Characterization of oqxAB in Escherichia coli Isolates from Animals, Retail Meat, and Human Patients in Guangzhou, China

The purpose of this study was to investigate the prevalence and genetic elements of oqxAB among Escherichia coli isolates from animals, retail meat, and humans (patients with infection or colonization) in Guangzhou, China. A total of 1,354 E. coli isolates were screened for oqxAB by PCR. Fifty oqxAB-positive isolates were further characterized by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), S1-PFGE, genetic environment analysis, plasmid replicon typing, and plasmid sequencing. oqxAB was detected in 172 (33.79%), 60 (17.34%), and 90 (18.07%) E. coli isolates from animal, food, and human, respectively. High clonal diversity was observed among oqxAB-positive isolates. In 21 oqxAB-containing transformants, oqxAB was flanked by two IS26 elements in the same orientation, formed a composite transposon Tn6010 in 19 transformants, and was located on plasmids (33.3~500 kb) belonging to IncN1-F33:A-:B- (n = 3), IncHI2/ST3 (n = 3), F-:A18:B- (n = 2), F-:A-:B54 (n = 2), or others. Additionally, oqxAB was co-located with multiple resistance genes on the same plasmid, such as aac(6′)-Ib-cr and/or qnrS, which were identified in two F-:A18:B- plasmids from pigs, and blaCTX−M−55, rmtB, fosA3, and floR, which were detected in two N1-F33:A-:B- plasmids from patients. The two IncHI2/ST3 oqxAB-bearing plasmids, pHNLDF400 and pHNYJC8, which were isolated from human patient and chicken meat, respectively, contained a typical IncHI2-type backbone, and were similar to each other with 2-bp difference, and also showed 99% identity to the Salmonella Typhimurium oqxAB-carrying plasmids pHXY0908 (chicken) and pHK0653 (human patient). Horizontal transfer mediated by mobile elements may be the primary mechanism underlying oqxAB spread in E. coli isolates obtained from various sources in Guangzhou, China. The transmission of identical oqxAB-carrying IncHI2 plasmids between food products and humans might pose a serious threat to public health.

The purpose of this study was to investigate the prevalence and genetic elements of oqxAB among Escherichia coli isolates from animals, retail meat, and humans (patients with infection or colonization) in Guangzhou, China. A total of 1,354 E. coli isolates were screened for oqxAB by PCR. Fifty oqxAB-positive isolates were further characterized by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), S1-PFGE, genetic environment analysis, plasmid replicon typing, and plasmid sequencing. oqxAB was detected in 172 (33.79%), 60 (17.34%), and 90 (18.07%) E. coli isolates from animal, food, and human, respectively. High clonal diversity was observed among oqxAB-positive isolates. In 21 oqxAB-containing transformants, oqxAB was flanked by two IS26 elements in the same orientation, formed a composite transposon Tn6010 in 19 transformants, and was located on plasmids (33.3∼500 kb) belonging to IncN1-F33:A-:B-(n = 3), IncHI2/ST3 (n = 3), F-:A18:B-(n = 2), F-:A-:B54 (n = 2), or others. Additionally, oqxAB was co-located with multiple resistance genes on the same plasmid, such as aac(6 ′ )-Ib-cr and/or qnrS, which were identified in two F-:A18:B-plasmids from pigs, and bla CTX−M−55 , rmtB, fosA3, and floR, which were detected in two N1-F33:A-:B-plasmids from patients. The two IncHI2/ST3 oqxAB-bearing plasmids, pHNLDF400 and pHNYJC8, which were isolated from human patient and chicken meat, respectively, contained a typical IncHI2-type backbone, and were similar to each other with 2-bp difference, and also showed 99% identity to the Salmonella Typhimurium oqxAB-carrying plasmids pHXY0908 (chicken) and pHK0653 (human patient). Horizontal transfer mediated by mobile elements may be the primary mechanism underlying oqxAB spread in E. coli isolates obtained from various sources in Guangzhou, China. The transmission of identical oqxAB-carrying IncHI2 plasmids between food products and humans might pose a serious threat to public health.

