肺炎链球菌分离株的耐药性与毒力基因研究

Investigation of resistant genes and virulence genes of Streptococcus pneumonia

目的探讨新生儿肺炎链球菌分离株耐药性、毒力基因的携带状况与菌株间的亲缘性。方法选择2012年1月-2016年12月期间于医院分娩的未曾出院的新生儿肺炎患儿分离出的39株肺炎链球菌,采用pbp2B种特异聚合酶链反应(PCR)检测判定肺炎链球菌,采用纸片扩散法(K-B法)测定药物敏感性,采用PCR及序列分析的方法分析5种耐药基因(pbp2B突变型、ermB、mefA、tetM、intTn)与10种毒力基因(pspA、pspC、rrgA、nanA、pavA、iga、hysA、lytA、lytB、ply),并对检测结果作样本聚类分析。结果 39株肺炎链球菌对青霉素、红霉素、阿奇霉素、四环素的耐药率均>89.0%,对头孢噻肟、万古霉素、替考拉宁均敏感;39株菌5种耐药基因均有检出,且检出率非常高,大环内酯类药物耐药基因总检出率达100.0%,并检出7种毒力基因;样本聚类分析提示39株菌存在9个小克隆,但具有多样性,9个小克隆分别为1-34-39号株、6-35号株、3-10-19号株、13-16-14号株、15-17-18号株、23-30号株、36-37号株、24-25-26号株、4-28号株。结论本组菌株中检出pbp2B突变型、ermB、mefA、tetM、intTn等5种耐药基因是导致本组菌对青霉素、大环内酯类药物和四环素耐药率高的重要原因,耐药表型与基因型基本对应;分离株携带的6种毒力基因有黏附、侵袭、自溶、细胞毒、生物膜形成等毒力作用。

OBJECTIVE To investigate the distribution of resistant genes and virulence genes,and phylogenetic analysis of Streptococcus pneumonia（S.pneumonia）isolated from newborns.METHODS A total of 39 strains of S.pneumonia were collected from throat swabs of newborn before discharging from the Affiliated Hospital of School of Medicine of Ningbo University from Jan.2012 to Dec.2016.S.pneumonia was confirmed by PCR of species specific pbp2 B.K-B method was used to detect drug susceptibilities.Then,5 kinds of resistant genes（pbp2 B mutation,ermB,mefA,tetM,and intTn）and 10 kinds of virulence genes（pspA,pspC,rrgA,nana,pavA,iga,hysA,lytA,lytB,and ply）were analyzed by PCR,and these genes were used as molecular markers to perform sample cluster analysis.RESULTS Resistant rates of 39 strains of S.pneumonia to penicillin,erythromycin,azithromycin and tetracycline were more than 89%,while all strains were sensitive to cefotaxime,vancomycin,and teicoplanin.The 5 kinds of resistant genes were positive in 39 strains,the positive rates were high,and positive rates of resistant genes to macrolides were 100.0%.The 7 kinds of virulence genes were positive.Sample cluster analysis showed that 9 small clones existed in 39 strains（strain NO.1-34-39,strain NO.6-35,strain NO.3-10-19,strain NO.13-14-16,strain NO.15-17-18,strain NO.23-30,strain NO.36-37,strain NO.24-25-26,and strain NO.4-28）,but had diversity.CONCLUSION The 5 kinds of resistant genes（pbp2 B mutation,ermB,mefA,tetM,and intTn）in this group of S.pneumonia play key role in resistance to penicillin,macrolides and tetracycline.Resistant phenotypes match genotypes basically.The 6 kinds of virulence genes in the isolated strains have virulence of adhesion,invasion,autolysis,cytotoxicity,and biofilm formation.