Resistance Profiling and Molecular Characterization of Extended-Spectrum/Plasmid-Mediated AmpC β-Lactamase-Producing Escherichia coli Isolated from Healthy Broiler Chickens in South Korea

We aimed to identify and characterize extended-spectrum β-lactamase (ESBL)-and/or plasmid-mediated AmpC β-lactamase (pAmpC)-producing Escherichia coli isolated from healthy broiler chickens slaughtered for human consumption in Korea. A total of 332 E. coli isolates were identified from 339 cloacal swabs in 2019. More than 90% of the isolates were resistant to multiple antimicrobials. ESBL/pAmpC-production was noted in 14% (46/332) of the isolates. Six of the CTX-M-β-lactamase-producing isolates were found to co-harbor at least one plasmid-mediated quinolone resistance gene. We observed the co-existence of blaCMY-2 and mcr-1 genes in the same isolate for the first time in Korea. Phylogenetic analysis demonstrated that the majority of blaCMY-2-carrying isolates belonged to subgroup D. Conjugation confirmed the transferability of blaCTX-M and blaCMY-2 genes, as well as non-β-lactam resistance traits from 60.9% (28/46) of the ESBL/pAmpC-producing isolates to a recipient E. coli J53. The ISECP, IS903, and orf477 elements were detected in the upstream or downstream regions. The blaCTX-M and blaCMY-2 genes mainly belonged to the IncI1, IncHI2, and/or IncFII plasmids. Additionally, the majority of ESBL/pAmpC-producing isolates exhibited heterogeneous PFGE profiles. This study showed that healthy chickens act as reservoirs of ESBL/pAmpC-producing E. coli that can potentially be transmitted to humans.


Introduction
Escherichia coli is a commensal bacterium of the intestinal tract of humans and animals. It constitutes a reservoir of resistance genes for a wide range of pathogenic bacteria. The level of resistance in this bacterium is a good indicator of the selection pressure exerted by antimicrobial use and for the resistance problem to be expected in related pathogenic bacteria [1]. Therefore, investigation of the antimicrobial resistance profiles of indicator bacteria, such as E. coli, is essential to detect the spread of resistant bacteria between animals and humans [2].
Healthy food animals are frequently reported as reservoirs of extended-spectrum β-lactamase (ESBL) and plasmid-mediated AmpC β-lactamase (pAmpC)-producing E. coli, and have caught considerable attention worldwide [3,4]. The ESBL/pAmpC enzymes are known to hydrolyze the β-lactam ring of β-lactam antibiotics and cause the emergence of resistance to a considerable number of β-lactam antibiotics, including extended-spectrum cephalosporins [5].
Besides, sequenced using previously-described primers. Additionally, a multiplex PCR assay was conducted to detect genes encoding for six AmpC families and positive isolates were amplified using specific primers. The bla CTX-M and AmpC-positive strains were further screened for plasmid-mediated quinolone resistance (PMQR) genes: qnrA, qnrB, qnrC, qnrD, qnrS1, qnrV, qepA, and aac (6 ) Ib-cr genes. Sequence analysis was performed using ABI3730XL DNA sequence analyzer (SolGent, Daejeon, Korea) and comparison with known sequences was performed with the Basic Local Alignment Search Tool (BLAST) programs at the National Center for Biotechnology Information website (www.ncbi.nlm.nih. gov/BLAST). The primers and their PCR conditions used for the detection of resistance genes are listed in Table S1.

Conjugation Experiment
The broth-mating experiment was performed to determine the transferability of bla CTX-M genes to sodium azide-resistant E. coli J53 [22]. Transconjugants were selected on Muller-Hinton agar, supplemented with sodium azide (150 µg/mL) and cefotaxime (2 µg/mL). The antimicrobial susceptibility profiles and β-lactamase gene carriage of the transconjugants were also determined, as described above.

Molecular Characterization of ESBL/pAmpC-Producing E. coli
A PCR-based replicon typing kit (DIATHEVA, Fano, Italy) was used to determine the replicon types of the transconjugants following the manufacturer's protocol. The genetic environment of the bla CTX-M/CMY-2 genes was investigated using PCR and Sanger sequencing, as described previously [23,24]. A combination of IS26 or ISEcp1 forward primers, and a CTX-M reverse consensus primer (MA2) were used to investigate regions upstream of the bla genes. A MA1 primer and reverse primers of IS903 or orf477 were used to characterize downstream regions of the bla genes. The primers and their PCR conditions used for the detection of the bla CTX-M and bla CMY-2 genetic environments are listed in Table S1. Additionally, pulsed-field gel electrophoresis (PFGE) analysis of ESBL/pAmpC-producing E. coli strains was also performed following XbaI digestion of chromosomal DNA (Takara Bio Inc., Shiga, Japan), as described previously [25]. Then, PFGE bands were analyzed using Bionumerics software (UPGMA) and relatedness of the isolates was calculated using the unweighted pair group method with the arithmetic average algorithm based on the Dice similarity index. Further, a multiplex PCR assay targeting chuA, yjaA, and the DNA fragment TspE4.C2 was used to determine the phylogenetic characteristics of the ESBL/pAmpC-producing strains [26].

