Molecular Characterization of Plasmids Encoding CTX-M β-Lactamases and their Associated Addiction Systems Circulating Among Escherichia coli from Retail Chickens, Chicken Farms, and Slaughterhouses in Korea

Abstract

Extended-spectrum β-lactamases (ESBLs), particularly those of the CTX-M types, are the predominant resistance determinants of Escherichia coli that are rapidly spreading worldwide. To determine CTX-M types, E. coli isolates were collected from retail chickens (n = 390) and environmental samples from chicken farms (n = 32) and slaughterhouses (n = 67) in Korea. Fifteen strains harboring bla_CTX-M genes were isolated from 358 E. coli isolates. The most common CTX-M type was eight of CTX-M-15, followed by six of CTX-M-1 and one of CTX-M-14. The bla_CTX-M genes were identified in the isolates from retail chickens (n = 9), followed by feces, water pipes, floors, and walls. Conjugations confirmed the transferability of the plasmids carrying bla_CTX-M genes to the recipient E. coli J53 strain. Furthermore, eight addiction systems carried by the replicons in CTX-M types were confirmed. The dominant system was identified as ccdAB, vagCD, and pndAC in donor strains and transconjugants. The clonal relationship between the two strains carrying bla_CTX-M genes indicates that E. coli may transmit from the farm to retail chickens, suggesting a possible public health risk. Our findings demonstrate that the detection of CTX-M types in E. coli isolates is important for tracking ESBL production in animals, and suggest linkage of multiple addiction systems in plasmids bearing bla_CTX-M genes.

Introduction

Extended-spectrum β-lactamases (ESBLs) are among the most important of acquired resistance determinants disseminated in the Enterobacteriaceae [3,8]. ESBLs confer resistance to oxyimino-cephalosporins, which severely limits treatment options for a variety of illnesses, including urinary tract and gastrointestinal infections [8,29]. Early ESBLs evolved from TEM- and SHV-type β-lactamases in Klebsiella pneumoniae, [20] whereas CTX-M-type ESBLs that are frequently associated with Escherichia coli have rapidly increased in the past decade [4,17,25]. The CTX-M family can be clustered into five groups (CTX-M-1, -2, -8, -9, and -25) based on their amino acid sequence similarities [29]. Recent studies have identified CTX-M-1 and CTX-M-9 family groups in Enterobacteriaceae from Korea [7,12,33]. In particular, the CTX-M family in E. coli is widespread worldwide and poses a major health concern in both communities and hospitals [25].

Currently, a genetic survey is being performed nearly worldwide to identify the dissemination of CTX-M β-lactamases [4,17,25]. Some studies have shown a relationship between blaCTX-M genes on conjugative plasmids and bacterial clones. Additional reports have suggested that such plasmids exploit the plasmid addiction system, which tracks toxin-antitoxin gene pairs. The addiction system may contribute to killing plasmid-free cells, allowing for the dissemination of intra- and inter-species plasmids [11,13]. Researchers have revealed that E. coli plasmids bearing blaCTX-M genes may contribute to the development of multiple addiction systems in their host [21]. In support of these findings, studies in European countries have demonstrated a high incidence of blaCTX-M genes in E. coli strains among healthy animals [1,14]. Furthermore, a Korean study has shown that E. coli clones with a high frequency of virulence determinants and addiction systems influence the prevalence of CTX-M β-lactamases among E. coli from cattle, farm workers, and the farm environment [30].

Although studies have identified the association of addiction systems and blaCTX-M genes in E. coli among humans, it is necessary for researchers to perform such studies focused on animals, especially for animals that serve as a food source for human consumption. Therefore, the aim of this study was to characterize CTX-M β-lactamases in E. coli isolated from retail chickens, chicken farms, and slaughterhouses in Seoul, Gyeonggi-do and Jeollabuk-do, Korea and to investigate the association between E. coli CTX-M β-lactamases and their plasmid addiction systems.

