Bacterial isolates

A total of 696 non-duplicate E. coli isolates (318 avian including 177 from ducks, 110 from chickens and 31 from geese, and 378 from pigs) were isolated from diseased food-producing animals between March 2002 and August 2012. The animals were from more than 80 farms all over Guangdong province. Animals we chose liver or heart tissues to obtain isolates were infected with E. coli, and the other animals showed diarrhea. Further information about these animals, the underlying disease and possible antimicrobial pretreatment were unfortunately not available. Cotton swabs of the liver and heart tissues or faeces from these animals were streaked onto MacConkey agar. After 16h incubation at 37°C, one colony with typical E. coli morphology was selected and purified on MacConkey agar. One colony was selected from each sample and all E. coli isolates were identified by classical biochemical methods and confirmed using the API 20E system (bioMe ´rieux). All identified isolates were stored at -80°C in Luria–Bertani broth containing 30% glycerol. E. coli C600, resistant to streptomycin, was used as the recipient strain in the conjugation experiments.

Antimicrobial susceptibility testing

Susceptibilities to enrofloxacin, ciprofloxacin, levofloxacin, nalidixic acid, amikacin, gentamicin, florfenicol, chloramphenicol, ampicillin, ceftiofur, cefotaxime, doxycycline, and tetracycline of the 696 isolates were assayed by the agar dilution method, according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [19]. Escherichia coli ATCC 25922 was used as the control strain. Isolates were classified as either susceptible or resistant according to the interpretative standards recommended by the CLSI [19]. As there are no CLSI breakpoints for florfenicol and ceftiofur that are applicable to E. coli of animal origin, the breakpoints of florfenicol (≥16mgL^-1) and ceftiofur (≥ 8mgL^-1) were sourced from the Danish Integrated Antimicrobial Resistance Monitoring and Research Program [20] and a previous report [21]. ESBL-producing isolates were screened by double-disk synergy test using both cefotaxime and ceftazidime in the presence or absence of clavulanic acid as recommended by the CLSI.

Detection of PMQR determinants

PMQR genes including qnrA, qnrB, qnrS, qnrC, qnrD, aac(6’)-Ib-cr, qepA, oqxA, and oqxB among the 696 clinical E. coli isolates were analyzed by PCR amplification using previously described primers [2,5,16,21,22]. The association of IS26 with oqxAB was investigated by PCR as reported previously [23], using forward primer IS26-F (5’-GCTGTTACGACGGGAGGAG-3’) and reverse primer oqxA-R (5’-GAGGTTTTGATAGTGGAGGTAGG-3’). The DNA sequences obtained after direct sequencing of the amplification products were confirmed using the BLAST algorithm available through the National Center for Biotechnology Information (NCBI).

Detection of ESBL-encoding genes

ESBL-encoding genes (bla_TEM, bla_SHV, bla_CTX-M-1G, bla_CTX-M-9G, bla_CTX-M-2G, and bla_CTX-M-25G) among the ESBL-producing isolates were analyzed by PCR amplification using previously published primers and protocols [24,25,26]. Among the 136 isolates harboring bla_CTX-M-9G, 89 were selected randomly to be directly sequenced using the PCR products, and 33 out of the 63 bla_CTX-M-1G-positive isolates were randomly selected to be sequenced. The DNA sequences obtained were compared with genes in GeneBank (http://www.ncbi.nlm.nih.gov/) to confirm the subtypes of ESBL-encoding genes.

Conjugation experiment

Isolates harboring oqxAB and genes encoding ESBLs, were selected for conjugation experiments by the broth-mating method using E. coli C600 as the recipient [27]. Transconjugants were selected on MacConkey agar plates containing streptomycin (1,000mg/L) and cefotaxime (2mg/L). The transconjugants harboring oqxAB and ESBL-encoding genes mentioned above were confirmed by PCR as previously and antimicrobial susceptibility testing for the transconjugants, recipient, and donors.

Plasmids analysis of transconjugants

Incompatibility (Inc) groups were assigned by PCR-based replicon typing of transconjugants [28]. To better clarify IncF plasmids, replicon sequence typing of IncF plasmids was carried out according to the method reported previously [29]. Alleles were assigned by submitting the amplicon sequence to the plasmid multilocus sequence typing (pMLST) database (http://www.pubmlst.org/plasmid).

