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

【作者】 杨虹； 王辉； 彭黎明； 杨启文； 孙宏莉； 徐英春； 陈民钧；

【机构】 北京大学深圳医院检验科；

【摘要】 目的了解近年来发现的质粒介导耐药机制在肠杆菌临床株对喹诺酮类耐药中所起的作用及2006年qnr和aac(6′)- Ib-cr在中国的流行情况。方法按筛选条件:环丙沙星(CIP)≥0.25ug/ml、头孢噻肟(CTX)≥2.0ug/ml、头孢曲松(CRO)≥2.0ug/ ml的弗劳地枸橼酸杆菌(cfr)、阴沟肠杆菌(ecl)、大肠埃希菌(e- co)、肺炎克雷伯菌(kpn),从全国九家教学医院收集的974株非重复的革兰氏阴性杆菌中共筛选出197株,其中cfr有30株,ecl有35株,eco有77株,kpn有55株。用PCR扩增方法检测筛选菌株的qnrA、qnrB、qnrS和aac(6′)-Ib质粒介导耐药基因;并以内切酶BtsCI的酶切消化aac(6′)-Ib PCR阳性产物确定aac(6′)-Ib- cr。以质粒接合试验(E.coli J53AZ为受体菌,叠氮钠和磺胺作为质粒选择药)了解喹诺酮耐药的可转移性;用琼脂稀释法测定结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,以了解qnr和aac (6′)-Ib-cr引起的喹诺酮类耐药水平及其他的药敏信息。结果在197株筛选出的肠杆菌中,qnr共检出83株,总阳性率为42.1%(83/197),其中qnrA有17株,qnrB有46株,qnrS有24株;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,总阳性率为45.7%(90/197)。aac(6′) -Ib-cr共检出36株,总阳性率为18.3%(36/197);在aac(6′)-Ib阳性株中,aac(6′)-Ib-cr阳性率高达40%(36/90)。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65.7%、65.5%、63.3%、6.5%,aac (6′)-Ib-cr阳性检出率分别为8.6%、21.8%、26.7%、16.9%。质粒接合试验获得了13株结合子,环丙沙星对接合子的MIC范围为0.125～1ug/ml,相差8倍;结合子与受体菌相比,CIP的耐药性提高了16-125倍,LEV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临床分离株中高度流行;质粒介导的喹诺酮耐药可转移;qnr和aac(6′)-Ib-cr能介导喹诺酮低水平耐药,有的甚至已达到折点,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。更多还原

【Abstract】 Objective To investigate the importance of recent- lydiscovered plasmid—mediated quinolone resistance and the prevalence of qnr and aac(6’)-Ib-er in China in 2006.Methods A total of 197 clinical isolates of enterobacteriaceae(Citrobaeter fre- undii,Enterohacter cloacae,Escheriehia coil and Klebsiella pneu- moniae with ciprofloxacin≥0.25ug/ml,cefotaxime≥2.0ug/ml,and ceftriaxone≥2.0ug/ml)were screened from the 974 non-repetitive clinical isolates collected from the nine teaching hospitals in China, and among these,30 isolates were Citrobaeter freundii,35 isolates Enterobacter cloacae,77 isolates Escherichia coli and 55 isolates Klebsiella pneumoniae. All the screened isolates were detected for the plasmid—medi- ated quinolone resistance gene of qnr(qnrA,qnrB and qnrS)and aac (6’)-Ib by PCR,and all positive isolates for aac(6’)-Ib were further analyzed by digestion with BtsCI(New England Biolabs)and/or di- rect sequencing of the PCR products to identify aac(6’)-Ib-cr.Con- jugation experiments were done with azide-resistant E.coli J53 as a recipient,with selection for plasmid-encoded antimicrobial resistance (Sulfamethoxazoh),and azide counterselection.This test proved the plasmid—mediated quinolone resistance is transferable.The minimal inhibitory concentration(MICs)of ciprofloxacin and other antibacte- rial agents were determined by agar dilution method for the conju- gants,the donors and the recipient,so as to research the quinolone resistance level resulted from the qnr and aac(6’)-Ib-cr and the oth- er information.Results Qnr was present in 42.1%(83/197)of isolates ,and among these,17 isolates carried qnrA,46 isolates carried qnrB ,24 isolates carried qnrS,2 isolates carried qnrA and qnrB,and 2 isolates carried qnrB and qnrS;aac(6’)-Ib was present in 45.7%(90/ 197)of isolates,40%(36/90)of which carried the cr variant responsi- ble for low-level ciprofloxacin resistance;18 isolates carried qnr and aac(6’)-lb-cr.Qnr was present in 65.7% of Enterobaeter cloacae isolates,65.5% of Klebsiella pneumoniae isolates,63.3% Citrobacter freundii isolates,and 6.5% of Escherichia coli isolates respectively; aac(6’)-Ib-cr was present in 8.6% Enterobacter cloacae isolates, 21.8% KlebsieUa pneumoniae isolates,26.7% Citrobacter freundii isolates,16.9% Escherichia coli isolates respectively.The 13 transconjugants showed 16-to 250-fold increases in the MICs of ciprofloxacin and 16-to 31-fold increases in the MICs of lev- ofloxacin relative to that of the recipient.Conclusion Transferable plasmid—mediated low level quinolone resistance associated with qnr and aac(6’)-Ib-cr widely existed in the entembacteriaceae strains and perhaps this contributes to the rapid increase of bacterial resistance to quinolones in China.更多还原