耐多药结核分枝杆菌对喹诺酮药物的耐药特性及分子机制的研究
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摘要
目的研究喹诺酮药物对临床分离耐多药结核分枝杆菌(MDRTB)的最小抑菌浓度(MIC)及耐药分子机制,探索使用喹诺酮类药物治疗MDRTB感染的可行性。方法采用微量孔Alamar Blue显色法检测氧氟沙星、左氧氟沙星、莫西沙星和加替沙星对MDRTB的MIC;PCR方法扩增MDRTB的gyr基因并测序。结果 40株氧氟沙星敏感MDRTB对氧氟沙星、左氧氟沙星、莫西沙星和加替沙星均敏感;未检测到gyr基因发生突变。40株氧氟沙星耐药MDRTB对氧氟沙星、左氧氟沙星、莫西沙星和加替沙星敏感率分别为0%、30.0%、42.5%、40.0%;检测到33株(82.5%)gyrA基因和2株(5%)gyrB基因发生突变,突变类型为Ala90Val、Ser91Pro、Asp94Gly、Asp94His、Asp94Asn、Asp94Ala、Asn477Thr和Thr478Asn。结论 MDRTB对喹诺酮类药物的耐药机制以gyrA基因Ala90Val、Ser91Pro、Asp94Gly突变类型为主;莫西沙星是治疗氧氟沙星低水平耐药MDRTB的首选方案
Objective To investigate the resistance of multidrug-resistant Mycobacterium tuberculosis(MDRTB) to quinolones and its molecular mechanism. Methods The minimum inhibitory concentrations(MICs) of ofloxacin(OFX), levofloxacin(LFX),moxifloxacin(MFX) and gatifloxacin(GAT) for MDRTB were detected by the microplate alamar blue assay. The gyr genes of MDRTB were amplified by PCR and sequenced. Results All 40 MDRTB strains sensitive to OFX were also sensitive to LEV, MAX and GAT; and no gyr gene mutations were detected. In 40 OFX-resistant MDRTB strains, the sensitivity rates to OFX, LEX, MAX and GAT were0.0%, 30.0%, 42.5% and 40.0%, respectively; the gyrA gene mutations were detected in 33 strains(82.5%) and the gyrB mutations were detected in 2 strains(5.0%). The mutation types were Ala90 Val, Ser91 Pro, Asp94 Gly, Asp94 His, Asp94 Asn, Asp94 Ala, Asn477 Thr and Thr478 Asn. Conclusion The Ala90 Val, Ser91 Pro and Asp94 Gly of the gyrA mutation types may be the molecular mechanisms of the resistance to quinolone in MDRTB, and MFX can be the first choice for the treatment of OFX-resistant RMDRTB.
Objective To investigate the resistance of multidrug-resistant Mycobacterium tuberculosis(MDRTB) to quinolones and its molecular mechanism. Methods The minimum inhibitory concentrations(MICs) of ofloxacin(OFX), levofloxacin(LFX),moxifloxacin(MFX) and gatifloxacin(GAT) for MDRTB were detected by the microplate alamar blue assay. The gyr genes of MDRTB were amplified by PCR and sequenced. Results All 40 MDRTB strains sensitive to OFX were also sensitive to LEV, MAX and GAT; and no gyr gene mutations were detected. In 40 OFX-resistant MDRTB strains, the sensitivity rates to OFX, LEX, MAX and GAT were0.0%, 30.0%, 42.5% and 40.0%, respectively; the gyrA gene mutations were detected in 33 strains(82.5%) and the gyrB mutations were detected in 2 strains(5.0%). The mutation types were Ala90 Val, Ser91 Pro, Asp94 Gly, Asp94 His, Asp94 Asn, Asp94 Ala, Asn477 Thr and Thr478 Asn. Conclusion The Ala90 Val, Ser91 Pro and Asp94 Gly of the gyrA mutation types may be the molecular mechanisms of the resistance to quinolone in MDRTB, and MFX can be the first choice for the treatment of OFX-resistant RMDRTB.
引文
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[15]刘志广,郭倩,魏剑浩,等.结核分枝杆菌gyrA基因突变与氧氟沙星耐药水平的相关性研究[J].实用预防医学,2015,22(6):641-644.DOI:10.3969/j.issn.1006-3110.2015.06.001.
