using contaminated water affects the health of people who drink it. To help
with this study, they need to talk to people who have had their well water tested. Because you have
submitted a sample for testing, they would like to talk with you about taking part in their study.
I am phoning to ask if you would agree to allow the Safe Water Unit to give your name and phone
number to these researchers. They would then contact you to explain the study and ask if you would be
willing to participate. Would it be okay if they called you to explain the study?
[NO]
Thank you for your time. Good bye.
[YES] I have a few other questions I need to ask to see if you are eligible to take part in the study. Do
you use the water that was sent for testing on <FILL: Date Collected>? (Note: i.e. not for real estate,
conservation area, or municipal building)
[NO real estate, moved] The researchers need to talk with someone who can answer questions about
the water source and possible sources of contamination, so you are not eligible for the study. Thank you
very much for your time. Good bye.
[NO use UV/other filter/bottled or YES] Is the water from a well or other water source in Southern
Ontario?
(Note: i.e. not their cottage in North Bay.)
[NO] The researchers are only interested in talking with people who are using water from Southern
Ontario, so you are not eligible for the study. Thank you very much for your time. Good bye.
[YES] Have you submitted this water for testing before or since the test on <FILL: Date Collected>?
[NO/DONT KNOW] Good (okay). The Ontario Well Water Study will be calling you in the next week
or two to explain the study and ask if you will agree to participate. They will also be asking you if other
members of your household would be willing to take part in the study. Thank you for your time and good
bye.
[YES] And, has this water source ever tested positive for E. coli? (Note: some people send in water
samples from before and after a treatment system, e.g. UV lights. If this sample is a post-treatment one
and the same water source tested positive for E. coli, we cant accept them as a B control. Similarly,
water tests may vary from time-to-time, so if this water source tested positive in the past year, we cant
accept them.)
members of your household would be willing to take part in the study. Thank you for your time and good
bye.
107
Appendices
[YES] Was the positive test within the past year?

members of your household would be willing to take part in the study. Thank you for your time. Good
bye.
[YES] The researchers are only interested in talking with people who have had uncontaminated water
for a year or longer, so you are not eligible for the study. Thank you very much for your time. Good bye.
Southern Ontario:
Brant county & Brantford (and surrounding areas in county)
Bruce & Grey counties & Owen Sound, Walkerton, Durham, Southampton.
Elgin county & St. Thomas, Aylmer
Haldimand & Norfolk counties & Simcoe, Dunnville, Caledonia
Halton region & Oakville
Hamilton city & region
Huron county & Clinton, Wingham
Lambton county & Sarnia, Forest
Middlesex county & London, Strathroy
Niagara region & St. Catherines, Welland
Oxford county & Woodstock
Perth county & Stratford, Listowel
Waterloo city & region, Cambridge
Wellington & Dufferin counties - Guelph, Mount Forest, Orangeville, Shelburne, Palmerston
NEW EXPANDED AREA:
Ottawa-Carelton (and surrounding areas in county)
Eastern Ontario Cornwall, Rockland, Hawkesbury, Casselman.
Renfrew County Arnprior, Deep River, Barrys Bay.
Kingston, Frontenac, Lennox & Addington Napanee, Cloyne, Sharbot Lake
Hastings and Prince Edward Belleville, Bancroft, Picton, Trenton
Leeds, Grenville & Lanark Brockville, Gananoque, Kemptville, Almonte, Smiths Falls
Haliburton, Kawartha, Pine Ridge Lindsay, Campbellford, Brighton
Durham Region Whitby, Ajax, Oshawa, Pickering, Uxbridge
Toronto Scarborough, North York, Etobicoke
York region Newmarket, Unionville, Richmond Hill
Peterborough county and city Cochrane, Hearst, Hornepayne, Moosonee, Matheson
Muskoka-Parry Sound Huntsville, Burks Falls
Peel Brampton, Mississauga
Simcoe county - Barrie, Orillia, Midland, Collingwood, Cookstown
108
Appendices
Appendix F

Telephone Script for Initial Study Contact
Hello, my name is <FILL First and last name >. I am calling for the Ontario Well Water Study.
May I please speak with <FILL: Name of submitter >.
Im calling for a group of researchers at the University of Western Ontario and the Ministry of
Health who are studying bacteria in well water. A few days ago, the Safe Water Unit called you and
asked for permission for me to contact you about your participation in the study. You many also
have seen information about the study attached to the water sample bottle you submitted for testing.
The researchers are studying why the water in some wells becomes contaminated with bacteria that
are resistant to antibiotics while water in other wells does not. They are also looking at whether
using contaminated water affects the health of the people who drink it. To help with this study we
need to talk to people like you who have had their water tested. It is important that we talk to
people whose water tested positive for E. coli as well as people whose water tested negative so we
can see whether there are differences between the groups.
May I take a few minutes to explain what your participation in the study would include?
[NO] Arrange for a date/time to return call.
[YES] To understand why some wells become contaminated and why some people carry antibiotic
resistant bacteria, we are going to interview 900 households in Southern Ontario. If you agree to
participate in the study, a trained interviewer will come to your home to interview you about your
well and things that might affect whether it becomes contaminated with bacteria. The interviewer
will also ask you, and other members of your household, questions about your health and other
things that might affect the bacteria in your intestines. Also, to find out which bacteria live in your
intestines, we will ask you to give us a simple rectal swab.
All personal information will be kept confidential and participation in this study is completely
voluntary. However, we think this research is very important, especially considering the growing
concern around antibiotic resistant bacteria and we hope you will agree to take part.
Do you have any questions about the study?
Would you be willing to take part in this study?
[NO] Thank you very much for your time. Good bye.
[YES] I would like to set up a time when I can visit your home to talk with you. We would also
like to ask others in your household if they would agree to take part in the study.
109
Appendices
Would it be okay with you if I ask others in your household if they would agree to take part in the
study?
[NO] That is fine, thank you. (Continue with appointment)
[YES] Thank you, I will ask them if they are interested in participating at that time.
[ALL] I am in your area on <FILL: First choice of day, date, and morning, afternoon, or evening >.
Would this be a good time to catch yourself [and others in your household] at home?
[NO] How about <FILL: Second choice: Day, date, and morning, afternoon, or evening?
[YES] Thank you, the interview will take about <FILL: 1 hour for household interview plus 30
minutes per personal interview>. I should arrive about <FILL: time >. If Im going to be late, I will
call to let you know.
Would you give me your address and directions to your home?
_______________________________________________________________________________
_______________________________________________________________________________
Do you have any questions?

