UvA-DARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl)

UvA-DARE (Digital Academic Repository)

Antimicrobial drug resistance at the human-animal interface in Vietnam

Nguyen, V.T.

Link to publication

Creative Commons License (see https://creativecommons.org/use-remix/cc-licenses):Other

Citation for published version (APA):Nguyen, V. T. (2017). Antimicrobial drug resistance at the human-animal interface in Vietnam.

General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s),other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).

Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, statingyour reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Askthe Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam,The Netherlands. You will be contacted as soon as possible.

Download date: 21 Sep 2020

CHAPTER 6 CONTRIBUTION OF NON-INTENSIVE CHICKEN FARMING TO

EXTENDED-SPECTRUM BETA-LACTAMASE PRODUCING ESCHERICHIA COLI COLONIZATION IN HUMANS IN SOUTHERN

VIETNAM

CHAPTER 6

Chapter 6: Contribution of non-intensive chicken farming to extended-spectrum beta-lactamase producing Escherichia coli colonization in humans in southern Vietnam

Nguyen Vinh Trung1,2,3, Juan J Carrique-Mas3,4, Willemien van Rooijen1, Nguyen Thi Nhung3, Hoang Ngoc Nhung3, Ha Thanh Tuyen3, Pham Van Minh3, James Campbell3,4, Ho Huynh Mai5, Thai Quoc Hieu5, Nguyen Thi Nhu Mai6, Anita Hardon7, Roderick Card8, Muna Anjum8, Jaap A.Wagenaar9,10, Ngo Thi Hoa3,4, Constance Schultsz1,2,3

1 Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands 2 Department of Global Health-Amsterdam Institute for Global Health and Development, The Netherlands 3 Oxford University Clinical Research Unit, Centre for Tropical Medicine, Ho Chi Minh City, Vietnam 4 Centre for Tropical Medicine, Nuffield Department of Medicine, University of Oxford, United Kingdom 5 Sub-Department of Animal Health, My Tho, Tien Giang, Vietnam 6 Preventive Medicine Center, My Tho, Tien Giang, Vietnam 7 Center for Social Science and Global Health, University of Amsterdam, The Netherlands 8 Animal and Plant Health Agency, United Kingdom 9 Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, The Netherlands 10 Central Veterinary Institute of Wageningen UR, Lelystad, The Netherlands

(Manuscript submitted)

88

CHAPTER 6

Abstract

Overuse of antimicrobial substances in agriculture in Asia has been reported, but the risk of

acquisition of extended spectrum beta-lactamase (ESBLs) in humans through non-intensive

chicken farming still remains unclear. To study the potential contribution of chicken farming to

colonization with ESBL-producing Escherichia coli (ESBL-Ec) in humans, we collected faecal

samples from 204 randomly selected farmers and their chickens, and from 306 community-based

individuals who did not raise poultry. ESBL-Ec was isolated from MacConkey agar with and

without antimicrobials supplemented. ESBL genes were characterized using microarray, PCR,

and sequencing. The prevalence of ESBL-Ec colonization was 20.0% in chicken farms, 31.1% in

chicken farmers, 49.5% in rural individuals and 38.3% in urban individuals. Multivariable

analysis showed that colonization with ESBL-Ec in humans was associated with the human

usage of antimicrobial drugs (OR=2.10, 95% CI= 0.97 – 4.55) but not with involvement in

chicken farming (OR= 0.46, 95% CI=0.30 – 0.70). CTX-M-55 was identified as the most

common ESBL encoding gene in chicken (12/20, 60.0% versus 8/60, 13.3% in human isolates,

p<0.05), whilst CTX-M-27 was the most prevalent in human isolates

(33/60, 55.0% versus 2/20, 10.0%; p<0.05). In 3/204 (1.5%) of the farms, identical ESBL genes

were detected in ESBL-Ec isolated from farmers and their chickens. These findings suggest that

non-intensive chicken farming is not a major source of ESBL-Ec colonization in humans and that

human antimicrobial drug usage appears as a more important driver of ESBL-Ec colonization in

community-based individuals in southern Vietnam.

Keywords: ESBL-producing, Escherichia coli, antimicrobial use, antimicrobial resistance,

farmer, poultry

89

CHAPTER 6

Introduction

The spread of extended-spectrum beta-lactamases (ESBLs) in nosocomial and community-

acquired Enterobacteriaceae is a challenge since therapeutic options for infections with these

organisms are limited [1]. Colonization with ESBL-producing Escherichia coli (ESBL-Ec) has

been documented in healthy humans as well as in food-producing animals, including chickens

[2], and the prevalence has increased significantly during the last decades [3, 4]. High and

inappropriate antimicrobial drug usage in humans and in food animals is an important driving

force for this increased prevalence [5].

It has been suggested that transmission of bacteria and/or mobile genetic elements carrying

ESBL encoding genes from food-producing animals to humans may contribute to human

infection with ESBL-Ec. Recent studies comparing ESBL genes and resistance

plasmids in isolates of poultry and human origin suggest that a significant proportion of human

extra-intestinal ESBL-Ec infections may originate from poultry because poultry and human

isolates appeared genetically related or carried similar resistance genes ESBL-Ec [6]. However,

in the majority of these studies human E. coli isolates came from invasive infections and were

compared with isolates from commercially purchased chicken meat samples. Bacterial

contamination of such samples may not represent the situation on the farm of origin and can

therefore not be directly attributed to chicken farming practices. In addition, human and chicken

samples were collected in different time frames whilst accompanying data on relevant

antimicrobial drug usage, which may explain observed resistance characteristics better than

transmission between host populations, were lacking. All of these comparative studies were

carried out in developed countries where industrial farming systems are predominant. The risk of

human colonization with ESBL-Ec resulting from farming practices in developing countries,

where poultry farms are often small and farming is practiced with low-levels of biosecurity and

high usage of antimicrobial drug [7] has not been addressed.

The Mekong Delta is a region in Vietnam where, as in large parts of Asia, a significant

proportion of the population raises poultry at home, typically in small numbers [8]. In addition,

available data indicate high rates of carriage of antimicrobial drug resistant E. coli in this region

[9, 10], where antimicrobial drugs for use in both animals and humans are available over the

90

CHAPTER 6

counter [11]. We, therefore, aimed to study the potential contribution of transmission of ESBL-

Ec from poultry to ESBL-Ec colonization in humans by determining the prevalence and

similarities in resistance encoding gene content of ESBL-Ec colonizing chickens and humans

and to relate these to antimicrobial drug usage.

Methods

Study population

A total of 204 chicken farms and 204 chicken farmers, defined as an adult person (≥ 18 years

old) responsible for raising the chickens, who were not hospitalized in the previous 4 weeks,

were randomly selected as described previously [9, 12]. In brief, sampling was stratified by farm

size (10 – 200 chickens, ‘household farms’; 201 – 2000 chickens, ‘small size farms’) and by

district (My Tho city, Cho Gao district and Chau Thanh district) (total 6 strata).

Age- and sex-matched individuals who were not involved in poultry farming were randomly

selected from the same district as the farmers (N=204), as well as from the provincial capital

(N=102) using the population census provided by the Preventive Medicine Centre (PMC) in Tien

Giang [13].

Written informed consent was obtained from all participants prior to recruitment. The study was

approved by the Peoples’ Committee of Tien Giang Province, the Department of Health in Tien

Giang and the Oxford University Tropical Research Ethics Committee (OxTREC, No. 48/11).

Data and sample collection

Data and samples were collected from March 2012 to April 2013. Data on antimicrobial drug

usage in both chickens and humans was collected using structured questionnaires

(Supplementary Materials 1 and 2).

Faecal samples from chickens were collected using boot-swabs or hand-held gauze swabs, as

described previously [9]. Rectal swab samples from human participants were obtained using

Faecalswab (Copan, Italy).