[2]许榕青,李丹,林银霞,等.基因芯片技术检测结核分枝杆菌利福平和异烟肼耐药性临床应用评价[J].中国人兽共患病学报,2017,33(1):43-48.DOI:10.3969/j.issn.1002-2694.2017.01.008.
[3]Ahmad N,Javaid A,Sulaiman SAS,et al.Resistance patterns,prevalence,and predictors of fluoroquinolones resistance in multidrug resistant tuberculosis patients[J].The Brazilian Journal of Infectious Diseases,2016,20(1):41-47.DOI:10.1016/j.bjid.2015.09.011.
[4]Yu X,Wang G,Chen S,et al.Wild-type and non-wild-type mycobacterium tuberculosis MIC distributions for the novel fluoroquinolone antofloxacin compared with those for of loxacin,levofloxcian,and moxifloxacin[J].AntimicrobialAgents and Chemoterapy,2016,60(9):5233-5237.DOI:10.1128/AAC.00393-16.
[5]Willby M,Sikes RD,Malik S,et al.Correlation between GyrA substitutions and ofloxacin,levofloxacin,and moxifloxacin crossresistance in Mycobacterium tuberculosis[J].Antimicrob Agents Chemother,2015,59(9):5427-34.DOI:10.1128/AAC.00662-15.
[6]Pang Y,Zong Z,Huo F,et al.In Vitro Drug Susceptibility of Bedaquiline,Delamanid,Linezolid,Clofazimine,Moxifloxacin,and Gatifloxacin against Extensively Drug-Resistant Tuberculosis in Beijing,China[J].Antimicrob Agents Chemother,2017,61(10):900-917.DOI:10.1128/AAC.00900-17.
[7]Bablishvili N,Tukvadze N,Shashkina E,et al.Impact of gyrB and eis Mutations in Improving Detection of Second-Line-Drug Resistance among Mycobacterium tuberculosis Isolates from Georgia[J].Antimicrobial Agents and Chemotherapy,2017,61(9):1921-1916.DOI:10.1128/AAC.01921-16.
[8]王志锐,谢彤.氟喹诺酮耐药与结核分枝杆菌中gyrA和gyrB基因突变的研究进展[J].医学综述,2017,23(13):2516-2521.DOI:10.3969/j.issn.1006-2084.2017.13.005.
[9]李雨晴,万李,陈杏,等.中国西北四省(区)结核分枝杆菌分离株一线药物耐药状况及其影响因素分析[J].中国人兽共患病学报,2017,33(5):(下转第528页398-402.DOI:10.3969/j.issn.1002-2694.2017.05.003.
[10]Disratthakit A,Prammananan T,Tribuddharat C,et al.Role of gyrB mutations in pre-extensively and extensively drug-resistant tuberculosis in Thai clinical isolates[J].Antimicrobal Agents and Chemotherapy,2016,60(9):5189-5197.DOI:10.1128/AAC.00539-16.
[11]张玉娇,李晓静,米凯霞.结核分枝杆菌耐氟喹诺酮类药物的分子机制研究进展[J].遗传,2016,38(10):918-927.DOI:10.16288/j.yczz.16-136.
[12]Chien JY,Chien ST,Chiu WY,et al.Moxifloxacin Improves Treatment Outcomes in Patients with Ofloxacin-Resistant MultidrugResistant Tuberculosis[J].Antimicrob Agents Chemother,2016,60(8):4708-4716.DOI:10.1128/AAC.00425-16.(上接第524页)
[13]Rodwell TC,Valafar F,Douglas J,et al.Predicting extensively drug-resistant Mycobacterium tuberculosis phenotypes with genetic mutations[J].J Clin Microbiol,2014,52(3):781-789.DOI:10.1128/JCM.02701-13.
[14]Bernard C,Veziris N,Brossier F,et al.Molecular Diagnosis of Fluoroquinolone Resistance in Mycobacterium tuberculosis[J].Antimicrob Agents Chemother,2015,59(3):1519-1524.DOI:10.1128/AAC.04058-14.
[15]刘志广,郭倩,魏剑浩,等.结核分枝杆菌gyrA基因突变与氧氟沙星耐药水平的相关性研究[J].实用预防医学,2015,22(6):641-644.DOI:10.3969/j.issn.1006-3110.2015.06.001.