革兰阴性菌qnrA基因介导的喹诺酮耐药分子机制研究

目的了解革兰阴性菌质粒介导qmA耐药基因的发生率、分子遗传学背景及其阳性株的耐药谱。方法收集2004年4月-2006年4月对萘啶酸耐药的临床分离无重复株共629株，采用特异引物PCR结合测序进行qnrA阳性株的识别，表型确认试验结合PCR检测识别产ESBL或AmpC酶的qnrA阳性株，Kirby-Bauer法和Etest法进行qnrA阳性株的药敏检测，质粒接合转移及Southem杂交检测进行qmA基因的质粒定位，PCR策略克隆携带qnrA基因整合子基因结构并进行引物步移测序。结果qnrA阳性株的总检出率为1.9％（12／629）。菌种分布为肺炎克雷伯菌2．2％（3／138），阴沟肠杆菌17．1％（6／35），产气肠杆菌9．1％（1／11），枸橼酸杆菌属12．5％（1／8），沙门菌属14．3％（1／7）。qnrA基因定位在80～180kb大小质粒上的su／1型Ⅰ类整合子基因结构中。其中4株菌qnrA基因定位在整合子In37上，另外8株菌qnrA基因定位在一种新型的整合子InX上。所有qnrA阳性株均产ESBL，并具有可转移多重耐药的特征。结论广东地区喹诺酮抗菌药耐药株中存在着质粒介导的耐药机制，但发生率较低；其耐药基因qnrA的水平传播能力有可能导致细菌耐药性的播散。

Mechanisms of qnrA-mediated quinolone resistance in gram-negative bacteria in southern China

Objective To investigate the prevalence of the plasmid-encoded quinolone resistance gene qnrA in epidemical gram-negative bacteria, and its molecular genetic background as well as the resistance profile in the isolates harbouring qnrA gene. Methods A total of 629 nalidixic-acid resistant isolates of nonrepetitive gramnegative bacteria were collected in southern China between April 2004 and April 2006. qnrA-positive strains were screened by PCR using specific primers combining with DNA sequencing. ESBL or AmpC-producing isolates were distinguished by the phenotypic confirmatory test combining with DNA sequencing. Antibiotic susceptibility testing for qnrA-positive strains was carried out by Kirby-Bauer and E-test method. Plasmid conjugation and Southern hybridization was performed to determine the location of qnrA gene. The integron structures containing qnrA gene were cloned by PCR strategy and sequenced by primer walking. Results The incidence of qnrA-positive strains in nalidixic-acid resistant bacteria was 1.9%(12/629). qnrA gene was identified in 2.2%(3/138) K. pneumoniae, 17.1%(6/35) E. cloacae, 9.1%(1/11) E.aerogenes, 12.5%(1/8) C. freundii and 14.3%(1/7) S. chol eraesuis . The qnrA gene was embedded in complex sul1-type integrons located on plasmids that varied in size (from 80 to 180 kb, respectively). Among them, four qnrA -positive isolates carried integron In37, other eight ones carried a new integron InX. All qnrA-positive isolates were ESBL producing and transferable multiple drug-resistance strains. Conclusion The qnrA gene was uncommon among clinical isolates in Guangdong, but its identification in twelve isolates from southern China indicated the emergence of this quinolone resistance mechanism. In the future, horizontal gene transfer may become a more important means of conferring resistance to quinolones and other antibiotics.