INTRODUCTION
The efflux pump OqxAB, originally identified in the conjugative IncX1 plasmid pOLA52 from a porcine Escherichia coli isolate in 2003, belongs to the resistance-nodulation-division family, and is encoded by the oqxA and oqxB genes, which are located in the same operon (Sørensen et al., 2003;Hansen et al., 2004). OqxAB mediates resistance, or reduces susceptibility to multiple antimicrobials, including quinoxalines, chloramphenicol, trimethoprim, and quinolones, and is recognized as a plasmid-mediated quinolone resistance (PMQR) determinant (Hansen et al., 2007;Ruiz et al., 2012).
Recently, the oqxAB genes have been identified as the most prevalent PMQR genes in E. coli isolates from food-producing animals in China (Liu et al., 2012Xu et al., 2015), as well as from animal-derived food products (Xu et al., 2014). This could be due to the widespread use of olaquindox as a growth promoter for pigs weighing below 35 kg and mequindox against enteropathogenic E. coli infections in swine and poultry (He T. et al., 2015). Our previous study demonstrated high oqxAB prevalence in E. coli isolates from animals, farm environment, and farm workers, and clonal transmission of oqxAB-carrying isolates between swine and farm workers was observed (Zhao et al., 2010). Additionally, oqxAB was observed to be relatively prevalent in E. coli isolates from pigs, ducks, chickens, meat (pork and chicken meat), and healthy humans (55.7, 40.6, 25.8, 16.2, and 7.2%, respectively;Yang T. et al., 2014). However, oqxAB has been rarely reported in human clinics and animals in Europe, where olaquindox has been banned as an animal feed additive since 1999 (Hansen et al., 2005), with detection rates of 0.46% in E. coli isolates from patients in the UK and Ireland (Ciesielczuk et al., 2013) and 1.62% from porcine E. coli strains from Denmark and Sweden (Hansen et al., 2005). Altogether, oqxAB might possibly be transmitted from animals to humans via the food chain or close contact, in China. Furthermore, IS26 plays an important role in oqxAB dissemination (Hansen et al., 2004;He T. et al., 2015). IncHI2 plasmids have been recently demonstrated to mediate oqxAB spread in S. Typhimurium and S. Indiana among animals in China, as well as in clinical S. Typhimurium isolates in Hong Kong Wong et al., 2016). Thus, this study was aimed at determining the distribution and genetic elements (IS26 and plasmids) of oqxAB among E. coli isolates from different sources (animals, animal-derived food products, and human clinics) in Guangzhou, China, determining the complete nucleotide sequence of two IncHI2 plasmids carrying oqxAB and comparing them with those previously reported from different sources in China, and outlining the possible routes of oqxAB transmission via the food chain.

Bacterial Strains
A total of 1,354 individual E. coli isolates were collected from Guangzhou, Guangdong province, China between July 2011 and May 2013, including 509 animal strains (372 pig and 137 chicken strains) isolated from fecal samples of healthy animals from one hog market and one live poultry market, 346 strains from retail meat (247 pork and 99 chicken meat samples) recovered from fresh or chilled pork and chicken samples purchased from supermarkets and farmers' markets, and 498 strains from patients (71 E. coli strains from urine samples from inpatients with urinary tract infection, 427 E. coli strains from fecal samples of inpatients and outpatients) from four hospitals. The samples were inoculated onto MacConkey agar, and suspected E. coli colonies (one isolate per sample) were selected and identified by standard biochemical testing. All the isolates were tested for the presence of oqxAB by PCR, and 100 amplified products were randomly selected for sequencing (Table S1).

