摘要目的 研究脓肿分枝杆菌复合群亚种鉴定的有效方法.方法 将19株克拉霉素敏感的脓肿分枝杆菌临床分离株扩增hsp65和rpoB基因相关序列,分别用Mega软件和PhyML软件单独及联合对其构建系统进化树以鉴定其亚种,对鉴定结果不一致的菌株进行16S-23S间隔序列测序加以判定.扩增分析rrl和erm(41)基因特征,采用微量肉汤稀释法对菌株进行克拉霉素诱导耐药检测,分析各亚种的基因表型模式.结果 19株脓肿分枝杆菌复合群中,hsp65和rpoB基因鉴定结果一致的有Mycobacterium massiliense(M.massiliense)亚种10株、Mycobacterium abscessus(M.abscessus)亚种4株和Mycobacterium bolletii(M.bolletii亚种1株,其余4株采用hsp65基因分类为M.massiliense,采用rpoB基因分类为M.abscessus,最终采用间隔序列测序鉴定为M.massiliense.rrl基因分析19株均未发现与克拉霉素耐药相关的突变.erm(41)基因分析结果表明,M.abscessus的-35位启动子序列与M.bolletii和M.massiliense不同,第28位碱基存在多态性(T28或C28),M.bolletii和M.massiliense的-35位启动子序列及第28位碱基分子特征一致.14株M.massiliense菌株均存在erm(41)基因276 bp长片段和2 bp短片段缺失,4株M.abscessus和1株M.bolletii均携带完整的erm (41)基因.克拉霉素诱导耐药试验结果显示,3株携带T28的M.abscessus(菌株号:CI02、CI04和CI12)和1株M.bolletii均可诱导产生克拉霉素高水平耐药,其余14株M.massiliense和1株携带C28多态性的M.abscessus(菌株号:CI17)对克拉霉素始终保持敏感.暂未发现-35位启动子序列差异与克拉霉素诱导耐药相关.结论 hsp65基因适用于脓肿分枝杆菌复合群亚种分子鉴定,且脓肿分枝杆菌复合群的erm(41)基因分子特征及相关克拉霉素诱导耐药表型特征均有亚种特异性,hsp65基因和erm(41)基因检测可用于脓肿分枝杆菌复合群亚种鉴定.

abstractsObjective To identify the subspecies of Mycobacterium abscessus (M.abscessus) group.Methods The corresponding genes (hsp65 and rpoB) in 19 clinical isolates (CIs) of M.abscessus group susceptible to clarithromycin were amplified by PCR.Phylogenetic analyses and subspecies identification of the hsp65 gene and rpoB gene were conducted separately and jointly by using the Mega program and PhyML program,and the conflicting species were further identified by the internally transcribed spacer (ITS) sequencing.The rrl and erm(41) of M.abscessus group detection were performed by PCR sequencing,and MICs of inducible resistance to clarithromycin were determined by the broth microdilution method,and then the phenotypic patterns of 19 isolates were analyzed.Results Comparisons between hsp65 and rpoB sequences of the 19 clinical isolates led to the identification of 10 CIs as Mycobacterium (M).massiliense,4 CIs as Mycobacterium (M).abscessus,and 1 CI as Mycobacterium (M).bolletii,while the other 4 isolates were identified as M.massiliense by hsp65 gene sequencing and as M.abscessus by rpoB gene sequencing.Ultimately the 4 conflicting isolates were identified as M.massiliense by ITS sequencing.Nomutations in the rrl gene with clarithromycin resistance were found.The-35 sequence of the erm (41)promoter of M.abscessus was different from that of M.bolletii and M.massiliense,and the nucleotide at position 28 was polymorphic (T28 or C28) ;-35 sequence and the nucleotide at position 28 were the same in erm(41) for M.bolletii and M.massiliense.Fourteen M.massiliense strains shared 100％ (14/14) homology for erm(41) with 276 bp deletions and 2 bp deletions,and no deletions were found in 4 CIs as M.abscessus and 1 CIs as M.bolletii.The elarithromyein inducible resistance test showed that 3 M.abscessus with T28 (CI02,CI04,CI12) and 1 M.bolletii (CI18) strains were highly resistant,and the other 14 M.massiliense and 1 M.abscessus with polymorphic C28 (CI17) strains remained susceptible.No correlations between-35 sequence of erm(41) promoter and clarithromycin inducible resistance were found.Conclusions Hsp65 is applicable to the identification of the subspecies of M.abscessus group,and due to the fact that subspecies of M.abscessus group shows distinct genotytpcally feature in erm(41) sequencing and phenotypic feature in clarithromycin susceptibility,hsp65 and erm(41) can be applied to the identification of subspecies of M.abscessus group,and the resulting data may be useful for clinical diagnosis and treatments.