Carriage of carbapenemase‐ and extended‐spectrum cephalosporinase‐producing Escherichia coli and Klebsiella pneumoniae in humans and livestock in rural Cambodia; gender and age differences and detection of bla OXA‐48 in humans

Abstract Objectives This study investigates the frequency and characteristics of carbapenemase‐producing Escherichia coli/Klebsiella pneumoniae (CPE/K) and extended‐spectrum cephalosporinase‐producing E. coli/K. pneumoniae (ESCE/K) in healthy humans and livestock in rural Cambodia. Additionally, household practices as risk factors for faecal carriage of ESCE/K are identified. Methods Faecal samples were obtained from 307 humans and 285 livestock including large ruminants, pigs and poultry living in 100 households in rural Cambodia in 2011. Each household was interviewed, and multilevel logistic model determined associations between household practices/meat consumption and faecal carriage of ESCE/K. CPE and ESCE/K were detected and further screened for colistin resistance genes. Results CPE/K isolates harbouring bla OXA‐48 were identified in two humans. The community carriage of ESCE/K was 20% in humans and 23% in livestock. The same ESBL genes: bla CTX‐M‐15, bla CTX‐M‐14, bla CTX‐M‐27, blaCTX‐M‐55, bla SHV‐2, bla SHV‐12, bla SHV‐28; AmpC genes: bla CMY‐2, bla CMY‐42, bla DHA‐1; and colistin resistance genes: mcr‐1‐like and mcr‐3‐like were detected in humans and livestock. ESCE/K was frequently detected in women, young children, pigs and poultry, which are groups in close contact. The practice of burning or burying meat waste and not collecting animal manure indoors and outdoors daily were identified as risk factors for faecal carriage of ESCE/K. Conclusions Faecal carriage of E. coli and K. pneumoniae harbouring extended‐spectrum cephalosporinase genes are common in the Cambodian community, especially in women and young children. Exposure to animal manure and slaughter products are risk factors for intestinal colonization of ESCE/K in humans.


| BACKG ROU N D
Escherichia coli and Klebsiella pneumoniae can cause a variety of severe infections, which are increasingly difficult to treat due to acquired resistance to critically important antibiotics (WHO, 2012). Resistance to broad-spectrum beta-lactam antibiotics in E. coli and K. pneumoniae is commonly due to the production of enzymes, which are characterized as carbapenemases, extended-spectrum beta-lactamases (ESBLs) and plasmid-borne AmpC beta-lactamases (AmpCs), and the latter two may also be collectively referred to as extended-spectrum cephalosporinases (ESCs; Padmini, Ajilda, Sivakumar, & Selvakumar, 2017).
Genes encoding carbapenemases, ESBLs and pAmpCs are often located on mobile genetic elements, for example plasmids, in E. coli and K. pneumoniae, enabling dissemination of antibiotic resistance genes between bacteria (Padmini et al., 2017). The emergence of carbapenemase-producing E. coli/K. pneumoniae (CPE/K) and extended-spectrum cephalosporinase-producing E. coli/K. pneumoniae (ESCE/K) in livestock populations, environment and the community shows that transmission and persistence of such bacteria occur also outside of clinical settings (Guenther, Ewers, & Wieler, 2011).
To date, there are no published reports of CPE/K in humans or livestock in Cambodia or of community carriage of ESCE/K in Cambodia. However, ESCE/K isolates have been detected as causative pathogens in Cambodian patients (Caron et al., 2018;Emary et al., 2015;Moore et al., 2016;Rammaert et al., 2012;Vlieghe et al., 2015). Genetic characterization of ESBL-producing E. coli Results: CPE/K isolates harbouring bla  were identified in two humans. The community carriage of ESCE/K was 20% in humans and 23% in livestock. The same ESBL genes: bla  , bla  , bla  , bla CTX-M-55 , bla SHV-2 , bla  , bla SHV-28 ; AmpC genes: bla CMY-2 , bla CMY-42, bla DHA-1 ; and colistin resistance genes: mcr-1-like and mcr-3-like were detected in humans and livestock. ESCE/K was frequently detected in women, young children, pigs and poultry, which are groups in close contact. The practice of burning or burying meat waste and not collecting animal manure indoors and outdoors daily were identified as risk factors for faecal carriage of ESCE/K.