Conjugation/Transformation Experiments and Plasmid Characterization
The 50 oqxAB-positive E. coli strains subjected to PFGE were further analyzed by performing conjugation experiments with streptomycin-resistant E. coli C600 as the recipient strain following a previously described protocol (Chen et al., 2007). Transconjugants were selected using 32 µg/mL olaquindox and 3,000 µg/mL streptomycin.
The presence of oqxAB in the transconjugants/transformants was confirmed via PCR and sequencing. Other resistance genes, including floR, bla CTX−M , rmtB, and fosA3 were also screened using the primers listed in Table S1. Antimicrobial susceptibility of all the transformants and E. coli DH5α recipient strain were determined using the agar dilution or broth microdilution method (limited to colistin). All the transformants were characterized by PCR-based replicon typing and were screened IncX plasmids as described previously (Carattoli et al., 2005;Johnson et al., 2012). Replicon sequence typing and plasmid double locus sequence typing were performed to further characterize IncFII and IncHI2 plasmids according to previously described protocols Villa et al., 2010). S1-PFGE (Barton et al., 1995), and Southern blot hybridization were performed to determine the number of plasmids and the sizes of oqxAB-carrying plasmids in all the FIGURE 1 | Comparison of antimicrobial resistance among oqxAB-positive E. coli isolates from food-producing animals, food products, and human patients. AMP, ampicillin; CTX, cefotaxime; AMI, amikacin; GEN, gentamycin; NEO, neomycin; APR, apramycin; TET, tetracycline; FLR, florfenicol; CL, colistin; SXT, sulfamethoxazole/trimethoprim; CIP, ciprofloxacin; OLA, olaquindox. transformants, using a non-radioactively labeled oqxAB-specific probe. The genetic context of oqxAB was determined by PCR mapping (Table S2). Ten transformants with a single oqxABcarrying plasmid were further analyzed by restriction fragment length polymorphism (RFLP) using the endonuclease ApaLI, TZC215-1, and AHH13-1 were excluded since their sizes were significantly different from other oqxAB-carrying plasmids.

Nucleotide Sequences Accession Numbers
The nucleotide sequences of plasmids pHNLDF400 and pHNYJC8 have been deposited in the GenBank database under the accession numbers KY019258 and KY019259, respectively.
Among the 50 oqxAB-positive E. coli isolates from different sources analyzed using XbaI-PFGE, 44 oqxAB-carrying E. coli isolates from animals (n = 19), animal-derived food products (n = 6), and patients (n = 19) exhibited 43 distinct PFGE patterns (Figure 2). Notably, the isolates belonging to the same STs identified in our study showed different PFGE patterns; however, the ST410 E. coli isolates BYMP20 and YZHF29, which were obtained from pork and human sources from different districts  Frontiers in Microbiology | www.frontiersin.org in Guangzhou within 12 km, showed similar PFGE patterns (E1 and E2; Figure 2).
MLST and PFGE demonstrated the molecular diversity of the oqxAB-positive E. coli isolates, and suggested that clonal transfer might not be the main mechanism underlying oqxAB dissemination among E. coli isolates in Guangzhou, China.

Analysis of oqxAB-Carrying Plasmids
Surprisingly, none of the 50 randomly selected oqxAB-positive E. coli isolates transferred oqxAB to E. coli C600 via conjugation, which is consistent with the results reported for S. Typhimurium (Wong et al., 2014). Therefore, transformation assays were performed, and 21 transformants were obtained successfully. All the transformants were subjected to S1-PFGE and Southern blotting. The results demonstrated that the transformants carried one to four plasmids of various sizes. oqxAB was detected in plasmids with sizes ranging from ∼33.3 to ∼500 kb. Four transformants (SNJ23-1, SNJ41-1, ZYTM154-1, and LDHF159-1) showed two hybridization signals, whereas no hybridization signal was observed in SNX19-2 ( Table 1). Thirteen transformants contained IncFII (n = 4), IncFIA (n = 2), IncFIB (n = 3), IncHI2 (n = 3), and IncN (n = 5) replicons. Notably, three transformants carried both IncN and IncFII replicons, which were classified as N1-F33: A-: B- (Table 1). Interestingly, the three transformants were obtained from three original E. coli isolates exhibiting different PFGE patterns from the same hospital, of which two carried only one oqxAB-bearing plasmid with similar sizes (Table 1 and Figure S2). Three IncHI2 plasmids from food product and patients were assigned to ST3 via plasmid double locus sequence typing ( Table 1). PCR-based replicon typing could not determine the replicon types of the remaining eight transformants. Although rare, an untypable oqxAB-carrying plasmid was described previously . Ten transformants with single plasmid were selected for RFLP with ApaLI. Interestingly, two N1-F33:A-:B-plasmids from the patients of the same hospital (ZYTM118-1 and ZYTF32-1) showed identical patterns, as well as two F-:A18:B-plasmids from pigs (TZC152-6 and TZC212-1). Additionally, three ∼76.8 oqxAB-bearing plasmids obtained from pig, chicken meat, and human, exhibited identical patterns ( Figure S3).
Sequences of the regions surrounding oqxAB were determined via PCR mapping and sequencing. oqxAB was flanked by two IS26 elements in the same orientation, and formed a composite transposon Tn6010 (IS26-oqxA-oqxB-oqxR-IS26) in 19 transformants. Additionally, one IS26 element was present in upstream or downstream of oqxAB in the remaining transformants TZC338-4 and SNX19-2. Since its first identification in the plasmid pOLA52 from porcine E. coli in 2003 (Sørensen et al., 2003), Tn6010 is believed to play a vital role in oqxAB transmission among Enterobacteriaceae isolates He T. et al., 2015). Our results further support that horizontal transfer mediated by mobile elements seems to be the main mechanism for oqxAB transmission in E. coli from different sources.