Thank you very much for agreeing to take part in this important study. I will be there on <FILL:
Date and time >.
Would you like to take down my name and phone number in case you need to change this meeting
or if you have any [further] questions about the study?
110
Appendices
111
Appendices
112
Appendices
113
Appendices
114
Appendix H
Appendices
Ontario Well
Water Study
Information and Consent Form

You are invited to participate in a research study looking at bacterial resistance to antibiotics in well water
and whether it is linked to antibiotic resistant bacteria in the human gut.
Purpose of the study
Several doctors and scientists from the London Health Sciences Centre, Mount Sinai Hospital, and the public
health system are studying the public health impact of drinking, or being exposed to, well water
contaminated with bacteria that are resistant to antibiotics. They want to find out whether well water is a
potential source of bacteria that are resistant to antibiotics. The study investigators will compare the
environment surrounding wells contaminated with bacteria that are antibiotic resistant to wells that are not
contaminated to determine what factors may lead to contamination with antibiotic resistant bacteria.
The doctors and scientists are also studying gut bacteria. Some gut bacteria strains cause no harm and can be
found in most healthy humans. Once acquired, humans may carry these bacteria in their gut for a long time.
However, these bacteria are among the most common causes of infections in humans and they may carry
genes linked with antibiotic resistance.
How the study works
This study will collect information from over 1,500 people living in Ontario who use private well water and
have sent a sample for testing to a public health laboratory between May 1, 2005 and May 31, 2006. Only
people who are residents of the household for three months or more before the test was sent, who are 12
years and older, and speak English are eligible to participate in this study.
Risks of participating in the study
Although it is highly unlikely, there is a small risk of rectal perforation while performing the rectal swab.
Benefits of participating in the study
Although there is not any known benefit for any individual participating in the study, the results of this
investigation may help find out what factors are linked with contamination of private wells with antibioticresistant bacteria so it may be prevented in future. This study may also help determine what things are linked
with humans carrying antibiotic-resistant bacteria in their gut so they can be avoided.
What you will be asked to do if you agree to participate
The study consists of two separate parts. The first is a visit to your home to interview you about your
property, household, and well. The interviewer will also tour your property to note the location of your well,
septic system, open water, pastures, and farm buildings. This should take about one hour.
The second part is a personal interview about your health and, to find out which bacteria live in your gut, we
will ask you to give us a rectal swab. A rectal swab is a good way to find out which bacteria live in a
persons gut. A rectal swab is a Q-tip that is inserted about 1 to 2 cm (less than 1) into a persons own
anal area to sample stool. If you consent to provide a rectal swab, you also agree to have the sample tested
for antibiotic resistant bacteria at the study lab, and to have the bacteria grown from your rectal swab frozen
in the lab for up to one year.
We would also like to talk to other members of your household for this study. However, we ask you to
inquire whether other members of your household would be willing to be contacted by the researchers before
giving us their name or contact information.
Page of 2
115
Participant initials: _________

101
Appendices
Confidentiality
Participation in this study is completely voluntary. You may refuse to participate. You are free to withdraw
from the study at any time during the study. You may refuse to answer any question or to complete any
interview, even if you have already started it.
All personal information will be kept confidential and only grouped data will be published or released to the
public. Personal information, such as names, will be replaced by a number on all surveys and specimens to
protect privacy.
Other things you should know
If you are already participating in another study at this time, please inform the study person right away to
determine if it is appropriate for you to participate in this study.
You may call or write the study co-ordinator or the primary investigator listed on this form (at the number
below) if you have questions at any time.
You do not waive any legal rights by signing this information/consent form.
You will be given a copy of this consent form.
I have read the information/consent form, have had the nature of the study explained to me, and I agree to
participate. All questions have been answered to my satisfaction.
Signature ______________________________________
Date ________________________
Name (please print) _______________________________
I have read the information/consent form, have had the nature of the study explained to me, and I agree to
give a rectal swab. All questions have been answered to my satisfaction.
Signature ______________________________________
Date ________________________
Signature of person obtaining informed consent ____________________________________________

Contact information
Study Coordinator and Co-investigator
Brenda Coleman, PhD candidate
b.coleman@utoronto.ca
(519) 631-3159 ext. 265
Primary Investigator
Dr. Marina Salvadori
London Health Sciences Centre
(519) 685-8500 ext. 52255
If you have any questions about the conduct of this study or your rights as a
research subject you may contact Dr. J. Gilbert, VP Research and Development,
at London Health Sciences Centre at (519) 685-8500 ext. 76649.
Page of 2
116
Participant initials: _______
Appendices
Appendix I

Household Questionnaire
Date of interview:
(dd/mm/yyyy)
Interviewer: ________________________________________
Household ID:
Consent acquired
I am going to start with a general household questionnaire. It should take about 10
minutes and covers things about people who live here, your water supply and septic
system, and even your pets.
You are free to refuse to answer any question and to stop the interview at any time.
However, your answers are all important and I hope you are able to answer all of the
questions I ask you.
I am going to start with a few questions about you.
1. The respondent is:
Male
Female
2. How old are you?
years
(999 for dont know/refused)
3. How long have you lived at this address? (using this well)
months
years
(999 for dont know/refused)
I am going to ask about people who currently live in your home. For these
questions, I would like to know about people who live in your home, whether or not
they are related to you, but who live at this address four or more days per week.
4. Including yourself, how many adults, that is people 20 years and older, currently live in
your home?
(99 dont know/refused)
5. How many youths 12 to 19 live here?

