新疆喀什地区结核分枝杆菌分离株氧氟沙星耐药基因gyrA突变位点分析

Detection and analysis of sites of mutations in the ofloxacin resistance gene gyrA from Mycobacterium tuberculosis isolated in Kashi

目的了解新疆喀什地区、石河子地区及华东地区结核分枝杆菌临床分离株对氧氟沙星的耐药性,分析耐药相关基因gyrA喹诺酮耐药决定区(QRDR)突变的位置及类型,以比例法药敏试验为参照,评价DNA测序技术检测氧氟沙星耐药的效率。方法采用比例法药敏试验检测191株结核分枝杆菌临床分离株对氧氟沙星药物的耐药性;对喹诺酮类药物耐药相关基因gyrA的QRDR区进行扩增与测序,分析耐药相关突变位点。以药敏试验结果为标准,通过Kappa值评价DNA测序和药敏试验在结核分枝杆菌临床分离株氧氟沙星耐药检测的一致性。结果 191株临床分离株结核分枝杆菌中16株为氧氟沙星耐药株,氧氟沙星总耐药率为8.37%。对gyrA基因QRDR区域扩增后测序,共发现4个位点存在突变;分别是第56、74、90、94位密码子。其中,第56位密码子处由CTC→CTG,为同义突变。74位密码子突变均发生在喀什分离株,由GCC→GTA,氨基酸由Ala突变为Val;90位密码子由GCG→GTG,氨基酸由Ala突变为Val;94位密码子突变占临床分离株耐药位点突变的71%,共发现GAC→TAC、GAC→GCC、GAC→GGC3种突变类型,第94位氨基酸Asp分别突变为Thr、Ala、Gly。与药敏试验结果比较,DNA测序对结核分枝杆菌临床分离株氧氟沙星耐药检测具有高度一致性(Kappa=0.895)。结论 gyrA基因第74、90、94位密码子的突变与结核分枝杆菌对氧氟沙星药耐药有关,喀什分离株存在74位新突变位点。用DNA测序技术分析结核分枝杆菌gyrA基因氧氟沙星耐药相关突变对判断结核分枝杆菌氧氟沙星类药物耐药性具有较高的灵敏度和特异性,可为结核分枝杆菌氟沙星耐药性诊断提供依据。

Objectives To investigate the ofloxacin（OFX）resistance of Mycobacterium tuberculosis isolated in Kashi,Shihezi,and East China and to analyze the location and types of mutations in the quinolone resistance-determining region（QRDR）of the gyrAgene.Culture-based phenotypic drug susceptibility testing was used as the gold standard to evaluate the efficiency with which DNA sequencing detected the OFX resistance of M.tuberculosis. Methods The OFX resistance of 191 clinical isolates was analyzed using a traditional drug susceptibility test（DST）.The QRDR in the gyrAgene of each isolate was amplified with PCR and then sequenced.With DST as the standard,the software DNAMAN was used to align sequences to evaluate the consistency with which DST and DNA sequencing detected OFX resistance in clinical isolates of M.tuberculosis based on Kappa values. Results The total OFX resistance of the 191 isolates was 8.37%（16/191）.Four nucleotide substitutions were detected in codons 56,74,90,and 94.Three strains（21.4%）have double-site mutations,including two strains with mutations at codons 90 and 94 and one strain with mutations at codons 56 and94.A mutation from CTC→CTG at codon 56 was a synonymous mutation.Clinical isolates from Kashi had a mutation from GCC→GTA at codon 74,changing the amino acid from alanine（Ala）to valine（Val）.A mutation at codon 90 from GCG→GTG changed the amino acid from alanine（Ala）to valine（Val）.Mutations at codon 94 accounted for 71% of the mutations in clinical isolates.Three mutations from GAC→TAC,from GAC→GCC,and from GAC→GGC respectively changed amino acid 94 from Asp（aspartic acid）to Thr（threonine）,Ala（alanine）,and Gly（glycine）.Kappavalues were analyzed using the software SPSS Statistics23.In comparison to the results of drug sensitivity testing,the results of DNA sequencing were highly consistent in terms of the detection of OFX-resistant strains of clinical isolates of M.tuberculosis（Kappa=0.895）. Conclusion Mutations at codons 74,90,and 94 in the gyrAgene are related to the OFX resistance of M.tuberculosis,and a new mutation at codon 74 was found in isolates from Kashi.DNA sequencing had a high level of sensitivity and specificity and a high degree of consistency in terms of detecting the OFX resistance of clinical isolates of M.tuberculosis.DNA sequencing saves time and it provides a basis for rapid diagnosis of OFX-resistant M.tuberculosis.