Prevalence and characteristics of extended-spectrum β-lactamase genes in Escherichia coli isolated from piglets with post-weaning diarrhea in Heilongjiang province, China

Objectives: The purpose of this study was to investigate the prevalence of extended spectrum β-lactamase (ESBL) genes in Escherichia coli isolated from post-weaning diarrhea (PWD) piglets in Heilongjiang province, China. Methods: Of 458 E. coli isolated from 589 fecal samples from PWD piglets, a total of 198 isolates were confirmed as ESBL producers by the double-disk synergy test (DDST). Polymerase chain reaction (PCR) and sequencing were performed to identify genes for ESBL, plasmid-mediated quinolone resistance (PMQR), and integrons. Results: Of the 198 isolates, blaCTX−M and blaTEM were detected in 191 and 149 isolates, respectively. Sequencing revealed that 10 blaCTX−M subtypes were detected, and blaCTX−M−14 was the most prevalent, followed by blaCTX−M−55 and blaCTX−M−65. Of the 149 TEM-positive strains, four were blaTEM−52 and the rest were blaTEM−1. Among the 198 ESBL-positive isolates, 173 isolates were found to harbor at least one PMQR gene, with oqxAB, qnrS, qnrB, qepA, and aac(6′)-Ib-cr being detected alone or in combination in 125, 114, 26, 24, and 45 strains, respectively. One hundred and fifty-five ESBL-positive isolates were also positive for class I integron (int1), and eight different gene cassette arrays were confirmed in 110 isolates by restriction fragment length polymorphism (RFLP) and DNA sequencing analyses, with predominance of dfrA17-aadA5, dfrA12-orfF-aadA2, and dfrA1-aadA1 arrays. Conclusion: To the best of our knowledge, this is the first report of the blaTEM−52 gene in pig E. coli isolates in China and this is also the first description of the coexistence of the qnrB, qnrS, aac(6′)-Ib-cr, qepA, and oqxAB genes in one E. coli strain.


INTRODUCTION
Colibacillosis, caused by pathogenic Escherichia coli, is one of the most significant diseases in the swine industry. Diarrhea is the most common symptom of colibacillosis in pig livestock, and it is classified into two types: neonatal diarrhea (ND), which occurs in sucking piglets, and post-weaning diarrhea (PWD), which usually occurs within 2 weeks of weaning (Luppi et al., 2015). In general, ND and PWD infections can be prevented effectively by passive colostral and lactogenic immunity obtained by vaccination of the sow (Truszczynski and Pejsak, 2014). When immunity treatment fails, the antimicrobials such as cephalosporins, fluroquinolones, and co-trimoxazole are one of effective measures for the treatment of diarrhea. Accompanying the wide use of these antimicrobials in treating piglets diarrhea over the past decades, resistant isolates, especially multidrugresistant isolates have been observed (Seiffert et al., 2013). The emergence and spread of antimicrobial resistance poses a serious challenge in controlling diarrhea in swine production.
The major mechanisms conferring resistance to cephalosporins rely on the production of extended-spectrum β-lactamases (ESBLs; Perez et al., 2007). ESBLs are a group of enzymes mediating resistance to most β-lactams approved in human and veterinary medicine, including extended-spectrum cephalosporins and monobactams but excluding carbapenems and cephamycins. These enzymes are now widely distributed worldwide in Gram-negative bacteria, which mainly exist in Enterobacteriaceae, especially in E. coli. A large surveillance of antimicrobial resistance has shown that the detection rate of ESBLs has increased significantly not only in human clinical E. coli isolates (Jones et al., 2013), but also in E. coli isolates from food-producing animals and food products, which has been raising concern about their possible transmission through the food chain. ESBLs are categorized into three types, which are TEM, SHV, and CTX-M, while the CTX-M β-lactamases have been divided into five groups known as CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25 which have become the most prevalent in the world (Bonnet, 2004). ESBL-encoding genes are usually located on plasmids which are highly mobile and can harbor resistance genes to several other unrelated classes of antimicrobials, such as fluroquinolones, and co-trimoxazole (Wang et al., 2014).
The emergence of plasmid-mediated quinolone resistance (PMQR) has been reported since 1998, indicating that quinolone resistance can also be acquired through horizontal gene transfer (Strahilevitz et al., 2009). To date, four known PMQR determinants have been identified include Qnr proteins, aminoglycoside acetyltransferase AAC(6 ′ )-Ib-cr, and the quinolone efflux pumps proteins QepA and OqxAB (Ruiz et al., 2012). Although the PMQR determine relatively small increases in the MICs of quinolones, these changes are sufficient to facilitate the selection of mutants with higher levels of resistance. Integrons, which are capable of capturing, excising and expressing genes cassettes that encode determinants of antimicrobial-resistance, play important roles in the horizontal dissemination of antibiotic resistance genes in bacteria (Cambray et al., 2010). Class 1 integrons were widespread in ESBL-producing isolates and play an important role in multi-drug resistance . As a matter of growing concern, PMQR genes and integrons have often been found to co-exist on the same plasmid with genes encoding ESBLs and to be co-transferred to recipients. Food animals colonized with ESBL-positive E. coli have been considered as potential sources of resistant E. coli causing infection in the community. E. coli antimicrobial drug resistance is strongly related to phylogenetic grouping (Barguigua et al., 2013). It has been observed that E. coli strains fall into four main phylogenetic groups (A, B1, B2, and D). Virulent extraintestinal strains belong mainly to group B2 and, to a less extent, to group D, whereas most commensal strains belong to group A and B1.
In the past few years, a rapid emergence and dissemination of ESBL-positive E. coli isolates have been increasingly reported in food animals in different countries and have gained considerable attention worldwide (Smet et al., 2010). Although there are many studies describing the molecular characteristics of ESBL genes among healthy food-producing animals, but limited data are available for the distribution and characteristics of ESBL genes in E. coli isolated from diseased animals (Liu et al., 2013a). Therefore, the main purpose of this study was to investigate the prevalence of ESBL genes in E. coli isolates collected from PWD piglets in the Heilongjiang province during 2010-2013 and assess the relatedness of PMQR genes and integrons with ESBL genes within the same strain.