(If none, enter 0, do NOT leave blank)
117
Appendices
6. And how many children 4 to 11 years?
7. How many children under 4 years of age live in your household?

(If zero, skip to Q=8)
7a. Are any of the children still in diapers? (Includes pull-ups)

Yes
No
Dont know / refused
7b. Do any children in your household go to a day care centre?

(5 or more children in centre; child in care 1 or more days/week)
Yes
No
Dont know
8.
Does anyone in the household work at any of the following

Yes
No
Day care centre or babysitting service

Hospital, nursing home or residential home
Sewage treatment plant
Any other job where they are in contact with
human waste: ______________________
Farm with livestock (any type)
Abattoir, butcher shop, or meat processing
Animal feed processing plant
Nursery or landscaping service
Any other job where they are in contact with
meat, animals, or animal waste:___________
9. What township and county is this residence a part of?

Township: ___________________________________ (Write dont know/refused as required)
County: _____________________________________
10. Do you have a swimming pool? (not a pond or swimming hole)

Yes
No
Dont know
11. Do you have a hot tub or spa?

No
Yes
12. Have many washrooms do you have in your home?

(With toilet; include outhouse; 99 for dont know/refused)
118
Appendices
Now I would like to ask a few questions about your pets.

13. Do you have any pets?
Yes
No (Skip to Q=14)
Dont know/refused
13a. What kind of pets do you have?

Dog(s)
Cat(s)
Bird(s)
Other: specify: ________________________________
14. In the past 3 months, have any animals spent more than a few minutes inside the
house? (Several hours per week. Also include animals that live in house e.g. hamster)
Yes
No (Skip to Q=17)
Dont know/refused
14a. What kind of animals have spent time inside the house?
Dog(s)
Cat(s)
Bird(s) - (Skip to Q=16)
Other: specify: ________________________________ - (Skip to Q=16)
________________________________ - (Skip to Q=16)
15. How often would you say you give your <FILL: cat and/or dog> any of the following.
Would you say your pet(s) often, sometimes, rarely or never get(s)
(Read list, if more than one pet, indicate the more extreme measure for each item)
Often
Some
Rarely
Never
times
Commercial dry or canned food
Commercial biscuits or dry treats
Raw meat (any kind)
Cooked meat
Raw hide treats
16. Were any of these animals on antibiotics in the past three months?
Yes
No (Skip to Q=17)
Dont know/dont remember
16a. Do you recall what kind of antibiotic were they given?

__________________________________
__________________________________
__________________________________
__________________________________
119
Appendices
Im going to ask a few questions about your drinking water now.

17. Where do you get the water you use for drinking? Is it from a private well, a well used
by 6 or more households, a cistern, a municipal system, or some other source?
Private well
Communal well
(6 or more households)
Cistern
Municipal (or town) water
Bottled (bulk or individual)
Other: _____________________________
Dont know
17a. And where do you get the water you use for bathing, dental care, and other
household uses?
Same as above (Q=17)
Private well
Communal well
(6 or more households)
Cistern (Skip to Q=21)
Municipal (or town) water (Skip to Q=21)
Other: _____________________________
Dont know
18. What type of well do you have? Is it drilled, dug, bored, or driven, which is also called
a sand point or well point?
Drilled
Dug or bored
Driven (sandpoint or wellpoint)
Other: ________________________
Dont know
19. How deep is your well?

feet (9999 for dont know)
metres
20. How old is it?

months (999 for dont know)
years
21. Have any repairs or maintenance been done on your well or water lines in the
past 12 months?
Yes
No skip to Q=22
Dont know
21a. And in the past 3 months have any repairs or maintenance been done on your
well or water lines?
Yes
No
Dont know
120
Appendices
22. Why did you submit your water for bacteriological testing this most recent time?
Do it regularly / routinely
Off colour / cloudy
Bad / different taste
Odour
Heavy rain
People ill with stomach illness / diarrhoea
E. coli in previous test
Coliforms in previous test
Other: _____________________________________________________
No specific reason
Dont know
23. How many times did you send your water for bacteriological testing in the past
12 months?
Number
Many times (dont know exactly, but more than 10)
Dont know
24. How many times has your well water tested positive for E. coli in the past
12 months? (Use results sheets if available)
Number
Many times (dont know exactly but more than 10)
Dont know
24a. And do you recall how many times it tested positive for coliforms?
Number
Many times (dont know exactly but more than 10)
Dont know
25. Do you currently treat the water you use for drinking? By treating, I mean boiling,
adding chlorine or some other treatment to remove bacteria and other contaminants?
Yes
No (Skip to Q=26)
Dont know
25a. How do you treat it?

Boil
Chlorine Did you shock treat it
or use a chlorinator
Filtration
Brita or other filter system
Ultraviolet (UV)
Ozone
Other: ______________________________
Dont know
121
Appendices
25b. When did you start treating it?
(dd/mm/yyyy)
(Year only if several years ago)
25c. And do you treat the water you use for dental care, bathing, and other
household uses?
Yes
No (Skip to Q=26)
Dont know
25d. How do you treat it?

