Molecular Characterization of Extended-Spectrum β-Lactamase-Producing Multidrug Resistant Escherichia coli From Swine in Northwest China

Objectives: The aim of the present study was to explore the prevalence and molecular characterization of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli collected from pig farms in Northwest China. Methods: Between May 2015 and June 2017, a total of 456 E. coli isolates were collected from fecal samples of healthy and diarrheal pigs in Northwest China to screen the ESBL producers. The β-lactamases, plasmid-mediated quinolone resistance (PMQR) genes and virulence genes among ESBL producers were corroborated by PCR and sequencing. Finally, ESBL producers were further grouped according to phylogenetic background and genetic relatedness. Results: Forty-four (9.6%) out of the 456 E. coli isolates were identified as ESBL-producing isolates. All ESBL producers exhibited multidrug resistance (MDR) phenotype, and more than 90% of the ESBL producers were resistant to amoxicillin, amoxicillin-clavulanic acid, oxytetracycline, enrofloxacin and sulfamethoxazole/trimethoprim. All ESBL producers harbored at least one type of β-lactamase, with blaCTX−M, blaTEM, blaSHV, blaOXA−48, and blaKPC−2 being detected in forty, thirty, seven, four, two and one isolates, respectively. Sequencing revealed the most common blaCTX−M subtype was blaCTX−M−14 (n = 24), followed by blaCTX−M−15 (n = 14), blaCTX−M−64 (n = 11), blaCTX−M−9 (n = 10) and blaCTX−M−123 (n = 9). qnrS (n = 23) was the predominant PMQR gene, and all PMQR genes were detected in co-existence with β-lactamase genes. estA (n = 18) and F4 (n = 18) were the most prevalent enterotoxin and fimbrial adhesin, respectively, and 27 different virotypes were found with respect to the association of enterotoxins and fimbrial adhesins. Twenty-four different sequence types (STs) were identified among 44 ESBL producers, and clones ST405, ST10 and ST648 were strongly present in more than one-third (34.1%) of ESBL producers. Conclusion: All ESBL-producing E. coli isolates exhibited MDR phenotype, and showed high prevalence of β-lactamase and PMQR genes. Especially, one isolate harbored ESBL genes blaTEM, blaSHV, blaCTX−M−9, blaCTX−M−14, blaCTX−M−64, and carbapenemase gene blaOXA−48 and blaKPC−2, as well as PMQR genes qnrS, qnrB, qnrD, qepA and aac(6')-Ib-cr.


INTRODUCTION
Escherichia coli (E. coli) is both a ubiquitous commensal bacterium in intestinal tract and an important pathogen of diarrhea or extraintestinal infections of humans and animals, and both commensal and pathogenic isolates usually share the same environment (Wu et al., 2013). Cephalosporins are effective for gram-negative bacterial infections, especially for infections caused by multidrug resistant (MDR) E. coli (Silva-Sanchez et al., 2013). At present, extended-spectrum β-lactams are not the first-line treatment in food animals, whereas the resistance to β-lactams, especially to the thirdand fourth-generation cephalosporins has increased markedly accompanying their massive or inappropriate use over the past decades, and it is also considered as an important public health challenge (Agersø and Aarestrup, 2013). Nowadays, one of the most worrisome resistance mechanisms to β-lactams is the emergence of extended-spectrum β-lactamases (ESBLs), which could inactivate oxyimino-β-lactams like third-generation cephalosporins and aztreonam (Liu et al., 2015). Moreover, ESBLs are generally located on the transmissible plasmids, and could be acquired between bacteria by conjugation mechanism (Cantas et al., 2015). A recent study has further suggested that ESBL-producing E. coli isolate, along with their antibiotic resistance genes, can spread from food animals and animals-derived foods to humans via food-chain (Geser et al., 2012). Additionally, plasmid-mediated AmpC β-lactamase bla CMY−2 , carbapenemases bla NDM−1 , bla OXA−48 and bla KPC−2 are also increasingly described (Conceição-Neto et al., 2017;Subirats et al., 2017). As a result, the dissemination of ESBL-producing isolates poses a serious risk to both animal and human health. Furthermore, ESBL producers have been associated with resistance to non-β-lactam antimicrobials, such as fluoroquinolones, aminoglycosides and sulfonamides, which are often used long term to treat and prevent diseases on pig farms in China (Tian et al., 2009(Tian et al., , 2012Yuan et al., 2009). Especially, plasmid-mediated quinolone resistance (PMQR) genes are thought to be linked with ESBL production, and spread of E. coli co-expressing PMQRs and ESBLs could contribute to growing concerns about resistant E. coli isolates .
The prevalence of ESBL-producing E. coli isolates in food animals has been increasing worldwide, and they pose a serious challenge in controlling bacterial diarrhea in swine industry. However, very little data have been reported on the occurrence and various types of β-lactamases among E. coli from swine in Northwest China. The main purpose of this study was to screen ESBL-producing E. coli isolates collected from pig farms in Northwest China, and further analyze ESBL producers based on genetic relatedness, virulence profiles, and the occurrence and transferability of β-lactamase and PMQR genes.