Antimicrobial Resistance of Indicator E. coli
We identified 332 E. coli isolates from 339 fecal samples obtained from 34 different broiler farms. Resistance to nalidixic acid (92.5%) was the highest, followed by resistance to ampicillin (86.4%), ciprofloxacin (78.3%), and tetracycline (71.7%) ( Table 1). Resistance to amoxicillin/clavulanic acid, cefepime, cefoxitin, ceftazidime, and colistin was low (0.6-3.6%). We observed ceftiofur resistance in 13.9% (46/332) of the isolates. However, resistance to meropenem was not detected. All isolates were resistant to at least one antimicrobial agent, and MDR was noted in 94.3% of the isolates (Table 2). Besides, about 34% of the isolates exhibited resistance to at least eight antimicrobials. Among 103 different resistance patterns observed in this study, resistance to ampicillin, chloramphenicol, ciprofloxacin, nalidixic acid, streptomycin, sulfisoxazole, tetracycline, and trimethoprim/sulfamethoxazole (12.5%) was the most frequent MDR pattern.

Molecular Characteristics of ESBL/pAmpC-Producing E. coli
The ESBL/pAmpC-producing isolates exhibited resistance to several antimicrobial classes, such as aminoglycoside, tetracycline, quinolones, and folate pathway inhibitors ( Table 4). Six of the CTX-M β lactamase-producing isolates were found to co-harbor at least one PMQR gene, with qnrS1, qnrS2, and aac (6 )-Ib-cr being detected alone or in combination. Notably, the bla CTX-M-65 , qnrS2, and aac (6 )-Ib-cr genes were found to be carried together in one isolate. Additionally, one isolate from farm E co-carried bla CMY-2 and mcr-1 genes.
The transconjugants carrying the bla CTX-M-55 gene presented distinct types of genetic environments, namely bla CTX-M-55 -orf477 (n = 9) and ISEcp1-bla CTX-M-55 -orf477 (n = 4) elements (Table 5). ISEcp1-bla CTX-M-1 -orf477 and bla CTX-M-14 -IS903 elements were identified in three and five transconjugants, respectively. The bla CMY-2 and bla CTX-M-55+CMY-2 gene expression was driven by the ISEcp1 insertion sequence, but IS903 and orf477 elements were not detected downstream of bla CMY-2 and bla CTX-M-55+CMY-2 genes. PFGE analysis of 46 E. coli strains carrying bla CTX-M and bla CMY-2 genes from 21 different farms demonstrated 34 arbitrary pulsotypes ( Figure S1). In general, most of the isolates were heterogeneous. We observed identical PFGE profiles in bla CTX-M-14, bla CTX-M-55 , and bla CTX-M-65 -carrying strains from farms AC, AE, AG, and I. Similarly, the two bla CTX-M-14 -carrying isolates from farms AC and AE exhibited identical PFGE profiles. However, DNA from five strains was constantly auto-digested. Consequently, a cluster formed by these strains was excluded from the analysis.