Same as above (Q=25)
Boil
Chlorine Did you shock treat it
or use a chlorinator
Filtration
Brita or other filter system
Ultraviolet (UV)
Ozone
Other: ______________________________
Dont know
25e. When did you start treating it?
(dd/mm/yyyy)
(Year only if several years ago)
Now a few questions about your septic system. Remember that everything you tell
me during this survey is confidential. Your name will not be connected to anything
you tell me and it will never be shared with anyone outside this study.
26. How is your domestic sewage handled? Do you have a (Read list)
Septic tank and weeping bed (aka: field or leaching bed)
Field tank
Holding tank
Lagoon
Surface discharge (Skip to Q=28)
Municipal system (Skip to Q=28)
Other: _____________________________________
Dont know
***Do NOT read***
27. When was the last time you had the tank [lagoon] pumped?
years (888 for never)
28. How old is your septic system?

If Q=26 is municipal system ask How long have you been on the municipal sewage
system? (Note: Oldest part if renovations completed)
years
122
Appendices
29. Have any upgrades or maintenance been done on your sewage system in the past 12
months?
Yes
No Skip to Q-30
Dont know
29a. And in the past 3 months have any repairs or maintenance been done on your
sewage system??
Yes
No
Dont know
Next I would like to ask a few things about your property.

30. How would you describe the soil on your property. Would you say it is predominantly
(Read list)
Gravel
Sand
Loam, or
Clay
Dont know
***Do NOT read***
31. How many acres of property do you own or rent at this location?
acres (9999 for dont know)
hectares
32. Would you describe your property as being (Read list)

Farm
Non-farm rural
Village or hamlet (<1,000 people)
Small town (1,000 to 10,000 people)
Other: ________________________________
33. Have livestock been housed on this property in the past 12 months? This includes
animals owned and/or cared for by your family or housed here and cared for by other
people. (Include pony, chickens, pigeons, etc. but not cats, dogs unless it is a kennel)
Yes
No (Skip to Q=37)
Dont know
33a. What type of livestock have been on this property in the past 12 months?
(Check all that apply.)
Dairy cattle
Beef cattle
Sheep (lambs)
Goats
Other: _________________________________
Other: _________________________________
123
Pigs
Horses or ponies
Chickens
Turkeys
Appendices
33b. What is the largest number of <FILL: type of livestock> that have been housed
on this property in the past 12 months?
____________________________ (type)
____________________________ (type)
____________________________ (type)
____________________________ (type)
34. Are livestock currently housed on the property?
Yes
No
Dont know
35. Do [or did] you care for the livestock on this property?
Yes
No (Skip to Q=37)
Dont know
36. Have you used antibiotics as a feed supplement for your livestock in the past 12
months?
Yes
No (Skip to Q=37)
Dont know
36a. What types of antibiotics have you used as a feed supplement?

________________________________ (Write in dont know if applicable)
________________________________
________________________________
36b. When did you start using antibiotics in your feed?
(mm/yyyy)
(Year is sufficient if several years ago)
36c. Are you still using antibiotics in your feed?

Yes (Skip to Q=37)
No
Dont know
36d. When did you stop using it?
(mm/yyyy)
37. Has manure been stored on your property or spread on your fields in the past 12
months?
Yes
No (Skip to Q=39)
Dont know
124
Appendices
37a. Where, in relation to your well, has manure been stored or spread over past
year? Would you say it is stored or spread (Read list)
(Includes liquid or solid; stored in any way)
Within 15 metres (50) of your well
Within 30 metres (100) of the well (i.e. 16 to 30 metres)
Within 100 metres (330) of your well (31 to 100 metres)
More than 100 metres (330) from the well
Dont know ***Do NOT read***
37b. And within the past 3 months, where, in relation to your well, has manure been
stored or spread? Would you say it is stored or spread (Read list)
Within 30 metres (100) of the well
More than 100 metres (330) from the well (Skip to Q=38)
37c. When was the last time manure was stored or spread on fields within 30
metres (100) of your well? Would you say (Read list)
Within the past month
Within the past 3 months
More than 12 months ago, or
Never [not since s/he lived in house]
38. How soon is manure usually worked into the ground when it is spread? Would
you say it is worked in (Read list)
Same day (includes injected)
Within 1 to 3 days
Within 4 to 7 days
More than one week after it is spread
39. Has a neighbour bordering your property had livestock on their land in the past 12
months? By bordering, I mean a neighbour that shares a fence line with you.
No (Skip to Q=40)
Yes
Dont know
39a. What type of livestock were on that property within the past 12 months?
(Check all that apply)
Dairy cattle
Beef cattle
Sheep (lambs)
Goats
Other: _________________________________
Other: _________________________________
125
Pigs
Horses or ponies
Chickens
Turkeys
Appendices
40. Has a neighbour bordering on your property stored manure on their property or spread
manure on their fields in the past 12 months? (Stored in any way including piled, barnyard, tank..)
Yes
No (Skip to Q=42)
Dont know
40a. Where, in relation to your well, have neighbours stored or spread manure over
past 12 months? Would you say it is spread (Read list)
Within 15 metres (50) of well
More than 100 metres (330) from well
Not stored/spread in past 12 months (Skip to Q=42)
Dont know
***Do NOT read***
40b. Where, in relation to your well, have neighbours stored or spread manure
over past three months? Would you say it is spread (Read list)
Within 100 metres (330) of well (Skip to Q=41)
More than 100 metres (330) from well (Skip to Q=41)
Not spread in past 3 months (Skip to Q=41)
Dont know
***Do NOT read***
40c. When was the last time manure was stored or spread on fields within 30
metres (100) of your well? Would you say (Read list)
Within the past month
Within the past 12 months ago
More than 12 months ago, or
Never
Dont know
***Do NOT read***
41. How soon is manure usually worked into the ground when it is spread? Would
you say (Read list)
Same day (includes injected)
Within 1 to 3 days
Within 4 to 7 days
More than one week
Not spread (stored only)
Dont know
***Do NOT read***
42. Do you fertilize your vegetable or flower gardens or fruit orchards with animal manure?
This includes manure purchased in bags at a store or garden centre.
Yes
No
Dont know
126
Appendices
43. Has sludge from human waste been spread on fields within 90 metres (300 feet) of
your well in the past 12 months?
Yes
No
Dont know
44. And in the past 12 months, has waste from meat processing been spread within 90
metres (or 300 feet) of your well?
Yes
No
Dont know
I am going to ask a few questions that will help us group your information with other
households most like your own. Remember that nothing about you, as an
individual, will ever be released and you are identified by number in this study.
45. First, what is the highest level of education that has been attained by any adult in the
household? Would that be (read list)
Less than grade 9
Some high school
University
Not stated ***Do NOT read***
46. What is your best estimate of the total off-farm income, before taxes and deductions,
of all household members combined, from all sources, in 2005? Was that total household
income (Read list include income from government sources)
Less than $20,000
$20,000 to less than $40,000
$40,000 to less than $60,000
$60,000 to less than $80,000
$80,000 or more
Or do you not have off-farm income
Not stated
***Do NOT read***
For farming households only (Q=32=farm)

