Meat and Fish as Sources of Extended-Spectrum β-Lactamase–Producing Escherichia coli, Cambodia

We compared extended-spectrum β-lactamase–producing Escherichia coli isolates from meat and fish, gut-colonized women, and infected patients in Cambodia. Nearly half of isolates from women were phylogenetically related to food-origin isolates; a subset had identical multilocus sequence types, extended-spectrum β-lactamase types, and antimicrobial resistance patterns. Eating sun-dried poultry may be an exposure route.

We performed whole-genome sequencing for 1 ESBL -producing E. coli isolate from each food sample and all human-origin ESBL-producing E. coli isolates (Appendix sections 1.4-1.6) and compiled genetic and phenotypic characteristics of these 196 isolates (Appendix Tables 6, 7). We also determined the distribution of multilocus sequence types (MLSTs) encoding predominant ESBL-or carbapenemase-gene types ( Figure 1).
Phylogenetic analysis of ESBL-producing E. coli genomes revealed 3 distinct clans ( Figure 2, panel A). Clan I/ B2&D (n = 53) comprised mostly human-origin isolates, including isolates from colonized persons and most infected patients. Clans II/A (n = 69) and III/B1 (n = 47) included isolates from colonized persons and from food but not from infected patients. Each clan comprised an exclusive subset of sequence types (STs); clan I/B2&D included ST131 and clonal complex (CC) 38, clan II/A included CC10, and clan III/B1 included CC58 and CC156. Approximately half (21/39) of isolates in clans II/A and III/B1 from colonized patients belonged to STs detected in both humans and meat (Appendix Table 8).
We determined the distributions of ESBL-encoding genes and resistance patterns among isolates from colonized persons by clan ( Figure 2, panels B and C). The bla CTX-M-55 gene was more common among colonization isolates belonging to clan II/A than to clan I/B2&D (p<0.05). Amphenicol resistance was more common among colonization isolates belonging to clan II/A than clan I/B2&D (p<0.05) and was most often encoded by floR (Appendix Table 7).
Our genomic and epidemiologic findings suggest that ESBL-producing E. coli that contaminates meat and fish in Phnom Penh may be disseminating to the community. ESBL-producing E. coli were highly prevalent among the meat and fish we sampled. More than 80% of food-origin isolates were amphenicol resistant, and two thirds produced CTX-M-55. When food-origin isolates were compared Figure 2. Genomic comparisons of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli from humans, fish, pork, and chicken from Cambodia and differences in human colonization isolates by phylogenetic clan. All isolates were phenotypically resistant to third-generation cephalosporins (data not shown). A) Whole-genome sequence-based phylogenetic tree of 195 ESBL-producing E. coli genomes comprising 87 human colonization isolates, 15 human clinical isolates, and 93 isolates from fish, pork, and chicken meat and resulting phylogenetic clans I/B2&D (n = 53), II/A (n = 69), and III/B1 (n = 47). B) ESBL-encoding genes of human colonization E. coli isolates, by phylogenetic clan. C) Phenotypic resistance of human colonization ESBL-producing E. coli isolates to antimicrobial drugs of 8 classes, by phylogenetic clan. Clinical isolates are not included in panels B or C. Of 87 human colonization genomes, 13 did not group into a phylogenetic clan and thus are excluded from panels B and C. Prevalence of outcome differed significantly (p<0.05, indicated by *) between 2 indicated clans by post hoc Tukey test. Only statistically significant differences are depicted. 1, quinolone; 2, co-trimoxazole; 3, tetracycline; 4, aminoglycoside; 5, macrolide; 6, amphenicol; 7, carbapenem; 8, colistin.
with human-origin isolates, ≈40% of ESBL-producing E.coli from healthy persons grouped into the same phylogenetic clans that comprised most food-origin isolates. Approximately half of these colonization isolates had MLSTs detected among food, and a substantial portion were more likely to produce CTX-M-55 and be amphenicol resistant than colonization isolates that grouped separately. The fact that chloramphenicol has not been used in human medicine for almost 20 years in Cambodia, yet chloramphenicol analogs (e.g., florfenicol, thiamphenicol) are administered to food animals (5,7), suggests a food origin for these colonizing isolates.
Healthy women colonized with amphenicol-resistant ESBL-producing E. coli were more likely to eat poultry meat prepared by sun drying, a process that may not eliminate bacteria (8). Although we did not test dried meat samples for ESBL-producing E. coli contamination, our finding is consistent with those of other studies (8,9). Women reported having prepared dried poultry at home. Especially in lowresource households, sun-dried meat may become crosscontaminated by raw meat, dust, animals, and flies (8).
Our findings are concerning because of growing interest in using chloramphenicol as a drug of last resort for panresistant strains of bacteria (10). In the early 2000s, the Cambodia government stopped purchasing chloramphenicol because of concerns about side effects. Since restriction of this drug, infections in the hospital setting have reverted to a chloramphenicol-susceptible phenotype (11). Nevertheless, our findings suggest that amphenicol resistance genes are circulating in the  3 (0.4-4.5) 3 (12) 10 (16) 0.7 (0.2-2.7) *Blank cells indicate variable not included in multivariate models. aOR, adjusted (for age) OR; CHL, chloramphenicol; ESBL, extended-spectrum lactamase; OR, odds ratio. †Not reported for 4 women (missing data). All 4 were colonized with CHL-susceptible ESBL-producing Escherichia coli. One woman was colonized with CTX-M-55-type E. coli, whereas the other 3 were colonized with other CTX-M-encoded isolates. ‡With persons in other households.
community, potentially because amphenicol use in food animals has selected for resistant bacteria that can spread to humans (12). This possibility is concerning because physicians in Cambodia are often unable to assess the resistance of infectious agents before prescribing antimicrobial drugs (4).
Our study had several limitations. First, for logistical reasons, we sampled meat and fish during only 1 season. Contamination rates may have differed had we sampled across seasons (13). Second, although we included colonization samples from healthy women, all women had recently given birth in healthcare settings. However, more than half were colonized with ESBL-producing E. coli phylotypes A and B1, supporting community-associated, rather than healthcare-associated, acquisition. Third, we were unable to include clinical isolates from the same population that contributed colonization isolates. Thus, differences in colonization and clinical isolates could have resulted from population differences. Fourth, we did not sample food animals, which could have helped confirm that CTX-M-55-type and amphenicol-resistant ES-BL-producing E. coli circulate among them. Last, we did not investigate additional potential pathways for ESBLproducing E. coli transmission to colonized women, such as contact with persons employed at farms or slaughterhouses or proximity to such operations.

Conclusions
This study, which integrated epidemiologic and genomic methods to characterize community, clinical, and environmental data, supports concerns that the dissemination of antimicrobial drug-resistant bacteria from food animals to humans may be more likely in low-and middle-income countries (14,15). This finding is concerning because meat consumption is projected to drastically increase in these countries, and animal production that relies on routine antimicrobial drug use is being promoted to meet this demand (14). Particularly for low-and middle-income countries such as Cambodia, implementation of multisectoral strategies to combat antimicrobial resistance from a One Health perspective must be supported, and food safety should be prioritized.