91

CHAPTER 6

Data and sample collection were conducted by combined sampling teams from Tien Giang

SDAH and PMC. Samples were stored and transported at 4oC to the laboratory at the Oxford

University Clinical Research Unit in Ho Chi Minh City and cultured within 24 hours after

collection

ESBL-producing E. coli isolation

E. coli isolation and identification were performed using MacConkey agar with and without

antimicrobials (ceftazidime, nalidixic acid and gentamicin) supplemented as described

previously [9]. Susceptibility testing and potential production of ESBL was confirmed using a

double disk diffusion test in accordance with CLSI guidelines [14]. Quality controls for

identification and sensitivity testing were performed on a weekly basis.

All isolates with a unique phenotypic antimicrobial susceptibility pattern in each subject were

stored for further analyses. Strains with an intermediate sensitive result were considered

resistant. A chicken farm or participant was defined as ‘positive’ for ESBL-Ec if at least one

ESBL-Ec isolate was cultured from any of the MacConkey plates used.

Identification of antimicrobial resistance genes and sequence types

To characterize beta-lactamase gene families in the ESBL-Ec isolates and other antimicrobial

resistance genes, a miniaturized micro-array (AMR-08, Alere, Jena, Germany) was used as

described previously [15]. The list of genes included in the AMR-08 microarray is shown in the

Supplementary Material 3. ESBL gene families detected from the array were further

characterized using PCR amplification and sequencing (Supplementary Material 2).

Multilocus sequence typing was performed on 43 E. coli isolates using the scheme described

previously [16].

Sample size and data analyses

The chosen sample size of 204 chicken farmers and 306 unexposed individuals is sufficient to

detect a difference in prevalence of colonization with ESBL-Ec, from 50.5% (unexposed

individuals) to 65.0% (exposed individuals) with 80% power and 95% confidence interval.

92

CHAPTER 6

As there is no method for determining sample size for genomic microarray analyses of bacterial

isolates, we randomly selected 80 ESBL-Ec isolates (20 isolates each study group from the total

of 40, 101, 173 and 57 ESBL-Ec isolates in chicken flocks, farmers, rural and urban individuals,

respectively) to study the distribution of ESBL encoding genes across groups. In addition, to

assess the similarity of ESBL encoding genes between chicken and farmer isolates of the same

farm, we analyzed all pairs of ESBL-Ec from the 16 farms where both chicken and farmer were

phenotypically ESBL-positive (‘matched isolates’). We used discriminant analysis of principal

components (DAPC) to compare overall antimicrobial resistance gene profiles distribution, not

limited to ESBL genes, between the different study groups [17].

The prevalence of colonization with ESBL-Ec was adjusted for the stratified survey design by

assigning a stratum-specific sampling weight (Supplementary Material 2).

We built a logistic regression model to investigate risk factors associated with the outcome

presence of phenotypically positive ESBL-Ec among all human participants (Supplementary

Material 4). Based on their biological plausibility and a p-value < 0.15 in the univariable

analyses, variables were considered for multivariable analysis and were included using a step-

wise forward approach [18]. Variables were retained in the final models if their p-value was <

0.05. All interactions between final significant variables were tested. All statistical analyses were

performed using R packages ‘epicalc’, ‘survey’ and ‘adegenet’ (http://www. r-project.org).

Results

Prevalence of colonization with ESBL-Ec in chickens and humans

Among 510 enrolled human participants, the median age was 46 [interquartile range 39 – 54

years] and 63.9% were male. The adjusted prevalence of ESBL-Ec colonization was 20.0% in

chicken farms, 31.1% in chicken farmers, 49.5% in rural individuals and 38.3% in urban

individuals (Table 1)

93

CHAPTER 6

Table 1. Prevalence of colonization with ESBL-producing Escherichia coli in chickens and humans in southern Vietnam

Subject Number of ESBL-Ec positive subjects (Prevalence; %)

Adjusted Prevalence (95% CI)

Chicken (N=204) 30 (14.7) 20.0 (10.8 – 29.1) Farmer (N=204) 65 (31.9) 31.1 (24.3 – 37.8) Rural (N=204) 101 (49.5) 49.5 (42.6 – 56.4) Urban (N=102) 39 (38.2) 38.3 (28.8 – 47.7)

Risk factors associated with ESBL-Ec colonization in humans

Chicken farmers were at lower risk of colonization with ESBL-Ec than rural individuals not

involved in poultry farming (OR= 0.46; 95% CI= 0.30 – 0.70) (Table 2). However, human usage

of any antimicrobial drug during the month prior to the study visit was associated with ESBL-Ec

colonization in humans (OR= 2.1; 95% CI= 0.97 – 4.55).

Table 2. Risk factor analyses for colonization with ESBL-producing Escherichia coli in human individuals (N=510) in southern Vietnam

aIntercept: - 0.068 (SEM±0.14) bnot involved in chicken farming cduring the month prior to the study visit

Distribution of ESBL genes and other antimicrobial resistance genes among ESBL-Ec

Microarray analysis of 80 randomly selected ESBL-Ec isolates demonstrated that CTX-M,

including CTX-M-1 group and CTX-M-9 group, was the predominant gene, found in 16/20

(80.0%) of chicken- and 60/60 (100%) of human isolates (Table 3). In 4 chicken ESBL-Ec

isolates, no ESBL gene could be detected by our microarray. Distribution of CTX-M genes

across isolates from farmers and unexposed individuals was similar. However, distribution of

CTX-M genes across chicken and human isolates was different. CTX-M-1 group genes were

commonly prevalent in both chicken (12/20, 60.0%) and human isolates (24/60, 40.0%), whilst

Variable Total number of participants

No. of ESBL-Ec positive participants

ORa 95% CI p-value

Participant group Rural individualb 204 101 ref ref ref Urban individualb 102 39 0.64 0.39 – 1.04 0.07 Chicken farmer 204 65 0.46 0.30 – 0.70 <0.001 Use of any antimicrobial drugsc 34 18 2.1 0.97 – 4.55 0.061

94

CHAPTER 6

CTX-M-9 group genes were more prevalent in isolates from humans (37/60, 61.7% versus 4/20,

20.0%) (p<0.05). One isolate from a chicken farmer was positive with both CTX-M-1 group and

CTX-M-9 group ESBL genes.

Table 3. Distribution of CTX-M genes in ESBL-producing Escherichia coli from chickens, farmers and individuals not involved in poultry farming in southern Vietnam

CTX-M group/gene

No. of chicken isolates (%)

No. of farmer isolates (%)

No. of rural individuals isolates (%)

No. of urban individual isolates (%)

No. of humana isolates (%)

(N=20) (N=20) (N=20) (N=20) (N=60) CTX-M-1 group 12 (60.0) 6 (30.0) 10 (50.0) 8 (40.0) 24 (40.0) CTX-M-15 0 2 (10.0) 8 (40.0) 6 (30.0) 16 (26.7) CTX-M-55 12 (60.0) 4 (20.0) 2 (10.0) 2 (10.0) 8 (13.3) CTX-M-9 group 4 (20.0) 15 (75.0) 10 (50.0) 12 (60.0) 37 (61.7) CTX-M-14 1 (5.0) 1 (5.0) 2 (10.0) 0 0 CTX-M-27 2 (10.0) 13 (65.0) 8 (40.0) 12 (60.0) 33 (55.0) CTX-M-65 1 (5.0) 0 0 0 0 Novel CTX-Mb 0 1 (5.0) 0 0 0

a Humans including farmers, rural and urban individuals b The DNA sequence of this novel gene differed from CTX-M-27 by one amino acid (Q7L).

A total of 76 PCR amplicons were DNA-sequenced to identify the specific CTX-M variants and

the results are shown in Table 3. Among CTX-M-1 group isolates, CTX-M-55 was the only

variant found in chicken isolates whereas two variants of the CTX-M-1 group, both CTX-M-55

and CTX-M-15, were detected in 24 human isolates. Among CTX-M-9 group isolates, CTX-M-

27 was the most prevalent variant detected in human isolates, followed by CTX-M-14 and CTX-

M-65. One novel variant within the CTX-M-9 group was identified from a farmer.