Discussion
Our observations revealed that most of E. coli isolated from healthy broilers were resistant to multiple antimicrobials and possessed diverse ESBL-encoding genes that could be readily spread to humans. Although CTX-M-15 is considered the predominant ESBL type in the Korean poultry industry [27], we observed CTX-M-14 and CTX-M-55 type ESBLs in most of the isolates.
Consistent with previous findings in Korea [28,29] and other countries [30][31][32][33], E. coli isolates exhibited high rates of resistance to ampicillin, nalidixic acid, tetracycline, and sulfisoxazole. However, it was lower than those described in recent reports in Asia and Africa [34][35][36]. Additionally, the proportion of MDR isolates in this study corresponded with previous reports [28,33]. The isolates exhibited more than 100 different resistance patterns and most of these patterns were associated with quinolones, penicillins, and tetracyclines. High antimicrobial resistance rates and diverse resistance patterns observed in this study coincide with the marked increase in the use of antimicrobials, including penicillins, fluoroquinolones, phenicols, and tetracyclines in the Korean poultry industry [37]. The variations in antimicrobial resistance among countries might be because of differences in geographical region, locally approved antimicrobials, and farm management systems.
Fluoroquinolones are considered critically important antimicrobials for both humans and animals [38]. About 80% of the isolates were resistant to ciprofloxacin, a finding which is consistent with previous reports in Poland [32], Korea [39], and Vietnam [40]. However, it was higher than those reported in several Asian countries [31,35,[41][42][43]. Although ciprofloxacin is not approved for animal uses, the continuous utilization of enrofloxacin in food animals, especially chickens in Korea, could be contributing to the increase in ciprofloxacin resistance [37].
Third-generation cephalosporin-resistant isolates are often resistant to multiple antimicrobials and are considered a potential threat to animal and human health [44]. The ceftiofur resistant rate in this study was slightly higher than previous reports in Korea (12%) [45] and the US (7%) [46]. Nevertheless, it was lower than Lee et al. (22%) [47] and Zhang et al. (47%) [33] in Korea and China, respectively. Various authors reported the relationship between ceftiofur use and resistance to third-generation cephalosporins in poultry production [48][49][50]. Therefore, although information on the use of this antimicrobial in farms was not available, the frequent application of ceftiofur in food animals could lead to the emergence of ceftiofur-resistant E. coli isolates.
A variety of ESBL/pAmpC genes have been identified in bacteria isolated from food animals worldwide. Most noteworthy of these are the bla CTX-M-14 , bla CTX-M-15 , bla CTX-M-27 , and bla CTX-M-55 variants, which have been associated with the global spread of β-lactam antibiotic resistance in humans and food animals [51,52]. In Korea, β-lactam antibiotics resistance in chicken [14,16,28] and human [53][54][55] isolates is commonly associated with bla CTX-M-1 , bla CTX-M-14 , and bla CTX-M-15. However, bla CTX-M-55 was the most frequent ESBL gene observed in this study. Our finding concurred with a recent report in E. coli strains from retail chicken meat in Korea [17]. CTX-M-55 is a CTX-M-15 variant that possesses enhanced β-lactamase-hydrolyzing activity and structural stability [56]. Since its first detection in ESBL-producing E. coli in 2004 and 2005 in Thailand, it has been widely reported in E. coli isolated from food animals and humans in many countries [17,48,[57][58][59][60]. The observation suggests that CTX-M-55 may be supplanting CTX-M-15.
E. coli harboring bla CTX-M-14 has been frequently detected in food animals in Korea [14,16,22] and other countries [57,60]. In this study, bla CTX-M-14 (26.1%) was the second most frequent ESBL gene. Similarly, Park et al. [17] and Seo et al. [28] detected bla CTX-M-14 in 22% and 14% of ESBL-producing broiler chicken E. coli isolates in Korea, respectively. Additional studies have also observed bla CTX-M-14 -carrying E. coli isolates in food and companion animals, as well as in humans in several Asian countries [52-54, 61,62], indicating its widespread distribution and the potential threat to public health.
In this study, only a few isolates were positive for bla CTX-M-1 and bla CTX-M- 65 . bla CTX-M-65 was frequently detected in ESBL-producing E. coli isolated from chicken in Korea [16,17] and China [52]. Although bla CTX-M-1 was detected in ESBL-producing strains recovered from chickens and farm environments in Korea [14,22,28], it is among the most frequent ESBL-encoding gene reported in Europe [61,63,64]. blaCTX -M is known to spread between animals and humans through the food chain and isolates of humans and foods of animal origin commonly shared dominant CTX-M genotypes. Thus, broiler chickens may serve as an important reservoir and source of human infection [51].
PMQR genes were commonly associated with low-level fluoroquinolone resistance and promoted the selection of high-level resistant strains [73]. In this study, the PMQR genes were identified in association with bla CTX-M-1 , bla CTX-M-55 , and bla CTX-M-65 genes. Most of the PMQR genes were associated with bla CTX-M-55. The bla CTX-M-55 genes commonly co-localize with other resistance genes, such as PMQR genes and genes encoding 16S rRNA methyltransferases [74,75]. The co-existence of PMQR and ESBL genes in Enterobacteriaceae have been reported in many countries, including Korea [73,[76][77][78]. The widespread use of quinolones and third-generation cephalosporins in food animals has led to the emergence of PMQR and ESBL-producing E. coli. The co-occurrence of these genes in chicken isolates constitutes a public health concern.
