结核分枝杆菌gyrA和gyrB基因突变与莫西沙星耐药水平关系

Relationship between mutations of gyrA and gyrB genes and moxifloxacin resistance in Mycobacterium tuberculosis

目的 研究莫西沙星耐药菌株的耐药相关基因突变特征,以及不同突变类型与莫西沙星最低抑菌浓度(minimum inhibitory concentration,MIC)的关系。方法 选取2019—2021年鄂尔多斯市第二人民医院检验科(162株)和首都医科大学附属北京胸科医院细菌免疫室(103株)保存的结核分枝杆菌,采用微孔板稀释法检测这些菌株对莫西沙星的MIC,同时对莫西沙星耐药菌株的DNA拓扑异构酶亚基A (gyrase subunit A,gyr A)和DNA拓扑异构酶亚基B (gyrase subunit B,gyr B)基因进行测序,统计莫西沙星耐药率的差异。结果 本研究总计纳入265株结核分枝杆菌,所有菌株中对莫西沙星耐药82株,耐药率为30.9%。耐多药结核分枝杆菌(multidrug resistant tuberculosis,MDR-TB)和非MDRTB菌株中,莫西沙星耐药率分别为46.1%(70/152)和10.6%(12/113),MDR-TB菌株耐药率显著高于非MDR-TB菌株(P<0.01)。在82株莫西沙星耐药菌株中,总计有59株(72.0%)和3株(3.7%)分别检测到携带gyr A基因突变或/和gyr B基因突变,最常见的突变在gyr A基因的94位点密码子(50.0%)。在gyr A和gyr B突变中,结核分枝杆菌的现代型(30.4%)和古代型(32.0%)差异无统计学意义;gyr A基因的90位点突变株、94位点天冬氨酸突变为酪氨酸以及丙氨酸菌株和gyr B基因突变株大部分表现为对莫西沙星低水平耐药(MIC<2μg/ml);而gyr A基因91位点突变株和94位点由天冬氨酸突变为甘氨酸以及丙氨酸引起高水平莫西沙星耐药,分别有75.0%、53.6%和46.2%的突变菌株(MIC≥2μg/ml)。结论 gyr A基因的94位点突变是莫西沙星耐药菌株最主要的耐药机制,不同gyr A和gyr B突变类型导致对莫西沙星不同的耐药水平。MDR-TB菌株中有46%的菌株对莫西沙星耐药,提示在使用氟喹诺酮前有必要开展药敏试验,以期制订合理的治疗方案。

Objective To investigate the mutation characteristics of drug-resistant genes among moxifloxacin-resistant Mycobacterium tuberculosis isolates,and the relationship between different mutant types and minimal inhibitory concentrations(MICs)against moxifloxacin.Methods The M.tuberculosis isolates were enrolled from clinical laboratory of The Second People􀆳s Hospital of Ordos(162 strains)and Department of Bacteriology and Immunology,Beijing Chest Hospital,Capital Medical University(103 strains)included during 2019-2021.Microporous plate dilution method was used to detect these strains of minimum bacteriostatic concentration(MIC)against moxifloxacin.At the same time,moxifloxacin-resistant isolates were sequenced for genetic mutations in gyrase subunit A(gyrA)and gyrB.The difference of drug resistance rate of moxifloxacin was statistically significant with P<0.05.Results A total of 265 Mycobacterium tuberculosis isolates were enrolled in this study,of which 82 were resistant to moxifloxacin,with a drug resistance rate of 30.9%.In multidrug-resistant tuberculosis(MDR-TB)and non-MDR-TB strains,the drug resistance rate of moxifloxacin was 46.1%(70/152)and 10.6%(12/113),respectively,and the drug resistance rate of MDR-TB strains was significantly higher than that of non-MDR-TB strains(P<0.01).Of the 82 isolates resistant to moxifloxacin,a total of 59(72.0%)and 3(3.7%)were detected to carry gyrA gene mutations or/and gyrB gene mutations,respectively,with the most common mutations at gyrA gene codon 94(50.0%).Among gyrA and gyrB mutations,there was no statistical difference between the modern(30.4%)and ancient types(32.0%)of Mycobacterium tuberculosis.gyrA gene mutant strain 90,aspartic acid mutant tyrosine at 94,alanine strain and gyrB gene mutant strain mostly showed low resistance to moxifloxacin(MIC<2μg/ml).However,high levels of moxifloxacin resistance were caused by mutation at gyrA gene 91 and aspartic acid mutation to glycine and alanine at gyrA gene 94,with 75.0%,53.6%and 46.2%of the mutant strains MIC≥2μg/ml,respectively.Conclusions Locus 94 mutation of gyrA gene is one of the main moxifloxacin-resistant strains of resistant mechanism,and different gyrA and gyrB mutations lead to different resistant level for moxifloxacin.46%of strains with MDR-TB were resistant to moxifloxacin,suggesting the need for drug sensitive test before the use of fluoroquinolone in order to formulate a reasonable treatment regimen.