| The quinoxaline-di-N-oxide olaquindox (OQX) has been widely used as a growth promoter in pig farming, which has been considered as a relatively safe antibiotic. Since the drug has been used in the aquaculture for a long tine, drug resistance problem has become increasingly serious. In2003, a conjugative plasmid, pOLA52, conferring resistance to olaquindox was isolated from Escherichia coli of porcine origin from Denmark. The OqxAB efflux pump reduced susceptibility towards chloramphenicol and quinolones. Because the OqxAB efflux pump reduced susceptibility towards quinolones, it was recognized as a plasmid-mediated quinolone resistance (PMQR) determinant in2009. Until now, the data on the epidemiology and resistance mechanism of OqxAB were limited.In this study, it aimed to investigate the epidemiology and genetic elements of the efflux pump gene, oqxAB in different sources of E.coli strains. It determined the mequindox and olaquindox minimum inhibitory concentration (MIC) of the isolates and defined a resistance breakpoint. It analysed the associativity among the oqxAB genotype and mequindox and olaquindox resistance phenotype. Resistance of mequindox and olaquindox in different sources of E.coli strains was also investigated and evaluated. Conjugation experiments were carried out between oqxAB-positive E.coli strains and receptor strain E.coli J53. It also determined the mequindox and olaquindox minimum inhibitory concentration of the transconjugants and the resistance to other kind of antimicrobial agents to evaluate the effect of oqxAB gene and the cotransfer of the other kinds of antibiotical resistance. The prevalence and distribution of oqxAB in different sources of E.coli strains in2011and2012were also been investigated. Testing for chromosomal and/or plasmidic location was carried out using genomic DNA and subsequent hybridization. The full-length of oqxAB and flanking DNA were sequenced and identified the genetic elements.1. Prevalence of the oqxAB gene in Escherichia coli isolates from humans, animals and the environmentIt determined the mequindox and olaquindox minimum inhibitory concentration of the1017E.coli strains. The MICs of mequindox and olaquindox for only oqxAB-positive E.coli strains were256μg/ml or128μg/ml. When the MICs of mequindox and olaquindox were64μg/ml, the rate of oqxAB-positive E.coli strains (95.4%,76.8%) was higher than oqxAB-negative E.coli strains (4.6%,23.2%). The MIC50s (64μg/ml,64μg/ml) of mequindox and olaquindox for oqxAB-positive E.coli strains were higher than MIC5os(4μg/ml,16μg/ml) of mequindox and olaquindox for oqxAB-negative E.coli strains. From what had been discussed above, resistance of mequindox and olaquindox was defined with a breskpoint of64μg/ml. In different sources of E.coli strains,92.4%and99.1%oqxAB-positive E.coli strains conferred resistance or intermediate to mequindox and olaquindox respectively.99.4%and95.2%oqxAB-negative E.coli strains conferred susceptibility or intermediate to mequindox and olaquindox respectively. It implied that the oqxAB genotype is very consistent with the mequindox and olaquindox resistance phenotype in E. coli isolates. The resistance of mequindox and olaquindox was higher in pig isolates (33.3%,41.5%) than in chicken and environmental isolates. In addition to animal E. coli isolates,2.6%human E. coli isolates showed resistance to the two drugs. The resistance of mequindox and olaquindox was higher in animal intestinal E. coli isolates (16.8%,23.2%) than in extraintestinal isolates (11.2%,13.9%). Environmental isolates (36.4%) appeared the highest resistance to olaquindox.Thirty five transconjugants were successfully obtained from210oqxAB-positive E.coli strains by conjugation experiments. The33transconjugants conferred resistance to mequindox and olaquindox, and the MICs of mequindox and olaquindox for the other2transconjugants were32μg/ml. Five classes of antimicrobial agents which include ampicillin (68.6%), tetracycline (85.7%), chloromycetin (74.3%), nalicixic acid (65.7%) and gentamicin (71.4%) were able to cotransfer with oqxAB gene together with resistance. OqxAB conferred resistance to mequindox and olaquindox and contributed to different level decreases in susceptibility to other antibiotics. For the other antibiotics, the transconjugants showed4-256and2-128fold increases in the MICs of chloromycetin and nalicixic acid respectively, when compared with the recipient.This study evaluated the prevalence of PMQR genes in150E. coli isolates from different sources between2011and2012. PMQR determinants were present in74(49.3%) isolates. qnrB, qnrS, qepA and oqxAB were detected alone or in combination in0.7%,7.3%,1.3%and35.3%, and the prevalence of oqxAB gene was the highest among these genes. Prevalence of oqxAB gene was higher in pig isolates (63.6%,7/11) than in chicken isolates (40%,48/120). It detected the occurrence of oqxAB in isolates from pigeon for the frist time. The oqxAB gene was detected in20.2%of1022Escherichia coli isolates which collected between1993and2010in China. The prevalence of oqxAB in E.coli strains isolated from2011to2012was higher than those previously studied. However, a surprisingly high prevalenc of oqxAB (41.6%) was recently detected in E. coli isolates from different sources in2002-2004.2. The chromosomal and/or plasmidic location for oqxAB gene in the different strainsTesting for chromosomal and/or plasmidic location was carried out using genomic DNA and subsequent hybridization in35transconjugants and35donators and26isolates which transfer of the plasmid was not successful. The sizes of oqxAB plasmids from35transconjugants and35donators were varying from40kb to360kb. There were34donators that oqxAB signals only in large plasmid. And there was another one donator that oqxAB signals in both chromosomal and large plasmid (350kb,310kb). The sizes of oqxAB plasmids from26isolates were varying from43kb to310kb. There were25isolates that oqxAB signals only in large plasmid. And there was another one that oqxAB signals in chromosomal.The full-length of oqxAB and flanking DNA matched completely the sequences in pOLA52by DNA sequencing. Moreover, a BLAST search indicated that OqxA and OqxB sequences from2E. coli isolates in this study showed96%-99%similarity with the corresponding sequences from the chromosomal segment of Klebsiella pneumoniae MGH78578, NTUH K2044and342. Up to now,17oqxAB sequences from E. coli were available in GenBank (accession numbers1X412478, JX469117, JX442218, JX429863, JX294475, JF912901, HQ674771, HQ674770, AB601773, AB601772, AB601771, AB601770, GQ497565, GQ120634, EU370913, JF912902, GU477622), sharing identical amino acid sequences.4oqxAB genes from E. coli which were available in GenBank (accession numbers JX469117, JX429863, EU370913, GU477622) were flanked by IS26. The oqxAB gene in this study flanked by IS26was identical to the chromosome segment (composite transposon Tn6010) of K. pneumoniae MGH78578, which suggested that the dissemination of oqxAB among different E. coli strains might be mediated by the mobile element. |
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