Rifampicin Reduces Susceptibility to Ofloxacin in Rifampicin-resistant Mycobacterium tuberculosis through Efflux

Abstract

Rationale-Central dogma suggests that rifampicin resistance in Mycobacterium tuberculosis develops solely through rpoB gene mutations.Objective-To determine whether rifampicin induces efflux pumps activation in rifampicin resistant M. tuberculosis strains thereby defining rifampicin resistance levels and reducing ofloxacin susceptibility.Methods-Rifampicin and/or ofloxacin minimum inhibitory concentrations (MICs) were determined in rifampicin resistant strains by culture in BACTEC 12B medium. Verapamil and reserpine were included to determine their effect on rifampicin and ofloxacin susceptibility. RTqPCR was applied to assess expression of efflux pump/transporter genes after rifampicin exposure. To determine whether verapamil could restore susceptibility to first-line drugs, BALB/c mice were infected with a MDR-TB strain and treated with first-line drugs with/without verapamil. Measurements and Main Findings-RifampicinMICs varied independently of rpoB mutation and genetic background. Addition reserpine and verapamil significantly restored rifampicin susceptibility (p = 0.0000). RT-qPCR demonstrated that rifampicin induced differential expression of efflux/transporter genes in MDR-TB isolates. Incubation of rifampicin monoresistant strains in rifampicin (2 μg/ml) for 7 days induced ofloxacin resistance (MIC> 2 μg/ml) in strains with an rpoB531 mutation. Ofloxacin susceptibility was restored by exposure to efflux pump inhibitors. Studies in BALB/c mice showed that verapamil in combination with first-line drugs significantly reduced pulmonary CFUs after 1 and 2 months treatment (p < 0.05).Correspondence and requests for reprints should be addressed to Robin M. Conclusion-Exposure of rifampicin resistant M. tuberculosis strains to rifampicin can potentially compromise the efficacy of the second-line treatment regimens containing ofloxacin, thereby emphasising the need for rapid diagnostics to guide treatment. Efflux pump inhibitors have the potential to improve the efficacy of anti-tuberculosis drug treatment. KeywordsMycobacterium tuberculosis; drug resistance; rifampicin; efflux pumps; cross resistance Rifampicin is one of the most important anti-tuberculosis (anti-TB) antibiotics; it exerts its bactericidal activity by inhibiting the early steps of gene transcription by binding to the β-subunit of RNA polymerase (RpoB) encoded by the rpoB gene (1). This activity is responsible for shortening the treatment period and reducing the proportion of recurrent TB cases. The currently accepted paradigm suggests that resistance to rifampicin develops through a process of spontaneous mutation (nonsynonymous single nucleotide polymorphisms [nsSNPs]) in the rpoB gene (2), followed by antibiotic selection during periods of poor adherence or monotherapy. Luria-Delbrück fluctuation tests show that rifampicin resistance appears spontaneously at a rate of 10 −9 to 10 −8 mutations per cell division (3, 4). These nsSNPs largely occur in an 81-bp region in the rpoB gene known as the rifampicin resistance-determining region (...

Acceleration of Tuberculosis Treatment by Adjunctive Therapy with Verapamil as an Efflux Inhibitor

“…These were compared with the AUC achieved with human doses to model human dosing in mice. Verapamil (6.25 mg/kg) had an AUC(0-8) of 255 ng$h/ml, which is close to the human AUC(0-8) of 234 ng$h/ml and is a dose used in previous studies (9,24).…”

“…Interestingly, it has been shown previously that blocking calcium channels in vivo in M. tuberculosis-infected mice using specific antibodies significantly reduced bacterial loads (6). Induction of efflux pump expression or mutations enhancing pump activity may explain the newly attained multidrug resistance and tolerance induced by antimicrobial drug exposure (7)(8)(9)(10)(11)(12). Furthermore, several mycobacterial efflux pumps, including Rv1258c, and their regulators are induced during macrophage infection (10,(13)(14)(15)(16)(17)(18).…”

The epidemiology, pathogenesis, transmission, diagnosis, and management of multidrug-resistant, extensively drug-resistant, and incurable tuberculosis

“…[171][172][173]196,197 In this theory of drug resistance (figure 5) several initiating factors exist such as low drug dosage due to poor dosing practices, pharmacokinetic variability, or inadvertent monotherapy with, for example, quino lones for bacterial pneumonias and other conditions that turn out to be tuberculosis. As part of the bacterial stress reaction to the suboptimal antibiotic concentrations-and to effective monotherapy-efflux pumps in the bacilli are upregulated within hours.…”

“…The demonstration of co-occurrence of canonical mutations in drug target genes and MICs that decrease in the presence of an efflux pump inhibitor, as well as recent studies in clinical isolates, seem to support this new idea. 6,197,198,[199][200][201] Some phylogenetic lineages of M tuberculosis (genotypes) and the strains of these lineages have greater propensity to cause acquired drug resistance-this theory has been especially recognised since the Beijing strain started gaining notoriety in the MDR tuberculosis epidemic. 202 Ford and colleagues 203 did Luria-Delbrück fluctuation analysis on a panel of laboratory and clinical isolates from lineage 2 and lineage 4, to which the Beijing strain belongs, and found the number of mutants per cell plated in a single culture ranged from 2·42 × 10 − ⁹ for the CDC-1551 strain (lineage 2) to 1·94 × 10 − ⁸ for the HN878 strain (lineage 4), which was a significant difference.…”

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“…Drug efflux pumps have been described in several mycobacteria till to date. Some examples, M. smegmatis LfrA and MFS transporter compatible to the QacA for a multidrug efflux pump of Staphylococcus aureus, where it was the first multidrug efflux pump reported 6 . When expressed on a plasmid, LfrA mediates low level resistance to fluoroquinolones and other toxic compounds such as ethidium bromide.…”