Bacterial Isolates
From May 2010 to August 2013, a total of 589 fecal samples from piglets (<2 months) with PWD were collected from 21 pig farms located in different geographic areas of Heilongjiang province, in the Northeastern China (Table 1). These fecal samples were collected from individual piglets using a sterile swab that was placed into an Eppendorf tube and transported to the laboratory within 12 h. The samples brought to the laboratory were immediately inoculated on MacConkey agar, and five randomly selected colonies with typical E. coli morphology were selected from each sample. The bacterial strains were identified using classing biochemical methods and confirmed as E. coli using the API-20E Bacterial Identification System (bioMérieux, France). All confirmed E. coli isolates were stored in Luria-Bertani (LB) broth, containing 20% glycerol at −80 • C for further studies.

Antimicrobial Susceptibility Testing and ESBL Determination
The minimum inhibitory concentrations (MICs) of ampicillin, ceftiofur, cefotaxime, amikacin, gentamicin, tetracycline, chloramphenicol, florfenicol, nalidixic acid, ciprofloxacin, enrofloxacin, trimethoprim/sulfamethoxazole, and colistin were determined by the agar dilution method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI; Clinical Laboratory Standards Institute, 2012). The antimicrobial concentrations were from 0.06 to 256 µg/ml. E. coli ATCC25922 was used as the quality control strain. All E. coli isolates were screened for the production of ESBLs by the double-disk synergy test (DDST) using both cefotaxime and ceftazidime, alone and in combination with clavulanic acid as recommended by the CLSI. The DDST was considered positive when the inhibition zone produced by the combined effects of either ceftazidime or cefotaxime plus clavulanic acid was ≥5 mm larger than that produced by either ceftazidime or cefotaxime alone.

Integrons Analysis and Characterization of Inserted Gene Cassettes
PCR was performed to check the presence of class 1, 2, and 3 integrons in all 198 ESBL-positive E. coli isolates using primers specific for the integron integrase genes intI1, intI2, and intI3 listed in Table 2 (Dillon et al., 2005). Strains that was positive for intI1 gene were subsequently subjected to PCR for amplification of the variable region of class 1 integrons using primer listed in Table 2 (White et al., 2000). The PCR products of class 1 integrons variable regions with identical amplicon lengths were analyzed by restriction fragment length polymorphism (RFLP) using HinfI (TaKaRa, Japan) according to the method of previous study (Gu et al., 2008). PCR products with identical RFLP profiles were regarded as the same gene cassette array. PCR products with different gene cassette arrays were sequenced and analyzed.

Phylogenetic Genotyping
Phylogenetic grouping of the E. coli isolates was determined using a triplex PCR for the genes chuA, yjaA, and TspE4C2 as described previously (Clermont et al., 2000).

Statistical Analysis
Frequency of antimicrobial resistance profiles between ESBLpositive and ESBL-negative E. coli isolates were compared using the Pearson Chi-square test with the software SPSS 17.0, and the level of significance was set at p < 0.05.

