ABSTRACT
Multidrug-resistant tuberculosis (MDR-TB) patients not cured at the time of stopping treatment are exposed to Minimum Inhibitory Concentration (MIC) and sub-MIC levels for many months after discontinuing bedaquiline (BDQ) or clofazimine (CFZ) treatment. In vitro cultures treated with BDQ and CFZ sub-MIC concentrations clearly showed enrichment in the Rv0678 mutant population, demonstrating that pre-existing Rv0678 mutants can be selected by sub-MIC concentrations of BDQ and CFZ if not protected by an alternative MDR-TB treatment.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis, Multidrug-Resistant , Humans , Mycobacterium tuberculosis/genetics , Clofazimine/pharmacology , Antitubercular Agents/pharmacology , Antitubercular Agents/therapeutic use , Diarylquinolines/pharmacology , Tuberculosis, Multidrug-Resistant/drug therapy , Tuberculosis, Multidrug-Resistant/microbiology , Microbial Sensitivity TestsABSTRACT
Objectives: Resistance-associated variants (RAVs) in Rv0678 , a regulator of the MmpS5-MmpL5 efflux pump, have been shown to lead to increased MICs of bedaquiline (2- to 8- fold) and clofazimine (2- to 4-fold). The prevalence of these Rv0678 RAVs in clinical isolates and their impact on treatment outcomes are important factors to take into account in bedaquiline treatment guidelines. Methods: Baseline isolates from two bedaquiline MDR-TB clinical trials were sequenced for Rv0678 RAVs and corresponding bedaquiline MICs were determined on 7H11 agar. Rv0678 RAVs were also investigated in non-MDR-TB sequences of a population-based cohort. Results: Rv0678 RAVs were identified in 23/347 (6.3%) of MDR-TB baseline isolates. Surprisingly, bedaquiline MICs for these isolates were high (> 0.24â mg/L, n = 8), normal (0.03-0.24 mg/L, n = 11) or low (< 0.03â mg/L, n = 4). A variant at position -11 in the intergenic region mmpS5 - Rv0678 was identified in 39 isolates (11.3%) and appeared to increase the susceptibility to bedaquiline. In non-MDR-TB isolates, the frequency of Rv0678 RAVs was lower (6/852 or 0.7%). Competition experiments suggested that rifampicin was not the drug selecting for Rv0678 RAVs. Conclusions: RAVs in Rv0678 occur more frequently in MDR-TB patients than previously anticipated, are not associated with prior use of bedaquiline or clofazimine, and in the majority of cases do not lead to bedaquiline MICs above the provisional breakpoint (0.24â mg/L). Their origin remains unknown. Given the variety of RAVs in Rv0678 and their variable effects on the MIC, only phenotypic drug-susceptibility methods can currently be used to assess bedaquiline susceptibility.
Subject(s)
Antitubercular Agents/pharmacology , Clofazimine/pharmacology , Diarylquinolines/pharmacology , Mycobacterium tuberculosis/drug effects , Tuberculosis, Multidrug-Resistant/microbiology , Anti-Inflammatory Agents/pharmacology , Antibiotics, Antitubercular/therapeutic use , Antitubercular Agents/therapeutic use , Clinical Trials as Topic , Clofazimine/therapeutic use , Diarylquinolines/therapeutic use , Humans , Microbial Sensitivity Tests , Mycobacterium tuberculosis/genetics , Prevalence , Rifampin/therapeutic use , Sequence Analysis, DNA , Tuberculosis, Multidrug-Resistant/epidemiologyABSTRACT
Efflux pumps are membrane proteins capable of actively transporting a broad range of substrates from the cytoplasm to the exterior of the cell. Increased efflux activity in response to drug treatment may be the first step in the development of bacterial drug resistance. Previous studies showed that the efflux pump Mmr was significantly overexpressed in strains exposed to isoniazid. In the work to be described, we constructed mutants lacking or overexpressing Mmr in order to clarify the role of this efflux pump in the development of resistance to isoniazid and other drugs in M. tuberculosis. The mmr knockout mutant showed an increased susceptibility to ethidium bromide, tetraphenylphosphonium, and cetyltrimethylammonium bromide (CTAB). Overexpression of mmr caused a decreased susceptibility to ethidium bromide, acriflavine, and safranin O that was obliterated in the presence of the efflux inhibitors verapamil and carbonyl cyanide m-chlorophenylhydrazone. Isoniazid susceptibility was not affected by the absence or overexpression of mmr. The fluorometric method allowed the detection of a decreased efflux of ethidium bromide in the knockout mutant, whereas the overexpressed strain showed increased efflux of this dye. This increased efflux activity was inhibited in the presence of efflux inhibitors. Under our experimental conditions, we have found that efflux pump Mmr is mainly involved in the susceptibility to quaternary compounds such as ethidium bromide and disinfectants such as CTAB. The contribution of this efflux pump to isoniazid resistance in Mycobacterium tuberculosis still needs to be further elucidated.
