PCR Ribotyping And Toxin Gene Polymorphism Of Clostridium Difficile In China

Objective To understand the PCR-ribotype distribution of clinic toxigenic Clostridium difficile strains in four areas, and obtain sequence of tcdA and tcdB. In our study, we make analysis of polymorphism, and discuss the possible phylogeny characteristics based on the sequence of tcdA and tcdB. At the same time, to find the suitable genie locus, we combine the sequence of tcdA and tcdB and PCR-ribotype. Our study offer data for the further research on epidemiology, source tracing, and developing suitable molecular method for detecting.Methods A total of 104 strains from Beijing, Guangdong, Shandong, Henan were collectd. All strains were performed toxin genotype and PCR-ribotyping. We download Clostridium difficile 630(A+B+ strain) and M68(A-B+ strain) from genbank, and primer design was taken according to template of Clostridium difficle 630 and M68, and contigs were determined by DNAStar v7.1 software. Analysis of data including base pair matching and grouping, single nucleotide polymorphism, and phylogeny were made based on sequences. Furthermore, we performed bioinformatics and genome comparison analysis on sequence of tcdA and tcdB, and the numers of SNPs on the site of various PCR-ribotypes of tcdA and tcdB were figured out manually. At last, we conducted a statistical analysis based on clinical data. Results In this study,we obtained PCR-ribotypes of 104 clinical Clostridium difficile strains from four different areas of China, and divided into 20 types. Genetic characteristics of Clostridium difficile strains were different between western country and China.In some areas of China, RT017 was the dominant strain (21.15%), followed by RT046/043 (15.53%), RT001 (14.56%). This study also received a different type of representative strains tcdA and tcdB sequence,50 sequences respectively. The results found that, the gene polymorphism of toxin A and toxin B were obvious, nonsynonymous SNP mutations and deletions in tcdA base sequence occurs mainly in the receptor binding domain, suggesting that the region experienced intense positive evolutionary selection pressure. There was no large fragment base deletion phenomenon in tcdB sequence, but the SNP was significantly higher than tcdA, and strains between the mutation rate should be greater than tcdA, a faster rate of evolution, which focuses on non- synonymous mutations SNP glucosyltransferase domain and auto-protease domain. This phenomenon prompted the presence or HGT recombinant toxin genes or entire PaLoc area is conducive to the evolution of the strain preserve their dominant gene sequences. After analysis, PCR-ribotypes do exist some relationship with the toxin gene sequences, such as 12 strains were RT001 and the sequences all were A05B04,11 strains were RT017 and 9 of them were A11B07,9 strains were RT046/043 and 7 of them were A02B02,3 strains were RT012 which were A08B01.Conclusion The predominated type of Clostridium difficile was RT017 (20.39%), followed by RT046/043 (15.53%), RT001 (14.56%), and there was a regional phenomenon. It was popular with RT017 in Beijing and Guangdong, RT046/043 in Henan and RT001 in Shandong. This study showed that we should establish appropriate detection methods for different regions. The main toxin type of Clostridium difficile was A+B+. Also tcdA and tcdB had high genetic variation and polymorphism. Single nucleotide polymorphism (SNP) and large fragment nucleotide sequence deletions of tcdA occurred in the 3’ end of the receptor binding domain. SNP of tcdB was higher than tcdA, and it mainly occurred in glucosyltransferase domian. This study explored the relationship between the gene sequences of toxin and PCR-ribotypes, and found that there was some relationship between them. For example,12 strains were RT001 and the sequences all were A05B04,11 strains were RT017 and 9 of them were A11B07,9 strains were RT046/043 and 7 of them were A02B02,3 strains were RT012 which were A08B01. We can build an appropriate method which can show not only the toxin type of Clostridium difficile but also the PCR-ribotype, and which was important implications for the rapid detection and treatment of Clostridium difficile infection.