Integrons and Gene Cassettes
The class 1 integron integrase gene intI1 was detected in 78.3% (155/198) ESBL-positive E. coli isolates. intI2 and intI3 genes were not detected in any of the isolates. The variable regions of class 1 integron was amplified using primers complementary to the conserved regions flanking the inserted resistance genes. One hundred and ten of the 155 intI1-positive E. coli isolates were positive for the variable region and the inserted gene cassette sizes varied in size from 0.75 to 1.9 kb. BLAST analysis of sequenced products of variable region of class 1 demonstrated the presence of eight different cassette combinations for 10 different genes. These genes encode for the resistance to trimethoprim (dfrA1, dfrA5, dfrA7, dfrA12, dfrA17, and dfrA27) and aminoglycosides (aadA1, aadA2, aadA5, and aad22). Out of 110 intI1-positive isolates 57 carried dfrA17-aadA5 gene cassette; 28 harbored dfrA12-orfF-aadA2 gene cassette; 12 harbored dfrA1-aadA1 gene cassette. The sequences of the dfrA1-aadA1, dfrA17-aadA5, and dfrA12-orfF-aadA2 gene cassettes have been deposited in the GenBank database under the accession numbers KT692980, KT692981, and KT692982, respectively. The distribution of integron among ESBL-positive E. coli was shown in Table 6.

Phylogenetic Groups
All ESBL-positive isolates were allocated to one of the four phylogenetic groups A, B1, B2, and D. The results showed that 53.5% (106/198) of these isolates belonged to group A, 24.2% (48/198) to group D, and 21.2% (42/198) to group B1. Only two strains were found in group B2.

