Emergence and Comparative Genomics Analysis of Extended-Spectrum-β-Lactamase-Producing Escherichia coli Carrying mcr-1 in Fennec Fox Imported from Sudan to China

The extended-spectrum-β-lactamase (ESBL)-producing members of the Enterobacteriaceae family are a global concern for both animal and human health. There is some information indicating a high prevalence of ESBL producers in food animals. Moreover, there have been an increasing number of reports on ESBL-producing strains resistant to the last-resort antibiotic colistin with the global dissemination of the plasmid-mediated mcr-1 gene, which is believed to have originated in animal breeding. However, little is known regarding the burden of ESBL-producing Enterobacteriaceae on wild animals. No data were available on the prevalence of antimicrobial resistance (AMR) among wild animals imported into China. This is the first study to investigate the microbiological and genomics surveillance investigation of ESBL colonization among fennec fox (Vulpes zerda) imported from Sudan to China, and we uncovered a high prevalence of ESBL-EC. Furthermore, the underlying mechanism of colistin resistance in an isolate that harbored mcr-1 was also investigated. Results of characterization and analysis of 29 ESBL-producing E. coli may have important implications on our understanding of the transmission dynamics of these bacteria. We emphasize the importance of improved multisectoral surveillance for colistin-resistant E. coli in this region.