47. What is your best estimate of the net income from your farm, before taxes, in 2005?
Was that net income (Read list)
Less than $20,000
$20,000 to less than $40,000
$40,000 to less than $60,000
$60,000 to less than $80,000
$80,000 or more
Not stated
***Do NOT read***
Not applicable: Not a farming property
127
Appendices
Thank you. That is all of the questions I have about the household.
I would like to take a few measurements around your property to show where things
are in relation to your well. However, I would also like to ask you a few questions
about your personal water use, use of medicine, travel, and some other things that
might influence whether you carry antibiotic resistant bacteria.
Would you prefer I do the personal interview(s) now and do the measurements after
we are through with it?
48. GPS coordinates from well:
.
.
Longitude
(heading/degrees/minutes/seconds) Latitude
(heading/degrees/minutes/seconds)
49. Note the distance between the well and each of the following
Distance
Septic tank
Weeping tile
House
Vegetable or flower garden
Area used to store manure in
past 12 months
Stables or kennels
Land used as pasture in the
past 12 months
Field where manure applied
in past 12 months
Fields tilled/worked in past 12
months
Open water
Forest/wooded area
Sanitary land fill site
Nearest property line
Neighbours septic system
Municipal sewage tile
if within 1000 metres
Other:
* 9999 = dont know
8888 = not applicable
Thank you very much for your time.

128
Km
Ft
Yd
Mile
NA
DK
Appendices
Appendix J

Personal Questionnaire
Date of interview:
(dd/mm/yyyy)
Interviewer: _________________________________
Personal ID:
Consent acquired
This interview should take about 10 minutes and covers things about your
medical, work, and travel history, as well as some questions about your use of water
and some personal habits.
You are free to refuse to answer any question and to stop the interview at any
time. However, your answers are all important and I hope you are able to answer all of
the questions I ask you.
First, I am going to ask a few questions about yourself so we can group you with
others like you.
1. What month and year were you born? (Birthday this month: record next month if not passed)
(mm-yyyy Enter 99-9999 for dont know/refused)
2.
Male
Female
3. How long have you lived at this address? (meaning at the house with this well)
months (Enter 999 for dont know/refused)
years
Now I am going to ask a few questions about your health.

4. Has a doctor ever told you that you have(Read list)
Yes
No
Diabetes (type I or type II)
Cancer
Arthritis or rheumatism
Heart disease or high blood pressure
Asthma, bronchitis, or emphysema
Autoimmune disease like lupus or Graves disease
Migraines
Kidney disease
Crohns disease, ciliac disease, colitis, ileitis, or IBS
Ulcers
Any other digestive problems*: __________________
Any other chronic conditions: ____________________
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129
D.K.
Appendices
(*e.g. diverticulitis, recurring heart burn, etc.)
5. Have you been hospitalized, for at least one night, in the past 12 months? (Ref: calendar)
Yes
No (Skip to Q=6)
Dont know/remember
5b. When were you admitted to hospital? (Probe: any other admissions in past year?)
5c. And how many nights did you stay in hospital in <FILL: month>?
Date of admission
dd/mm/yyyy
# of nights
admitted
**Enter 99-99-9999 for dont know/dont remember
Now Im going to ask a few questions about medications and other medical treatments
you may have used in the past three months.
6. Within the past three months, have you taken any of the following medications or
treatments? Have you taken.(Read list Refer to calendar)
Yes
No
Steroids like prednisone or cortisone
Immunosuppressive drugs like cyclosporine
Chemotherapy (for cancer)
Radiation therapy
Aspirin or ASA for more than a day or two at a time
7. How about antibiotics like penicillin, tetracycline, gentamycin, and other prescriptions for
infections. Have you taken any antibiotics in the past three months?
Yes
No (Skip to Q=9 if NO to all medications and treatments.Skip to Q=8 if no only to Q=7)
Dont remember
7a. What antibiotics have you taken in the past three months? (Probe: any others?)
7b. How long were you on <FILL: name of antibiotic>?
days/weeks (Circle)
______________________________________
______________________________________
days/weeks (Circle)
______________________________________
days/weeks (Circle)
______________________________________
days/weeks (Circle)
______________________________________
days/weeks (Circle)
(Enter dont know if applicable)
8. Are you currently taking any of the medications or treatments I asked about?
Yes
No (Skip to Q=9)
Dont remember

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130
Appendices
8a. What medication(s) [treatments] are you taking right now?