Conclusions:
Faecal carriage of E. coli and K. pneumoniae harbouring extended-spectrum cephalosporinase genes are common in the Cambodian community, especially in women and young children. Exposure to animal manure and slaughter products are risk factors for intestinal colonization of ESCE/K in humans.

K E Y W O R D S
AmpC, Cambodia, carbapenemase, colistin, ESBL, risk factors, rural population, zoonoses Impacts • Multidrug-resistant Escherichia coli and Klebsiella pneumoniae harbouring cephalosporinase genes are common in rural Cambodian communities, especially in women, small children, poultry and pigs.
• The presence of cephalosporinase, carbapenemase and colistin resistance genes in bacteria from humans and livestock in Cambodian communities is worrying as such genes reduce the effectiveness of critically important antibiotics.
• Contact with animal manure and animal slaughter products enhance the risk of faecal colonization of multidrug-resistant E. coli and K. pneumoniae in humans.
isolates in bloodstream infections in Cambodia revealed that 96% were of CTX-M-type, mainly bla CTX-M-15 and bla CTX-M-14 (Vlieghe et al., 2015). Little is known about the situation in Cambodian livestock, but one study detected E. coli isolates harbouring bla  and bla CMY-2 from faecal samples from five pigs in a Cambodian slaughterhouse (Trongjit, Angkittitrakul, & Chuanchuen, 2016).
Interviews with pig farmers have revealed that antibiotic use was common in pig farms . ESBL-producing Salmonella has recently been isolated from retail meat in Phnom Penh, Cambodia, and most isolates were harbouring bla CTX-M-55 (Nadimpalli et al., 2018).
The objectives of this study were to determine the detection frequency of carbapenem-and third-generation cephalosporin-resistant E. coli and K. pneumoniae in humans and livestock and to analyse whether household practices and meat consumption are potential risk factors associated with community carriage in rural Cambodia.
Furthermore, we wanted to characterize the specific carbapenemase/extended-spectrum cephalosporinase gene-variants found and screen for colistin resistance genes and antibiotic susceptibility in CPE/K and ESCE/K isolates.

| Interviews
On day 1, the female head of the household was interviewed using a questionnaire, as previously described (Osbjer et al., 2015). Questions focused on eight household practices: (a) livestock home slaughter, (b) livestock access to sleeping and food preparation areas, (c) consumption of unsafe water (untreated well or pond water), (d) hand wash with soap after handling animals, (e) bury or burn meat waste products, (f) daily collection of animal manure indoors and outdoors, (g) hand wash with soap before and after cooking and (h) consumption of undercooked meat and meat consumption: the number of days each month that the household consumed pork, beef, fish, poultry and wild animals. Antibiotic use was not investigated in this study.

| Detection frequency
Pearson's chi-square tests were performed using GraphPad Prism version 8 to analyse differences in detection frequencies of ESCE/K and CPE/K between hosts. To control for multiple chi-square tests on the livestock data (Ruminants vs. Pigs, Pigs vs. Poultry and Ruminants vs. Poultry), a Bonferroni correction (p ≤ 0.02) was used. In the human data (Adult females vs. Adult males and Children 0-5 years vs. Children 6-15 years), multiple tests were not performed, and thus, p-values ≤0.05 were considered significant.

| Risk factors
Statistical analysis was performed in SAS for Windows 9.3 (SAS Institute Inc.).