 

Materials and Methods

Bacterial Strains

A total of 489 samples were obtained from Seoul, Gyeonggi-do and Jeollabuk-do, Korea in January, February, and July 2013, which included retail chickens (n = 390) and environmental samples from chicken farms (feces, feed, water pipe, and wall) (n = 32) and slaughterhouses (production line, floor, and guts) (n = 67). Retail chicken samples from 19 traditional markets and swab samples were collected from two farms in Gyeonggi-do and two farms in Jeollabuk-do. For E. coli isolation, chicken samples (25 g) were pre-enriched in 225 ml of buffered peptone water for 18-24 h at 37℃. In order to assay for bacterial growth, the environmental samples were streaked onto Eosin Methylene Blue agar (BBL; Becton Dickinson Micro System, Cockeysville, MD, USA) and incubated at 37℃ for 16-18 h. Based on colony morphology and color, the bacterial strains were identified by using the Vitek 2 microbial identification system (bioMérieux Vitek, Hazelwood, MO, USA).

Antimicrobial Susceptibility Testing

The antimicrobial susceptibility was determined by using the disc diffusion method, in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines [6]. The antimicrobial agents (BBL) and their corresponding concentrations were as follows: ampicillin (AM, 10 μg), piperacillin (PIP, 100 μg), amoxicillin-clavulanic acid (AmC, 20/10 μg), cephalothin (CF, 30 μg), cefazolin (CZ, 30 μg), cefoxitin (FOX, 30 μg), cefamandole (MA, 30 μg), cefotaxime (CTX, 30 μg), ceftazidime (CAZ, 30 μg), cefepime (FEP, 30 μg), imipenem (IPM, 10 μg), amikacin (AN, 30 μg), aztreonam (ATM, 30 μg), streptomycin (S, 25 μg), gentamicin (GM, 10 μg), ciprofloxacin (CIP, 30 μg), nalidixic acid (NA, 30 μg), trimethoprim-sulfamethoxazole (SXT, 1.25/23.75 μg), chloramphenicol (C, 30 μg), and tetracycline (TE, 30 μg). The double-disc synergy test was performed in order to confirm CTX-M-type isolates. The minimum inhibitory concentrations (MICs) of selected antimicrobials (CTX and CAZ) were determined using E-test strips (AB BIODISK; Solna, Sweden). E. coli strain ATCC 25922 was used as a quality control for the susceptibility test.

Identification of blaCTX-M Genes

The blaCTX-M gene was detected by polymerase chain reaction (PCR) using primers as previously described by Tofteland et al. [36]. To confirm the presence of blaCTX-M β-lactamases, the sequences of the CTX-M-1, 8, 9, and 10 families were used for specific grouping. CTX-M-type families were detected by the PCR method. In order to subgroup the CTX-M isolates identified, sequence analysis was performed by Macrogen Inc. (Seoul, Korea). The sequences were confirmed to those in the GenBank nucleotide database using the Basic Local Alignment Search Tool (BLAST) program available through the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/BLAST).

Conjugations

The transferability of blaCTX-M genes in E. coli isolates to E. coli J53 by conjugation was determined by broth-mating experiments at 37℃, as described in Tamang et al. [31]. Transconjugants were selected on MacConkey agar plates containing sodium azide (100 μg/l; Sigma) and cefotaxime (2 μg/ml). All the transconjugants were tested for antimicrobial susceptibility and the presence of β-lactamase genes, as described above.

PCR-Based Replicon Typing of Plasmids

Plasmid DNAs were isolated from transconjugants using a miniprep plasmid kit in accordance with the manufacturer’s instructions (Life Technologies, USA). Plasmid replicon typing of E. coli strains carrying the CTX-M-type genes was conducted by PCR using 18 primer pairs (HI1, HI2, I1-Iγ, L/M, FIA, FIB, FIC, F, N, P, W, T, A/C, K, B/O, X, Y, and FII) [5]. Positive PCR products matching the amplified replicon sizes were sequenced and the sequences were confirmed by BLAST search [5].

