Overexpression of efflux pump genes is one of the mechanisms causing drug resistance in Mycobacterium tuberculosis

Abstract

ABSTRACT Currently, drug resistance is a formidable obstacle to tuberculosisprevention globally. A deep understanding of the drug-resistance mechanism of Mycobacterium tuberculosis is helpful to deal with this problem. In this study, 46 clinical isolates of Mycobacterium tuberculosis from Zigong, Sichuan, China, including 5 sensitive isolates, 5 rifampicin (RIF) mono-resistant isolates, 18 isoniazid mono-resistant isolates, and 18 multi-drug-resistant isolates, were selected. We evaluated the impact of drug induction on the relative expression levels of 10 putative efflux pump genes, namely, Rv3065 , Rv2836c , efpA , Rv1410c , Rv1250 , Rv0876c , Rv1819c , Rv0933 , Rv1217c , and Rv1218c . Meanwhile, we assessed the effect of verapamil, an efflux pump inhibitor, on the drug resistance of the isolates. The results showed that 100% (5/5) of the RIF mono-resistant isolates, 44.4% (8 of 18) of the isoniazid mono-resistant isolates, and 88.9% (16 of 8) of the multi-drug-resistant isolates overexpressed anyone of the 10 efflux pump genes; however, none of the 10 efflux pump genes were overexpressed on the sensitive isolates. Among drug-resistant strains, Rv1250 (51.2%) and Rv0933 (53.7%) were the genes with the highest frequency of overexpression. Only the rifampicin mono-resistant isolates showed a significant increase in the number of overexpressed efflux pump genes after drug induction, while the change trends of the numbers of overexpressed efflux pump genes in isoniazid mono-resistant isolates and multi-drug-resistant isolates after drug induction were not definite. Verapamil did not have a significant effect on the minimum inhibitory concentrations (MICs) of isoniazid mono-resistant isolates but reduced the MICs of all sensitive isolates and some multi-drug-resistant isolates. We also investigated the correlation between polymorphic loci of mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing, mutations in drug resistance-associated genes, and overexpression of the efflux pump genes. The results indicated that there was no significant association between the two most polymorphic loci, MIRU26 and MIRU31, and the incidence of overexpression of the efflux pump genes. However, isolates with mutations in drug resistance-associated genes exhibited a higher rate of overexpression of the efflux pump genes compared to those without mutations. In conclusion, we found that drug-resistant isolates had a higher background expression level of the efflux pump genes compared to sensitive isolates. This suggests that the overexpression of the efflux pump genes could be one of the mechanisms causing drug resistance in Mycobacterium tuberculosis . We also found that verapamil was able to reduce MICs in some drug-resistant isolates, indicating that inhibition of the efflux pump could be used as an adjuvant treatment option for drug-resistant tuberculosis patients. IMPORTANCE Gene mutations cannot explain all drug resistance of Mycobacterium tuberculosis , and the overexpression of efflux pump genes is considered another important cause of drug resistance. A total of 46 clinical isolates were included in this study to analyze the overexpression of efflux pump genes in different resistant types of strains. The results showed that overexpression of efflux pump genes did not occur in sensitive strains. There was no significant trend in the overexpression of efflux pump genes before and after one-half of MIC drug induction. By adding the efflux pump inhibitor verapamil, we can observe the decrease of MIC of some drug-resistant strains. At the same time, this study ensured the reliability of calculating the relative expression level of efflux pump genes by screening reference genes and using two reference genes for the normalization of quantitative PCR. Therefore, this study confirms that the overexpression of efflux pump genes plays an important role in the drug resistance of clinical isolates of Mycobacterium tuberculosis .