中国九家教学医院肠杆菌科细菌质粒介导的喹诺酮耐药机制研究

目的 研究质粒介导的喹喏酮类的耐药基因qnr和aac(6')-Ib-cr在我国肠杆菌科临床株中的分布状况.方法 从全国9家教学医院收集的421株非重复的4种肠杆菌科细菌[大肠埃希菌(eco)、肺炎克雷伯菌(kpm)、阴沟肠杆菌(ecl)、弗劳地柠檬酸杆菌(cfr)]中,按环丙沙星(CW)≥0.25μg/ml、头孢噻肟(CTX)≥2.0μg/ml、头孢曲松(cno)≥2.0μg/ml的条件筛选出197株,其中cfr30株,ecl 35株,eco 77株,kpm 55株.采用PCR检测筛选菌株的qnrA、qnrB、qnrS和aac(6')-IB质粒介导耐药基因;并以内切酶BtsCI酶切消化aac(6')-Ib PCR阳性产物以确定aac(6')-Ib-cr.接合试验确定喹诺酮耐药的可转移性;用琼脂稀释法测定接合子及其供体菌和受体菌对环丙沙星及其他抗菌药的MIC值.结果 在197株筛选出的肠杆菌科细菌中,qnr共检出83株,总阳性率为42%(83/197),其中qnrA有17株(9%),qnrB有46株(23%),qnrS有24株(12%);qnrA和qnrB同时阳性有2株,qnrB和qnrs同时阳性有2株.aac(6')-Ib共检出90株(46%),aac(6')-Ib-cr共检出36株(18%).qnr和aac(6')-Ib-cr同时阳性的有18株.在ecl、kpn、cfr、eco中,qnr的阳性率分别为66%、66%、63%、6%,aac(6')-Ib-cr阳性检出率分别为9%、22%、27%、17%.qnr和aac(6')-Ib-cr在所有的4种肠杆菌科细菌中的阳性率分别为20%(83/421)和9%(36/421).质粒接合试验获得了13株接合子,环丙沙星对接合子的MIC范围为0.125～1μg/ml,相差8倍;接合子与受体菌相比,CIP的耐药性提高了16～125倍,左氧氟沙星的耐药性提高了16～31倍.结论 在中国,qnr和aac(6')-Ib-cr相关的质粒介导喹诺酮类耐药性在肠杆菌科临床分离株中分布广泛;qnr和aac(6')-Ib-cr能介导喹诺酮低水平耐药,这可能是我国细菌对喹诺酮类耐药性上升迅速的重要原因.

The mechanism study of plasmid-mediated quinolone resistance in clinical isolates of Enterobacteriaceae from nine teaching hospitals in China

Objective To investigate the prevalence of plasmid-mediated quinolone resistance qnr and aac(6')-Ib-cr in Enterobacteriaceac in Chiha.Methods A total of 197 clinical isolates with ciprofloxacin≥0.25μg/ml,cefotaxime≥2.0μg/ml and ceftriaxone≥2.0 μg/ml were screened from the 421 non-repetitive clinical isolates of Enterobac teriaceae(Escherichia coli,Klebsiella pneumoniae,Citrobacter freundii,and Enterobacter cloacae)from the nine teaching hospitals in China.qnrA,qnrB,qnrS and aac(6')-Ib gene were detected by PCR.aac(6')-Ib-cr gene was further identified by the digestion with BtsCI followed by sequencing.Conjugation experiments were done.The MIC of ciprofloxacin and other antibacterial agents in donor strain and acceptor strain were determined by agar dilution.Results Qnr was present in 42%(83/197)of isolares,and among these,17 isolates carried qnrA(9%),46 isolates carried qnrB(23%),24 isolates carried qnrS(12%),2 isolates carried qnrA and qnrB,and 2 isolates carried qnrB and qnrS.aac(6')-Ib was present in 46%(90/197)of isolates,40%(36/90)of which carried the cr variant responsible for low-level ciprofloxacin resistance.18 isolates carried qnr and aac(6')-Ib-cr.Qnr wag present in 66% of Enterobacter cloacae isolates,66% of Klebsiella pneumoniae isolates,63% of Citrobacter freundii isolates,and 6% of Escheriehia coil isolates,respectively,aac(6')-Ib-cr was present in 9% of Enterobacter cloacae isolates,22% of Klebsiella pneumoniae isolates,27% of Citrohacter freundii isolates,and 17% of Escherichia coil isolates,respectively,qnr and aac(6')-Ib-cr were present in 20% (83/421)and 9% (36/421) of all isolates respectively. The 13 transconjugants showed 16 to 125 fold increases in the MICs of ciprofloxacin and 16 to 31 fold increases in the MICs of levofloxacin relative to that of the recipient Conclusion Transferable plasmid-medlated low level quinolone resistance associated with qnr and aac(6')-Ib-cr widely exists in the enterobacteriaceae strains and perhaps this may contribute to the rapid increase of bacterial resistance to quinolones in China.