肠杆菌科细菌喹诺酮耐药基因qnrB的研究  

Study on qnrB-mediated quinolone resistance among Enterobacteriaceae species

作　　者：马晓波[1] 吕晓菊[1] 陈慧莉[1] 高燕渝[1] 母丽媛[1] 俞汝佳[1] 杜蓉[1] 牛菲菲[1] 

机构地区：[1]四川大学华西医院感染性疾病中心,成都610041

出　　处：《中国抗生素杂志》2009年第2期114-119,共6页Chinese Journal of Antibiotics

摘　　要：目的对2005年10月～2006年4月临床分离的120株肠杆菌科细菌检测qnrB的分布,同时研究qnr阳性菌株与blaSHV、blaCTX-M或质粒型AmpCβ-内酰胺酶基因的关系,为进一步研究质粒介导的喹诺酮类耐药机制(PM QR)提供实验依据。方法采用碱裂解变性法提纯质粒DNA,PCR法检测qnrA和qnrB的存在,接合实验验证qnr的转移性。扩增qnrB全序列、测序。对所有qnr阳性菌株,采用nitrocefin法检测qnr阳性菌产β-内酰胺酶情况,多重PCR法检测blaSHV、blaCTX-M及多种质粒型AmpCβ-内酰胺酶基因。结果120株肠杆菌科细菌中,qnrA和qnrB的检出率分别为30.8%与27.5%,其中13株细菌同时检出qnrA和qnrB;肠杆菌属、克雷伯菌属细菌及大肠埃希菌qnr的检出率分别为52.9%、59.3%和50.0%。17株细菌可扩增出681bp的全序列片段。测序结果显示,部分菌株包含qnrB1;另有4株细菌的qnrB序列相同,但与qnrB3相比存在3个位点核苷酸序列突变(201位A→T,271位T→G,498位G→A)而导致编码蛋白的氨基酸存在2个位点差异,为新的qnrB亚型(qnrB6),GenBank注册号为:EF520349。所有qnr阳性菌均可使nitrocefin纸片变红色。qnr阳性菌中,49株(89.1%)包含blaSHV或blaCTX-M;约70.0%可检测到质粒型AmpC相关的β-内酰胺酶基因,分别有18和16株细菌可扩增到blaDHA及blaEBC。结论我院临床存在qnr阳性菌株的流行,发现一种新的qnrB亚型(qnrB6)。Objective To investigate the prevalence of qnrB among 120 unique strains of Enterobacteriaceae species isolated from Oct 2005 to Apr 2006 in our Hospital and to study the relationship between blaSHV, blaCTX-M or plasmid-mediated AmpC β-1actamase gene and qnr-positive bacteria. Method Plasmid DNA were extracted with E. Z. N.A. Plasmid Mini kit and screening of qnrA and qnrB was carried out by PCR amplification. Transferrance of qnr in vitro was determined by conjugation experiments. The NCBI BLAST program was utilized for sequence comparisons. The existence of β-1actamase in qnr-bearing strains was evaluated by nitrocefin test. Among qnr-positive bacteria, detection of the blaSHV, blaCTX-M-3-like, blaCTX-M-4-like and bla gene for plasmid-mediated AmpC β-1actamase were done by multiplex PCR. Results qarA, qnrB were detected in 37（30. 8% ） and 33（27.5% ） isolates, respectively, and 13 isolates carried both genes. The prevalence of qnr in Enterobacter sp., Klebsiella sp. and E. coli was 52.9% 59.3% and 50. 0%, respectively. The complete sequence of 618bp was amplified in 17 isolates carried qnrB. The sequence of qnrB in some strains was perfectly identical to that of qnrB-1, while a new qnrB-like gene was found among 4 isolates, which differs from qnrB3 by 3 nucleotides （201 A→T,271T→G,498G→A） resulting in mutation of 2 amino acids （Lys67Asn,Ser91Ala）, named as qnrB6（Accesion No. EF520349）. blaSHV or blaCTX-M were present in 49 strains of qnr-bearing Enterobacteriaceae, and almost 70% of qnr-positive isolates mainly blaEBC, blaDHA were detected to harbor gene for plasmid-mediated AmpC β-1aetamase. Conclusion Evidences suggested the emergence of qnrB -borne Enterobacteriaceae species in West China, and qnrB6, a new subtype of qnrB was also described.

关 键 词：质粒介导的喹诺酮类药物耐药 qnrB bla基因 水平传播 

分 类 号：R978.19[医药卫生—药品]