猪源艰难梭菌的分离鉴定与生物学特性分析  

Isolation and Biological Characterization of Clostridioides difficile from Pigs

作　　者：张悦 艾伟诚 王斐 梁婉 谢思思 华琳 李春辉[3] 彭忠[1] 吴斌[1] ZHANG Yue;AI Weicheng;WANG Fei;LIANG Wan;XIE Sisi;HUA Lin;LI Chunhui;PENG Zhong;WU Bin(College of Veterinary Medicine,State Key Laboratory of Agricultural Microbiology,The Cooperative Innovation Center for Sustainable Pig Production,Huazhong Agricultural University,Wuhan 430070,China;Animal Husbandry and Veterinary Institute,Key Laboratory of Prevention and Control Agents for Animal Bacteriosis,Ministry of Agriculture and Rural Affairs,Hubei Academy of Agricultural Science,Wuhan 430070,China;Infections Control Center,Xiangya Hospital of Central South University,Changsha 410008,China)

机构地区：[1]华中农业大学动物医学院农业微生物学国家重点实验室生猪健康养殖协同创新中心,武汉430070 [2]湖北省农业科学院畜牧兽医研究所农业农村部畜禽细菌病防治制剂创制重点实验室,武汉430070 [3]中南大学湘雅医院感染与控制中心,长沙410008

出　　处：《中国动物传染病学报》2022年第2期27-33,共7页Chinese Journal of Animal Infectious Diseases

基　　金：湖北省创新行动计划项目(2018skjcx05)。

摘　　要：为了了解我国猪源艰难梭菌(Clostridioides difficile,CD)的病原学及分子生物学特征,本研究收集湖北和广东地区疑似患有肠炎的病猪粪便样品,提取基因组DNA,利用已经报道的5重PCR方法对艰难梭菌的16S rDNA基因、毒素A的编码基因tcdA、毒素B的编码基因tcdB以及二元毒素编码基因cdtA和cdtB进行检测,同时采用厌氧培养法,对PCR检测毒素编码基因为阳性的粪便样品进行艰难梭菌的分离培养,并对所分离到的菌株进行药物敏感性测试和全基因组重测序。结果发现:本研究所采集到的97份粪便样品经PCR检测显示有80份样品为16S rDNA阳性,其中仅有2份样品为tcdA和tcdB阳性。从这两份样品中分离出一株tcdA和tcdB阳性的艰难梭菌,命名为HuB01。药敏试验发现,HuB01对头孢菌素(FOX)、氯霉素(CHL)、克林霉素(CLI)、氨苄西林(AMP)、头孢曲松钠(CRO)耐药,对莫西沙星(MXF)和万古霉素(VAN)表现出中度耐药,而对利福西明(RFX)、甲硝哒唑(MTZ)、非达霉素(FDX)敏感。全基因组重测序发现HuB01的全基因组大小为3.93 Mb,平均GC含量为28.31%,编码3691个蛋白基因及63个tRNA和10个rRNA;3691个基因中含有31个耐药基因和164个毒力相关基因。多序列位点分型(MLST)结果显示HuB01为ST11。由于目前国内对于艰难梭菌的研究大多集中在人医领域,因此本研究可为兽医领域艰难梭菌的相关研究奠定基础。To understand the etiological and genetic characteristics of swine Clostridioides difficile,fecal samples were collected from pigs with suspected enteritis in Hubei and Guangdong provinces for DNA isolation and detection for the 16S rDNA,the toxin A encoding gene tcdA and the toxin B encoding gene tcdB as well as the binary toxin encoding genes cdtA and cdtB using a documented 5-plex PCR method.Bacterial isolation was performed on fecal samples positive for the toxin-encoding genes through anaerobic cultures.Antimicrobial susceptibility testing and whole genome sequencing were also performed on the bacteria isolated herein.The results showed that 80 out of 97 fecal samples were positive for the 16Sr DNA gene and 2 samples were also positive for tcdA and tcdB.Bacterial isolation was performed on these 2 fecal samples carrying tcdA and tcdB.As a result,one strain of C.difficile was isolated and designated as HuB01,which was confirmed positive for tcdA and tcdB but negative for cdtA and cdtB.Antimicrobial susceptibility testing showed that HuB01 was resistant to cephalosporin,chloramphenicol,clindamycin,ampicillin and ceftriaxone,moderately resistant to moxifloxacin and vancomycin but susceptible to rifaximin,metronidazole and fidaximicin.Whole genome sequencing revealed that the genome of HuB01 was approximately 3.93 Mbp in length with an average GC content of 28.31%.This genome contained a total of 3691 protein coding genes,63 tRNAs and 10 rRNAs.These protein coding genes contained 31 resistance genes and 164 virulence related genes.Mutilocus sequence typing using the whole genome sequence showed that HuB01 was a ST11 clone.As most of the studies on C.difficile currently concentrated on the field of human medicine,the results of this study laid a foundation for future study in the field of veterinary medicine.

关 键 词：艰难梭菌 分离鉴定 PCR检测 药敏试验 全基因组重测序 

分 类 号：S858.61[农业科学—临床兽医学]