Sample Collection and Bacterial Culture
During May 2015 to June 2017, 456 E. coli isolates (270 from healthy pigs, 186 from diarrheal pigs) were isolated from fecal samples of different swine in ten pig farms, which are widely dispersed across Shaanxi and Gansu provinces. Fecal samples were collected from individual pigs using a sterile cotton swab and transported to laboratory within 12 h. All samples were immediately seeded on MacConkey agar (Beijing Land Bridge Technology Co., Ltd, Beijing, China). After incubation at 37 • C for 18 to 24 h, three colonies with typical E. coli morphology (bright pink with a dimple) were randomly selected and transferred to Eosin Methylene Blue agar (Qingdao Hope Bio Technology Co., Ltd, Qingdao, Shandong, China) for further purification. Finally, the suspect E. coli isolates on Eosin Methylene Blue agar (green colonies with a metallic sheen) were subjected to biochemical tests (indole, methyl red, oxidase, citrate, and triple sugar iron) as described previously (Liu et al., 2017). All confirmed E. coli isolates were stored at −80 • C in Tryptic Soy broth medium containing 30% glycerol for later study.

Antimicrobial Susceptibility Testing
The minimum inhibitory concentrations (MICs) of ampicillin, amoxicillin-clavulanic acid, ceftiofur, cefotaxime, ceftrizxone, ceftazidime, meropenem, enrofloxacin, ciprofloxacin, florfenicol, sulfamethoxazole/trimethoprim, gentamicin, amikacin, oxytetracycline, and colistin were determined by a standardized microdilution method following CLSI guidelines (CLSI, 2013). All MIC determinations were performed in triplicate, with E. coli ATCC 25922 serving as a quality control. Meanwhile, double-disk diffusion method was used to screen for the ESBL production among all isolates with cefotaxime and ceftazidime alone and in combination with clavulanic acid by using the guidelines recommended by CLSI (2013). Initial screening analyses indicated that 44 (9.6%) E. coli isolates were identified as phenotypic ESBL producers, which were further investigated for molecular characterization.

Conjugation Experiments
In order to analyze the horizontal transferability of β-lactamase and PMQR genes, especially bla OXA−48 gene, conjugation experiments were performed with eight ESBL-producing E. coli isolates, including four bla OXA−48 positive isolates, from different pig farms in seven different regions. Conjugation experiments were conducted by broth mating method using E. coli J53 AZ r as a recipient (Shaheen et al., 2011). Transconjugants were selected on Tryptic Soy agar plates containing sodium azide (150 µg/ml) and cefotaxime (2 µg/ml). All transconjugants, recipient and donors were subjected to antimicrobial susceptibility testing. PCR and sequencing were performed to verify the transferability of PMQR and β-lactamase genes.

Multilocus Sequence Typing (MLST)
MLST of ESBL-producing E. coli isolates was performed as described previously (Wirth et al., 2006). A detailed scheme of gene amplification, allelic type and sequence type assignment methods is available on the MLST website (http://mlst.warwick. ac.uk/mlst/dbs/Ecoli).

Statistical Analysis
Significance was determined by Pearson's Chi-squared test with Yates continuity correction using "R" software (version 3.0.1), and the level of significance was set at P < 0.05.

Conjugation Experiments
Five out of eight ESBL producers successfully transferred the β-lactamase genes to recipient strain E. coli J53 AZ r . PCR analysis showed that the presence of respective β-lactamase genes, including one bla OXA−48 -carrying plasmids from all transconjugants. Accordingly, PMQR genes qnr and aac-(6 ′ )-Ib-cr were co-transferred with β-lactamase genes ( Table 3).
Antimicrobial susceptibility patterns showed that all donors and their transconjugants were resistant to amoxicillin-clavulanic acid, ampicillin, ceftiofur, cefotaxime, and all transconjugants exhibited an increase of at least 8-fold in MICs compared to the recipient, E. coli J53 AZ r . The enrofloxacin MICs for four transconjugants harboring aac-(6 ′ )-Ib-cr ranged from 0.125 to 0.5 mg/L, representing an increase of 4-fold to 8-fold compared with the recipient (Table 3). Additionally, the transconjugants remained susceptible to meropenem, enrofloxacin, florfenicol, oxytetracycline, gentamicin, sulfamethoxazole-trimethoprim and colistin, whereas one bla OXA−48 positive transconjugant reduced meropenem susceptibility.