Detection of Addiction Systems

For the donor strains and transconjugants bearing blaCTX-M genes, the following eight plasmid addiction systems were detected and confirmed by PCR: PemK-PemI (pemKI) (plasmid emergency maintenance), CcdA-CcdB (ccdAB) (coupled to cell division), RelB-RelE (relBE) (relaxed control of stable RNA synthesis), ParD-ParE (parDE) (DNA replication), VagC-VagD (vagCD) (virulence-associated protein), Hok-Sok (hok-sok) (host killing), PndA-PndC (pndAC) (promotion of nucleic acid degradation), and SrnB-SrnC (srnBC) (stable RNA) [20].

Analysis of Genetic Relatedness by Pulse-Field Gel Electrophoresis (PFGE)

The clonality of the CTX-M-producing E. coli isolates was determined by performing PFGE using XbaI-digested (enzyme was purchased from Takara Bio Inc., Shiga, Japan) genomic DNA. The DNA fragments were separated on a 1.4% agarose gel (pulsed-field certified agarose (Bio-Rad)) in accordance with the Centers for Disease Control and Prevention PulseNet standardized protocol [9]. Electrophoresis was performed using 0.5× Tris-borated-EDTA buffer under the following conditions: gradient, 6 V/cm; run time, 18 h; pulse times, 2.2-54.2sec; temperature, 14℃; and angle, 120°. In order to identify the similarities between CTX-M types, restriction fragment length polymorphism analysis was performed and results were analyzed using InfoQuest FP software ver. 4.5 (Bio-Rad) to construct a dendrogram. For comparison, a cluster analysis was performed using the Pearson correlation similarity index. The relationship was calculated by the unweighted-pair group method using average linkages.

 

Results

Antimicrobial Resistance

In total, 358 E. coli strains were isolated from retail chicken (n = 302) and environmental samples from Korean chicken farms (n = 20) and slaughterhouses (n = 36). Among the strains, 15 CTX-M-producing E. coli isolates were identified. All of the blaCTX-M-positive isolates were resistant to AM, CF, CZ, MA, and CTX, whereas 93.3%, 93.3%, 53.3%, and 46.7% were resistant to FEP, ATM, CAZ, and IPM, respectively. Moreover, some of blaCTX-M-positive strains were resistant to other classes of antibiotics, including PIP (100%), NA (86.7%), CIP and TE (80%), S (60%), and C and SXT (46.7%). However, all the strains were susceptible to AN, AmC, and FOX.

Detection of blaCTX-M Genes

A total of 15 E. coli isolates carrying blaCTX-M genes were determined. The predominant CTX-M type identified was CTX-M-15 (n = 8), followed by CTX-M-1 (n = 6) and CTX-M-14 (n = 1). Table 1 shows the prevalence of the 15 isolates carrying blaCTX-M genes from the sources in retail chickens, chicken farm, and slaughterhouses. The blaCTX-M genes were identified predominantly in E. coli isolates from retail chickens (n = 9), followed by feces (n = 2) and water pipes (n = 2), floor (n = 1), and wall (n = 1).

Table 1.aStage of processing conveyer belt.

Transferability of blaCTX-M Genes

The transfer of blaCTX-M gene by conjugation was confirmed in the recipient strain E. coli J53 from 15 blaCTX-M gene-positive isolates. PCR analysis verified that the respective blaCTX-M genes were present in all the transconjugants. Table 2 shows the characteristics of the conjugation-positive E. coli isolates carrying blaCTX-M genes.

Table 2.aEtest, Epsilometer test; NT, not typeable by PCR-based replicon typing; CT, cefotaxime; TZ, ceftazidime. bGG, Gyeonggi-do; JB, Jeollabuk-do; BC, Bucheon-si; GR, Guri-si; GY, Goyang-si; HS, Hwaseong-si; IS, Iksan-si; UJ, Uijeongbu-si; YJ, Yangju-si.

