Molecular analysis of toxin genes and tcdC genotypes among clostridium difficile isolates in King Chulalongkorn Memorial Hospital

Abstract

Clostridium difficile, a Gram-positive, anaerobic spore-forming bacterium, is a major cause of antibiotic-associated diarrhea and pseudomembranous colitis known as C. difficile-associated disease (CDAD). Major virulence factors contributing to the diseases are toxins A (TcdA) and B (TcdB) produced by toxigenic strain. With the increased frequency and severity of CDAD, this study aimed to characterize Clostrdium difficile isolates in King Chulalongkorn Memorial hospital for the presence of genes encoding toxins A and B, binary toxin and mutation in TcdC gene. From August 2011 to September 2012, 1,114 stool samples from suspected CDAD patients were cultured anaerobically. Suspected C. difficile colonies characterized by cell morphology, odor, and fluorescence under 365-nm UV illumination were isolated from 242 samples. Toxigenic C. difficile cultures were confirmed by multiplex polymerase chain reaction (PCR) targeting a species-specific internal fragment of triose phosphate isomerase (tpi) gene, toxin A gene (tcdA) and toxin B gene (tcdB). Of 242 C. difficile culture, 235 were confirmed to be C. difficile which 149(63.40%) were toxigenic and 86(36.60%) were non-toxigenic. Of 149 toxigenic C. difficile; toxins A and B-positive (A+B+) C. difficile were found in 84 (56.38%) samples and toxin A-negative, toxin B-positive (A-B+) C. difficile were found in 65 (43.63%) samples. Toxin A- positive, toxin B- negative (A+B-) C. diffcile was not found. Binary toxin investigated by multiplex PCR targeting cdtA and cdtB revealed that binary toxin-positive C. difficile (No.38) was found in one sample. C. diffcille No.38 which also harbor TcdA and TcdB genes was subjected to tcdC sequencing and the result showed 18 bp in frame deletion resulting in the truncate peptide of 232 amino acids. Clinical data available from 176 patients revealed that toxigenic C. difficile isolates were recovered from patients aged 60 years (69.81%), corresponding with the finding that advanced age is the risk factor of toxigenic C. difficile infection. In conclusion, the prevalence of toxigenic C. difficile in suspected CDAD patients was 13.37% and the hypervirulent strain was not found in this study.