8b. And when did you start taking <FILL: name of medication>?
________________________________________
/
/
/
/
________________________________________
________________________________________
/
/
________________________________________
/
/
(dd/mm/yyyy)
(dd/mm/yyyy)
(dd/mm/yyyy)
(dd/mm/yyyy)
(Use prescription bottle when available - Enter dont know if applicable)
9. When you are prescribed antibiotics, how often do you take the medication exactly as
prescribed? By that I mean, taking the right number of pills at the right time of day. Would you
say you always, usually, sometimes, rarely or never take it exactly as prescribed?
Always
Usually
Sometimes
Rarely or never
Have never been prescribed an antibiotic (Skip to Q=10a)
Dont know
10. When you use antibiotics, how often do you finish the full prescription? Would you say
you always, usually, sometimes, rarely, or never finish all of the prescription?
Always (Skip to Q=11)
Usually
Sometimes
Rarely or never
Have never been prescribed an antibiotic
Dont know
10a. When you do not finish all medication or when it goes out of date or expires,
how do you usually dispose of what is remaining? (List all that apply)
Return to pharmacy / drug store
Throw in garbage for land fill
Throw in the toilet / down sink
Always complete medications
Other (specify) ______________________________
Dont know
11. We define diarrhoea as three or more loose stools or bowel movements in any 24-hour
period. Have you suffered from diarrhoea in the past three months?
Yes
No (Skip to Q=12)
Dont remember
11a. Have you had diarrhoea in the past month?

Yes
No (Skip to Q=12)
Dont remember
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Appendices
11b. How many times have you had diarrhoea in the past month?
number (99 for dont know. If zero, use 0, do not leave blank)
11c. How many days did the last episode of diarrhoea last?
hours (99 for dont remember/dont know)
days
11d. How many days of school or work - including work at home - did you miss
because of it?
hours (99 for dont remember/dont know)
days (If zero, use 0, do not leave blank)
The next set of questions are about some of the foods you eat.
12. Did you drink raw or unpasteurized milk, or eat diary products made from raw milk, in the
past three months? (includes cream, butter, yoghurt, cheese, or ice cream)
Yes
No (Skip to Q=13)
Dont know
12a. How often did you drink raw milk or eat dairy products made from raw milk?
Would you say most days of the week, a few times a month, or less often?
Most days
A few times a month
Less often
Dont know
13. Have you drank unpasteurized cider in the past three months?
Yes
No
Dont know
Now I am going to ask you a few questions about your use of water.
14. While at home, approximately how many 8 ounce (240 mL) glasses of water do you drink
every day? This would also include water in hot drinks like tea or coffee and for cold drinks
like orange juice or Kool Aid.
How many 8-ounce glasses do you think you drink every day?
glasses (Refer to glass - enter 99 for dont know but probe for estimate)
15. Do you regularly use bottled water at home? By regularly, I mean most days of the week.
(Individual and bulk)
Yes
No (Skip to Q=16)
Dont know
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132
Appendices
15a. Including water used for hot and cold drinks, about how many 8 ounce
(240 mL) glasses of bottled water do you drink every day?
glasses (99 for dont know)
15b. Do you use bottled water to (Read list)

Yes
No
Wash vegetables or fruit

Brushing teeth
Washing hands
15c. How long have you been using bottled water at home?
days (99 for dont know)
months
years
This section is about meal preparation and some of your personal practices.
16. How often are you involved in meal preparation in your household? Would you say you
always, usually, sometimes, rarely or never prepare meals?
Always
Usually
Sometimes
Rarely (Skip to Q=17)
Never (Skip to Q=17)
Dont know
16a. When preparing meals, do you touch raw beef, pork, or poultry with your bare
hands?
Yes
No
Dont know
17. In general, how often do you wash your hands with soap and water for each of the
following. Would you say you always, usually, sometimes, rarely, or never wash your hands
with soap and water (Read list)
Always
Usually
Before preparing food

After handling raw meat/poultry
Before eating meals
After using the toilet
After playing with pets/animals
After changing diapers
After handling garbage
After caring for sick people
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133
Some
times
Rarely
Never
Not
applic.
Appendices
Now Im going to ask about your work and leisure activities.

18. Have you travelled outside Canada in the past 12 months? (Includes USA if overnight)
Yes
No (Skip to Q=19)
Dont know
18a. To what country or countries did you travel? (Probe: any others? If many, focus on the
past 3 months. If several to same country, focus on most recent trips)
18b. What dates did you travel to return from <FILL: country>?
______________________________
dd/mm/yyyy
______________________________
dd/mm/yyyy
______________________________
dd/mm/yyyy
(Enter dont know if applicable)
18c. Did you get diarrhoea while you were travelling or within a few days of
returning from your trip(s)?
Yes
No
Dont know
19. Over the past three months have you been swimming in an ocean, lake, river, or pond?
(Include foreign and Canadian)
Yes
No
Dont know
20. Have you been swimming in a pool in the past three months? (public or private)
Yes
No
Dont know
21. And how about a hot tub? Have you used a hot tub in the past three months?
Yes
No
Dont know

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Appendices
I am going to read you a list of activities that might be a part of your day-to-day life.
This includes things you might do at work, at home, or during leisure time.
22. Over the past three months, have you been in personal contact with human waste
including diapers or bedpans, or while doing plumbing repairs or working at a sewage
treatment plant?
Yes
No (Skip to Q=23)
Dont know
22a. Would you say you were in contact with human waste several times per week,
several times per month, or less often?
Several times per week
Several times per month
Less often
23. Have you been in personal contact with antibiotics for either human or animal use over
the past three months? This might include at a pharmacy, veterinary clinic, or on a farm.
Yes
No (Skip to Q=24)
Dont know
23a. Would you say you were in contact with antibiotics several times per week,
Less often
24. Have you been in personal contact with animal or pet food, either at home or at work, in
the past three months?
Yes
No (Skip to Q=25)
Dont know
24a. Would you say you were in contact with animal or pet foods several times per
week, several times per month, or less often?
Less often
25. Have you touched raw beef, pork or lamb with your bare hands in the past three months?
Yes
No (Skip to Q=26)
Dont know