Sudan to China, and we uncovered a high prevalence of ESBL-EC. Furthermore, the underlying mechanism of colistin resistance in an isolate that harbored mcr-1 was also investigated. Results of characterization and analysis of 29 ESBL-producing E. coli may have important implications on our understanding of the transmission dynamics of these bacteria. We emphasize the importance of improved multisectoral surveillance for colistin-resistant E. coli in this region. KEYWORDS ESBL, Escherichia coli, MCR-1, fennec fox, CTX-M-55 T he increased use of antimicrobial agents in human and veterinary medicine has resulted in an emergence of antimicrobial resistance (AMR) in clinical settings, as well as animal hospitals at large (1). The global spread of extended-spectrum ␤-lactamases (ESBLs) is of great concern to human and animal health, and CTX-M ESBLs are the most predominant ESBL enzymes worldwide (2,3). There is increasing evidence that the dissemination of ESBL producers is an issue that is no longer restricted to the clinical service system but represents a growing problem involving animal and environmental integrity (4). In the past few years, ESBL-producing Escherichia coli (ESBL-EC) strains have been reported in healthy animals (5,6), but the burden of ESBL-producing members of the Enterobacteriaceae family on wild animals remain largely undetermined. The dissemination of ESBL genes among wild animals is important to human health because wildlife may act as potential reservoirs and vectors for the spread of AMR (4). No data were available on the prevalence of AMR among wild animals imported into China. Here, we performed a microbiological and genomics surveillance investigation of ESBL colonization among fennec fox (Vulpes zerda) imported from Sudan to China.
A total of 168 nonduplicate fresh fecal samples from wild fennec fox imported from Sudan to China were collected from the quarantine yard between August 2017 and May 2018. Permission was obtained from the Entry-Exit Inspection and Quarantine Bureau of China. Individual fresh fecal samples were collected using a sterile swab and subsequently diluted in sterile physiological saline solution as described previously (7). The diluted fecal samples were plated on MacConkey agar supplemented with 1 mg/ liter cefotaxime and 1 mg/liter meropenem and incubated for 18 h at 37°C. The resulting strains were then isolated and identified by matrix-assisted laser desorption ionizationϪtime of flight mass spectrometry (MALDI-TOF MS) (Bruker, Bremen, Germany) as described previously (5). Confirmation of ESBL-EC was further performed using a double-disk diffusion method, according to the protocol described by the Clinical and Laboratory Standards Institute (CLSI) (https://clsi.org/). The genes encoding CTX-M type enzymes were screened by PCR followed by Sanger sequencing as reported previously (8).
A total of 104 Gram-negative isolates resistant to broad-spectrum cephalosporins were cultured by selective medium plates. Of these isolates, 29 isolates from 168 fennec fox fecal samples were identified to be ESBL-EC, giving a carriage rate of 17.3% (29/168). We did not identify carbapenemase-producing E. coli from any sample. Only a few reports have described the antimicrobial use patterns and antimicrobial-resistant bacteria among fur animals (12,13). To the best of our knowledge, this study reports for the first time the occurrence of ESBL-EC in fennec fox. Previous surveillance data on AMR among Enterobacteriaceae from wild animals underscored a wide dissemination of multidrug-resistant (MDR) bacteria, exhibiting an acquired resistance to multiple antibiotic categories (4). On the other hand, previous investigations showed that wildlife act as important role for spreading AMR across the globe (14). The outcome could be explained by the rapid transmission routes and the occurrence of MDR bacteria in potential ecological niches across national boundaries. The risk of zoonotic potential for ESBL-encoding bacteria from importing animals deservedly garners considerable attention.
Among these ESBL-EC strains, total resistance to cefotaxime was observed (100%), followed by very high incidences of resistance to tetracycline (86.2%), ciprofloxacin (72.4%), and trimethoprim-sulfamethoxazole (72.4%) (see Fig. S1 in the supplemental material). All isolates were susceptible to piperacillin-tazobactam, tigecycline, imipenem, and meropenem. Interestingly, only one isolate (EcFF273) was observed to be resistant to colistin. PCR and sequencing further confirmed the presence of the mcr-1 gene in isolate EcFF273. In this study, the bla CTX-M gene was detected in all ESBL-EC strains. The most prevalent genotypes observed in this study were bla CTX-M-55 and bla CTX-M-14 gene (n ϭ 10 for each), followed by bla CTX-M-15 (n ϭ 8), and bla CTX-M-64 (n ϭ 1). CTX-M-55 became one of the most common ESBL types detected in humans and animals and in the environment in Asian countries (3,8,15,16). Very recently, the occurrence of CTX-M-55-producing E. coli has been increasingly reported in the environment and diverse animal species in Europe (17,18). Our findings further highlighted that wild animals from North Africa may act as the reservoir of CTX-M-55.
WGS and in silico analysis found two types of genetic context surrounding bla CTX-M-14 (Fig. 2a). In addition, CTX-M-15-producing isolates were genetically diver-gent, and the genetic structure of bla CTX-M-15 gene could be categorized into two groups (Fig. 2b). Of note, four ST93 isolates have the same structure of bla CTX-M-55encoding fragment (Fig. 2c), which is consistent with the phylogenetic analysis results (Fig. 1). These observations suggest the clonal spread of ST93 among our isolate collection. Interestingly, plasmidSPAdes analysis generated the assembled sequence of the 65-kb IncI2 plasmid pMCR-EcFF273, which is in line with the plasmid size confirmed by the Southern blot and S1 PFGE experiments (Fig. S2). The assembled sequence was subjected to a BLAST search against the nr/nt database. An overall identical identity (99.9%) query showed coverage of 99% to p1108-MCR (GenBank accession no. MG825380) from E. coli of chicken origin in China. Annotation of the plasmid sequence revealed a typical structure surrounding the mcr-1 gene (nikA-nikB-mcr-1-pap2) in these plasmid contigs (Fig. 2d). Conjugation analysis confirmed that the mcr-1 gene in EcFF273 was transferable to the recipient cells (data not shown). Although the use of colistin in food animals is widespread, it is rarely connected to wild animals. These data clearly demonstrated that colonized E. coli strains potentially contribute to the dissemination and transfer of mcr-1 to pathogenic bacteria from fennec fox. Of note, Southern blot and S1-PFGE revealed that 11 ESBL-EC carried ESBL genes on the plasmid and most of the isolates (17/28) carried bla CTX-M genes on the chromosome (Fig. S2). Our results are in line with the previous observations of chromosomally integrated ESBL genes occurring in Enterobacteriaceae isolates from animals (22,23).
This study is limited by the relatively small number of fecal samples tested. Further studies involving more fennec fox samples are urgently needed to study the dissem-  ination dynamics of mcr and ESBL genes. In addition, it is not yet clear how MCR producers make their way into fennec fox, although this study supports recent findings that MCR-1-producing E. coli appears to be colonizing new hosts. The introduction of mcr-1-positive bacteria in fennec fox may be explained by the food chain-based dissemination pathway, and further comprehensive investigation on the transmission of MCR-1 in quarantine yard with "one health" perspective is warranted.
Collectively, this study reports for the first time the occurrence of ESBL-EC in fennec fox. The high prevalence of ESBL producers and the occurrence of MCR-1 producer in fennec fox imported into China from Sudan are unexpected. In addition, it clearly demonstrated that commensal E. coli strains can be reservoirs of bla CTX-M and mcr-1, potentially contributing to the dissemination of such genes. Our results support the implication of fennec fox as a biological vector for ESBL-EC.
Data availability. The Whole Genome Shotgun BioProject for the ESBL-EC isolates has been deposited at DDBJ/EMBL/GenBank under BioProject accession no. PRJNA562084.

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Supplemental material for this article may be found at https://doi.org/10.1128/ mSphere.00732-19. We declare that we have no conflicts of interest relevant to this paper.