Liu Jiayun, Shi Wanliang, Zhang Shuo, Hao Xiaoke, Maslov Dmitry A, Shur Kirill V, Bekker Olga B, Danilenko Valery N, Zhang Ying
Department of Clinical Laboratory, Xijing Hospital, Fourth Military Medical University, Xi'an, China.
Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, United States.
Front Microbiol. 2019 Feb 19;10:216. doi: 10.3389/fmicb.2019.00216. eCollection 2019.
Although drug resistance in is mainly caused by mutations in drug activating enzymes or drug targets, there is increasing interest in the possible role of efflux in causing drug resistance. Previously, efflux genes have been shown to be upregulated upon drug exposure or implicated in drug resistance in overexpression studies, but the role of mutations in efflux pumps identified in clinical isolates in causing drug resistance is unknown. Here we investigated the role of mutations in efflux pump Rv1258c (Tap) from clinical isolates in causing drug resistance in We constructed point mutations V219A and S292L in Rv1258c in the chromosome of and the point mutations were confirmed by DNA sequencing. The susceptibility of the constructed Rv1258c mutants to different tuberculosis drugs was assessed using conventional drug susceptibility testing in 7H11 agar in the presence and absence of efflux pump inhibitor piperine. A C14-labeled PZA uptake experiment was performed to demonstrate higher efflux activity in the Rv1258c mutants. Interestingly, the V219A and S292L point mutations caused clinically relevant drug resistance to pyrazinamide (PZA), isoniazid (INH), and streptomycin (SM), but not to other drugs in While V219A point mutation conferred low-level drug resistance, the S292L mutation caused a higher level of resistance. Efflux inhibitor piperine inhibited INH and PZA resistance in the S292L mutant but not in the V219A mutant. The S292L mutant had higher efflux activity for pyrazinoic acid (the active form of PZA) than the parent strain. We conclude that point mutations in the efflux pump Rv1258c in clinical isolates can confer clinically relevant drug resistance, including PZA resistance, and could explain some previously unaccounted drug resistance in clinical strains. Future studies need to take efflux mutations into consideration for improved detection of drug resistance in and address their role in affecting treatment outcome .
虽然结核分枝杆菌中的耐药性主要由药物激活酶或药物靶点的突变引起，但人们越来越关注外排作用在导致耐药性方面可能发挥的作用。此前，在外排基因的研究中发现，药物暴露后外排基因会上调，或者在过表达研究中与耐药性有关，但临床分离株中鉴定出的外排泵突变在导致耐药性方面的作用尚不清楚。在此，我们研究了临床分离株中外排泵Rv1258c（Tap）的突变在结核分枝杆菌中导致耐药性的作用。我们在结核分枝杆菌染色体上的Rv1258c中构建了点突变V219A和S292L，并通过DNA测序确认了这些点突变。使用常规药敏试验，在含有和不含有外排泵抑制剂胡椒碱的7H11琼脂中评估构建的结核分枝杆菌Rv1258c突变体对不同抗结核药物的敏感性。进行了一项C14标记的吡嗪酰胺摄取实验，以证明结核分枝杆菌Rv1258c突变体具有更高的外排活性。有趣的是，V219A和S292L点突变导致对吡嗪酰胺（PZA）、异烟肼（INH）和链霉素（SM）产生临床相关耐药性，但对结核分枝杆菌中的其他药物没有耐药性。虽然V219A点突变导致低水平耐药，但S292L突变导致更高水平的耐药。外排抑制剂胡椒碱可抑制S292L突变体中的INH和PZA耐药性，但不能抑制V219A突变体中的耐药性。S292L突变体对吡嗪酸（PZA的活性形式）的外排活性高于亲本菌株。我们得出结论，临床分离株中外排泵Rv1258c的点突变可导致临床相关耐药性，包括PZA耐药性，并可解释临床菌株中一些先前无法解释的耐药性。未来的研究需要考虑外排突变，以改进结核分枝杆菌耐药性的检测，并探讨它们在影响治疗结果方面的作用。