Plasmid Sequencing and Comparative Analysis
Two oqxAB-bearing IncHI2 plasmids in this study, namely pHNYJC8 from YJMC8 of food origin and pHNLDF400 from LDHF400 of human origin, were selected for sequencing. Plasmids pHNLDF400 and pHNYJC8 have sizes of 249,152 and 249,153 bp, respectively, and both have a GC content of 46.51%. They contain a typical IncHI2-type backbone, which encodes genes for plasmid replication, conjugative transfer, maintenance, and stability. Similar to other IncHI2 plasmids, such as pEC5207 (KT347600, porcine E. coli, China), pHNLDF400 and pHNYJC8 also carry a set of tellurite resistance determinants (terZABCDEF).
cassette array, which is flanked by the 3 ′ -conserved segment (3 ′ -CS) and interrupted by a Tn21-like transposon Tn chrA , itself truncated by IS26. Most importantly, pHNSHP45-2 also harbors the colistin resistance gene, mcr-1, which is associated with ISApl1, and is inserted in the plasmid backbone (Figure 3). Similarly, a 6,760-bp fragment containing fosA3 and bla CTX−M−65 is identified in pHNAH67, whereas pHNLDF400 and pHNYJC8 contain a ∼28.3-kb fragment with multiple resistance genes oqxAB, sul1, aadA2, dfrA12, aphA1, sul3, aadA1a, cmlA1, and aadA2, which is absent in pHNAH67 (Figure 2). The absence/presence of these regions might be explained by IS26-mediated homologous recombination or replicative transposition (He S. et al., 2015). Furthermore, the oqxAB-associated composite transposon Tn6010 and the upstream fragment (3,678-bp) containing Tn2, IS26 (which interrupts the Tn2 segment), and the bleomycin resistance genes blms, orf63, and ISEnca1 (which is truncated by IS26 of Tn6010), were identical to the corresponding regions in the originally identified IncX1 oqxAB-carrying plasmid pOLA52, as well as pHXY0908, pHK0653, and pHNSHP45-2. The above mentioned results suggest that these plasmids may have originated from the same IncHI2-type plasmid, acquiring or losing various regions containing resistance genes, and spread among Enterobacteriaceae species in livestock, food products, and humans in different regions in China.

CONCLUSION
In conclusion, our findings suggested that oqxAB dissemination among E. coli isolates from various sources could be due to horizontal transfer mediated by mobile elements, such as Tn6010, N1-F33:A-:B-, and IncHI2 plasmids. Identical oqxABcarrying IncHI2 (ST3) plasmids were detected in the retail meat samples and human patients, similar to the previously described oqxAB-bearing IncHI2 plasmids pHXY0908 and pHK0653 from S. Typhimurium from chicken and patient. Thus, the transmission of similar oqxAB plasmids between animals, animal-derived food, and humans, and further human-tohuman contact in communities and hospitals requires continued monitoring.

ETHICS STATEMENT
This study was carried out in accordance with the recommendation of ethical guidelines of South China Agricultural University. Individual written informed consent for the use of fecal or urine samples was obtained from all the patients and animal owners.

AUTHOR CONTRIBUTIONS
JHL, ZZ, and JW conceived the study. CZ, XC, ZG, JL, JW, WL, XH, LZ, JH, YX, MY, and TH carried out the experiments. JW, CZ, and XC analyzed the data. JW wrote the manuscript. JHL and ZZ revised the manuscript. All authors read and approved the final manuscript.