Effect of efflux pump inhibitors on drug susceptibility of ofloxacin resistant Mycobacterium tuberculosis isolates
Effect of efflux pump inhibitors on drug susceptibility of ofloxacin resistant Mycobacterium tuberculosis isolates
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2011-05
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通讯作者：
Mradula Singh;G. Jadaun;Ramdas;K. Srivastava;Vipin Chauhan;Ritu Mishra;K. Gupta;S. Nair;D. Chauhan;V.D. Sharma;K. Venkatesan;V. Katoch
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作者：
Mradula Singh;G. Jadaun;Ramdas;K. Srivastava;Vipin Chauhan;Ritu Mishra;K. Gupta;S. Nair;D. Chauhan;V.D. Sharma;K. Venkatesan;V. Katoch
背景与目的在耐药结核病，尤其是耐多药结核病（MDR）中，氟喹诺酮类药物（FQs）被用作二线药物。然而，耐药结核分枝杆菌的发病率正在迅速增加，这可能是由于FQs在治疗各种其他疾病中的广泛使用。最重要的已知机制，即，在FQ抗性M中未观察到显著比例的FQ抗性中的gyrA突变。结核病分离株表明耐药性可能是由于其他机制，如主动药物外排泵。本研究通过使用不同的抑制剂，如羰基氰间氯苯腙（CCCP）、2，4-二硝基苯酚（DNP）和维拉帕米，在临床分离的M.结核方法55株M.用刃天青微量滴定法（REMA）检测45株氧氟沙星（OFL）耐药株和10株氧氟沙星敏感株，观察外排抑制剂对氧氟沙星最低抑菌浓度（MIC）的影响。结果CCCP可使OFL的MIC降低2-8倍（16/45; 35.5%），维拉帕米（24/45; 53.3%）和DNP（21/45; 46.6%），而在鉴定为OFL敏感的分离株的情况下，这些对氧氟沙星MIC没有显示出任何影响。所有三种抑制剂均观察到24.5%的分离株MIC水平发生变化。总体上，30株（66.6%）分离株在用这些抑制剂治疗后OFL MIC降低。共对8株分离株进行gyrA基因测序，其中7株（87.5%）存在已知突变。在8个测序的分离株中，7个（87.5%）在存在外排抑制剂的情况下显示MIC变化2至8倍。解释与结论我们的研究结果表明，主要易化超家族（MFS）家族的主动外排泵（CCCP和DNP抑制）和ATP结合盒（ABC）转运蛋白（维拉帕米抑制）参与了M.结核病分离株。这些结果的流行病学意义需要在前瞻性研究中使用适当数量的样本/分离株来确定。
Background & objectives In drug resistant, especially multi-drug resistant (MDR) tuberculosis, fluoroquinolones (FQs) are used as second line drugs. However, the incidence of FQ-resistant Mycobacterium tuberculosis is rapidly increasing which may be due to extensive use of FQs in the treatment of various other diseases. The most important known mechanism i.e., gyrA mutation in FQ resistance is not observed in a significant proportion of FQ resistant M. tuberculosis isolates suggesting that the resistance may be because of other mechanisms such as an active drug efflux pump. In this study we evaluated the role of the efflux pumps in quinolone resistance by using various inhibitors such as carbonyl cyanide m-chlorophenyl hydrazone (CCCP), 2,4-dinitrophenol (DNP) and verapamil, in clinical isolates of M. tuberculosis. Methods A total of 55 M. tuberculosis clinical isolates [45 ofloxacin (OFL) resistant and 10 ofloxacin sensitive] were tested by Resazurin microtitre assay (REMA) to observe the changes in ofloxacin minimum inhibitory concentration (MIC) levels in presence of efflux inhibitors as compared to control (without efflux inhibitor). Results The MIC levels of OFL showed 2-8 folds reduction in presence of CCCP (16/45; 35.5%), verapamil (24/45; 53.3%) and DNP (21/45; 46.6%) while in case of isolates identified as OFL sensitive these did not show any effect on ofloxacin MICs. In 11 of 45 (24.5%) isolates change in MIC levels was observed with all the three inhibitors. Overall 30 (66.6%) isolates had reduction in OFL MIC after treatment with these inhibitors. A total of eight isolates were sequenced for gyrA gene, of which, seven (87.5%) showed known mutations. Of the eight sequenced isolates, seven (87.5%) showed 2 to 8 fold change in MIC in presence of efflux inhibitors. Interpretation & conclusions Our findings suggest the involvement of active efflux pumps of both Major Facilitator Super Family (MFS) family (inhibited by CCCP and DNP) and ATP Binding Cassette (ABC) transporters (inhibited by verapamil) in the development of OFL resistance in M. tuberculosis isolates. Epidemiological significance of these findings needs to be determined in prospective studies with appropriate number of samples / isolates.