The distribution of other antimicrobial resistance genes in ESBL-Ec isolated from farmers, rural

individuals and urban individuals, as detected by microarray, is shown in the Supplementary

Material 5. The DAPC of antimicrobial resistance gene profiles, including all antimicrobial

resistance genes detected by microarray, indicated similarity between isolates from human

sources (Figure 1). Isolates from chickens exhibited profiles that were overall distinct from those

from the human groups. However, ESBL-Ec in urban population was most distinct and the

genotypes of the chicken isolates were closer to the rural and farmer isolates than to isolates from

the urban population.

95

CHAPTER 6

Figure 1. Discriminant analysis of principle components of genotypic antimicrobial resistance profiles applied to 80 randomly selected ESBL-producing Escherichia coli isolates from chicken and humans in southern Vietnam

Characteristics of ESBL-Ec isolated from chicken and farmer on the same farm

On 16/204 farms (6.9%; 95% CI= 3.4 – 10.3%) ESBL-Ec were detected phenotypically in both

the farmers and their chickens. Microarray, PCR and sequencing results revealed that in 10

farms, isolates from chickens and farmers showed different ESBL genes. On three other farms,

ESBL genes could not be detected by microarray or by PCR for detection of any CTX-M gene,

in three isolates from chickens (farm 10, farm 12 and farm 13). Hence a comparison of ESBL

genes between the farmers and their chickens on these 3 farms was impossible. In the remaining

three farms (1.5%; 95% CI= 0 – 3.1%), ESBL genes of ESBL-Ec isolated from three farmers and

their chickens were identical (farm 3, farm 11 and farm 16, Table 4). However, analysis of genes

encoding for resistance against other classes of antimicrobial drugs showed differences between

chicken and farmer isolates in all three farms (Supplementary Material 5). In addition, multi-

locus sequence typing of these isolates showed identical sequence types for chicken and farmer

isolates in one of these three farms only (farm 11, Table 4)

96

CHAPTER 6

Table 4. Comparison of ESBL-producing Escherichia coli isolated from farmer and chicken on the same farm

a These isolates showed different antimicrobial resistance phenotype NT: Non-typeable NI: Non-identified Underlining depicts identical ESBL genes and sequence type of ESBL-Ec isolated from chicken and farmer on the same farm

Farm Subject Isolate Number Sequence Type

ESBL gene Other resistance genes

1 Chicken 1 156 CTX-M-55 TEM, aadA1, strB, mrx, tetA, dfrA12, dfrA14, sul2, sul3 Farmer 1a 38 CTX-M-27 strB, mrx, tetA, dfrA17, sul1, sul2 Farmer 2a 38 CTX-M-27 strA, strB, mrx, tetA, dfrA17, sul1, sul2

2 Chicken 1 206 CTX-M-55 TEM, aadA1, aadA2, aadA4, qnrS, tetA, cmlA1, floR, dfrA12, sul1, sul3 Farmer 1 131 CTX-M-14 blaACC, TEM, strA, strB, mphA, mrx, tetA, dfrA17, sul1, sul2

3 Chicken 1 NT CTX-M-55 qnrS, tetA, floR Farmer 1a 155 CTX-M-55 TEM, aadA1, strB, qnrS, tetA, floR, dfrA14, dfrA17, sul2, sul3 Farmer 2a 155 CTX-M-55 TEM, aphA, aadA1, strA, strB, qnrS, tetA, floR, dfrA14, sul2, sul3

4 Chicken 1a 617 CTX-M-55 TEM, aphA, aadA1, aadA2, aadA4, tetM, cmlA1, floR, dfrA12, sul2, sul3 Chicken 2a 156 CTX-M-55 TEM, aac6-Ib, aadA1, strA, strB, tetA, tetB, catA, floR, catB3, sul1, sul2 Chicken 3a 1114 CTX-M-105 TEM, aphA, aadA1, strA, strB, tetA, floR, sul2, sul3 Farmer 1 38 CTX-M-27 TEM, strA, strB, ermB, mrx, tetA, dfrA17, sul1, sul2 Farmer 2 38 CTX-M-27 TEM, strA, strB, ermB, mrx, tetA, dfrA17, sul1, sul2

5 Chicken 1 349 CTX-M-55 aadA1, tetA, dfrA14, sul3 Farmer 1a 1163 CTX-M-27 TEM, ermB, mrx, tetB, dfrA17 Farmer 2a 1193 CTX-M-15 TEM, strB, mrx, tetA, dfrA17, sul2 Farmer 3a 1163 CTX-M-27 TEM, ermB, mrx, tetB, dfrA17, sul1

6 Chicken 1 448 CTX-M-55 aadA1, tetA, floR, dfrA14, sul3 Farmer 1 131 CTX-M-24 TEM, strB, mrx, tetA, dfrA17, sul1, sul2

7 Chicken 1 457 CTX-M-27 TEM, aphA, aadA1, strA, strB, ermB, catIII, cmlA1, dfrA14, sul2, sul3 Farmer 1 394 CTX-M-15 TEM

8 Chicken 1 NT CTX-M-14 TEM, aphA , tetA, floR, dfrA14, sul2, sul3 Farmer 1a 31 CTX-M-27 TEM, aadA1, ermB, mrx, tetA, catA, dfrA17, sul1 Farmer 2a 31 CTX-M-27 TEM, aadA1, mrx, tetA, catA, dfrA17 Farmer 3a 31 CTX-M-27 TEM, aadA1, ermB, mrx, tetA, catA, dfrA17, sul1

9 Chicken 1a 101 CTX-M-14 TEM, aphA, tetA, floR, dfrA14, sul2, sul3 Chicken 2a 101 CTX-M-55 TEM, aphA, aadA1, qnrS, tetA, tetM, floR, dfrA14, sul2, sul3 Farmer 1 NT CTX-M-27 strA, strB, ermB, mrx, tetA, dfrA12, sul1, sul2

10 Chicken 1 NT NI blaCMY, TEM, strA, strB, ermB, mrx, tetA, floR, dfrA17, sul1, sul2 Farmer 1 131 CTX-M-15 TEM, strA, strB, mrx, tetA, dfrA17, sul1, sul2

11 Chicken 1 226 CTX-M-65 TEM, aadA1, strB, mrx, tetA, cmlA1, floR, dfrA12, sul2, sul3 Farmer 1 226 CTX-M-65 aadA1, strA, strB, tetA, cmlA1, floR, dfrA12, sul2, sul3

12 Chicken 1 746 NI blaCMY, aadA1, aadA2, aadA4, tetA, catIII, cmlA1, floR, dfrA12, sul2, sul3 Farmer 1 69 CTX-M-27 mrx, tetA, dfrA17, sul2

13 Chicken 1 10 NI blaCMY, OXA-1, TEM, aadA1, aadB, strA, strB, tetA, floR, catB3, dfrA1, sul2 Farmer 1 131 CTX-M-15 OXA-1, aac6-Ib, mrx, tetA, dfrA17, sul1

14 Chicken 1 156 CTX-M-55 TEM, aphA, aadA1, tetA, floR, dfrA14, sul3 Farmer 1a 10 CTX-M-15 blaACC, TEM, aadA1, mphA, mrx, tetA, dfrA14, dfrA1, sul1, sul2 Farmer 2a 10 CTX-M-15 TEM, aadA1, mrx, tetA, dfrA14, sul2

15 Chicken 1 NT CTX-M-55 TEM, aphA, aadA1, strA, strB, qnrS, mrx, tetA, floR, dfrA14, sul2, sul3 Farmer 1 226 CTX-M-27 ermB, mrx

16 Chicken 1 162 CTX-M-55 TEM, strB, mrx, tetA, floR, dfrA17, sul2, sul3 Farmer 1 410 CTX-M-55 TEM, aphA, aadA1, strA, strB, tetA, floR, dfrA14, sul2, sul3

97

CHAPTER 6

Discussion

The prevalence of colonization with ESBL-Ec in chicken farms in Vietnam was almost 20%.

