辽宁省大连市耐亚胺培南铜绿假单胞菌产金属β-内酰胺酶基因检测及脉冲场凝胶电泳分型

Analysis on genetic homology and metallo-β-lactamase genes of imipenem-resistant Pseudomonas aeruginosa clinical isolates

目的了解辽宁省大连市分离的耐亚胺培南铜绿假单胞菌(PA)的金属β-内酰胺酶携带情况,探讨该地区临床分离产金属β-内酰胺酶PA的染色体多态性特征。方法选取2013年1月至2014年9月临床分离的400株PA进行菌种鉴定和药物敏感试验,聚合酶链反应(PCR)法扩增产金属β-内酰胺酶的基因,并对扩增阳性产物进行测序确认;脉冲场凝胶电泳(PFGE)检测分析其染色体多态性。结果药敏结果发现有89株PA对亚胺培南耐药,且多数为多重耐药菌株;金属β-内酰胺酶基因PCR扩增阳性11株,其中8株检出IMP-1基因,3株检出VIM-2基因,其余几种金属β-内酰胺酶基因均未检出。89株耐亚胺培南PA的PFGE图谱可分成15种型(A^O),其中A型46株、B型16株、C型4株、D型5株、E型4株、F型3株、G型2株、H型2株,I型至O型分别各有1株。PFGE图谱集中的A^G型各型中的菌株来源于4家不同医院,呈现多态性,每群均存在型别100%相同的克隆株。结论大连地区耐亚胺培南的89株PA多数是多重耐药菌株,产金属β-内酰胺酶的基因型只有2种,为IMP-1和VIM-2。PFGE结果提示该地区菌株虽具有基因多态性,但仍存在相似度较高的流行优势PFGE型别,具有高度的同源性。PFGE具有很强的菌株同源性分析能力,适用于细菌耐药传染源溯源。

Objective To investigate the genetic homology and carriage of metallo-β-lactamase genes of imipenem-resisitant Pseudomonas（ P.） aeruginosa isolated in Dalian,Liaoning province. Methods A total of 400 clinical isolates of P.aeruginosa were collected from four hospitals in Dalian during January 2013-September 2014. After the isolate identification,the drug susceptibility test was conducted following the standard protocol of Clinical and Laboratory Standards Institute（ CLSI）. E-test（ IPM/ EDTA） was dione to detect metallo-β-lactamase gene phenotype,PCR was applied to detect MBL genes,and pulsed-field gel electrophoresis（ PFGE） was performed for homology analysis. Results Eighty-nine imipenem-resistant P. aeruginosa strains were identified through drug susceptibility test,most of which showed multiresistance. Among the 89 strains, only 17 strains screened by E-test were proved to be metallo-β-lactamase positive（ 19. 11%）; 11 strains carried metallo-β-lactamase genes,in which 8 strains carried IMP-1 gene and 3 strains carried VIM-2gene; A total of 15 different PFGE types（ A-O） were identified with 46 strains in type A,16 in type B,4 in type C,5 in type D,4 in type E,3 in type F,2 in type G,2 in type H,1 each in type I-O. The P. aeruginosa belonging to PFGE type A-G were mainly collected from 4 hospitals,which were gene diversity,but the strains in each type shared 100%homology. Conclusion This study showed that the drug resistance of P. aeruginosa is serious in Dalian. The hydrolyzing effect of the metallo-β-lactamase is one of the main resistance mechanism of P. aeruginos to imipenem,the major genotypes were IMP-1 and VIM-2. The result of PFGE suggested that outbreak has not yet occurred in this area. PFGE can be used in the source tracing and molecular epidemiological study of drug resistance and homology of the pathogens.