脓肿分枝杆菌gyrA和gyrB基因突变与左氧氟沙星和莫西沙星耐药的相关性

Detection of Mutations in the gyrA and gyrB Genes Associated with Fluoroquinolone-resistance among Clinical Isolates of Mycobacterium abscessus in China

目的分析脓肿分枝杆菌gyrA和gyrB基因的突变形式及其与左氧氟沙星和莫西沙星耐药的相关性。方法纳入菌株为2008至2012年首都医科大学附属北京胸科医院分离的、且经16SrRNA、rpoB基因、hsp65和ITS（Internal Transcribed Spacer）测序鉴定为脓肿分枝杆菌临床分离株。用微孔板阿尔玛蓝染色方法测定脓肿分枝杆菌临床分离株对左氧氟沙星和莫西沙星的MIC。PCR扩增临床分离株的gyrA和gyrB基因，并对扩增产物进行测序和序列比对分析。结果70株脓肿分枝杆菌临床分离株，包括45株脓肿亚种和25株马赛亚种，其中68株对左氧氟沙星耐药（10株对莫西沙星耐药），交叉耐药率为14.3％（10株／68株）。不论是脓肿亚种还是马赛亚种，所有临床分离株gyrA基因的喹诺酮耐药决定区（QROR）序列第83位为丙氨酸，而gyrB基因QRDR第447位和464位分别是精氨酸和天冬酰胺。马赛亚种临床株的gyrB基因的第541位氨基酸不同于脓肿亚种临床株，存在苏氨酸置换成丝氨酸的亚种间氨基酸多态性，可用于脓肿分枝杆菌亚种鉴定，但未发现与左氧氟沙星和莫西沙星耐药相关的氨基酸突变。结论脓肿分枝杆菌gyrA和gyrB基因的突变形式与左氧氟沙星和莫西沙星耐药没有明确的相关性。

Objective To determine molecular mutations in gyrA and gyrB genes and their association with fluoroquinone-resistance among Mycobacterium abscessus isolates from China. Methods Antimicrobial susceptibility profile of Mycobacterium abscessus isolates was evaluated for resistance to levoftoxacin （LFX） and moxifloxacin （MFX）. The quinolone resistant determing regions （QRDR） of gyrA and gyrB genes of all the isolates was sequenced and analyzed. Results A total 70 Mycobacterium abscessus isolates were analyzed, including 45（64% ） M. abscessus subsp, abscessus and 25 （36%） M. abscessus subsp, massiliense. Analysis of antimicrobial susceptibility profile showed 97% （n=68） of isolates were resistance to LFX and 14.3% （n=10） of isolates were resistance to MFX. All the MFX-resistant strains were resistant to LFX. Cross-resistance to LFX and MFX was noted in 14.3% （n=10） of isolates. Sequencing analysis revealed that the Ser83Ala substitution in the gyrA gene and Lys447Arg and Ser464Asn substitutions in the gyrB gene were found in all the isolates from both M. abscessus subsp, abscessus and M. abscessus subsp, massiliense. No mutation was found to be associated with cross-resistance t）fM. abscessus to LFX and MFX in the entire gyrA and gyrb gene. Conclusions Our data suggest that there is no clear correlation between the type of mutation in the gene gyrA and gyrB, and the MIC levels of LFX and MFX for resistant M.abscessus strains.