Analysis of the blaCTX-M Plasmids

Plasmids from blaCTX-M-positive E. coli J53 transconjugants revealed a conjugative plasmid. Four of 18 plasmid replicon types, including Inc I1-Iγ, Inc FIA, IncF, and IncY, were present either alone or in combination. All the plasmids expressing blaCTX-M-1, -15 genes were either positive within two replicon types or were non-typeable, whereas plasmids harboring the blaCTX-M-14 gene showed three replicon types, Y, FIA, and F (Table 2). The predominant replicon type was confirmed as F (66.7%). However, only one strain carrying blaCTX-M-1 was determined to express I1-Iγ without the F replicon type. Among the 15 conjugation-positive plasmids, four were non-typeable for the 18 incompatibility groups. All of them harbored CTX-M-1 and CTX-M-15, except for one harboring CTX-M-14.

Distribution of Addiction Systems

Upon further analysis of the E. coli strains, eight kinds of addiction systems were identified: pemKI, ccdAB, relBE, parDE, vagCD, hok-sok, pndAC, and srnBC (Table 3). In total, 85 plasmid addiction systems were confirmed in the 15 CTX-M-producing E. coli donor strains. However, 30 addiction systems were detected among the transconjugants. Among the donor strains, the mean number of addiction systems was highest in CTX-M-15-producing E. coli isolates (5.4), followed by those producing CTX-M-1 (4.6) and CTX-M-14 (0.7). Although eight addiction systems were detected in donor strains, four systems (ccdAB, parDE, vagCD, and pndAC) were identified in the transconjugants. vagCD was the most common addiction system in both donor strains and transconjugants. In contrast to the donor strains, none of the addiction systems was detected in transconjugants expressing the blaCTX-M-14 gene. The mean number of addiction systems for both blaCTX-M-1- and blaCTX-M-15-positive transconjugants was 1.9, none of which harbored pemKI, relBE, hok-sok, or srnBC.

Table 3.Donor strain and transconjugant numbers of addiction systems in accordance with blaCTX-M genes detected in E. coli isolates.

PFGE Analysis

To determine the clonality of the blaCTX-M-positive E. coli isolates, molecular typing analysis was carried out by using XbaI-digested genomic DNA isolated from the original samples obtained from the following sites: 19 traditional markets, two chicken farms, and two slaughterhouses (each from Gyeonggi-do and Jeollabuk-do in Korea). PFGE patterns were analyzed for the 15 E. coli strains carrying blaCTX-M-15 (n = 8 ), blaCTX-M-1 (n = 6), and blaCTX-M-14 (n = 1) genes (Fig. 1). The results revealed that 13 of 15 isolates were not genetically related, whereas the remaining two strains (13-ECO-VE-338 and 13-ECO-VE-339) isolated from the floor and chicken feces in the same farm showed high similarity to 90%. Furthermore, these two strains showed the same characteristics in CTX-M type and antimicrobial resistance pattern.

Fig. 1.Dendrogram showing PFGE analysis of XbaI-digested DNA from the 15 CTX-M-producing E. coli strains isolated from retail chickens, chicken farms, and slaughterhouses in Korea.

 

Discussion

In this study, 15/358 (4.19%) CTX-M-producing E. coli strains were isolated from retail chickens, chicken farms, and slaughterhouses. This percentage is higher than that shown in a previous study focused on CTX-M-type-producing E. coli isolated from chicken fecal samples, which revealed that 3.6% and 6.7% of single and pooled samples were CTX-M positive, respectively [26].

Among the strains identified in the current study, 14 (3.91%) belonged to the CTX-M-1 cluster and one (0.28%) belonged to the CTX-M-9 cluster [23]. ESBL-producing E. coli strains harboring blaCTX-M genes of CTX-M-1 or CTX-M-9 clusters were previously detected among food animals in China and Europe [2,18,35]. Moreover, a previous study in Korea demonstrated the prevalence of ESBL-producing E. coli among farm workers, where there was a rapid increase in human clinical E. coli isolates of the CTX-M-1 or CTX-M-9 clusters [32].

