| As an important member of quinoxaline-1,4-dioxides (QdNOs), olaquindox has been widely used in farming because of its outstanding growth promoting effect. But the drug resistance problem has become serious. In 2004, the gene conferring resistance to olaquindox oqxAB was detected on the plasmid pOLA52. oqxAB is composed of oqxA and oqxB. It belongs to RND family, and encodes multi-resistance efflux pump. The OqxAB efflux pump not only reduces susceptibility towards QdNOs, but also chloromycetin and quinolones. In 2009, oqxAB was recognized as a plasmid-mediated quinolone resistance (PMQR) gene. Until now, the data about oqxAB is still limited.To investigate the prevalence of oqxAB in E.coli isolates and its resistance mechanism, the prevalence of oqxAB and other PMQR genes in E.coli strains isolated between 1993 and 2014 was investigated. To explore why oqxAB was so popular in China, PFGE analysis and Southern blot were also carried out. The minimum inhibitory concentration (MIC) to olaquindox and mequindox was determined. The associativity among the oqxAB genotype and the resistance phenotype was then analyzed. For special strains, hybridization and gene sequencing were performed to investigate the reason for special cases. The effect of oqxAB on different antibiotics was determined by measuring the MICs of donors and transconjugants to explore the resistance mechanism of oqxAB.1. The prevalence of oqxAB and other PMQR genes in E.coli isolatesThis study detected the prevalence of PMQR genes in 1523 E.coli strains isolated from different sources between 1993 and 2014. The rate of PMQR genes was found to be high. Among all these genes, the prevalence rate of oqxAB was the highest (26.1%), especially in isolates from pigs, then the aac(6’)-Ib-cr (7.6%) and qnrS (6.0%). No qnrC or qnrD positive strain was detected. oqxAB was also found to be with other PMQR genes in the same strain. oqxAB along with aac(6’)-Ib-cr in the same strain was the most common and qnrS was also often found to be with them. In isolates from swine and avian,23.0% and 24.5% oqxAB-positive strains were also positive for other PMQR genes.To explore the reason for the high prevalence rate of oqxAB, PFGE analysis of oqxAB-positive strains was performed and the result showed that the dissemination of oqxAB was not mainly due to the clonal dissemination of positive strains. Human isolates could acquire the resistant genes through different opportunities. The results of SI-PFGE and Southern blot assay illustrated that oqxAB was often found to be located on mobilization large plasmids, which could be an important reason for the popularity of oqxAB. Meanwhile, except oqxAB, other PMQR genes were also found on the same mobilization plasmid, which could brought great threat to the health of human beings.The changes of resistance rate and oqxAB positive rate of avian borne strains between 1993 and 2014 were analyzed. In the period of 1993-2004, there were no significant changes in the resistance rate and oqxAB positive rate, which were both stay low. In 2005, olaquindox was approved in farming and mequindox was also approved subsequently. So in the peiod of 2005-2008, they increased dramatically, and in 2014, have almostly reached the level of 50%. Although the approval number of olaquindox and mequindox had decreased, the effect of the two drugs on the prevalence of oqxAB should not be ignored.2. The resistance mechanism of oqxABThe MIC to olaquindox and mequindox of 776 E.coli isolates from avian was determined. When the MIC to olaquindox and mequindox was equal or greater than 256 μg/ml, there were only oqxAB-positive strains. When the MIC was 128 μg/ml, the strains were almost positive for oqxAB except for few strains without oqxAB. When olaquindox and mequindox MIC was 64 μg/ml, the rate of oqxAB-positive strain (78.2% and 69.6%) was higher than oqxAB-negative strains (21.8% and 23.2%). And when the MIC to the two drugs was 32 μg/ml, the rate of 0qxAB-negative strain (93.0% and 67.1%) was higher than oqxAB-positive strain (7.0% and 32.9%). According to the experimental results, resistance of olaquindox and mequindox was defined with a breakpoint of 64 μg/ml. On the whole,98.6% and 95.0% oqxAB-positive E.coli strains conferred resistance or intermediate to olaquindox and mequindox respectively, and 91.9% and 95.5% oqxAB-negative strains conferred susceptibility of intermediate to olaquindox and mequindox, and the MIC50 and MIC90 of oqxAB-positive strains were much higher than those of oqxAB-negative strains. The oqxAB genotype was almost consistent with the resistance phenotype in E.coli isolates. But there were 45 and 25 strains without oqxAB being resistant to olaquindox and mequindox respectively, and 3 and 11 oqxAB-positive strains being susceptible to the two drugs respectively.Further study about 3 special strains which were positive for oqxAB but susceptible to olaquindox and mequindox was carried out. oqxAB in all of the strains was found to be located on chromosomal. The full-length of oqxAB showed 96%-99% similarity with pOLA52 and the chromosomal segment of Klebsiella. The flanking sequence of all strains showed high similarity with Klebsilla chromosomal. The sequence near oqxA was similar to that of AraC-type transcriptional regulator, which was reported to confer regulation of the express of oqxAB. That might explain why the strains were positive for oqxAB but susceptible to olaquindox and mequindox.The MICs of oqxAB-positive donors and transconjugants to different antibiotics were determined, and the QRDR mutations were also detected. oqxAB made recipient strain resistant to olaquindox and mequindox, and the MICs could be equal to that of donors. Other PMQR genes had no effect on the resistance to the two drugs. For ciprofloxacin, oqxAB only conferred low level of quinolone resistance, which made the MIC raised 2-15 times. But the MIC was still far from the ciprofloxacin resistance breakpoint. Except olaquindox, mequindox and quinolones, the transfer of oqxAB could also cause the transfer of the resistance to ampicillin, tetracycline, chloramphenicol and gentamicin. |
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