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Appendices
25a. Would you say you touched raw beef, pork, or lamb several times per week,
Less often
26. Over the past three months, have you touched raw poultry with your bare hands? This
would include chicken, turkey, or other poultry.
Yes
No (Skip to Q=27)
Dont know
26a. Would you say you have touched raw poultry several times per week,
Less often
27. And over the past three months how often have you been in direct contact with any of
these animals, meaning actually touching them or their manure? Would you say you were in
contact several times per week, several times a month, less often, or not at all with(Read list)
Several
per week
Several
per month
Less often
Not at all
Dairy cattle
Beef cattle
Horses
Pigs
Sheep
Goats
Chickens
Turkeys
Other birds (including wild)
Dogs
Cats
Game animals:________________________
Other: _______________________________
28. Do you attend school or work away from home? (incl. unpaid/volunteer work and all applicable)
Attend school
Work
No (Skip to end)
28a. On average, how many hours per week do you attend school or work away from
hours
home?
That is all of the questions I have for you at this time. Thank you very much for your
help with this study. Do you have any questions for me?
***Provide instructions on how to collect rectal swab.
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136
Appendices
Appendix K

RECTAL SWAB COLLECTION INFORMATION
How to collect your swab
The easiest way to ensure a proper sample is if you take your own rectal swabs and send
the swabs for testing. To take the swab:
Open the plastic covering by peeling back the top edges.

Place the numbered label on the tube with the black gel in it.
Remove the top from the tube with black gel in it.
Pick up the cotton-tipped swab and roll it around the wrinkled skin of your
anus (the skin around your rectum).
Then insert the swab into your rectum (the cotton tip should be completely
inside your rectum).
Rub around the inside of your rectum twice.
Remove the swab and place in the black gel. Press the swab into the black
gel until the top is on tightly.
Place the swab into the Specimen Transport Bag. (One per bag, please.)
Send it by mail as described below.
Mailing instructions
Once the swab has been collected place it in the envelope addressed to S. Braithwaite,
Alberta Provincial Laboratory for Public Health. Send it to the lab by Canada Post
(the address is on the envelope along with correct postage).
Note: The swab can be stored at room temperature.
All swabs can be mailed in the same envelope.
Please send your signed consent forms in the envelope addressed to B. Coleman, c/o The
Ontario Well Water Study.
If you have questions or comments please call Brenda Coleman at 519-631-3159 ext. 265.
Thank you very much for your help with our study.
137
Appendices
Appendix L
Variable
Human carriage
of antimicrobialresistant E. coli
Use of untreated
water
contaminated
with
antimicrobialresistant E. coli
Days: water
collection to
interview
Use bottled water
Age
Sex
Farming property
Education
(household)
Variables Derived from Personal and Household Questionnaires

Data source
Item
Derivation
-Study laboratory
- Rectal swab isolate
analyses (screening contaminated with E. coli resistant
and NARMS testing) to one or more antimicrobial
agents in 2004 NARMS panel
1
-Public health
-Public health laboratory
-Water not contaminated or
laboratory analysis
bacteriological analysis of water
treated: no E. coli
2
-Study laboratory
-Laboratory analyses: at least one contamination for one year
analyses (screening water sample contaminated with
or longer1,3 -ORcontaminated with E. coli
and NARMS testing) E. coli resistant to one or more
susceptible to all
-Initial consent
antimicrobial agents in 2002 or
antibiotics1,2 OR-Household
2004 NARMS panel for enteric
contaminated with
questionnaire
bacteria
3
antimicrobial resistant E. coli
-Initial consent (eligibility for B
and treated* for one year or
controls: Has this water tested
longer**
positive for E. coli in the past
-Contaminated:
year?
contaminated with
-H: date of household interview
antimicrobial resistant E.
-H25: Do you currently treat the
coli1,2 and not treated*
water you use for dinking? By
treating I mean boiling, adding
*Treated: yes to H25 and
chlorine, or some other treatment
H26 = water boiled, treated
to remove bacteria and other
with chlorine, UV, or ozone
contaminants.
**One year from date of
-H25a: How do you treat it?
-H25b: When did you start treating interview (H: date of
household interview and
it?
H25b)
-MOHLTC-SWU
- Date recorded as water
Days between date of water
database
collection date
collection and date of
-Personal
- P: Date of personal interview
personal interview
questionnaire
-Personal
- P15: Do you regularly use bottled -Tap water only: no to P15
questionnaire
water at home? By regularly, I
-Bottled water only: yes to
mean most days of the week.
P15 and P14=P16
- P14: While at home, approx. how -Both tap and bottled water:
many 8 oz. (240 mL) glasses of
yes to P15 and P14>P16
water do you drink every day? This
would also include water in hot
drinks like tea or coffee and for
cold drinks like orange juice or
Kool Aid.
- P16: Including water used for hot
and cold drinks, about how many 8
oz. glasses of bottled water do you
drink every day?
-Personal
- P1: What month and year were
-Years: date of interview less
questionnaire
you born?
date of birth (day=1)
-Personal
- P2: Respondent sex
As stated
questionnaire
-Household
- H32: Would you describe your
-Farm (as stated)
questionnaire
property as being farm, non-farm -Non-farm: non-farm rural,
rural, village or hamlet, small
village or hamlet, small
town, or other
town, or other
-Household
- H45: What is the highest level of
As stated
questionnaire
education that has been attained
Not stated: refused + dont
by any adult in the household?
know
138
Appendices
Variable
Income
(household)
Data source
-Household
questionnaire
Household size
-Household
questionnaire
Lab region
-MOHLTC-SWU
database
Antibiotic use
-Personal
questionnaire
Hospitalization
-Personal
questionnaire
Travel outside
Canada
Child in day care
-Personal
questionnaire
-Household
questionnaire
Livestock, dog, or
cat contact
-Personal
questionnaire
Raw red meat