In this study, blaCTX-M-15 was the predominant gene, followed by blaCTX-M-1 and blaCTX-M-14. The blaCTX-M genes characterized in the samples obtained from healthy chickens, chicken farms, and slaughterhouses were previously observed in animals in Korea [16,31,32]. A previous study reported the detection of CTX-M-1 in E. coli isolated from food-producing poultry in France, which was consistent with our results [10]. Taken together, these results showed that diverse CTX-M types in food-producing animals are present in Korea.

All the strains bearing blaCTX-M genes were transferred to E. coli recipients by conjugation in this study. Plasmid replicon typing of the transconjugants revealed plasmid diversity, which was similar to blaCTX-M plasmids previously described in the United Kingdom [8]. In the current study, all replicon types demonstrated that IncF plasmids (4/8; 50%) were the most common among the blaCTX-M-15-carrying plasmids. In agreement with a previous report [13], our results showed that the blaCTX-M-14 gene was carried on IncY, IncF, and IncFIA plasmids. Moreover, IncF and IncI1-Iγ were the predominant replicon types in the plasmids carrying the blaCTX-M-1 gene. It means that the spread of blaCTX-M genes were associated with IncF plasmids [20]. Therefore, these findings suggest that all the replicon types in the strains isolated from chickens were similar to the genetic structure of the plasmids.

Plasmid addiction systems can contribute to the stability and maintenance of plasmids in their hosts and may affect their distribution in the absence of antibiotic selection [8,11,24,32]. In this study, the fact that the donor strains carried blaCTX-M genes confirmed the association betweenthe eight addiction systems and the IncF plasmid replicon type. A previous study has shown that the IncF plasmid replicon type has developed the ability to diffuse in E. coli, but its adherence in E. coli populations may contribute to the presence of addiction systems [20]. In addition, four addiction systems in the transconjugants would imply that addiction systems might be located on a non-conjugative plasmid or in the chromosome, suggesting that those located on conjugative plasmids harbor resistance markers [32]. Furthermore, among the transconjugants, the ccdAB, vagCD, and parDE systems were associated with the IncF, IncFIA, and IncFIB replicon types, but the pndAC system was associated with the IncI1-Iγ replicon type (data not shown here). Mnif et al. [21] reported the relation between the vagCD system and the IncFIA and IncFIB replicon types. Taken together, these findings suggest that there is a combination of specific plasmids with addiction systems.

PFGE analysis showed that most of the blaCTX-M-positive isolates were highly diverse, with the exception of specific clonal strains. Two E. coli isolates (13-ECO-VE-338 and 13-ECO-VE-339) were genetically homogeneous, indicating the possibility that such similar strains may contribute to clonal expansion and horizontal transmission. Moreover, the CTX-M-15-producing E. coli isolate obtained from a chicken farm floor sample was related to an isolate from chicken feces (Fig. 1). These findings suggest that it is possible to transfer the E. coli isolates from the chicken farms environment to the chickens themselves, which represents a potential public health threat for human consumption of retail chickens.

In conclusion, this study has reported the identification and characterization of CTX-M β-lactamases and their associated addiction systems. Further analysis illustrated that the detection of CTX-M-producing E. coli is not only associated with the spread of a single clone but also is affiliated with various IncF replicon-type plasmids harboring multiple addiction systems. Our findings suggest that the linkage between addiction systems and plasmids carrying blaCTX-M genes may contribute to the stability of their plasmids in host cells. It means that the spread of E. coli harboring CTX-M via foodborne transmission or environmental pathways can affect human health. Therefore, further analysis is required to assess the risk posed by these microorganisms to human health, with respect to a food safety perspective.