This prevalence is relatively low compared to the prevalence of colonization with ESBL-Ec in

chicken farms in other countries in Europe which range from about 40% to 100% [19-21]. Third-

generation cephalosporins usage in chickens was common in Europe [22]. The fact that we did

not find any cephalosporins usage in 204 chicken farms in our study (Supplementary Material 6)

is probably one of the explanations for the lower prevalence of colonization with ESBL-Ec in

chicken farms in our study. However, such comparisons should be interpreted with some caution

because the data were obtained in intensive farming settings in Europe as opposed to the

household and small-scale farm settings in Vietnam, and have used different sampling methods.

In contrast, the prevalence of colonization with ESBL-Ec in rural and urban individuals in this

study was much higher than reported for European countries [23] but was similar to the

prevalence of colonization with ESBL-Ec in the community in other Asian countries [24, 25].

This high prevalence of colonization with ESBL-Ec is in agreement with the high and

uncontrolled use of antimicrobial drugs in the community [11]. It probably have contributed to

ESBL-Ec colonization in chickens, for example through environmental contamination [26].

We did not find an apparent association between non-intensive chicken farming and ESBL-Ec

colonization in humans indicating that non-intensive chicken farming is not a major source of

ESBL-Ec colonization in humans in this setting. However, we observed an association between

antimicrobial use during the previous month and ESBL-Ec colonization; the risk of colonization

with ESBL-Ec was doubled among individuals who reported recent antimicrobial use. These

findings are in line with a recent publication where risk factors associated with faecal

colonization with ESBL-producing Enterobacteriaceae in healthy individuals were reviewed

[27].

Although cephalosporin usage was not reported in chicken farms, the prevalence of ESBL-Ec in

chickens was 20.0% (95% CI= 10.8 – 29.1). Therefore, we speculate that some human activities

or unknown practices, for example poor biosecurity or environmental contamination due to poor

sewage control [28], may contribute to ESBL-Ec colonization in chickens. However, it is not

necessarily through direct transmission of ESBL-Ec from humans to chickens or vice-versa, 98

CHAPTER 6

given the differences in sequence types and in resistance gene content between ESBL-Ec isolates

from the farmers and their chickens. Although we did not perform plasmid characterization, the

differences in antimicrobial resistance gene content also suggest that different plasmids are

potentially circulating in different host populations, concordant with the findings from a recent

study in northern Vietnam [29].

We are aware of the limitations of a cross-sectional study design, which precludes any inferences

on the dynamics of ESBL-Ec transmission between chickens and humans in Vietnam. In

addition, the number of isolates from chicken available for genotypic analyses was smaller than

expected due to the relatively low prevalence of colonization with ESBL-Ec in the chicken

farms. Despite these limitations, this study provides a comprehensive view on the prevalence of

colonization with ESBL-Ec in household-scale and small-scale chicken farms in the Mekong

Delta of Vietnam, and simultaneously sampled humans with and without direct chicken exposure

from the same geographic region. We found differences in prevalence of colonization with

ESBL-Ec in chickens and humans and this difference correlated with differences in usage of

cephalosporins. Antimicrobial usage thus emerged as the key factor that drives ESBL-Ec

colonization rather than direct transmission of ESBL-Ec from chicken to humans or vice versa.

Genotyping studies from the Netherlands support this notion, as whole genome sequencing from

chicken and human isolates with identical ESBL-genes showed clear differences between ESBL-

Ec from these different hosts supporting the hypothesis that ESBL-Ec selection is host specific

whilst horizontal gene transfer may contribute to the spread of resistance determinants [30].

Therefore, further studies are needed to compare E. coli populations in humans and chickens at

the whole genome level to better elucidate host specificity of ESBL-Ec colonization and the

drivers of horizontal gene transfer.

References

1. Paterson, D.L. and R.A. Bonomo, Extended-spectrum beta-lactamases: a clinical update. Clin MicrobiolRev, 2005. 18(4): p. 657-86.

2. Carattoli, A., Animal reservoirs for extended spectrum beta-lactamase producers. Clin Microbiol Infect,2008. 14 Suppl 1: p. 117-23.

3. Canton, R., et al., Prevalence and spread of extended-spectrum beta-lactamase-producingEnterobacteriaceae in Europe. Clin Microbiol Infect, 2008. 14 Suppl 1: p. 144-53.

4. Hawkey, P.M., Prevalence and clonality of extended-spectrum beta-lactamases in Asia. Clin MicrobiolInfect, 2008. 14 Suppl 1: p. 159-65.

99

CHAPTER 6

5. Levy, S.B., The 2000 Garrod lecture. Factors impacting on the problem of antibiotic resistance. JAntimicrob Chemother, 2002. 49(1): p. 25-30.

6. Lazarus, B., et al., Do human extraintestinal Escherichia coli infections resistant to expanded-spectrumcephalosporins originate from food-producing animals? A systematic review. Clin Infect Dis, 2015. 60(3):p. 439-52.

7. Carrique-Mas, J.J., et al., Antimicrobial usage in chicken production in the Mekong Delta of Vietnam.Zoonoses Public Health, 2015. 62 Suppl 1: p. 70-8.

8. Burgos S, et al., Characterization of poultry production systems in Vietnam. Int J of Poult Sci, 2007. 6(10):p. 709-712.

9. Nguyen, V.T., et al., Prevalence and risk factors for carriage of antimicrobial-resistant Escherichia coli onhousehold and small-scale chicken farms in the Mekong Delta of Vietnam. J Antimicrob Chemother, 2015.70(7): p. 2144-52.

10. Dyar, O.J., et al., High prevalence of antibiotic resistance in commensal Escherichia coli among childrenin rural Vietnam. BMC Infect Dis, 2012. 12: p. 92.

11. CDDEP. Situation Analysis: Antibiotic Use and Resistance in Vietnam. 2010; Available from:http://www.cddep.org/sites/default/files/vn_report_web_1_8.pdf.

12. Trung, N.V., et al., Non-Typhoidal Salmonella Colonization in Chickens and Humans in the Mekong Deltaof Vietnam. Zoonoses Public Health, 2016.

13. Anonymous. WHO Global Principles for the Containment of Antimicrobial Resistance in Animals Intendedfor Food. WHO, Geneva. 2000; Available from: http://www.who.int/foodsafety/publications/containment-amr/en/.

14. Dang, S.T., et al., Impact of medicated feed on the development of antimicrobial resistance in bacteria atintegrated pig-fish farms in Vietnam. Appl Environ Microbiol, 2011. 77(13): p. 4494-8.

15. Card, R., et al., Evaluation of an expanded microarray for detecting antibiotic resistance genes in a broadrange of gram-negative bacterial pathogens. Antimicrob Agents Chemother, 2013. 57(1): p. 458-65.

16. Hirai, I., et al., Characterization of Escherichia coli producing CTX-M-type extended-spectrum beta-lactamase carriage in healthy Vietnamese individuals. Antimicrob Agents Chemother, 2015.

17. Jombart, T., S. Devillard, and F. Balloux, Discriminant analysis of principal components: a new method forthe analysis of genetically structured populations. BMC Genet, 2010. 11: p. 94.

18. Hosmer, D., S. Lemeshow, and R. Sturdivant, Applied Logistic Regression. Third ed. 2004: Wiley.19. Smet, A., et al., Diversity of extended-spectrum beta-lactamases and class C beta-lactamases among

cloacal Escherichia coli Isolates in Belgian broiler farms. Antimicrob Agents Chemother, 2008. 52(4): p.1238-43.

20. Dierikx, C., et al., Extended-spectrum-beta-lactamase- and AmpC-beta-lactamase-producing Escherichiacoli in Dutch broilers and broiler farmers. J Antimicrob Chemother, 2013. 68(1): p. 60-7.

