深圳地区结核分枝杆菌耐药及广泛耐药分离株的分子特征

Molecular characterization of drug-resistant and extensive-drug resistant isolates of Mycobacterium tuberculosis in Shenzhen of China

目的研究2007--2008年深圳地区MTB耐药和广泛耐药分离株的分子特征。方法参照世界卫生组织和国际防痨与肺部疾病联盟的标准，使用改良罗氏药敏培养基，用1％比例法药敏试验，筛选出针对异烟肼、利福平、链霉素、氧氟沙星和卡那霉素5种药物耐药或敏感的临床分离株，通过PCR扩增筛选菌株的rpoB、katG、rpsL、rrs、gyrA／B和rrs基因的相关序列，运用DNAStar和Blastn进行序列分析。结果筛选出实验菌株123株，其中耐药株73株，全敏感株50株。异烟肼耐药株katG基因突变率为44／52，突变位点全部为S315T或S315N。利福平耐药株rpoB基因突变率为44／47，突变位点主要集中在S531L（30／44）、H526D（9／44）和H526R（1／44）。链霉素耐药株以rpsl基因突变为主，突变位点为K43R（19／28）和K88Q（6／28）；rrs基因突变较少见，仅有491c—T（2／28）和513A—C（1／28），2个基因突变率合计为28／41。氧氟沙星耐药株突变率为11／11，以gyrA基因突变为主，突变位点包括D94A（2／11）、S91P（4／11）和A90V（3／11），3个突变位点总突变率为9／11；S95T存在于所有氧氟沙星耐药株和部分全敏感株gyrA基因中；gyrB未发现突变。卡那霉素耐药株rrs基因突变率为11／18，突变位点为1400A→G（9／11）和1483G→T（2／11）。其他突变位点在全敏感株中未发现。结论MTB耐药基因突变存在一定的地域特性。

Objective To study the molecular characterization of drug-resistant and extensive-drug resistant isolates of Mycobacteriurn tuberculosis （MTB） in Shenzhen of China during 2007--2008. Methods According to the standards of WHO and International Union Against Tuberculosis and Lung Disease （IUATLD）, 136 strains of MTB were collected by performing drug sensitivity test （DST） to isoniazid, rifampicin, streptomycin, ofloxacin and kanamycin on Lowenstein-Jensen in 1% proportion method. Genetic mutations in the corresponding resistance genes （rpoB, katG, rpsL, ITS（1）, gyrA/B, rrs（2）） in these MTB isolates were identified by PCR, followed by DNA sequencing of the purified PCR products. Results A total of 123 isolates were collected. Seventy-three isolates were drug resistant, and 50 isolates were drug susceptible. Among the isolates that were resistant to isoniazid, rifampicin, streptomycin, ofloxacin and kanamycin, the proportion of isolates that harboured mutations in the respective genes was 44/52, 44/47, 28/41, 11/11, and 11/18, respectively. For katG gene, the mutation detected was s315T or s315N. For rpoB, the most frequently found changes were s531L （30/44） and H526D （9/44） or H526R （1/44）. For the reported mutations related with streptomycin -resistant strains, K43R and K88Q were found in the rpsL locus, and 491C→T and 513A→C were found in the rrs gene. For gyrA, all gyrA mutations were clustered in codons 90, 91, and 94 apart from the S95T that was natural polymorphism, accounting for 9/11 of the ofloxacin-resistant isolates, and condon 91 was the most frequently mutated. No mutations were found in gyrB. The most frequent substitutions were 1400A-~G （9/11） and 1483~T （2/11 ） in a specific region of the rrs gene related with kanamycin-resistant strains. No mutations except S95T of gyrA was detected in the drug- susceptible isolates. Conclusions The mutation characterization of drug-resistant and drug- susceptible isolates of MTB was shown to vary according to geographic regions, and these findings can help understanding the molecular characterization and establishing a novel method for the detection of drug-resistant MTB.