Establishment And Application Of Toxin Genes Profile And Standard Ribotyping Library Of Clostridium Difficile In China

Objective:The aim of this study is to establish the toxin profile and PCR-ribotyping standard library of Clostridium difficile in China,which providing guideline for the domestic epidemiological surveillance.Furthermore,the purpose of this study is to describe the molecular characteristics of using MLST and PCR-ribotyping.This work will help better understanding of the epidemiology of and support construct strategies of control and prevention of C.difficile in China.Methods:Using CCFA medium,latex agglutination,proline paper method,anaerobic biochemical identification system and CDIFF medium,C.difficile was isolated.The amplification of GDH gene,tcdB gene,and 16S rDNA sequencing was used for confirmation of suspected C.difficile isolates.Seven housekeeping genes(adk,atpA,dxr,gly,recA,sodA and tpi)were amplified and sequenced for the MLST of C.difficile.Then sequences were BLAST in the online database(http://pubmlst.org/cdifficile).Specific allele numbers,the sequence type(ST)and clade groups were determined.In this experiment,a total of 100 strains were analyzed by MLST.Construction of complete toxin genes profile:the toxin genes profile of all 212 C.difficile strains were constructed.According to the literature,the primers and PCR condition of toxins and regulation genes targets in PaLoc and CdtLoc of C.difficile were selected.PCR results were showed by QIAxcel Advanced Screening Cartridge.The establishment of ribotyping Library:This experiment was based on the standard strains from European Centre for Disease Prevention and Control Brazier Collection and ATCC to establish ribotyping library.Using QIAxcel Advanced DNA High Resolution Cartridge to analysis of the ribotyping PCR purification products.Results:1 A total of 100 strains of C.difficile were isolated.2 The ST types of 100 strains of Cdifficile were obtained.ST332 is a newly discovered ST type.Most of the isolates were single ST type.ST37,ST54 and ST35 were the most common types in this study.3 Toxin profiles of all the 212 strains of C.difficile were obtained,including the toxin genes and related regulatory genes.The A+B+CDT-toxin profile were the most popular types in China.4 In this study,54 standard strains of C.difficile were analyzed to establishment of.All the 212 strains were analyzed using the standard ribotyping library.RT017 strain is the most found,and RT001,RT012 are also common types in this experiment.One with ST1 type was found which was confirmed as RT027 by ribotyping.In addition,8 isolates of the ST11 type(RT078 type)were found in this study.Conclusion:1 This study comprehensively clarify the molecular epidemiology characteristics of C.difficile in China with a relative large time span and extensive geological areas.2 The standard ribotyping library of C.difficile were constructed in china and ribotyping procedure was standardized.3 The results of MLST showed that the ST types found in China were diverse and ST37,ST3,ST35 and ST54 were the most common 3 types.4 RT017 strain is the most found,and RT001,RT012 are also common types in this experiment.5 The toxin genes and regulatory genes of all the 212 strains of C.difficile in China were determined,which showing the A+B+CDT-profile were preponderance.6 The results showed that ribotyping and ST results were complemented,which emphasize the necessity of the establishment of ribotyping library in China.