四川地区猪源艰难梭菌分子分型调查

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

摘要:

[背景] 艰难梭菌是一种重要的人畜共患肠道病原菌，可引起人和多种动物抗生素相关性腹泻或假膜性肠炎。四川作为我国主要的生猪产区，还未有猪源艰难梭菌流行病学调查的相关报道，对猪源艰难梭菌的防控及保障猪肉安全带来挑战。[目的] 调查四川地区猪源艰难梭菌的感染、流行情况，并对分离出的艰难梭菌进行分子分型研究。[方法] 收集来自四川生猪主要产区6个养殖场中猪的粪便标本（n=110），采用厌氧培养技术在艰难梭菌鉴别培养基上进行分离培养；采用PCR方法扩增艰难梭菌4个毒素基因（tcdA、tcdB、cdtA、cdtB）和7个管家基因（adk、atpA、dxr、glyA、recA、sodA、tpi），对分离株进行毒素基因分型和多位点序列分型。[结果] 从110份样品中，经革兰氏染色镜检及PCR鉴定，共分离出20株艰难梭菌，分离率高达18.18%；毒素基因分型结果显示共获得3种毒素基因型，包括tcdA⁺tcdB⁺cdtA/cdtB⁺（n=3）、tcdA⁺tcdB⁺cdtA/cdtB^—（n=6）、tcdA^—tcdB^—cdtA/cdtB^—（n=11）；多位点序列分型结果显示获得5个ST型，包括ST11（n=3）、ST3（n=1）、ST35（n=2）、ST36（n=4）、ST109（n=10）；进化树结果显示，所有分离株聚集为2个大群，分别为3个分支和17个分支。[结论] 四川主要生猪产区猪群存在艰难梭菌感染，分离株的分子分型呈多样性，主要流行型为ST11、ST3、ST35、ST36、ST109型，并且存在ST11型高毒力菌株流行的风险。

关键词:四川地区;猪;艰难梭菌;毒素基因分型;多位点序列分型

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)