利福平耐药结核分枝杆菌对氟喹诺酮类药物表型耐药与其基因突变的一致性研究

doi:10.19982/j.issn.1000-6621.20240133

摘要/Abstract

摘要：

目的: 研究利福平耐药结核分枝杆菌(Mycobacterium tuberculosis,MTB)对氟喹诺酮类药物(fluoroquinolones,FQs)耐药基因突变特征与FQs最低抑菌浓度(minimum inhibitory concentration,MIC)的关系。方法: 选取2016—2021年北京市结核病防治机构及定点医院收治的利福平耐药结核病(rifampicin resistant tuberculosis,RR-TB)患者分离培养阳性菌株进行微孔板法药物敏感性检测,总结RR-TB患者菌株对左氧氟沙星(levofloxacin,Lfx)和莫西沙星(moxifloxacin,Mfx)的MIC值;同时进行一代测序。分析FQs耐药相关基因gyrA和gyrB突变特征与FQs MIC之间的关系;以表型药物敏感性试验(phenotypic drug susceptibility testing,pDST)结果为参照标准,评价基因型药物敏感性试验(genotypic drug susceptibility testing,gDST)对FQs耐药的检测效能;探讨RR-MTB对FQs表型耐药与基因型耐药差异的原因。结果: 303株RR-TB患者菌株中,FQs pDST耐药率为27.7%(84/303),gyrA基因突变检出率为25.1%(76/303),未检测到gyrB基因突变。以pDST结果为参照标准,gDST检测RR-MTB的FQs耐药性的敏感度和特异度分别为84.5%(71/84;95%CI:74.6.1%~91.2%)和97.7%(214/219;95%CI:94.5%~99.1%)。两种方法结果不一致的菌株有25株,不一致率为8.3%(25/303)。FQs耐药菌株的MIC主要为2μg/ml,最常见的突变位点发生在第94位点(53.9%,41/76),gyrA基因在第88和94位点突变与pDST耐药完全一致,而第90和91位点突变的pDST耐药一致率分别为95.8%(23/24)和3/5。第88位点的突变与Lfx pDST耐药相关,与Mfx pDST高浓度耐药相关;第90位点的突变以丙氨酸转变为缬氨酸为主(92.3%,24/26),该突变发生的Lfx和Mfx最低MIC为临界浓度(1μg/ml和0.25μg/ml)。第94位点天冬氨酸突变为天冬酰胺均为Lfx和Mfx高浓度耐药(1/1),该位点天冬氨酸突变为酪氨酸与Lfx耐药(1/1)相关,与Mfx高浓度耐药相关(1/1)。结论: 北京地区RR-MTB的FQs耐药的主要机制是gyrA基因突变,不同gyrA基因突变提示FQs耐药水平存在差异。FQs pDST和gDST检测RR-MTB的结果高度一致,可及早应用gDST检测RR-TB患者的FQs的耐药性,以指导临床制定合理治疗方案。

关键词: 分枝杆菌,结核, 荧光喹诺酮类, 微生物敏感性试验, DNA突变分析, 抗药性,细菌

Abstract:

Objective: To elucidate the mutation profiles of fluoroquinolones (FQs) resistance genes in Mycobacterium tuberculosis (MTB) and to analyze the correlation between specific genetic mutations and the minimum inhibitory concentrations (MICs) of FQs. Methods: Positive strains isolated and cultured from rifampicin-resistant tuberculosis (RR-TB) patients, who were admitted to tuberculosis prevention and control institutions and designated hospitals in Beijing between 2016 and 2021, were selected for drug sensitivity testing using the microplate method. The MICs of levofloxacin (Lfx) and moxifloxacin (Mfx) for these RR-MTB strains were determined and summarized. Concurrently, first-generation sequencing was performed. The relationship between the mutation characteristics of the gyrA and gyrB genes, which were associated with FQs resistance, and the MICs of FQs was analyzed. Phenotypic drug sensitivity testing (pDST) results were used as a reference standard to evaluate the performance of genotypic drug sensitivity testing (gDST) in detecting FQs resistance. Additionally, the reasons for discrepancies between phenotypic and genotypic resistance in RR-MTB strains were investigated. Results: Among the 303 RR-MTB strains analyzed, the pDST resistance rate to FQs was 27.7% (84/303), and the detection rate of gyrA gene mutations was 25.1% (76/303). No gyrB gene mutations were detected. Using pDST results as the reference standard, the sensitivity and specificity of gDST for detecting FQ resistance in RR-MTB were 84.5% (71/84; 95%CI: 74.6%-91.2%) and 97.7% (214/219; 95%CI: 94.5%-99.1%), respectively. There were 25 strains with inconsistent results between the two methods, resulting in an inconsistency rate of 8.3% (25/303). The MIC of FQs-resistant strains was predominantly 2 μg/ml, with the most common mutation site located at codon 94 (53.9%, 41/76). The gyrA gene mutations at codons 88 and 94 were completely consistent with pDST resistance, while the consistency rates of pDST resistance at codons 90 and 91 were 95.8% (23/24) and 3/5, respectively. The mutation at position 88 was linked to resistance to Lfx in pDST and high-level resistance to Mfx. The mutation at codon 90, predominantly an alanine-to-valine substitution (92.3%, 24/26), resulted in MICs at the critical concentrations for Lfx (1 μg/ml) and Mfx (0.25 μg/ml). The aspartic acid mutation at codon 94 was associated with high-level resistance to Lfx and Mfx when mutated to asparagine (1/1). Additionally, this aspartic acid mutation, when altered to tyrosine, was linked to resistance to Lfx (1/1) and high-level resistance to Mfx (1/1). Conclusion: The primary mechanism of FQs resistance in RR-MTB in Beijing is attributed to mutations in the gyrA gene. Different gyrA mutations are indicative of varying levels of FQs resistance. The concordance between pDST and gDST for detecting FQs resistance in RR-MTB is high. Consequently, gDST can be implemented early to identify FQs resistance in RR-TB patients, facilitating the clinical development of effective treatment plans.