DISCUSSION
Over the past few years, with the development of an intensive and large-scale pig industry in China, the occurrence and epidemic of porcine diseases have taken on a lot of new characteristics (Liu et al., 2014). In particular, PWD caused by pathogenic E.
coli is one of the most serious threat for the swine industry. During PWD outbreaks in pig farms, it was certainly thought that antimicrobial therapy would be an important control measure (Amezcua et al., 2002). However, commonly used animal antimicrobial agents did not appear to be effective for curing PWD (Moredo et al., 2015). Disturbingly, only thirdgeneration cephalosporins such as ceftiofur and cefquinome are effective for controlling PWD (Hornish and Kotarski, 2002). The frequent use of third-generation cephalosporins as therapy or feed additives in pigs has selected high antimicrobial resistance in bacteria, which is becoming a serious issues in pig industry (Lei et al., 2010). All E. coli isolates in the current study were tested for their susceptibility to 13 antimicrobial agents. Similar to previous studies (Smith et al., 2010), we found a very high frequency of resistance to most of the conventional antimicrobial agents such as gentamicin, tetracycline, chloramphenicol, and trimethoprim-sulphamethoxazole normally used to treat PWD infections (Luppi et al., 2015). Certainly, these antimicrobial agents could not be the good choice to treat piglets with PWD in these farms. The remarkable finding in the present study is that 92.8% isolates were resistant to more than five antimicrobial agents, and 62.9% isolates were resistant to more than nine antimicrobial agents, which is much higher than our previous studies (Zhang et al., 2009). However, all isolates remained susceptible to colistin, which has been shown to be a safe and effective candidate to treat infections caused by E. coli in China pig farms (Wu et al., 2012). Comparing the frequency of resistance to these 13 antimicrobial agents, higher resistance rates were present in ESBL-positive strains than in negative ones, which suggested the former may acquire resistance easier than the latter. The prevalence of ESBL-positive E. coli isolates in foodproducing animals has been increasing worldwide (Liebana et al., 2013). In this study, 43.2% (198/458) of the E. coli isolates screened from PWD piglets were ESBL producers, which is  higher than the detection rates (12.6%, 41/326) in E. coli from healthy pigs (P < 0.001; Zheng et al., 2012). These results suggest that ESBLs are more frequently present in isolates from sick animals compared to commensal E. coli from the healthy foodproducing animals. The high prevalence rate of ESBL-positive E. coli in diarrhea piglets may be explained by the fact that the thirdgeneration cephalosporins are used more commonly in swine for treatment of ND and PWD diseases (Lei et al., 2010;Barton, 2014). Nevertheless, this constitutes a potential public health concern since the recovered sick animals carrying ESBL resistant gene may enter the food chain. The inappropriate use and/or overuse of third-generation cephalosporins has been associated with the emergence and spread of ESBL-positive E. coli in pigs (Cavaco et al., 2008). For that reason, in many countries, thirdgeneration cephalosporins have been forbidden for use in pig production, as a result of which, the occurrence of ESBL-positive E. coli has been significantly reduced (Agersø and Aarestrup, 2013). In china, however, third-generation cephalosporins are still approved for food-producing animals with no limitation. CTX-M-type ESBLs have been isolated increasingly from human, companion, and food-producing animals E. coli isolates in different countries in the last several years (Zhao and Hu, 2013). A 2003-2012 survey in China showed that CTX-Mtype ESBLs accounted for 87.1% of ESBL-positive E. coli strains isolated from food animals . CTX-M-14 remains the most abundant genotype, although the detection rate of CTX-M-55 and CTX-M-65 have shown a continuously increasing trend in recent years Zheng et al., 2012). Similar findings were obtained in the present study, the proportion of CTX-M-producing E. coli had increased to 96.5%, and CTX-M-14 remained the most common genotype of ESBLs, followed by CTX-M-55 and CTX-M-65. In addition, we also found the bla genes in these isolates, indicating a high diversity of bla CTX−M genes in E. coli isolates from PWD piglets in Heilongjiang province. In the past, bla TEM−52 has only been detected in E. coli isolates from chicken in China (Li et al., 2010), but now it has been found in four E. coli isolates from piglets of different farms. To our knowledge, this is the first report of bla TEM−52 in E. coli from pigs in China.
Emergence of PMQR gene has been reported worldwide and is being documented in ESBL producers in recent days (Poirel et al., 2012). In the present study, we found that the detection rate of PMQR genes was 87.4% among a collection of 198 ESBLpositive E. coli isolates. Somewhat similar finding has also been reported in previous study in China, where 83.8% (160/191) of ESBL-positive E. coli from swine contained the PMQR genes (Liu et al., 2013b). However, the detection rate of PMQR among ESBLpositive E. coli in other countries was low, with 50% of the E. coli isolates from human in Tunisian and 13.6% E. coli isolates from human in Mexico (Silva-Sánchez et al., 2013;Ferjani et al., 2015). Over the past 10 years, PMQR determinants have emerged as an important issue. Their detection among ESBL-positive E. coli from human and food-producing animals has been widely investigated and different rates have been reported depending on the country, origin of isolates, and the number of E. coli included (Seiffert et al., 2013). The widespread dissemination of PMQR determinants may lead to the rapid development of fluoroquinolone resistance because they can confer low-level resistance to fluoroquinolone and promote the selection of high-level resistant strains with mutations on the chromosome. In the present study, 52 of the 125 oqxAB positive strains possessed bla CTX−M−14 , and a significant association between the bla CTX−M−14 and oqxAB genes have been found,which validated the finding further that isolates with the oqxAB gene often possess a bla CTX−M−14 -producing plasmid (Liu et al., 2013b). This suggests that spread of the oqxAB gene might occur concurrently with the bla CTX−M−14 gene. Notably, the bla CTX−M−55 , qnrB, qnrS, aac(6 ′ )-Ib-cr, qepA, and oqxAB genes were found to be carried together in three isolates. Only a few previous studies have found bacteria harboring more than three PMQR genes (Yang et al., 2014). Whether multiple PMQR determinants have an additive effect on the MIC of fluoroquinolones is unknown and warrants further investigation.
Integrons are important genetic elements responsible for carriage and spread of antibiotic resistant determinants including ESBL enzymes. ESBL genes located on integrons-like structures are being increasingly reported worldwide (Bonnet, 2004). Hence, we examined the presence of class 1, class 2, and class 3 integrons. In this study, 78.3% (155/198) of ESBLpositive E. coli isolates harbored class 1 integron while none of the isolates contained class 2 and class 3 integron as found in previous studies (Sun et al., 2013). Sequence analysis of the amplified variable region of class 1 integron revealed that most of the integrons possessed dfrA and aadA gene cassettes conferring resistance to trimethoprim and streptomycin as reported by several researchers previously (Kar et al., 2015). This kind of gene cassette arrangement may be responsible for emergence of multiple drug resistance among ESBL producers isolated in this study. However, further study is needed to investigate the association between class 1 integron production and dissemination of ESBL type genes in E. coli from China.
Several authors have analyzed the distribution of the main phylogenetic groups among E. coli strains isolated from human and animals. We showed that E. coli clinical isolates from PWD piglets carrying ESBLs, collected over 3 years, belonged mainly to phylogroup A and, to a lesser extent, to phylogroups D and B1, whereas B2 strains were very little. This is in agreement with other studies on pathogenic E. coli of pig origin Abraham et al., 2014).
In our study we have detected a high prevalence of ESBL-positive E. coli isolated from PWD piglets. Future studies will be performed with the ESBL-positive isolates to find possible association among serotypes, virulence factors, resistance mechanisms, and transmission mechanisms that will be useful for epidemiological research.
In conclusion, in this study a high prevalence of ESBL-positive E. coli has been observed in fecal samples of PWD piglets collected from Heilongjiang province, and the high prevalence of PMQR genes and integrons were also widely detected among the ESBL-positive E. coli strains. The bla CTX−M−14 was found to be the dominant ESBL-encoding gene in this study, while oqxAB and qnrS were the dominant PMQR genes in the ESBL-positive E. coli. This is the first report of TEM-52 ESBL in bacterial isolates from pig in China. To our knowledge, this is also the first description of the coexistence of the qnrB, qnrS, aac(6 ′ )-Ib-cr, qepA, and oqxAB genes in one E. coli strain. More studies should be carried out in the future in order to ensure that the six PMQR genes were located on the same plasmid or not.