结核分枝杆菌rpsL和rrs基因突变与氨基糖苷类和多肽类抗结核药物耐药的关系

Relationship between drug-resistance of Mycobacterium tuberculosis to aminoglycoside and polypeptide and gene mutations of rpsL and rrs

目的测定结核分枝杆菌（MTB）临床分离株对链霉素（Am）、卡那霉素（Km）、阿米卡星（Am）、卷曲霉素（Cm）的敏感性，了解其耐药程度和交叉耐药水平，并进一步探讨耐药及交叉耐药与MTB的rrs和rpsL基因突变的关系。方法采用微量平板Alamar Blue检测（MABA）法，检测64株选自北京胸科医院的MTB临床分离菌株的Sin、Km、Am和Cm的最小抑菌浓度（minimum inhibitory concentration，MIC）值；并且对这些菌株rrs和rpsL基因进行序列测定。结果64株MTB临床分离株中，52株对Sm耐药，其中有42株（80．8％）发生rpsL基因突变，包括32株（76．2％）为128A→G（Lys43Arg），其MIC值均≥128／μg／ml；9株（21．4％）263A→G（Lys88Arg），1株（2．4％）263A→T（Lys88Met），MIC值介于4-64μg／ml。9株菌rrs基因514A→C突变，其中，8株MIC值介于4-8μg／ml，1株MIC值为2μg／ml。在2株Sm耐药株（MIC值是4〉g／m1）中没有发现rpsL基因、rrs基因530区及rrs基因1400区突变。18株菌检测到rrs基因1401A→G突变，均对Km和Am高度耐药。其中，15株为rrs基因1401A→G单突变，3株合并rrs基因其他位点突变。18株菌对Sm、Km、Am均耐药，且对Km和Am均高度耐药。18株rrs基因1401A→G突变菌株中，13株菌对Km、Am、Cm均耐药，且均为Km、Am高度耐药耐合并Cm低度耐药；5株菌对Cm敏感（MIC值均为2．5tzg／m1）。64株菌株均有rpsL基因363A→G突变，相应的密码子为121位AAA→AAG突变，其编码的氨基酸均为赖氨酸（Lys）。结论MTB的rpsL基因Lys43Arg、Lys88Arg／Met突变分别可能与Sm高度和中度耐药相关；rrs基因514A→C突变可能与Sm低度耐药相关。rrs基因1401A→G联合514A→C突变，或联合rpsL基因突变可能与Sm、Km、Am均耐药相关。rrs基因1401A→G是Km高度耐药及交叉耐药的分子基础，也可能是Km和Am与Cm部分交叉耐药的分子基础。

Objective To observe the drug resistance and cross-resistance of streptomycin （Sm）, kanamycin （Kin）, amikacin （Am）, capreomycin （Cm） for Mycobacteriurn tuberculosis （MTB） clinical isolates by drug suscepti-bility testing （DST）, and to investigate the association of mutation in MTB gene rpsL and rrs and the resistance and cross-resistance of these drugs. Methods The liquid dilution method Microdilution alamar blue assay （MABA） was performed to detect minimum inhibitory concentrations （MICs） of Sin, Kin, Am, Cm of 64 MTB clinical isolates obtained from Beijing Chest Hospital. Gene rrs and rpsL of 64 MTB clinical isolates were sequenced to analyze the relationship of the mutation in gene rrs and rpsL and the resistance and cross-resistance of Sin, Km, Am, Cm. Results Of the 64 isolates, 52 isolates were resistant to Sm, mutation in eodon 43 or 88 of rpsL gene were observed in 42 （80.8%） isolates, including 32 isolates （76.2%） with mutation in codon 43 （Lys43Arg） with Sm MIC≥128 9g/ml, 9 isolates （21.4%） with mutation in rpsL 263A→G （Lys88Arg） and I isolate （2.4%） with mutation in rpsL 263A→T （Lys88Met）, the Sm MICs of these isolates were 4-64μg/ml. Gene rrs 514A→C were observed in 9 isolates, the MICs of Sm were 4-8 μg/ml in 8 isolates and 2μg/ml in 1 isolate. Gene rrs 1401A→G mutation were observed in 18 isolates which were high-level resistant to both Km and Am, 15 isolates were observed only in Gene rrs 1401A→G mutation, and 3 isolates were combined with other mutation. All these isolates were resistant to Sm, Kin, Am three drugs, and were high-level resistant to Km and Am. Of 18 isolates with gene mutation in rrs 1401A→G, 13 isolates were high-level resistant to Km and Am, and low-level resistant to Cm, 5 isolates were sus-ceptible to Cm （MIC 2. 5μg/ml）. All these 64 isolates were observed 363A→G mutation in gene rpsL, with codon AAA→AAG, they all encode Lys. Conclusion Mutation in codon 43 of rpsL （Lys43Arg） and codon 88 （Lys88Arg/Met） of MTB was likely associated with high-level and moderate-level resistance to Sm, mutation in rrs 514 A→C was likely associated with low-level resistance to Sin. Combination of mutations in gene rrs 1401A→G and gene rpsL, rrs 514 may be the molecular mechanism of resistant to Sm, Km and Am. Mutation in rrs 1401A→G were the molecular basis of cross resistance between Km and Am, maybe also the molecular basis of partly cross resistance between Km/Am and Crn.