| Objective To make analysis of polymorphism and evolution of tcdA and tcdB data from sequencing clinic toxigenic Clostridium difficile strains in China and reference strains, together with data of other sequences of tcdA and tcdB from genBank, and discuss the possible phylogenic characteristics based on the sequence genetic feature of tcdA and tcdB from varying strains of types, all of which offering data for the further research on epidemiology, source tracing, and developing technology of molecular diagnosis.Methods 70 tcdA and tcdB sequences were gained from 70 clinic isolates of clostridium difficile (66 from various geographic sources in China and 4 references) by sanger sequencing after toxin genotyping and MLST, and primer design was taken according to template of Clostridium difficle 630 and M68, and contigs were determined by DNAStar v 7.1 software. Analysis of data including base pair matching and grouping, single nucleotide polymorphism, and phylogeny were made based on sequences gained before and others downloaded from genBank. Furthermore, we performed bioinformatics and genome comparison analysis on primers using for detecting tcdA and tcdB from literature, and the numers of SNPs on the site of various types of tcdA and tcdB were figured out manually. At last,2 primers were designed for detecting tcdA and tcdB in view of analysis of polymorphism and primer compare, followed by verification of clinic isolates.Results A total of 85 tcdA and 95 tcdB sequences were gained from 66 clinic isolates in China and 29 isolates abroad. Diversity exists obviously both in tcdA and tcdB sequences.10 TST tcdA and 5 TST tcdB were newfound compared with that in genBank. Events of nonsynonymous mutation of SNP and deletion mainly occurred in receptor binding domain of tcdA, which indicated strongly positive evolutionary selection here. There is no deletion in tcdB, however, the mutation rate in tcdB was higher than that of tcdA. Event of nonsynonymous mutation of SNP primarily located in glucosyltransferase domain (B12, B16) and receptor binding domain (B13, B14, B15, B16), providing clues of gene recombination or horizontal gene transfer in toxin genes, even the whole Paloc, which are beneficial for retention of favorable sequence in the process of evolution. Different from Europe and USA, the dominant strain was A15B16 (ST37, A-B+) in China, and this type was speculated as a potential threat in clinic and public control area in line with the characteristic of genetic sequence and its drug resistance.Conclusion This is a systematic analysis for tcdA and tcB of Clostridium difficile, which also is a supplement for gene library of tcdA and tcdB. We reinforced the high diversity in the two genes, and different epidemic strains possessed distinctive profiles of sequence, of which is not the same between China and Western. Considering the complex situation of epideology of Clostridium difficile in China, a optimized and suitable molecular method for detecting in short time was urgent for strains identification and collection on a large scale, which may take good advantage of the polymorphism research. In addition, it is necessary for us to promote basic research about toxigenic Clostridium difficile, like toxin virulence in different isolates, for further discussion and verification. |