21. Costa, D., et al., Prevalence of extended-spectrum beta-lactamase-producing Escherichia coli isolates infaecal samples of broilers. Vet Microbiol, 2009. 138(3-4): p. 339-44.

22. Collignon, P., et al., Human deaths and third-generation cephalosporin use in poultry, Europe. EmergInfect Dis, 2013. 19(8): p. 1339-40.

23. Woerther, P.L., et al., Trends in human fecal carriage of extended-spectrum beta-lactamases in thecommunity: toward the globalization of CTX-M. Clin Microbio Rev, 2013. 26(4): p. 744-58.

24. Li, B., et al., High prevalence of CTX-M beta-lactamases in faecal Escherichia coli strains from healthyhumans in Fuzhou, China. Scand J Infect Dis, 2011. 43(3): p. 170-4.

25. Sasaki, T., et al., High prevalence of CTX-M beta-lactamase-producing Enterobacteriaceae in stoolspecimens obtained from healthy individuals in Thailand. J Antimicrob Chemother, 2010. 65(4): p. 666-8.

26. Guenther, S., C. Ewers, and L.H. Wieler, Extended-Spectrum Beta-Lactamases Producing E. coli inWildlife, yet Another Form of Environmental Pollution? Front Microbiol, 2011. 2: p. 246.

27. Karanika, S., et al., Fecal Colonization With Extended-spectrum Beta-lactamase-ProducingEnterobacteriaceae and Risk Factors Among Healthy Individuals: A Systematic Review and Metaanalysis.Clin Infect Dis, 2016. 63(3): p. 310-8.

28. Van Minh, H., et al., Assessing willingness to pay for improved sanitation in rural Vietnam. Environ HealthPrev Med, 2013. 18(4): p. 275-84.

29. Ueda, S., et al., Limited transmission of bla(CTX-M-9)-type-positive Escherichia coli between humans andpoultry in Vietnam. Antimicrob Agents Chemother, 2015. 59(6): p. 3574-7.

100

CHAPTER 6

30. de Been, M., et al., Dissemination of cephalosporin resistance genes between Escherichia coli strains fromfarm animals and humans by specific plasmid lineages. PLoS Genet, 2014. 10(12): p. e1004776.

101

CHAPTER 6

Supplementary Material 1: Questionnaire to study antimicrobial use in humans in Tien Giang province, Vietnam

Name of interviewer: [_________________________________]

Interview date (dd/mm/yy) : [__|__]/[__|__]/[__|__]

We are conducting a study to investigate medicines used by Tien Giang farmers for their chickens and for their own health. You have received information about our study and you have agreed to participate. You gave us information on your medicine use for your chicken. We would now like to ask you some questions about your experience in the use of medicines for your own health and the health of your family members. For example, which medicines do you use, when and why.

Do you agree to do the interview now?

A. GENERAL INFORMATION 1. Age of respondent: [__|__] 2. Gender: Male Female 3. This question NOT APPLICABLE for chicken farmers.

Are there any animal species (not poultry) in your household now? Yes No If Yes, tick all that apply Pig Cattle/buffalo Dog Cat Fish Other

4. Who live in your household?No Relationship of family/household member with the

respondent 1. The respondent 2. Respondent’s partner3. Child (3A for child 1, 3B for child 2 and so on –from oldest to youngest) 4. Mother/Father5. Grandparents 6. Others (6A. Son/daughter-in-law, 6B. Grandchildren, 6C. other )

Age (in years, if less than 1 year

old, write 01)

Gender 1 - Male 2 - Female

Tick if the person was sampled

1 [_0|1_] [__|__] [__|__] [__]

2 [__|__] [__|__] [__|__] [__]

3 [__|__] [__|__] [__|__] [__]

4 [__|__] [__|__] [__|__] [__]

5 [__|__] [__|__] [__|__] [__]

6 [__|__] [__|__] [__|__] [__]

7 [__|__] [__|__] [__|__] [__]

8 [__|__] [__|__] [__|__] [__]

9 [__|__] [__|__] [__|__] [__]

10 [__|__] [__|__] [__|__] [__]

102

CHAPTER 6

B. MEDICINE REVIEW (ANTIBIOTIC USAGE ONLY) 5. What medicines does your family use to treat illnesses or to stay healthy? Do you keep them in a medicine cabinet? Can we see them?

a. Medicines seen: Yes No b. Antibiotics present: Yes No Don’t know c. Please fill in the following table starting with the antibiotics present in the cabinet, describe them in detail as well as the member in

your family treated with those. Then continue listing any other antibiotic not present in the cabinet used by your family member over the last month

Antibiotic

(commercial name) Present

in the cabinet

1.Yes

2.No

Commercial presentation

(Use code *)

Active ingredient per unit

(g or mg)

Who used the

antibiotic

(Use code**)

Illness

(Use code +)

How long ago was the last usage?

(Use code#)

How many units

per day?

Duration

(How many days)

Whose advice

(Use code ++)

1. [____] [__|__] [__|__] [____] [____] [__] [__|__] [__]

2. [____] [__|__] [__|__] [____] [____] [__] [__|__] [__]

3. [____] [__|__] [__|__] [____] [____] [__] [__|__] [__]

4. [____] [__|__] [__|__] [____] [____] [__] [__|__] [__]

5. [____] [__|__] [__|__] [____] [____] [__] [__|__] [__]

6. [____] [__|__] [__|__] [____] [____] [__] [__|__] [__]

7. [____] [__|__] [__|__] [____] [____] [__] [__|__] [__]

8. [____] [__|__] [__|__] [____] [____] [__] [__|__] [__]

9. [____] [__|__] [__|__] [____] [____] [__] [__|__] [__]

10. [____] [__|__] [__|__] [____] [____] [__] [__|__] [__]

Codes Lists: *: T: Tablet; C: capsules; SY: syrup; D: drops; I: injection; SA: sachets; SU: suppositorium; P: pomade (cream) **: 1.Respondent, 2. Respondent’s partner, 3. Child (3A for child 1, 3B for child 2 and so on – from oldest to youngest), 4.

Parents or Grandparents, 5. Others +: R: Respiratory symptoms/infections; G: Gastrointestinal symptoms/infections; M: Mouth and teeth symptoms/infections; S: wound/skin symptoms/infections; G: General malaise symptoms/infections; O: other symptoms/infections specify.#: 1. still used, 2. 1-7 days ago, 3. 1-4 weeks ago, 4. More than 1 month ago 5. Not use this antibiotic yet

++: 1. Drug sellers; 2. Doctor/Health professionals; 3. Friend/neighbor 4. Others

103

CHAPTER 6

C. EATING HABITS

6. How often do you eat chicken meat (include any dish with chicken)? (tick 1 only) Everyday or almost every day

3-4 times per week

1-2 times per week

2-3 times per month

Less than 2 times per month

Never

7. This question ONLY APPLICABLE for chicken farmersOf all chicken meat you eat (include eating out), please choose the statement that better reflects your situation (tick 1 only).

All of the chicken meat I eat is not reared in my farm Most of the chicken meat I eat comes from outside and some of it is reared in my farm Some of the chicken meat I eat comes from outside and some of them is reared in my farm Most of the chicken meat I eat comes from my farm and a some of it comes from outside All of the chicken meat I eat comes from my farm

8. How often do you eat chicken eggs (include any dish with eggs)? (tick 1 only) Everyday or almost every day 3-4 times per week 1-2 times per week 2-3 times per month Less than 2 times per month Never

9. This question ONLY APPLICABLE for chicken farmersOf all chicken eggs you eat (include eating out), please choose the statement that better reflects your situation (tick 1 only)

All of the chicken eggs I eat come from outside Most of the chicken eggs I eat come from outside and some of them are produced in my farm Some of the chicken eggs I eat come from outside and some of them are produced in my farm Most of the chicken eggs I eat come from my farm and some of them come from outside All of the chicken eggs I eat come from my farm

10. Source of water (tick all that apply)

Municipal supply Borehole/well Rain water

Pond River/stream/canal other, specify________________

11. Distance to the closest running water sources (in meter) [__|__|__|__|__]

D. QUESTION

12. Do you have any questions or comments? Yes No[_____________________________________________________________________] [_____________________________________________________________________]

Thank you, this is the end of the interview.

