高毒力艰难梭菌的基因型和毒力相关基因检测

摘要

Objective To investigate the genotype and variance of toxin associated genes of moxifloxacin‐resistant Clostridium difficile clinical isolates in Sydney .Methods Twenty‐two moxifloxacin‐resistant Clostridium difficile clinical isolates were collected from Sydney ,which were genotyped by using sequencer capillary gel electrophoresis based PCR‐ribotyping ,and toxin A and B cod‐ing gene tcdA and tcdB ,and binary toxin coding gene cdtA and cdtB were detected by using PCR method .Toxin regulator gene tc‐dC was analyzed by using PCR‐sequencing ,and was aligned with reference sequence of VPI 10463 (Genbank accession number :X92982) ,and the tcdC sequence types of all 22 isolates were identified by using blast tool in NCBI .Results Twenty‐one isolates were genotyped as hypervirulent PCR‐ribotypes 027 (RT027) ,and one isolate as RT078 ;all 22 isolates contained tcdA and tcdB for toxin A and B and cdtA and cdtB for binary toxin (tcdA+ tcdB+ cdtA+ cdtB+ ) .The tcdC sequence types of the 21 RT027 i‐solates belong to sc1 ,and that of the one RT078 isolate belongs to WA39 .Compared with tcdC reference sequence of VPI 10463 ,a consecutive 18 bp deletion (nt341 to 379) and one nucleotide deletion at position 117 were found in the 21 RT027 isolates ,and a consecutive 39 bp deletion (nt330 to 368) and one nucleotide mutation at position 184(C> T) were found in the one RT078 isolate . Conclusion Clostridium difficile hypervirulent RT027 was the common moxifloxacin resistant genotype ;Clostridium difficile hy‐pervirulent RT027 and RT078 clinical isolates contained genes for toxin A and B and binary toxin ,and contained gene sequence mu‐tation in toxin regulator gene tcdC .%目的：鉴定莫西沙星耐药的艰难梭菌临床菌株的基因型和毒素相关基因的携带情况。方法22株莫西沙星耐药的艰难梭菌临床菌株收集自澳大利亚悉尼地区，利用基于荧光毛细管电泳的聚合酶链反应（PCR）‐核糖体分型技术对其进行基因分型；利用PCR方法检测其毒素A、B编码基因 tcdA、tcdB和二元毒素编码基因cdtA 、cdtB；利用PCR‐基因测序方法检测毒素调节基因tcdC的基因序列，通过与参考菌株VPI 10463（Genbank收录号：X92982）比对了解其基因突变情况，通过与Genbank中的序列比对确定其序列型。结果21株菌株为高毒力核糖体型027（RT027），另1株为RT078；22株菌株毒力编码基因均为 tcdA＋tcdB＋ cdtA＋ cdtB＋；21株RT027 tcdC序列型为sc1，另1株RT078序列型为WA39，与参考株VPI 10463比较，RT027菌株tc‐dC序列均出现连续18个核苷酸（nt330‐347）的缺失和第117位单核苷酸的缺失，RT078菌株则出现连续39个核苷酸（nt341‐379）的缺失和第184位（C＞ T ）的突变。结论高毒力 RT027是最常见的莫西沙星耐药的艰难梭菌基因型；高毒力 RT027和RT078均携带毒素A、B和二元毒素编码基因，且毒素调节基因 tcdC均存在基因突变。