院内感染艰难梭菌的毒素检测及核糖体分型研究

Toxin detection and ribotyping of nosocomial Clostridium difficile strains

目的了解艰难梭菌临床分离株的毒力情况,并运用核糖体分型技术进行流行病学研究。方法采集住院腹泻患者的未成形粪便标本,无水乙醇处理后接种CDMN选择培养基行艰难梭菌分离培养,并通过革兰染色、耐氧试验和凝集试验行菌落鉴定;提取菌株DNA,选择特异性引物用PCR法扩增毒素基因tcdA和tcdB;核糖体分型针对细菌基因组16S～23S rDNA间区序列进行扩增,并根据电泳带型的多态性实现分型。结果共计分离得44株艰难梭菌,3种毒素型分别为A+B+型、A-B+型和A-B-型,分别占57%、34%和9%;18种核糖体型中以R8型和R4型为主,分别占20%和18%。结论本研究临床分离的艰难梭菌菌株中,毒素型以A+B+型为主,核糖体分型存在相对优势的型别;无证据提示存在院内艰难梭菌感染的暴发流行。

Objective To investigate the virulence of Clostridium difficile clinical isolates, and conduct an epidemiologic study by PCR-ribotyping for the strains. Methods The unformed stool samples from hospitalized patients with diarrhea were collected, and were inoculated onto CDMN selective culture media after pretreatment with dehydrated alcohol for the culture of Clostridium difficile. Isolates were identified by Gram staining, oxygen tolerance test and agglutination assay. Bacterial genome DNA was extracted from the Clostridium difficile strains, and toxin gent tcdA and tcdB were amplified by PCR with specific primers. Meanwhile, PCR-ribotyping was performed through amplification of the genomic 16S-23S rDNA intergenic spacer region sequence, followed by electrophoresis to distinguish different ribotypes according to the polymorphism of the bands. Results Forty-four Clostridium difficile strains were divided into 3 toxin types： A ＋ B ＋ strains, A - B ＋ strains and A - B - strains, which accounted for 57%, 34% and 9%, respectively. All the strains belonged to 18 ribotypes, mainly ribotype R8 （20%） and ribotype R4 （18%）. Conclusion In the study, the Clostridium difficile clinical isolates were mainly A ＋ B ＋ strains, and there existed relatively predominant ribotypes, but no evidence suggested nosocomial outbreak of Clostridium difficile infection.