CTX-M-14和CTX-M-24编码基因的检测及其功能表达

目的　了解上海华山医院临床分离大肠埃希菌和肺炎克雷伯菌中产生的超广谱 β 内酰胺酶 (ESBL)的基因型及其流行情况。方法　双纸片法和美国国家临床实验室标准委员会 (NCCLS)表型确定实验检测产ESBL菌株并对产ESBL菌株进行接合实验 ;提取产ESBL菌株及其转移接合子中的质粒 ,进行电泳分析 ;采用琼脂稀释法 ,测定各种菌株对多种抗菌药物的敏感性 ;TEM、SHV、CTX M 1组、CTX M 13组、Toho 1组通用引物检测产ESBL菌株及其转移接合子 ;编码框以外设计引物、聚合酶链反应 (PCR)扩增转移接合子中的CTX M 13组全编码基因 ,克隆入 pHSG398质粒、表达 ,并对PCR产物进行测序 ,分析其基因型 ;对这些产ESBL菌株进行脉冲场电泳 (PFGE)检测。结果　产ESBL菌株对多种 β 内酰胺类和非 β 内酰胺类抗生素耐药 ;产ESBL菌株可通过接合转移方式传递其耐药性 ,转移频率多为 10 -4～ 10 -5,多数非 β 内酰胺类抗生素的耐药性可以和ESBL耐药性发生共转移 ,琼脂糖电泳显示接合子从供体菌得到了一个 >2 3 1kb的质粒 ;转移接合子中仅CTX M 13组通用引物检测为阳性 ;4株转移接合子中CTX M 13组全编码基因序列与CTX M 14编码基因序列的同源性为 10 0 % ,其余 6株转移接合子中CTX M 13组全编码基因序列与CTX M 2 4编码基因序列的同源

Identification and expression of blaCTX-M-14 and blaCTX-M-24

OBJECTIVE: To identify the ESBL gene and the prevalence in Escherichia coli and Klebsiella pneumoniae strain isolated from Huashan Hospital, Shanghai. METHODS: Isolates were confirmed as an ESBL producing strain by double-disk synergy test and NCCLS Confirmatory Test. Antibiotic susceptibilities were determined by standard agar dilution procedure on Mueller-Hinton agar. To determine whether the resistance was transferable, the conjugation experiment was performed; plasmids were isolated from clinical isolates and transcojugants. The partial bla(gene) of ESBL producing isolates and their transcojugants were detected by PCR using universal primers for TEM, SHV, CTX-M-1group, Toho-1group, CTX-M-13group respectively. The entire bla(CTX-M-13) group were amplified by PCR using the primers outside the Open Reading Frame (ORF) of CTX-M-13group beta-lactamases; the PCR products of entire bla(CTX-M-13)group were cloned into vector and the recombinant plasmids were transformed into the recipient strain for expression; the PCR products were also directly sequenced and analyzed; the clinical isolates of ESBL producers were detected by PFGE. RESULTS: ESBL producers were resistant to most beta-lactams and non-beta-lactams. Most transconjugants were obtained at frequency of 10(-4) approximately 10(-5) and resistance to non-beta-lactams was cotransferred with the ESBL activity to the transconjugant. A plasmid of about > 23.1 kb was obtained from each tansconjugant by plasmid extraction. Partial gene amplification products of CTX-M-13 group gene were obtained from isolates and their transconjugants. The bla(CTX-M-13)group from 4 transconjugants were identified as bla(CTX-M-14), and other six were bla(CTX-M-24); those ESBLs were mediated by plasmids (> 23.1 kb); the transformants producing CTX-M-14 or CTX-M-24 were resistant to most beta-lactams, which were much more resistant to cefotaxime than to ceftazidine; PFGE patterns of those isolates were different. CONCLUSION: clinical isolate of Escherichia coli and Klebsiella pneumoniae isolated from Huashan Hospital, Shanhai produced CTX-M-14 or CTX-M-24, which caused the isolate resistant to most beta-lactams; no clone spread in those isolates was found.