DISCUSSION
The prevalence of ESBL-producing E. coli isolates in food animals has been increasing worldwide (Liebana et al., 2013). In China, diarrhea caused by pathogenic E. coli, especially ESBL-producing E. coli poses a serious threat to the swine industry and public health (Lei et al., 2010;Xu et al., 2015). The present study is the first contribution to explore the detailed characterizations of ESBL-producing E. coli isolates from pigs in Northwest China. Forty-four (9.6%) isolates were confirmed as ESBL producers, while it is noteworthy that 456 E. coli in this study isolated from feces of healthy and diarrheal pigs, and the prevalence of ESBL producer were significantly higher among isolates from diarrheal pigs than that form healthy isolates (20.4vs. 2.2%; P < 0.001).
The detectable rate of ESBL producer in diarrheal pigs was similar with the result in Sichuan (26.8%), a neighbor province of Shaanxi, while it was significantly lower than in Heilongjiang (43.2%), a province in the northeast China (Tian et al., 2009;Xu et al., 2015). Moreover, our results showed that ESBL producers mainly belonged to phylogenetic groups D and B2, and to a lesser extent to phylogenetic A, while the previous studies showed that E. coli from pigs or duck in China also mainly fell into phylogenetic groups A (Wang et al., 2010;Ma et al., 2012). It is further confirmed that the emergence of ESBL-producing E. coli has a geographic variation with respect to demographic, environmental, behavioral, socioeconomic and infectious risk factors with the extending of ESBL-producing isolates stage by stage.
All 456 E. coli isolates in this study were tested for their susceptibility to 15 antimicrobial agents. Overall, the number of resistant isolates in ESBL producers and isolates from diarrheal pigs were higher than that from healthy pigs (P > 0.001). It is suggested that the isolates from diarrheal pigs may be more likely to develop antibiotic resistance than that from healthy pigs because of the frequent use of antimicrobials in preventing and treating diarrhea. All ESBL producers were resistant to at least five antimicrobial agents, and vast majority of them (>93%) remained susceptible to meropenem and colistin, which are considered the effective candidates for treatment of serious infections caused by E. coli in pig farms of China. According to the virotypes, 86.4% of ESBL producers carried both enterotoxins and fimbrial adhesins. It is indicated that these isolates should be enterotoxigenic E. coli (ETEC), which are responsible for neonatal diarrhea and postweaning diarrhea in piglets. F4 fimbrial adhesin was present 40.9% of the ESBL producers, it is consistent with the previous studies that F4 adhesin gene is one of the most frequently found genes in E. coli isolates from suckling and weaning piglets (Vu Khac et al., 2006;Zhang et al., 2007). Furthermore, the gene combinations of F4+estA/estB were present in 34.1% of the isolates.
Since the early 2000s, CTX-M-type ESBLs have been increasingly reported, and they have now replaced TEM and SHV as the most common type of ESBL (Barguigua et al., 2011). The most predominant ESBL gene in this study was bla CTX−M (90.9%), and the similar findings showed that CTX-Ms accounted for 87.1% of ESBL-producing E. coli isolated from food animals based on a previous survey in China (Rao et al., 2014). bla CTX−M−14 remained the most common genotype, and followed by bla CTX−M−15 . It is surprising that no isolate contained bla CTX−M−1 , whereas it was detected in the ESBLproducing E. coli from dogs, retail pork and water bodies in Shaanxi province (Xi et al., 2015;Liu et al., 2016b). In regards to the linkage of phylogenetic group and β-lactamases, isolates of group D harbored more β-lactamases genes, and isolates of group A harbored less β-lactamases. Novel hybrid βlactamase gene bla CTX−M−123 was firstly discovered in E. coli from pig feces in China in 2013 (He et al., 2013), and it was detected in nine ESBL producers in this study. Moreover, four ESBL producers were commensal isolates from healthy pigs, it was further indicated that some commensal organisms in animals have acquired β-lactamase genes with the increasing use of β-lactams in animals. bla OXA−48 was detected in four ESBL producers from diarrheal pigs. As a globally emerging carbapenemase gene, bla OXA−48 could hydrolyze carbapenems and β-lactamase inhibitors but has no activity toward broadspectrum cephalosporins (Mathers et al., 2013). bla OXA−48 was firstly discovered in E. coli from dogs in Germany in 2013, and afterward it was reported in E. coli from companion animals in the United States in 2016 (Stolle et al., 2013;Liu et al., 2016a). In 2017, it was reported in pigs in Italy (Pulss et al., 2017). Most recently, it was detected in Enterobacteriaceae from river water in Algeria (Tafoukt et al., 2017). Considering the importance of bla OXA−48 gene in public health, it is necessary to further investigate the dissemination of bla OXA−48 producing E. coli isolates among different sources.