The co-existence of ESBL and mcr-1 genes in Enterobacteriaceae poses a serious public health threat. Despite several reports on the co-existence of mcr-1 and ESBL genes in E. coli strains isolated from humans, food animals, and fresh vegetables in various countries [52,[79][80][81][82][83], only a few reports are available on the co-existence of mcr-1 and bla CMY-2 in E. coli [84][85][86]. Notably, this is the first report on the co-existence of mcr-1 and bla CMY-2 in E. coli in Korea. Colistin is the last-resort antibiotic against multidrug-resistant E. coli, hence the co-existence of mcr-1 and bla CMY-2 poses a serious challenge to the application of antimicrobials in humans and animals.
Various plasmid replicon types, either alone or in combination, were identified in E. coli transconjugants. Several studies have reported the association between bla CTX-M-14 gene and different plasmid types, including IncF family plasmids, IncK, and IncI1-Iγ [12,87,88]. However, this study identified the bla CTX-M-14 gene predominantly on the IncHI2 plasmid. The bla CTX-M-55 gene was efficiently transferred to recipient E. coli from 72% of bla CTX-M-55 -carrying strains. This is presumably due to its frequent association with the IncF family of plasmids [27]. The IncF plasmid family is implicated in the dissemination of ESBLs because it is stably maintained in commensal E. coli [51]. In addition, the bla CTX-M-1 and bla CMY-2 genes predominantly belonged to IncI1α plasmid, a finding which concurred with Bevan et al. [51] and Carattoli, [7]. Further, the observation of diverse plasmid backbones in this study may reflect the co-occurrence of antimicrobial-resistant genes [27] and the dissemination of co-resistant bacteria [89].
ESBL-genes are often associated with insertion sequences (ISs), which are the smallest transposable elements capable of independent transposition in an organism [90]. The co-existence of ISEcp1 and ESBL/pAmpC genes in E. coli isolates is well documented [90][91][92]. Agreeing with this study, ISEcp1 is frequently found in the upstream regions of ESBL/pAmpC genes and plays an important role in the efficient capture, expression, and mobilization of bla CTX-M and bla CMY-2 genes [24,90]. Agreeing with previous reports [10,23,93], the orf477 element was found downstream of bla CTX-M-1 and bla CTX-M-55 genes, while IS903 was located downstream of bla CTX-M-55 .
PFGE analysis demonstrated that the majority of the bla CTX-M -carrying isolates were highly diverse, except for specific clonal strains from the same or different farms, whereas all bla CMY-2 -positive isolates showed different PFGE patterns. Therefore, clonal expansion and horizontal transmission within and between farms might contribute to the spread of ESBL/pAmpC-producing E. coli isolates. The proportion of subgroup D, which is considered pathogenic or an extraintestinal virulence-associated strain in our study (17.4%) was lower than Song et al. [27] (31%). The majority (82.6%) of ESBL/pAmpC-producing isolates in the current study mainly belonged to the commensal subgroups A or B1, which coincides with previous reports in Korea [27] and China [60]. Most of the pathogenic strains predominantly carried bla CMY-2 , suggesting the emergence of pathogenic strains of E. coli carrying quinolone resistance genes in the Korean poultry industry.
In conclusion, our study showed that healthy broiler chickens were a major reservoir of E. coli that are resistant to multiple antimicrobials, including those ranked as medically important. This study identified ESBL/pAmpC-producing E. coli strains carrying predominantly bla CTX-M-14 , bla CTX-M-55 , and bla CMY-2 genes. Notably, the majority of bla CMY-2 -carrying strains were pathogenic. This is the first report on the co-existence of mcr-1 and bla CMY-2 in pathogenic E. coli in Korea. Both horizontal and clonal spread could be implicated in the dissemination of ESBL/pAmpC-producing E. coli. However, the multilocus sequence types of the isolates remained unclear. Altogether, the results suggest that healthy chickens are a matter of concern in terms of transmission of ESBL/pAmpC-producing E. coli to humans through the food chain. Therefore, the prudent use of antimicrobials in food animals is needed to prevent the introduction of ESBL/pAmpC-producing isolates into the food chain. Additionally, long-term surveillance is needed to trace the evolution and dissemination of ESBL/pAmpC-producing E. coli in food animals and its possible association with human isolates.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-2607/8/9/1434/s1, Table S1: Lists of primer sequences and PCR conditions. Figure S1: Xbal-digested pulsed-field gel electrophoresis patterns of bla CTX-M and bla CMY-2 carrying E. coli strains isolated from healthy broiler chickens in Korea. Xbal macrorestriction analysis yielded no DNA banding patterns in five E. coli strain due to constant autodigestion of the genomic DNA during agarose plug preparation, and thus clusters formed by these strains were excluded (ND, not determined).