104

CHAPTER 6

Supplemental Material 2

Materials and Methods

Collection of antimicrobial usage data Data on human antimicrobial drug usage during the month prior to the study visit, including the product’s commercial name, packaging format, dosage, and duration of usage, was collected for all participants as well as for all household members by medicine cabinet surveys, using a structured questionnaire containing both open and closed questions (supplementary material 1). Data on antimicrobial usage for chickens was similarly collected during interviews with the farmers, using a questionnaire as published previously (1). The medicine cabinet survey has been shown to be efficient in getting data on antimicrobial drugs usage in the community (2). Usage of an antimicrobial drug was defined as the reported usage in the previous month and/or the presence of the antimicrobial drug in the medicine cabinet. All questionnaires were administered by staff from Sub-Department of Animal Health and Preventive Medicine Center in Tien Giang for chicken and human antimicrobial usage, respectively. PCR and sequencing of ESBL genes

Table 1: PCR and sequencing primers for detection of ESBL genes

Gene Name Sequence

CTX-M-1 CTX-M-1_seq_F1 5'-TCGTCTCTTCCAGAATAAGGA-3'

CTX-M-1_seq_F2 5'-GACGATGTCACTGGCTGAGC-3'

CTX-M-1_seq_R1 5'-GTTTCGTCTCCCAGCTGTC-3'

CTX-M-1_seq_R2 5'-AAACGGAATGAGTTTCCCCAT-3'

CTX-M-9 CTX-M-9_seq_F1 5'-CTGATGTAACACGGATTGACC-3'

CTX-M-9_seq_F2 5'-TGCAGTACAGCGACAATACC-3'

CTX-M-9_seq_R1 5'-AAAAGCCGTCACGCCTCC-3'

CTX-M-9_seq_R2 5'-CGTGATCTGATCCTTCAACTC-3'

TEM TEM_seq_F1 5'-CAATAACCCTGRTAAATGCTTCAA-3'

TEM_seq_F2 5'-TGGCATGACAGTAAGAGAATTAT-3'

TEM_seq_R1 5'-TCCTCCGATCGTTGTCAGAA-3'

TEM_seq_R2 5'-CAATCTAAAGTATATATGAGTAAACT-3'

ESBL gene families detected from the array were further characterized using PCR amplification and sequencing (see Table 1 for primers used). Sequences were obtained using the BigDye Terminator V.1.1 Cycle Sequencing Kit (Applied Biosystems, USA). Sequence data were analysed using Codoncode Aligner 1.3.4 (CodonCode Corporation, Dedham, MA). The sequences obtained were compared to those registered in GenBank and present in the ESBL database at http://www.lahey.org/studies/.

Adjustment of prevalence estimates for stratified study design

Since the study was designed as a stratified survey with a fixed number of farms and participants in each stratum, not all the study units (farms and participants in the 3 districts) had the same probability of being selected. The prevalence of fecal colonization with mcr-1–carrying bacteria in chickens and humans was adjusted for the stratified survey design by assigning a stratum-specific sampling weight (Wi) to each observation unit (farm or subject) and then by using the following equation: Wi = NT/Ni, where NT = the

105

CHAPTER 6

total number of chicken farms or humans in that study district and Ni = the number of farms or participants in each stratum sampled (i = 1. . .7) (Technical Appendix Tables 1 and 2). Standard errors were corrected to calculate the prevalence in each stratum. Sampling weight and sampling fraction of participants belonging to each study stratum were calculated under the assumption that chicken farmers accounted for 80% of the rural population.

Table 2. Sampling weight and sampling fraction of chicken farms, Tien Giang province, Vietnam, 2012–2013*

Stratum NT† Ni Fraction that should be sampled Fraction sampled Wi Chau Thanh household farm 10,762 34 0.3697 0.00117 317 Cho Gao household farm 16,101 34 0.5532 0.00117 474 My Tho household farm 2,026 34 0.0696 0.00117 60 Chau Thanh small farm 36 34 0.0012 0.00117 1 Cho Gao small farm 147 34 0.0051 0.00117 4 My Tho small farm 34 34 0.0012 0.00117 1 *Ni, no. of farms sampled per stratum; NT, no. of farms per stratum; Wi, sampling weight. † Tien Giang statistical office (3)

Table 3. Sampling weight and sampling fraction of participants, Tien Giang province, Vietnam, 2012–2013*

Stratum NT† Ni Fraction that should be sampled Fraction sampled Wi My Tho, rural 16,621 68 0.027 0.000111 244 My Tho, farmer 66,486 68 0.108 0.000111 978 Chau Thanh, rural 46,067 68 0.075 0.000111 677 Chau Thanh, farmer 184,266 68 0.301 0.000111 2,710 Cho Gao, rural 33,594 68 0.055 0.000111 494 Cho Gao, farmer 134,375 68 0.219 0.000111 1,976 My Tho, urban 131,650 102 0.215 0.000166 1,291 *Ni, no. of farms sampled per stratum; NT, no. of farms per stratum; Wi, sampling weight. † Tien Giang statistical office (3)

References

1. Nguyen VT, Carrique-Mas JJ, Ngo TH, Ho HM, Ha TT, Campbell JI, Nguyen TN, Hoang NN,Pham VM, Wagenaar JA, Hardon A, Thai QH, Schultsz C. 2015. Prevalence and risk factors forcarriage of antimicrobial-resistant Escherichia coli on household and small-scale chicken farms inthe Mekong Delta of Vietnam. J Antimicrob Chemother 70:2144-2152.

2. WHO. 2004. How to investigate the use of medicines by consumers.http://www.who.int/drugresistance/Manual1_HowtoInvestigate.pdf. Accessed

3. Anonymous. Statistical Office of Tien Giang Province. Statistical YearBook Tien Giang Province2011. Statistical Printing Factory, Ho Chi Minh city, Vietnam, 2012.

