广州地区儿童分离株青霉素耐药肺炎链球菌青霉素结合蛋白基因分型

Penicillin-binding proteins genotyping of penicillin resistance Streptococcus pneumonia isolated from children in Guangzhou area

目的了解广州地区儿童分离株青霉素耐药肺炎链球菌(PNSP)的分子流行病学,为肺炎链球菌感染性疾病防控提供实验依据。方法设计特异性引物,PCR扩增PNSP青霉素结合蛋白基因PBP1A、PBP1B、PBP2A、PBP2B、PBP2X、PBP3,进行测序分析。并使用Hinf I对PCR产物进行酶切,分析其限制性片段性长度多态性(RFLP)。结果成功提取PNSP的DNA、PCR结果显示:50株耐青霉素肺炎链球菌中:含PBP1A、PBP1B、PBP2A、PBP2B、PBP2X及PBP3的菌株阳性率分别为48.9%、64.4%、71.1%、31.1%、40.0%、31.1%。测序显示,它们与GenBank已知序列的同源性分别为99%、98%、100%、97%、95%、100%。使用Hinf I的RFLP分析显示:PBP1A、PBP1B、PBP2A、PBP3仅有一种基因型;PBP2B、PBP2X有两种基因型的,阳性率分别为71.4%、28.6%、66.7%、33.3%。结论广州肺炎链球菌儿童分离株耐青霉素基因的分布以PBP2A、PBP1B、PBP1A为主。使用Hinf I酶切,PBP2B、PBP2X具有两种基因型,其中优势型均大于65%。

Objective To understand the molecular epidemiology of penicillin resistance Streptococcus pneumonia（PNSP）isolated from children in Guangzhou area to provide the experimental basis for clinical prevention and control of Streptococcus pneumonia infectious diseases.Methods Specific primers were designed according to Genebank,penicillin binding protein（PBP）genes PBP1 A,PBP1B,PBP2 A,PBP2B,PBP2 X,PBP3were amplified by PCR.The sequencing analysis was performed.The PCR products were digested by Hinf I,and the restriction fragment length polymorphism（RFLP）was analyzed.Results DNA of PNSP was successfully extracted,the PCR results showed that in 50 strains of PNSP,the positive rates of bacterial strains containing PBP1 A,PBP1B,PBP2 A,PBP2B,PBP2 Xand PBP3were 48.9%,64.4%,71.1%,31.1%,40.0% and 31.1% respectively.The sequencing showed that their homologies with known sequences in GenBank were 99%,98%,100%,97%,95% and 100% respectively.Using RFLP in Hinf I showed that PBP1 A,PBP1B,PBP2 Aand PBP3only had one kind of genotype,PBP2 Band PBP2Xhad two kinds of genotypes,the positive rates were 71.4%,28.6%,66.7% and 33.3% respectively.Conclusion The gene distribution of PNSP strains among children in Guangzhou is dominated by PBP2 A,PBP1Band PBP1 A,there are two subtypes in PBP2 B,PBP2X when digested by Hinf I,in which the predominant subtype〉65%.