To analyze the location of the oqxAB gene and ESBL-encoding genes of transconjugants, S1 nuclease-PFGE and Southern blot analysis were performed. Briefly, whole-cell DNA of the transconjugants co-harboring oqxAB and ESBL- encoding genes embedded in agarose gel plugs was treated with S1 nuclease (TaKaRa, Dalian, China) and separated by PFGE alongside a standard lambda ladder PFG Marker (NEB, UK). Subsequently, Southern blot hybridization was performed with DNA probes specific for oqxB, bla_CTX-M-1G or bla_CTX-M-9G, which were non-radioactively labeled with a DIG High Prime DNA labeling and detection kit (Roche Diagnostics, Mannheim, Germany). Plasmid DNA extraction was performed using a QIAGEN Plasmid Midi kit (QIAGEN, Germany). Plasmids of transconjugants were digested with the endonuclease EcoRI (TaKaRa Biotechnology, Dalian, China) to analyze the restriction fragment length polymorphism (RFLP) profiles.

Molecular typing

To determine their genetic relatedness, chromosomal DNAs of 109 E. coli isolates randomly selected from the oqxAB-harboring isolates were digested with XbaI and subjected to pulsed-field gel electrophoresis (PFGE) according to a protocol described previously [30]. The DNA banding patterns were analysed using BioNumerics software version 2.5 (Applied Maths), and a cut-off value of 95% of the similarity values was chosen to indicate identical PFGE types. Salmonella enterica serotype Braenderup H9812 standards served as size markers.

Antimicrobial susceptibility testing

Almost all the 696 clinical E. coli isolates in this study were highly resistant to nadidixic acid (96.1%), ampicillin (95.1%) and tetracycline (96.3%). The antimicrobial resistance rates to other antibiotics were as follows: chloramphenicol (85.3%), enrofloxacin (82.6%), doxycycline (79.6%), ciprofloxacin (76.3%), streptomycin (74.0%) levofloxacin (73.6%), florfenicol (73.1%), gentamicin (64.4%) ceftiofur (46.3%), and amikacin (26.6%). Of the 696 isolates, 228 (32.8%) (97 from pigs, and 131 from avian) showed reduced susceptibility to cefotaxime (MIC≥2μg/mL), and all the 228 isolates were resistant to ceftiofur. ESBL production was detected by the screening method in 206 of the 228 isolates, representing 29.6% of the total 696 E. coli isolates.

Prevalence of PMQR genes

Four hundred and twenty-nine (61.6%) of the 696 isolates were found to have at least one PMQR gene by PCR and sequencing of the PCR products. oqxAB, found in 328 isolates (47.1% of the total), was the most prevalent PMQR gene, followed by qnrS (14.5%) and aac(6’)-Ib-cr (14.4%). The number of isolates harboring qnrB and qepA was 48 (7.0%) and 21 (3.0%), respectively, but no isolate was positive for qnrA, qnrC or qnrD. In addition, 180 of the 328 oqxAB-positive isolates were detected to be linked with IS26. The combination types of PMQR genes in E. coli of different origin were listed in Table 1.

ESBL-encoding genes detection

ESBL-encoding genes were detected in most E. coli isolates with a cefotaxime MIC ≥ 2 μg/mL. CTX-M-type genes were found to be dominant in the isolates with ESBL production, and 191 isolates carried one or two CTX-M genes, representing 27.4% of the total 696 clinical E. coli isolates. bla_SHV and bla_TEM type ESBL- encoding genes were not found in any of these isolates. Among the 191 bla_CTX-M-positive isolates, the number of isolates carrying bla_CTX-M-1G and bla_CTX-M-9G were 63 and 136, respectively, with 8 isolates carrying both bla_CTX-M-1G and bla_CTX-M-9G included. As the data of randomly sequenced bla_CTX-M that shown in Table 2, the most predominant CTX-M-encoding gene was bla_CTX-M-14 (n=36), followed by bla_CTX-M-55 (n=29). The most common CTX-M type in isolates from pigs was bla_CTX-M-14, whereas bla_CTX-M-27 was the most common type among isolates from avian (Table 2).

bla_CTX-M genes among the PMQR-positive isolates

The distribution of ESBL-encoding genes among the 429 PMQR-positive E. coli isolates was shown in Table 1. One hundred and sixty of the 429 PMQR-positive isolates were detected to harbor CTX-M type genes, whereas, bla_CTX-M genes were detected in 31 of the 267 PMQR-negative isolates (P<0.001). As shown in Table 1, the detection of CTX-M type genes in isolates carrying oqxAB (127/328) was significantly higher than that in oqxAB-negative isolates (64/368) (P<0.001). For avian, the number of isolates carrying oqxAB-bla_CTX-M-9G, oqxAB-bla_CTX-M-1G, and oqxAB-bla_CTX-M-9G-bla_CTX-M-1G were 48, 20, and 3, respectively. And there were 37, 15 and 4 isolates from pigs were found to harbor oqxAB-bla_CTX-M-9G, oqxAB-bla_CTX-M-1G, and oqxAB-bla_CTX-M-9G-bla_CTX-M-1G, respectively (Table 1).