| Detection of CPE/K and ESCE/K
The overall detection frequency of CPE/K was 1% in humans and 0% in livestock. The overall detection frequency of ESCE/K isolates was 20% in humans and 23% in livestock, with the detection frequency ranging from 5% to 62% in humans and 4% to 45% in livestock in the 10 villages (Figure 2). The detection frequency of ESCE/K isolates in adult females (n = 135) and adult males (n = 54) was 23% and 11%, respectively, Figure 3. There was a significant (p = 0.03) difference between the combined detection frequencies of CPE/K and ESCE/K isolates in adult females compared with the combined detection frequencies of CPE/K and ESCE/K in adult males. In Figure 3, the detection frequencies in children are grouped based on age. No CPE/K isolates were detected in children, and the detection frequency of ESCE/K isolates was significantly (p = 0.04) higher, 30%, in agegroup 0-5 years (n = 33) than in age-group 6-15 years (n = 85), 13%.
In livestock (n = 285), no CPE/K isolates were detected, but 23% carried ESCE/K. In ruminant (n = 103) and pigs (n = 39), the detection frequencies of ESCE isolates were 7% and 46%, respectively, and no ESCK isolates were detected. In poultry (n = 138), the detection frequency of ESCE/K isolates was 28% (Figure 4). The detection frequency was significantly higher in pigs and poultry compared with ruminants (both p < 0.0001), but the detection frequency was not significantly different between poultry and pigs (p = 0.10).

| Susceptibility to other antibiotics
Only CPE/K isolates and one ESCE expressed non-wild-type phenotypes to meropenem but resistance to other antibiotics was common ( Figure 5 and Table 1). In total, 96% of the isolates were characterized as multidrug resistant, that is expressed a non-wild-type phenotype to ≥3 antibiotic classes. According to the clinical breakpoints provided by CLSI, 92% of the isolates were characterized as multidrug resistant.