106

CHAPTER 6

Supplemental Material 3: Full list of genes included in the AMR-08 microarray No Probe Antibiotic Gene 1 hp_aac6_611 Aminoglycoside aac6'-Ib 2 hp_aac6_612 Aminoglycoside aac6'-Ib 3 hp_aac6_613 Aminoglycoside aac6'-Ib 4 hp_aac6_615 Aminoglycoside aac6'-Ib 5 hp_aac6_617 Aminoglycoside aac6'-IIc 6 hp_aadB-2_611 Aminoglycoside aadB 7 hp_aadB_611 Aminoglycoside aadB 8 hp_aphA_611 Aminoglycoside aac6'-aph2' 9 hp_armA_611 Aminoglycoside armA 10 hp_npmA_611 Aminoglycoside npmA 11 hp_rmtC_611 Aminoglycoside rmtC 12 prob_aac3IVa_1 Aminoglycoside aac3-IVa 13 prob_aac3Ia_1 Aminoglycoside aac3-Ia 14 prob_aac6Ib_1 Aminoglycoside aac6'-Ib 15 prob_aadA1_1 Aminoglycoside aadA1-like 16 prob_aadA2_1 Aminoglycoside aadA2-like 17 prob_aadA4_1 Aminoglycoside aadA4-like 18 prob_ant2Ia_1 Aminoglycoside aadB 19 prob_strA_611 Aminoglycoside strA 20 prob_strB_611 Aminoglycoside strB 21 hp_blaCMY_611 Beta-lactam blaCMY 22 hp_blaCMY_612 Beta-lactam blaCMY 23 hp_blaKHM-1_611 Beta-lactam blaKHM 24 hp_blaMOX-CMY9_613 Beta-lactam blaMOX, blaCMY 25 hp_ges-1_611 Beta-lactam blaGES 26 hp_gim1_611 Beta-lactam blaGIM-1 27 hp_imi3_611 Beta-lactam blaIMI3 28 hp_imp_611 Beta-lactam blaIMP 29 hp_imp_612 Beta-lactam blaIMP 30 hp_imp_613 Beta-lactam blaIMP 31 hp_imp_614 Beta-lactam blaIMP 32 hp_imp_615 Beta-lactam blaIMP 33 hp_imp_616 Beta-lactam blaIMP 34 hp_imp_617 Beta-lactam blaIMP 35 hp_kpc4_611 Beta-lactam blaKPC-4 36 hp_oxa_611 Beta-lactam blaOXA-23 37 hp_oxa_612 Beta-lactam blaOXA-40 38 hp_oxa_613 Beta-lactam blaOXA-48 39 hp_oxa_614 Beta-lactam blaOXA-51 40 hp_oxa_617 Beta-lactam blaOXA-58 41 hp_per1_611 Beta-lactam blaPER-1 42 hp_spm1_611 Beta-lactam blaSPM-1 43 hp_veb1_611 Beta-lactam blaVEB-1 44 prob_acc1_11 Beta-lactam blaACC-1 45 prob_acc2_11 Beta-lactam blaACC-1 46 prob_cmy_11 Beta-lactam blaCMY 47 prob_ctxM26_11 Beta-lactam blaCTX-M-26 48 prob_ctxM8_11 Beta-lactam blaCTX-M-8 49 prob_ctxM9_11 Beta-lactam blaCTX-M-9 50 prob_dha1_1 Beta-lactam blaDHA-1 51 prob_fox_11 Beta-lactam blaFOX 52 prob_mox_1pm Beta-lactam blaMOX 53 prob_oxa1_21 Beta-lactam blaOXA-1 54 prob_oxa2_11 Beta-lactam blaOXA-2 55 prob_oxa7_11 Beta-lactam blaOXA-7 56 prob_oxa9_11 Beta-lactam blaOXA-9 57 prob_pse1_1mm Beta-lactam blaPSE-1-like 58 prob_pse1_1pm Beta-lactam blaPSE-1-like 59 prob_shv1_11 Beta-lactam blaSHV 60 prob_tem1_1 Beta-lactam blaTEM 61 hpn_ampC_Eaer_611 Beta-lactam ampC (E. aerogenes) 62 hpn_blaCTXM1_611 Beta-lactam blaCTX-M-1 63 hpn_blaCTXM1_612 Beta-lactam blaCTX-M-1 64 hpn_blaCTXM1_613 Beta-lactam blaCTX-M-1 65 hpn_blaCTXM2_611 Beta-lactam blaCTX-M-2 66 hpn_blaMAL_611 Beta-lactam blaMAL

107

CHAPTER 6

67 hpn_blaOKP_A_611 Beta-lactam blaOKP 68 hpn_blaOKP_B_611 Beta-lactam blaOKP 69 hpn_cblA_611 Beta-lactam cblA 70 hpn_cepA_611 Beta-lactam cepA 71 hpn_blaNDM_612 Beta-lactam blaNDM 72 hpn_blaNDM_613 Beta-lactam blaNDM 73 hpn_cfxA_612 Beta-lactam cfxA 74 hpn_blaVIM_617 Beta-lactam blaVIM 75 hpn_blaVIM_618 Beta-lactam blaVIM 76 hpn_blaVIM_619 Beta-lactam blaVIM 77 hpn_blaVIM_620 Beta-lactam blaVIM 78 hpn_blaVIM_621 Beta-lactam blaVIM 79 hpn_blaVIM_622 Beta-lactam blaVIM 80 hpn_blaVIM_623 Beta-lactam blaVIM 81 hpn_blaVIM_624 Beta-lactam blaVIM 82 hpn_blaVIM_625 Beta-lactam blaVIM 83 hpn_blaVIM_626 Beta-lactam blaVIM 84 hpn_blaOXY_614 Beta-lactam blaOXY 85 hpn_blaOXY_615 Beta-lactam blaOXY 86 hpn_blaOXY_616 Beta-lactam blaOXY 87 prob_catA1_11 Chloramphenicol catA1 88 prob_catB8_12 Chloramphenicol catB8 89 prob_catIII_1 Chloramphenicol catA3 90 prob_cmlA1_11 Chloramphenicol cmlA1-like 91 prob_floR_11 Chloramphenicol floR-1 92 hpn_catB3_611 Chloramphenicol catB3-like 93 hp_ereA_611 Erythromycin ereA 94 hp_ereA_612 Erythromycin ereA 95 hp_ermB_611 Erythromycin ermB 96 hp_ermB_612 Erythromycin ermB 97 prob_intI1_1 Intergrase intl1 98 prob_intI2_11 Intergrase intl2 99 hp_mphA_611 Macrolide mphA

100 prob_qnr_11 Quinolone qnrA 101 hpn_qepA_612 Quinolone qepA 102 hpn_qepA_613 Quinolone qepA 103 hpn_qnrB_611 Quinolone qnrB 104 hpn_qnrB_612 Quinolone qnrB 105 hpn_qnrB_613 Quinolone qnrB 106 hpn_qnrB_614 Quinolone qnrB 107 hpn_qnrC_611 Quinolone qnrC 108 hpn_qnrS_611 Quinolone qnrS 109 hp_arr-1_611 Rifampin arr-1 110 hp_vatE_611 Streptogramin vatE 111 hp_vatE_612 Streptogramin vatE 112 prob_sul1_11 Sulfonamide sul1 113 prob_sul2_11 Sulfonamide sul2 114 prob_sul3_11 Sulfonamide sul3 115 hp_tetX_611 Tetracycline tetX 116 prob_tetA_11 Tetracycline tetA 117 prob_tetB_11 Tetracycline tetB 118 prob_tetC_11 Tetracycline tetC 119 prob_tetD_1 Tetracycline tetD 120 prob_tetE_11 Tetracycline tetE 121 prob_tetG_11 Tetracycline tetG 122 prob_tetG_12 Tetracycline tetG 123 hpn_tetM_Ecoli_611 Tetracycline tetM 124 hpn_tetQ_611 Tetracycline tetQ 125 prob_dfr12_11 Trimethoprim dfrA12 126 prob_dfr13_11 Trimethoprim dfrA13 127 prob_dfrA14_21 Trimethoprim dfrA14 128 prob_dfrA15_1 Trimethoprim dfrA15 129 prob_dfrA17_11 Trimethoprim dfrA17 130 prob_dfrA19_1 Trimethoprim dfrA19 131 prob_dfrA1_21 Trimethoprim dfrA01 132 prob_dfrA1_22 Trimethoprim dfrA01 133 prob_dfrA7_11 Trimethoprim dfrA07 134 prob_dfrA7_12 Trimethoprim dfrA07

108

CHAPTER 6

Supplementary Material 4: Univariable analyses for risk factors associated with ESBL-Ec colonization in humans (N=510)

Lower Upper No No. of ESBL (+) subject Total % OR 95%CI 95%CI P-value 1 Participant group

Chicken farmers 65 204 31.9% ref ref ref ref Rural individual not involved in chicken farming 101 204 49.5% 2.2 1.43 3.3 <0.001

Urban individual not involved in chicken farming 39 102 38.2% 1.4 0.83 2.28 0.22 2 Household location

Cho Gao district 71 170 41.8% 1.4 0.89 2.22 0.147 Chau Thanh district 61 170 35.9% ref ref ref ref

My Tho city 73 170 42.9% 1.6 1.02 2.52 0.043

3 Age of participant (median age was used asbreakpoint)

< 46 years old 100 248 40.3% 1 1 1 0.81

≥ 46 years old 105 262 40.1% ref ref ref ref 4 Male participant 140 326 42.9% 1.5 1 2.2 0.048 5 Presence of other animals 122 341 35.8% 0.6 0.39 0.83 0.004 6 Presence of pig(s) 32 104 30.8% 0.6 0.38 1 0.05

