脓肿分支枝杆菌embB基因耐乙胺丁醇的分析

目的 分析脓肿分支枝杆菌的embB基因,以探讨其耐乙胺丁醇的分子机制.方法 用16S rRNA基因序列分析法鉴定5株脓肿分枝杆菌临床株,测定乙胺丁醇对临床株及标准株( ATCC 19977)的最低抑菌浓度(MIC).PCR扩增embB基因的全序列,将所测序列进行生物信息学分析.结果 乙胺丁醇对5株脓肿分支杆菌标准株和临床株的MIC均为128μg/mL.,属高度耐药.从脓肿分支枝杆菌的标准株和临床株均扩增出约3200 bp片段,与GenBank中脓肿分支枝杆菌标准株的embB基因大小一致.5株临床株与标准株比较,其核苷酸序列存在9个点突变,在突变位点所编码的氨基酸序列中,仅第18位、87位、770位密码子编码与标准株不同的氨基酸.6株脓肿分支枝杆菌的embB基因与对EMB敏感的结核分支杆菌标准株(H37RV)的相应基因序列比较,第303-305位密码子的核苷酸序列存在差异,但仅第303、304位核苷酸编码的氨基酸序列不同,第306位密码子的核苷酸序列无差异.结论 脓肿分支杆菌对乙胺丁醇的耐药并非embB基因的突变所致,为embB基因天然存在结构的不同,属于天然耐药.结构的差异与第306位密码子无关,可能与第303、304密码子有关.

Analysis on the resistance of embB genes of Mycobacterium abscesses to ethambutol

Objective To investigate the molecular mechanism of the resistance of M.abscesses to ethambutol(EMB).Method 5 strains of M.abscesses isolated clinically were identified by analyzing their 16S rRNA gene sequence.The minimum inhibitory concentration(MIC) of EMB to clinical strains and standard strains(ATCC 19977) were detected.The complete sequences of embB gene was amplified by PCR,and then analyzed with bioinformatics.Result The 5 strains of clinical strains and standard strains of M.abscessus were highly resistant to EMB,the MIC of EMB to them all was 128 μg/mL.The fragments about 3 200 bp whose sizes were consistent with that of the embB gene of M.abscessus standard strain in GenBank were amplified from the standard strain and clinical isolateds of M.abscessus respectively.There were 9 point mutations in the corresponding nucleotide sequences of the clinical isolates comparing with the standard strain.In the amino acid sequences which were coded by the nucleotides of the point mutations,only those of the amino acid sequences coded by the 18th,87th,770th codons were different from standard strains.The 303th-305th codons except the 306th one in the corresponding nucleotide sequence of embB genes from the 6 strains of M.abscessus were different compared with the standard strains of M.tuberculosis(H37RV) which was sensitive to EMB,but only the 303rd and 304th amino acid sequences encoded by the nucleotide were different.Conclusion The resistance of M.abscessus to ethambutol was independent from the mutations and was dependent on the difference of the structure of embB gene.It belongs to natural resistance.The differences may be not related to the 306th codon,but to the 303rd and 304th codons of embB gene.