Abstract
Although drug resistance in Mycobacterium tuberculosis 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 M. tuberculosis. We constructed point mutations V219A and S292L in Rv1258c in the chromosome of M. tuberculosis and the point mutations were confirmed by DNA sequencing. The susceptibility of the constructed M. tuberculosis 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 M. tuberculosis 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 M. tuberculosis. 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 M. tuberculosis and address their role in affecting treatment outcome in vivo.
Highlights
Multidrug resistant (MDR) tuberculosis (MDR-TB), defined by resistance to at least the two frontline drugs isoniazid (INH) and rifampin (RIF), poses a significant challenge to effective treatment and control of the disease
To determine possible roles of efflux pumps in causing drug resistance, we performed a database search for mutations in the efflux pump Rv1258c (Tap) among clinical isolates whose whole genome sequences are deposited in the GenBank NCBI database
Several single nucleotide polymorphisms were found in the efflux gene Rv1258c (D23V, V219A, and S292L) in different clinical isolates of M. tuberculosis, which may affect the function of the efflux protein
Summary
Multidrug resistant (MDR) tuberculosis (MDR-TB), defined by resistance to at least the two frontline drugs isoniazid (INH) and rifampin (RIF), poses a significant challenge to effective treatment and control of the disease. The kanamycin-sensitive colonies were screened using colony-PCR followed by DNA sequencing to confirm the desired point mutation being constructed in the genome of M. tuberculosis H37Ra. Drug Susceptibility Testing and Isoniazid/Piperine Combination Studies
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