Key words: Mycobacterium tuberculosis, Fluoroquinolones, Microbial sensitivity tests, DNA mutational analysis, Drug resistance, bacterial

中图分类号:

于兰, 陈双双, 王嫩寒, 田丽丽, 赵琰枫, 樊瑞芳, 刘海灿, 李传友, 代小伟. 利福平耐药结核分枝杆菌对氟喹诺酮类药物表型耐药与其基因突变的一致性研究[J]. 中国防痨杂志, 2024, 46(8): 942-950. doi: 10.19982/j.issn.1000-6621.20240133

Yu Lan, Chen Shuangshuang, Wang Nenhan, Tian Lili, Zhao Yanfeng, Fan Ruifang, Liu Haican, Li Chuanyou, Dai Xiaowei. Consistency between phenotypic resistance to fluoroquinolones and genetic mutations in rifampicin resistant Mycobacterium tuberculosis strains[J]. Chinese Journal of Antituberculosis, 2024, 46(8): 942-950. doi: 10.19982/j.issn.1000-6621.20240133

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gyrA基因突变	菌株[株 (构成比,%)]	pDST		突变与耐药相关的可信度^[9]
gyrA基因突变	菌株[株 (构成比,%)]	敏感(株)	耐药(株)	突变与耐药相关的可信度^[9]
单位点突变	3(1.0)	2	1
88位点氨基酸(Gly88Cys)	1(0.3)	0	1	耐药性相关
90位点氨基酸(Ala90Ser)	1(0.3)	1	0	-
95位点氨基酸(Ser95Thr)	1(0.3)	1	0	-^a
双位点联合突变	73(24.1)	4	69
88+91位点氨基酸(Gly88Gly+Ser91Ser)	1(0.3)	1	0	-
88+95位点氨基酸(Gly88Cys+Ser95Thr)	1(0.3)	0	1	耐药性相关^a
90+95位点氨基酸(Ala90Gly+Ser95Thr)	1(0.3)	0	1	与暂定耐药性无关^a
90+95位点氨基酸(Ala90Val+Ser95Thr)	24(7.9)	1	23	耐药性相关^a
91+95位点氨基酸(Ser91Pro+Ser95Thr)	5(1.7)	2	3	耐药性相关^a
94+95位点氨基酸(Asp94Ala+Ser95Thr)	24(7.9)	0	24	耐药性相关^a
94+95位点氨基酸(Asp94Asn+Ser95Thr)	1(0.3)	0	1	耐药性相关^a
94+95位点氨基酸(Asp94Gly+Ser95Thr)	15(5.0)	0	15	耐药性相关^a
94+95位点氨基酸(Asp94Tyr+Ser95Thr)	1(0.3)	0	1	耐药性相关^a
无突变	227(74.9)	214	13

突变类型	合计 (株)	高水平耐药 [株(构成比,%)]	Lfx MIC(株)
突变类型	合计 (株)	高水平耐药 [株(构成比,%)]	0.25μg/ml	0.50μg/ml	1.00μg/ml	2.00μg/ml	4.00μg/ml	8.00μg/ml
Gly88Cys	2	1(50.0)	0	0	0	0	1	1
Ala90Val	24	3(12.5)	0	0	2	17	2	3
Ala90Ser	1	0(0.0)	1	0	0	0	0	0
Ala90Gly	1	0(0.0)	0	0	0	1	0	0
Ser91Pro	5	0(0.0)	1	0	2	1	1	0
Asp94Ala	24	3(12.5)	0	0	2	13	6	3
Asp94Asn	1	1(1/1)	0	0	0	0	0	1
Asp94Gly	15	7(46.7)	0	0	0	2	6	7
Asp94Tyr	1	0(0.0)	0	0	0	0	1	0

突变类型	合计 (株)	高水平耐药 [株(构成比,%)]	Mfx MIC(株)
突变类型	合计 (株)	高水平耐药 [株(构成比,%)]	0.06 μg/ml	0.12 μg/ml	0.25 μg/ml	0.50 μg/ml	1.00 μg/ml	2.00 μg/ml	4.00 μg/ml	8.00 μg/ml
Gly88Cys	2	2(2/2)	0	0	0	0	0	0	1	1
Ala90Val	24	11(45.8)	0	0	2	3	8	7	3	1
Ala90Ser	1	0(0.0)	0	1	0	0	0	0	0	0
Ala90Gly	1	0(0.0)	0	0	0	1	0	0	0	0
Ser91Pro	5	3(60.0)	2	0	0	0	0	1	2	0
Asp94Ala	24	19(79.2)	0	0	0	3	2	12	5	2
Asp94Asn	1	1(1/1)	0	0	0	0	0	0	1	0
Asp94Gly	15	11(73.3)	1	3	0	0	0	3	7	1
Asp94Tyr	1	1(1/1)	0	0	0	0	0	0	1	0