| D ISCUSS I ON
To the best of our knowledge, this is the first published report of ing Enterobacteriaceae is rare, but has been reported from humans in Lebanon (Beyrouthy et al., 2014) and Switzerland (Zurfluh et al., 2015). Bla OXA-48 harbouring isolates expressed a non-wild-type phenotype to meropenem, ciprofloxacin, sulfamethoxazole/trimethoprim, piperacillin/tazobactam, tetracycline and chloramphenicol but colistin resistance genes were not detected.
In this study, the combined detection frequency of CPE/K and ESCE/K in stool from adult females was significantly higher compared with adult males. This contrasts to community carriage in Western Europe where no difference between genders was observed (Ny et al., 2017;Valenza et al., 2014;Wielders et al., 2017).
Furthermore, ESBL colonization in male neonatal children was more common compared with female neonatal children in an Israeli hospital (Leikin-Zach et al., 2018). There are no obvious biological reasons for the observed difference in community carriage between sexes, and the explanation could be local gender-related behaviour leading to transmission between populations. In the current study population, women are often more responsible for the care of poultry and pigs (high level ESCE/K colonized livestock), while men generally take care of more valuable livestock such as ruminants (low level ESCE/K colonized livestock) (Osbjer et al., 2015). It has been previously shown that close contact with poultry increased community carriage of ESCE in Dutch humans (Huijbers et al., 2014). colonization in underweight newborns if the mother is a carrier (Denkel et al., 2014) (Guimaraes et al., 2009) and 49.6% in infants in Lebanon 2013 (Hijazi, Fawzi, Ali, & Abd El Galil, 2016a). The observed difference between children age 0-5 years (30%) and 6-15 years (13%) was statistically significant using the chi-square test, but due to a small sample size in the 0-5 years group (n = 33), the 95% confidence intervals are overlapping between the two groups, and our results should be interpreted with caution.
Our results show that certain household practices in rural  (Vlieghe et al., 2015). This indicates that the gut serves as a reservoir for extra-intestinal pathogenic E. coli, which has been previously suggested (Carlet, 2012). It is important to consider that the gene variation in the current study might be underestimated as only one colony was selected on each agar plate. In one E. coli and two K. pneumoniae, no extended-spectrum beta-lactamase gene could be identified and further molecular analysis is required to establish whether a less common or a novel ESBL gene can explain the phenotype.
The zoonotic potential of ESCE/K and CPE/K is of concern, and previous work has shown that the awareness of zoonotic risks of antibiotic-resistant bacteria is low in the current study population (Osbjer et al., 2015). Transmission of bacteria and/or mobile genetic elements between populations through contact and environmental exposure seem likely, as the same ESBL/pAmpC genes were detected in humans and livestock and the antibiotic resistance profile was similar in ESCE/K isolates. However, additional molecular work is needed to better understand relatedness between isolates in different hosts, but was not within the scope of this study.
The demonstrated community carriage of ESCE/K in humans and livestock in Cambodia (20% in humans and 23% in livestock) is similar to a recent report on ESBL-producing E. coli colonization in chicken farmers (20%) and chickens (35%) from Vietnam (Nguyen et al., 2019) but lower than in reports from Thailand (62%) and Vietnam/Laos (41%-70%) (Nakayama et al., 2015;Niumsup et al., 2018). The lower detection frequency in our study could be due to the rural habitat of the sampled population and that previous thawing of some samples has led to an underestimation of human carriage. The difference in ESCE/K detection between villages could partly be explained by skewed sampling. Poultry and pigs were sampled in high numbers in the high detection village number 4, as opposed to the low detection village number 7, which contained many samples from ruminants. In contrast, the detection frequency in humans was very high in village 7. Owning ruminants is associated with high wealth in Cambodia (Osbjer et al., 2015), and wealthy families might be more likely to travel and use medicine like antibiotics, both risk factors for acquiring antibiotic-resistant bacteria (Karanika et al., 2016). The high detection frequencies of ESCE/K in poultry and pigs could be related to transmission and inappropriate use of antibiotics. Pigs were often kept in crowded confinement (Osbjer et al., 2015), which allows for frequent transmission of bacteria. Previous studies have found that antibiotic use in the pig and poultry industry in Cambodia is widespread and uncontrolled (Om & McLaws, 2016;. ESCE is also frequent in poultry production in Europe, even in countries with low antibiotic resistance burden, and the prevalence is mainly related to vertical transmission (Blaak et al., 2015;Borjesson et al., 2016). The ESCE/K in pig manure is an environmental hazard, as pig farmers in Cambodia often dump the pig manure in the environment .
Colistin resistance genes were identified in poultry, pigs and humans. The mcr-1 gene has been previously detected in stool sample from a Cambodian child (Stoesser, Mathers, Moore, Day, & Crook, 2016) but to our knowledge this is the first finding of mcr-3-like gene in the country. Resistance to colistin is particularly worrisome as colistin can be the last available treatment for CPE/K (Falagas, Karageorgopoulos, & Nordmann, 2011). A study on backyard chicken farms in Vietnam concluded that detection of mcr-1-carrying bacteria in chicken samples was associated with colistin use and that detection in human samples was associated with exposure to mcr-1positive chickens (Trung et al., 2017). Through interviewing pig farmers in Cambodia, Ström, Boqvist, et al. (2018)) found that antibiotic use, including colistin, was common in pig farms and sometimes used as prophylactic treatment.

| CON CLUS IONS
Carbapenemase and colistin resistance genes were present in the Cambodian community to a low extent in 2011, but continuous surveillance is necessary as dissemination of multidrug-resistant bacteria is a dynamic process. Faecal carriage of E. coli and K. pneumoniae harbouring extended-spectrum cephalosporinase genes were common in rural Cambodia, with more frequent occurrence in women and young children. Environmental exposure and contact with animal manure and slaughter products were risk factors for intestinal colonization of ESCE/K, suggesting that farming households and animal health workers should be further educated on hygiene precautions to limit such exposure.

ACK N OWLED G EM ENTS
We thank the Cambodian families who participated in the study. We also extend our thanks to the commune, district and provincial veteri-

CO N FLI C T O F I NTE R E S T
None to declare.