PMQR genes were often found to be strongly associated with β-lactamase genes and even in the same plasmid, and they are not merely able to confer resistance against quinolones but also often related to ESBLs (Jeong et al., 2011). In this study, PMQR genes were present in 88.6% of ESBL producers, and the similar findings have been reported in ESBL-producing E. coli isolates form pigs in previous studies in China by Xu et al. (87.4%) and Liu et al (83.8%) Xu et al., 2015). Thirtyseven ESBL producers (84.1%) harbored at least one qnr gene, and qnrS was the predominant, whereas a low prevalence of qnr genes was detected among ESBL-producing E. coli isolates in France and Canada (1.6 and 1%, respectively). In addition, qepA gene was detected in combination with other PMQR and β-lactamase genes in four isolates (10%). The frequent combination of β-lactamases and PMQRs in this study further supported the previous studies that coproduction of β-lactamase and PMQR genes could contribute to the dissemination of MDR isolates, and also reflect the fact that genes encoding resistance to β-lactams and quinolones are located on the same plasmid.
Twenty-four different sequence types were identified, and three sequence types (ST405, ST10, and ST648) accounted for 34.1% of the ESBL producers. Sequence types ST10, ST38, ST131, ST648, and ST405 clones were documented in different sources according to MLST databases, and could favor the dissemination of CTX-M worldwide among E. coli isolates (Hernandez and Gonzalez-Acuna, 2016). In the present study, a few isolates belonging to different STs shared similar β-lactamase and PMQR gene profiles, whereas several isolates belonging to same ST exhibited different gene profiles. The similar results were observed among E. coli isolates from dogs and cats in previous studies (Liu et al., 2016a,b). The possible explanation is that the pig trade, personnel exchanges and water sources among adjacent regions may lead to the dissemination of isolates with same gene profiles or same ST types. Anyway, deeper analyses for such isolates are necessary in the future. It is noteworthy that bla OXA−48 gene were detected in four isolates with reduced susceptibility or resistance to meropenem. The bla OXA−48 positive isolates co-harbored variants of β-lactamase genes, and they also were associated with sequence types ST38, ST405, and ST131. Additionally, bla OXA−48 positive E. coli clone ST38 had been previously reported in France, Germany and Algeria (Poirel et al., 2011;Kaase et al., 2016;Bouaziz et al., 2017). In the current study, we firstly reported the occurrence of bla OXA−48 positive E. coli clone ST38 from a sucking piglet with diarrhea in Shaanxi. Clone ST38 has achieved notoriety as it is now rapidly and globally disseminated, and its potential to serve as a vehicle for spread of carbapenemases is profoundly alarming. bla NDM−1 producing E. coli isolate, emerging as a public health threat, has gained global attention as it could hydrolyze almost all β-lactams with the exception of aztreonam (Nordmann et al., 2012), and it has previously been detected in E. coli isolates from pigs (Fischer et al., 2012). Our results revealed that bla NDM−1 and other β-lactamase genes coexisted in one isolates, it is a potential public health concern as the pig carrying bla NDM−1 and other β-lactamase genes may enter the food chain.

CONCLUSION
In conclusion, all ESBL-producing E. coli isolates both from healthy and diarrheal pigs in Northwest China exhibited MDR phenotype. The bla CTX−M−14 and qnrS were the predominant β-lactamase gene and PMQR gene in ESBL producers, respectively. estA and F4 were the most prevalent enterotoxin and fimbrial adhesin, respectively. One ST131 isolate harbored β-lactamase genes bla TEM , bla SHV , bla CTX−M−9 , bla CTX−M−14 , bla CTX−M−64, and carbapenemase genes bla OXA−48 and bla KPC−2 , as well as PMQR genes qnrS, qnrB, qnrD, qepA and aac(6')-Ib-cr. The findings could provide useful information for a national monitoring of antimicrobial resistance in bacteria from food animals in China.

AUTHOR CONTRIBUTIONS
XL conceived and designed the experiments. HL and LW designed the experiment and drafted the manuscript. XL, HL, LW, HZ, and QP performed the experiments. XL, YL, and QL analyzed and explained the data for the work. All authors critically revised and approved the final manuscript.