7 Participants that used cephalosporins in the past month 11 20 55.0% 2.1 0.79 5.41 0.14

8 Participants that used antimicrobials in the pastmonth 18 34 52.9% 2.1 0.98 4.31 0.056

9 Chicken meat consumption Often (at least twice/week) 35 91 38.5% 1.1 0.44 2.75 0.84

Sometimes (at least twice/month) 158 392 40.3% 0.9 0.39 2.07 0.81

Never 12 27 44.4% ref ref ref ref 10 Egg consumption

Often (at least twice/week) 86 222 38.7% 0.9 0.44 1.82 0.76 Sometimes (at least twice/month) 101 247 40.9% 0.9 0.46 1.83 0.79

Never 18 41 43.9% ref ref ref ref

109

CHAPTER 6

Supplemental Material 5: Distribution of some common resistance genes (including ESBL genes) in ESBL-Ec isolated from chickens and humans

Gene Antimicrobial No. of isolates (%) Chicken Farmer Rural individual Urban Humana (N=20) (N=20) (N=20) (N=20) (N=60)

blaCTX-M-1-group Beta-lactam 12 (60.0) 6 (30.0) 10 (50.0) 8 (40.0) 24 (40.0) blaCTX-M-9-group Beta-lactam 4 (20.0) 15 (75.0) 10 (50.0) 12 (60.0) 37 (61.7)

blaTEMb Beta-lactam 16 (80.0) 12 (60.0) 10 (50.0) 7 (35.0) 29 (48.3) blaOXA-1 Beta-lactam 0 (0) 1 (5.0) 4 (20) 1 (5.0) 6 (10.0) blaOXA-7 Beta-lactam 1 (5.0) 0 (0) 0 (0) 1 (5.0) 1 (1.7) blaCMY Beta-lactam 4 (20.0) 0 (0) 0 (0) 0 (0) 0 (0)

blaACC-1 Beta-lactam 0 (0) 6 (30.0) 4 (20.0) 4 (20.0) 14 (23.3) catA1 Chloramphenicol 2 (10.0) 0 (0) 7 (35.0) 1 (5.0) 8 (13.3)

cmlA1-like Chloramphenicol 4 (20.0) 1 (5.0) 2 (10.0) 1 (5.0) 4 (6.7) floR-1 Chloramphenicol 13 (65.0) 3 (15.0) 1 (5.0) 2 (10.0) 6 (10.0)

catB3-like Chloramphenicol 1 (5.0) 1 (5.0) 0 (0) 0 (0) 1 (1.7) tetA Tetracycline 17 (85.0) 13 (65.0) 12 (60.0) 11 (55.0) 36 (60.0) tetB Tetracycline 3 (15.0) 4 (20.0) 6 (30.0) 7 (35.0) 17 (28.3)

dfrA12 Trimethoprim 3 (15.0) 0 (0) 1 (5.0) 0 (0) 1 (1.7) dfrA14 Trimethoprim 14 (70.0) 3 (15.0) 2 (10.0) 2 (10.0) 7 (11.7) dfrA17 Trimethoprim 5 (25.0) 14 (70.0) 11 (55.0) 13 (65.0) 38 (63.3) dfrA19 Trimethoprim 0 (0) 1 (5.0) 1 (5.0) 1 (5.0) 3 (5.0) dfrA1 Trimethoprim 0 (0) 1 (5.0) 0 (0) 0 (0) 1 (1.7) sul1 Sulfonamide 3 (15.0) 10 (50.0) 6 (30.0) 9 (45.0) 25 (41.7) sul2 Sulfonamide 15 (75.0) 13 (65.0) 11 (55.0) 12 (60.0) 36 (60.0) sul3 Sulfonamide 13 (65.0) 2 (10.0) 2 (10.0) 0 (0) 4 (6.7)

ermB Erythromycin 3 (15.0) 9 (45.0) 7 (35.0) 12 (60.0) 28 (46.7) mphA Macrolide 0 (0) 8 (40.0) 4 (20.0) 5 (25.0) 17 (28.3) mrx Macrolide 8 (40.0) 15 (75.0) 14 (70.0) 16 (80.0) 45 (75.0) qnrS Quinolone 5 (25.0) 2 (10.0) 0 (0) 1 (5.0) 3 (5.0) aphA Aminoglycoside 5 (25.0) 3 (15.0) 3 (15.0) 1 (5.0) 7 (11.7) aac6 Aminoglycoside 0 (0) 0 (0) 1 (5.0) 0 (0) 1 (1.7)

aac6'-Ib Aminoglycoside 1 (5.0) 1 (5.0) 5 (25.0) 1 (5.0) 7 (11.7) aadA1-like Aminoglycoside 15 (75.0) 3 (15.0) 3 (15.0) 2 (10.0) 8 (13.3) aadA2-like Aminoglycoside 1 (5.0) 0 (0) 1 (5.0) 0 (0) 1 (1.7) aadA4-like Aminoglycoside 1 (5.0) 0 (0) 1 (5.0) 0 (0) 1 (1.7)

aadB Aminoglycoside 0 (0) 1 (5.0) 1 (5.0) 0 (0) 2 (3.3) strA Aminoglycoside 6 (30.0) 9 (45.0) 5 (25.0) 6 (30.0) 20 (33.3) strB Aminoglycoside 11 (55.0) 12 (60.0) 11 (55.0) 10 (50.0) 33 (55.0) arr-1 Rifampin 5 (25.0) 1 (5.0) 0 (0) 1 (5.0) 2 (3.3)

a: humans including farmers, rural and urban individuals

b: any blaTEM. Sequencing of a random selection of 24 microarray blaTEM-positive isolates, including 4 ESBL-Ec isolates from chickens which were negative for any of the ESBL genes present on the microarray, showed that 100% of blaTEM genes were encoding narrow-spectrum beta-lactamase (TEM-1). Further sequencing of blaTEM genes was then abandoned.

110

CHAPTER 6

Supplemental Material 6: Usage of antimicrobial drugs in chickens and humans in southern Vietnam

Class of antimicrobial Chickens,* no. (%), N = 204 Humans,† no. (%)

Farmer, N = 204 Rural, N = 204 Urban, N = 102 Any antimicrobial drug 118 (57.8) 33 (16.2) 32 (15.7) 17 (16.7) 1st generation cephalosporin 0 (0) 12 (5.9) 17 (8.3) 7 (6.9) 2nd generation cephalosporin 0 (0) 1 (0.5) 2 (1.0) 1 (1.0) 3rd generation cephalosporin 0 (0) 5 (2.5) 6 (2.9) 1 (1.0) Penicillins 32 (15.7) 11 (5.4) 6 (2.9) 5 (4.9) Polymyxins 39 (19.1) 0 (0) 0 (0) 0 (0) Macrolides 38 (18.6) 3 (1.5) 4 (2) 4 (3.9) Quinolones 19 (9.3) 2 (1.0) 2 (1.0) 1 (1.0) Lincosamides 4 (2.0) 1 (0.5) 0 (0) 1 (1.0) Aminoglycosides 18 (8.8) 1 (0.5) 0 (0) 0 (0) Chloramphenicol 0 (0) 1 (0.5) 0 (0) 0 (0) Phenicols 12 (5.9) 0 (0) 0 (0) 0 (0) Sulfonamides/trimethoprim 12 (5.9) 0 (0) 0 (0) 1 (1.0) Tetracyclines 51 (25.0) 0 (0) 0 (0) 1 (1.0) Pleuromutilins 1 (0.5) 0 (0) 0 (0) 0 (0)

*Use during the previous 3 months for household-scale farms (≥10–200 chickens) or for the current flock for small-scale farms (>200–2000 chickens).†Use during the month before the survey visit.

111