健康鸡源肺炎克雷伯菌检测到质粒介导喹诺酮耐药qnrA基因

Plasmid-Mediated Quinolone Resistance Determinant qnrA Gene in Klebsiella pneumoniae Isolated from Health Chickens

通过PCR技术检测195株健康动物肠杆菌中qnrA基因的流行情况;琼脂稀释法对qnrA阳性菌株进行8种抗菌药物的药物敏感性试验;质粒接合转移试验研究qnrA的可转移性;Southern杂交对qnrA基因进行定位;PCR检测qnrA阳性菌中Ⅰ类整合酶intI基因的携带情况.结果表明,195株肠杆菌中检出1株qnrA阳性的肺炎克雷伯菌Klebsiella pneumoniae GDKA1,经Blast鉴定为qnrA1.药物敏感性试验表明GDKA1对磺胺甲恶唑、氨苄西林、大观霉素、氯霉素、四环素耐药,对环丙沙星(MIC=0.019 mg·L-1),头孢噻肟(MIC=0.047 mg·L-1)表现敏感,对萘啶酸(MIC=12 mg·L-1)表现为部分敏感.接合试验表明qnrA1位于可转移的质粒上,且可以通过接合试验水平传播.接合子TGDKA1对喹诺酮类药物的敏感性比大肠埃希菌Escherichia coli J53 AzR稍高,但远低于GDKA1·South-ern杂交进一步证实了qnrA1基因位于质粒上.TGDKA1的质粒扩增出intI1,表明qnrA1阳性菌株携带I类整合子.可见,qnrA1基因已在健康鸡源肺炎克雷伯菌中出现且同时携带I类整合子位于质粒上,整合子灵活多样的存在方式和传播方式为qnrA基因的传播提供了便利条件,整合子可能携带qnrA基因通过食物链传播给人,因此健康动物共生菌携带qnrA1基因值得注意.

All of the 195 clinical isolates of Enterobacteriaceae from chickens were screened for the qnrA by PCR.Susceptibility of qnrA-producing to 8 antimicrobial agents was determined by agar disc diffusion method.Conjugation experiment was performed by using Escherichia coli J53 AzR as the recipient strain.Southern hybridization was done with plasmid DNA and genetic DNA.IntIⅠwas screened from the qnrA positive strains.The results showed that a qnrA1 gene was identified in a Klebsiella pneumoniae GDKA1.GDKA1 showed resistance to sulfamethoxazole,ampicillin,spectinomycin,chloramphenicol,and tetracycline,susceptible to ciprofloxacin（MIC= 0.019 mg·L-1） and cefotaxime（MIC=0.047 mg·L-1） and intermediate to nalidixic acid（MIC= 12 mg·L-1）.A transconjugant was selected during the conjugation experiment and hybridization with qnrA1-specific probes revealed that the qnrA1 gene was located in plasmid DNA.Tranconjugant TGDKA1 showed a little higher quinolone MIC than E.coli J53 AzR but not as high as GDKA1.IntI1 was detected in qnrA gene positive strain.In conclusion,qnrA1 gene in K.pneumoniae isolated from health chicken might be transmitted together with class I integron to people through food chain.The presence of qnrA1 gene in health animal commensal bacteria should be noticed.