Molecular typing

Of the 109 isolates, 97 were successfully typed by PFGE, and a total of 88 different PFGE profiles were obtained, suggesting that most of the isolates in the study were from epidemiologically unrelated E. coli clones.

Co-transferability of oqxAB and bla_CTX-M genes and plasmid analysis

In this study, 84 transconjugants carrying bla_CTX-M were obtained from the 127 isolates with oqxAB-bla_CTX-M. Eighteen of the 84 transconjugants were found to be also positive for oqxAB. Among the 18 transconjugants, 8 carried oqxAB, bla_CTX-M-9G and aac(6’)-Ib-cr simultaneously (Table 3). The result of S1 nuclease-PFGE shown in Figure 1A revealed that 14 transconjugants except FS3Z3GT, FS9Y1CT, 70zuT and 5weiT, carried only one plasmid (FS3Z3GT also harboring a very small plasmid not detected using S1-PFGE). As listed in Table 3, the IncFII (5 different alleles) replicon types were detected in 13 transconjugants, 7 of them carrying two replicons (FII in combination with FIB, HI2, or N). HI2 replicon type was found in 6 transconjugants, with 3 carrying other replicons. All transconjugants showed extremely high-level resistance to ampicillin, ceftiofur and cefotaxime, at the same level as the donor strains. For quinolones, the transconjugants showed 2~16-, and 4~16-fold increases in the MICs of nalidixic acid, and ciprofloxacin, respectively, when compared with the recipient C600. As shown in Table 3, all the transconjugants were multidrug-resistant and showed resistance to more than two non-β-lactam antimicrobial agents. Notably, 13 transconjugants were found to show high-level resistance to florfenicol, a veterinary antibiotic commonly used in veterinary medicine and aquaculture. The floR gene, which confers resistance to florfenicol, was found in all the 13 transconjugants. The results of Southern blot hybridization revealed that oqxAB and bla_CTX-M were located on the same plasmid in the all 18 transconjugants (Figure 1). And floR was also located on these plasmids in the 13 transconjugants resistant to florfenicol. Interestingly, isolates FS3Z3C and FS3Z3G sharing the same PFGE pattern were both resistant to flofenicol, however, the two F18: A-: B1 plasmids of their transconjugants were different (floR in FS3Z3GT, none in FS3Z3CT) (Table 3 and Figure 1E)

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Figure 1. Plasmid analysis of transconjugants carrying oqxAB and bla_CTX-M.

(A) S1 nuclease-PFGE (B) Southern blot hybridization with the oqxAB probe. Lane 1-18: 45-6T, FS1Z4ST, a78T, CBJ3CT, a64T, 2YG4T, FS5E1DT, 42-2-2T, FS9Y1CT, A6T, 70zuT, FS3Z3GT, FS2Y1XT, FS6J2CT, 33-2T, 5weiT, FS3Z3CT, 7ganT; Lane M: lambda ladder PFG Marker. (C) Southern blot hybridization with the bla_CTX-M-9G probe (D) Southern blot hybridization with the bla_CTX-M-1G probe (E) Southern blot hybridization with the floR probe.

https://doi.org/10.1371/journal.pone.0073947.g001

As shown in Figure 2, the two F33:A-: B- plasmids p42-2 and pFS5E1DT shared the same EcoRI digestion profiles. This result could be confirmed by the positions of the bands in lane 7 and 8 in Figure 1. The EcoRI digestion profile of plasmid from FS2Y1XT was only one band different from that of p42-2 and pFS5E1DT (Figure 2). As shown in Figure 2, the 6 HI2 plasmids carrying both oqxAB and bla_CTX-M-9G also showed the same or highly similar EcoRI digestion profiles. The plasmids from the other 5 transconjugants harboring only one plasmid, shared very different EcoRI digestion profiles.

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Figure 2. EcoRI restriction digestion profiles of plasmids co-harboring oqxAB-bla_CTX-M group genes from transconjugants containing only one plasmid.

Lanes 1–14: A6T, 42-2T, FS5E1DT, FS2Y1XT, FS6J2CT, 7ganT, 33-2T, FS3Z3CT, 45-6T, 2YG4T, A64T, A78T, FS1Z4S, and CBJ3CT; Lane M1: λ-HindIII marker; Lane M2: DL15000.

https://doi.org/10.1371/journal.pone.0073947.g002