养殖龟鳖源气单胞菌耐药性与质粒介导喹诺酮类耐药基因分析

为了解养殖龟鳖源气单胞菌的耐药情况及质粒介导的喹诺酮类耐药(PMQR)、喹诺酮类耐药决定区(QRDR)与耐药表型之间的关系;实验采用K-B纸片法测定了1996—2013年从广东地区患病龟鳖分离的67株气单胞菌对23种常见抗菌药物的耐药性,并检测5种PMQR基因qnrA、qnrB、qnrS、qepA和aac(6')-Ib-cr,同时分析PMQR基因阳性菌株染色体上gyrA、parC基因QRDR的突变情况。结果显示,67株气单胞菌对氨苄西林、头孢噻吩和磺胺复合物的耐药率分别高达100%、92.54%和83.58%,对喹诺酮类药物呈现中等耐药,耐药率介于19.40%~64.18%,而对亚胺培南、呋喃妥因、阿米卡星、头孢噻肟敏感性较高,耐药率低于10%;79.10%(53/67)的菌株对3类或以上抗菌药物具有耐药性。19.40%(13/67)的菌株携带PMQR基因,其中,8.96%(6/67)携带qnrS1基因、5.97%(4/67)携带qnrS2基因、7.46%(5/67)携带aac(6')-Ib-cr基因[其中2株同时携带qnrS2和aac(6')-Ib-cr基因]。13株PMQR基因阳性菌株均分别携带1~4个质粒,大小介于0.8~15 kb;其中6株在gyrA基因及parC基因上均发生变异,3株仅在gyrA基因上发生变异,另外4株未发现QRDR的基因突变。研究表明,广东地区龟鳖源气单胞菌对多种抗菌药物耐药并存在多重耐药现象;而且PMQR机制的存在预示着喹诺酮类耐药性很可能会在水产临床上更加快速而广泛地传播,应引起重视。

Analysis of antimicrobial susceptibility and plasmid-mediated quinolone resistance genes in Aeromonas isolated from turtles

Aeromonas are known to cause several diseases in turtles.Antimicrobial agents,such as fluoroquinolones,are used to prevent and curtail bacterial infections.However,prolonged abuse of antimicrobials agents could result in the selection of fluoroquinolone-resistant Aeromonas.Quinolone resistance is due principally to chromosomal mutations or efflux pump.Mutations in the quinolone resistance-determining regions(QRDRs)of the gyrA and parC genes have been shown to be related to quinolone resistance in Aeromonas spp.Plasmid-mediated quinolone resistance(PMQR)determinants(qnrA,qnrB,qnrS and aac(6')-Ib-cr)have been identified exclusively in Enterobacteriaceae,but have very rarely been identified in Aeromonas.In order to determine antimicrobial susceptibility,the relationship of PMQR,QRDR and resistance profiles in clinical isolates of Aeromonas from turtles in Guangdong province,67 isolates were collected from diseased turtles in 1996-2013.All the isolates were tested for resistance to 23 antimicrobial agents by K-B agar disc diffusion method.The PMQR genes qnrA,qnrB,qnrS,qepA and aac(6')-Ib-cr in all these strains were screened by PCR.Then,mutations in QRDR of the gyrA and parC genes and the plasmids were detected in the PMQR positive strains.All the 67 strains were highly resistant to ampicillin,cephalothin and sulfonamides,the resistance rates of which were 100%,92.54% and 83.58%,respectively;it showed medium resistance to quinolones,whose rates were 19.40%-64.18%; the isolates were more susceptible to imipenem,nitrofurantoin,amikacin and cefotaxime,with the resistance rates below 10%.79.10%(53/67)strains showed multiple-resistance to at least three classes of agents.Of these 67 Aeromonas isolates,19.40%(13/67)harbored PMQR genes.PCR and DNA sequence results showed that 8.96%,5.97% and 7.46% were positive for genes qnrS1,qnrS2 and aac(6')-Ib-cr,respectively,and two strains carried both qnrS2 and aac(6')-Ib-cr.All the 13 PMQR positive strains carried 1-4 plasmids,the sizes of which were between 0.8 kb to 15 kb.Among the 13 isolates with PMQR genes,6 strains showed mutations in both gyrA and parC genes,3 strains carried point mutation in gyrA gene,and no mutations of both genes were found in 4 strains.These results indicated that Aeromonas isolated from turtles in Guangdong were of high level multiple-resistance to widely used drugs.The existence of PMQR genes implied a more widespread dissemination of resistance to quinolones in aquaculture,and this should attract more attention.