我国结核分枝杆菌gyrA不同突变类型对氟喹诺酮类药物耐药水平的相关性研究

Analysis of the relationship between different gyrA mutations and fluoroquinolone resistance levels in Mycobacterlum tuberculosis isolates from China

【摘要】目的研究gyrA基因在我国耐氟喹诺酮类药物结核分枝杆菌菌株中的突变特征，以及不同突变类型与氟喹诺酮类药物最低抑菌浓度（MIC）的关系。方法2007年我国开展结核病耐药基线调查，调查选取了70个调查点，共分离结核分枝杆菌4017株，其中145株为对氧氟沙星耐药的结核分枝杆菌菌株，有7株菌株在传代中发生污染或传代失败，本研究共纳入138株对氧氟沙星耐药的结核分枝杆菌菌株。对上述菌株的氟喹诺酮类药物耐药相关基因（gyrA）进行测序，分析耐药相关基因突变的特征；用微孔板稀释法检测这些菌株对氧氟沙星、左氧氟沙星和莫西沙星的MIC，统计不同突变类型菌株对氟喹诺酮类药物耐药率的差别。采用SPSSIi．0统计软件进行统计学处理，同种突变类型对不同氟喹诺酮类药物耐药率的比较采用Fisher确切概率法进行检验，以P〈O．05为差异有统计学意义。结果在138株氧氟沙星耐药菌株中，总计有90株（65．200）检测到氟喹诺酮类药物耐药相关基因gyrA发生突变，其中最常见的突变发生在第94位点（34．8％，48／138）。gyrA基因第88、89和91这3个位点的突变菌株（分别为2株、3株和5株），除莫西沙星有2株在91位点表现为高水平耐药，其余均表现为对3种氟喹诺酮类药物低水平耐药；第94位点突变类型较多，导致不同的耐药水平，第94位点天冬氨酸突变为天冬酰胺时氧氟沙星、左氧氟沙星和莫西沙星的高水平耐药比例分别为80．0％（8／10）、30．0％（3／10）和100．0％（10／10）；此外，1株第94位点天冬氨酸突变为半胱氨酸的菌株同时表现为对3种氟喹诺酮类药物的高水平耐药；而第94位点突变为丙氨酸时与低水平氟喹诺酮类药物耐药有关，其对氧氟沙星、左氧氟沙星和莫西沙星低水平耐药率分别为91．7%（11／12）、100．0％（12／12）和58．3％（7／12）。结论gyrA基因突变是我国人群对氟喹诺酮类药物耐药菌株最主要的耐药机制，不同gyrA突变类型导致对氟喹诺酮类药物不同的耐药水平。

Objective To investigate the prevalence of genetic mutations located in the gyrA gene among fluo- roquinolone （FQs）-resistant Mycobacterium tuberculosis isolates, and the relationship between different mutations and minimal inhibitory concentrations （MIC） against fluoroquinolones. Methods The national drug resistance baseline survey conducted in 2007 randomly enrolled 70 clusters from 31 provinces. A total of 4017 M. tuberculosis strains were collected, of which 138 were ofloxacin （Ofx）-resistant. All isolates were sequenced for genetic mutations in gyrA conferring FQ resistance. The broth dilution method was used to determine the MIC of these isolates against Ofx, levofloxacin （Lfx） and moxifloxacin （Mfx）. Fisher＇ s exact test was performed to compare the high-level resistance rates of different FQs. P〈0.05 was considered to be statistically significant. Results Of the 138 Ofx-resistant M. tuberculosis isolates, 90 （65.2%） isolates harbored gyrA mutations, the most frequent mutation being in codon 94 （34. 8% ,48/138）. Strains with mutations in codons 88, 89 or 91 of gyrA exhibited low-level FQ- resistance. Codon 94 mutations were diverse and were associated with different levels of FQ-resistance. When the mutation of codon 94 resulted in substitution of Asp by Asn, high-level Ofx-, Lfx- and Mfx-resistance was observed in 80.0% （8/10）, 30.0% （3/10） and 100.0% （10/10） of the isolates, respectively. In addition, one strain harbo ring a substitution of this Asp by Cys exhibited high-level resistance to Ofx, Lfx and Mfx In contrast, we found that strains harboring a substitution of this Asp by Ala had low level FQ resistance; 91.7% （11/12）, 100.0% （12/12） and 58.3% （7/12） of these isolates were resistant to low-levels of Ofx, Lfx and Mfx, respectively. Conelusien Mutation of gyrA is the most important mechanism that confers FQ resistance in China. Strains with different types of gyrA mutations are associated with different levels of FQ resistance.