contact
-Personal
questionnaire
Raw poultry
contact
-Personal
questionnaire
Item
- H46: What is your best estimate of
the total off-farm income, before
taxes and deductions, of all household
members combined, from all sources,
in 2005?
- H47: What is your best estimate of
the net income from your farm, before
taxes, in 2005?
- H4: Including yourself, how many
adults, that is, people 20 years and
older, currently live in your home?
- H5: How many youths 12 to 19 live
here?
- H6: How many children 4 to 11 years?
- H7: How many children under 4 years
of age live in your household?
(Note: not all water samples are
tested in the laboratory region of the
water source)
- P7: How about antibiotics like
penicillin, tetracycline, gentamicin,
and other prescriptions for infections.
Have you taken any antibiotics in the
past three months?
- P5: Have you been hospitalized, for
at least one night, in the past 12
months?
- P18: Have you travelled outside
Canada in the past 12 months?
- H7: How many children under 4 years
of age live in your household?
- H7b: Do any children in your
household go to a day care centre?
(defined as child in care 1 or more
days/week and centre with 5 or more
children)
- P27: Over the past 3 months, how
often have you been in direct contact
with any of these animals, meaning
actually touching them ore their
manure? Would you say you were in
contact several times per week,
several times a month, less often, or
not at all with
Cattle = Dairy cattle and/or beef cattle
Horses
Pigs
Sheep or goats = Sheep and/or goats
Poultry = Chickens and/or turkeys
Dogs
Cats
- P25: Have you touched raw beef,
pork, or lamb with your bare hands in
the past 3 months?
- P26: Over the past 3 months, have
you touched raw poultry with your
bare hands? This would include
chicken, turkey, or other poultry.
P: Personal questionnaire
H: Household questionnaire
MOHLTC-SWU: Ministry of Health and Long-Term Care (Ontario) Safe Water Unit
NARMS: National Antimicrobial Resistance Monitoring System
139
Derivation
-Household income: H46 +
H47.
-Dont know or refused in
either item was treated as
dont know / refused for
derived variable.
-Not stated: refused +
dont know
Household size: H4 + H5 +
H6 + H7
-Dont know or refused on
any item was treated as
refused for derived
variable
As recorded in database
*not eligible if water
source outside study area
As stated
As stated
As stated
Child in day care: yes to H7
+ H7b
Yes: several times per

week, several times per
month, or less often
No: not at all
Not stated: refused or not
stated
As stated
As stated

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THE ROLE OF DRINKING WATER

Brenda Lee Coleman

A thesis submitted in conformity with the requirements

Copyright by Brenda Lee Coleman, 2008

Drinking water as a source of antimicrobial resistance

The Role of Drinking Water as a Source of Transmission of Antimicrobial Resistant

Drinking water as a source of antimicrobial resistance

Drinking water as a source of antimicrobial resistance

Drinking water as a source of antimicrobial resistance

Drinking water as a source of antimicrobial resistance

Studies of E. coli contamination of private water sources, Ontario

Possible routes of transmission of E. coli to humans . . . . . . . . . . . . . .

Studies comparing E. Coli antimicrobial resistance rates: Canadian subjects

Drinking water as a source of antimicrobial resistance

1.2 Antimicrobial Resistance in E. coli

Drinking water as a source of antimicrobial resistance

Studies of E. coli Contamination of Private Water Sources, Ontario

Drinking water as a source of antimicrobial resistance

1.2.2 Epidemiology of Antimicrobial Resistance in E. coli

Drinking water as a source of antimicrobial resistance

Prevalence of antimicrobial resistant E. coli in Canadian studies

Prevalence across time

Prevalence across place

Drinking water as a source of antimicrobial resistance

Prevalence across persons

Factors affecting prevalence

Drinking water as a source of antimicrobial resistance

Possible Routes of Transmission of E. coli to Humans

Portal of entry: oral

* Focus of this research

Factors affecting prevalence: Person-to-person transmission.

Drinking water as a source of antimicrobial resistance

Factors affecting prevalence: Animal-to-human transmission.

Drinking water as a source of antimicrobial resistance

Factors affecting prevalence: Environmental and foodborne transmission.

Drinking water as a source of antimicrobial resistance

Factors affecting prevalence: Waterborne transmission.

Drinking water as a source of antimicrobial resistance

1.3 Objectives and Hypotheses

Drinking water as a source of antimicrobial resistance

Research hypotheses were:

2.1 Study Population

Drinking water as a source of antimicrobial resistance

E. coli positive water samples

Personal interview(s) & rectal swab(s)

2.1.1 Inclusion and Exclusion Criteria

Drinking water as a source of antimicrobial resistance

2.1.2 Subject Recruitment

Approvals and funding

2.2 Data Collection

Drinking water as a source of antimicrobial resistance

2.2.2 Ottawa, Kingston, Peterborough, Orillia, and Toronto Regions

Drinking water as a source of antimicrobial resistance

2.2.4 Laboratory Testing of Water and Rectal Swab Samples

Citrate, indole, malonate metabolism

2.3 Data Analyses

2.3.1 Objective 1: Prevalence of Antimicrobial Resistant E. coli

Drinking water as a source of antimicrobial resistance

Drinking water as a source of antimicrobial resistance

Potential effect modifier:

Drinking water as a source of antimicrobial resistance

Focal relationship analysis.

levels of a third variable (effect modifier). The inclusion of biologically-plausible combinations of