Molecular typing investigation of Clostridium difficile from pigs in Sichuan, China

Abstract:

[Background] Clostridium difficile is an important zoonotic enteric pathogen that can cause antibiotic-associated diarrhea or pseudomembranous colitis in human and various animals. As the main pig producing area in China, Sichuan has not yet reported on the epidemiological investigation of Clostridium difficile from pigs, which brings challenges to the prevention and control of Clostridium difficile from pigs and the guarantee of pork safety. [Objective] To investigate the infection and prevalence of Clostridium difficile from pigs in Sichuan province, and to study the molecular typing of the isolated Clostridium difficile. [Methods] Stool samples (n=110) of pigs suspected of being infected with Clostridium difficile from six farms in Sichuan were collected and isolated on the identification medium of Clostridium difficile by anaerobic culture. Four toxin genes (tcdA, tcdB, cdtA, cdtB) and seven housekeeping genes (adk, atpA, dxr, glyA, recA, sodA, tpi) of Clostridium difficile were amplified by PCR method. Toxin genotyping and multilocus sequence typing were performed on the isolates. [Results] In 110 samples, 20 strains of Clostridium difficile were isolated by Gram staining microscopy and PCR identification, and the isolation rate was as high as 18.18%. Toxin genotyping results showed that three toxin genotypes were obtained, including tcdA⁺tcdB⁺cdtA/cdtB⁺(n=3), tcdA⁺tcdB⁺cdtA/cdtB^—(n=6), tcdA^—tcdB^—cdtA/cdtB^—(n=11). The multilocus sequence typing result showed that five ST types, including ST11 (n=3), ST3 (n=1), ST35 (n=2), ST36 (n=4), and ST109 (n=10). The phylogenetic tree results showed that all the isolates were clustered into two groups with 3 branches and 17 branches, respectively. [Conclusion] Clostridium difficile infections were found in pig herds in the main pig producing areas in Sichuan, and the molecular types of isolates were diverse. The main epidemic types were ST11, ST3, ST35, ST36 and ST109, and there was the risk of ST11 strain with high virulence.

Key words:Sichuan;pig;Clostridium difficile;toxin genotyping;multilocus sequence typing (MLST)