Subject(s)
Antitubercular Agents/pharmacology , Isoniazid/pharmacology , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Mycobacterium tuberculosis , Acriflavine/pharmacology , Anti-Infective Agents, Local/pharmacology , Antineoplastic Agents/pharmacology , Bacterial Proteins , Cetrimonium , Cetrimonium Compounds/pharmacology , Drug Resistance, Bacterial/genetics , Drug Resistance, Bacterial/physiology , Enzyme Inhibitors/pharmacology , Ethidium/pharmacology , Gene Knockout Techniques , Microbial Sensitivity Tests , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Nitriles/pharmacology , Onium Compounds/pharmacology , Organophosphorus Compounds/pharmacology , Phenazines/pharmacology , Verapamil/pharmacologyABSTRACT
The Mycobacterium tuberculosis pandemic is a major health problem, further complicated by an increasing incidence of drug-resistant isolates and the existence of highly transmissible strains, such as those in the Beijing family. Streptomycin (STR)-resistant M. tuberculosis clinical isolates have been analyzed to look for mutations in the rpsL, rrs, and gidB genes. In addition, the Rv1258c gene, which encodes Tap, an efflux pump that transports STR, has been sequenced. Mutations affecting codons 43 and 88 of the rpsL gene were found in 44.4% of the strains, and 16.7% of the strains carried mutations in the rrs gene, both of which probably contribute to STR resistance. Many strains presented with mutations in the gidB gene, but the implication of those mutations in STR resistance remains unclear. Interestingly, a cytosine nucleotide insertion between positions 580 and 581 (denominated Tap(580)) in the Rv1258c gene has been found in all Beijing isolates included in this study, suggesting that it might be a novel polymorphism specific to the Beijing family of M. tuberculosis. A simple and fast restriction fragment length polymorphism (RFLP)-PCR method for detecting the Tap(580) insertion has been developed and used to screen a collection of 220 DNA samples obtained from cultures of M. tuberculosis isolates and 30 respiratory specimens. In all cases, the Beijing and non-Beijing representative samples were identified correctly. Tap(580) is a novel polymorphism specific to the highly transmissible Beijing family, which allows for fast detection of these strains even at the very early stages of infection.
Subject(s)
Drug Resistance, Bacterial , Genetic Markers , Molecular Typing/methods , Mutagenesis, Insertional , Mycobacterium tuberculosis/classification , Mycobacterium tuberculosis/genetics , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Genotype , Humans , Mycobacterium tuberculosis/drug effects , Polymorphism, Restriction Fragment Length , Sensitivity and Specificity , Streptomycin/pharmacologyABSTRACT
Drug-resistant mycobacterial infections are a serious global health challenge, leading to high mortality and socioeconomic burdens in developing countries worldwide. New innovative approaches, from identification of new targets to discovery of novel chemical scaffolds, are urgently needed. Recently, energy metabolism in mycobacteria, in particular the oxidative phosphorylation pathway, has emerged as an object of intense microbiological investigation and as a novel target pathway in drug discovery. New classes of antibacterials interfering with elements of the oxidative phosphorylation pathway are highly active in combating dormant or latent mycobacterial infections, with a promise of shortening tuberculosis chemotherapy. The regulatory approval of the ATP synthase inhibitor bedaquiline and the discovery of Q203, a candidate drug targeting the cytochrome bc1 complex, have highlighted the central importance of this new target pathway. In this review, we discuss key features and potential applications of inhibiting energy metabolism in our quest for discovering potent novel and sterilizing drug combinations for combating tuberculosis. We believe that the combination of drugs targeting elements of the oxidative phosphorylation pathway can lead to a completely new regimen for drug-susceptible and multidrug-resistant tuberculosis.
Subject(s)
Antitubercular Agents/pharmacology , Drug Discovery/trends , Energy Metabolism/drug effects , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/metabolism , Oxidative Phosphorylation/drug effectsABSTRACT
Bedaquiline (BDQ), an ATP synthase inhibitor, is the first drug to be approved for treatment of multi-drug resistant tuberculosis in decades. In vitro resistance to BDQ was previously shown to be due to target-based mutations. Here we report that non-target based resistance to BDQ, and cross-resistance to clofazimine (CFZ), is due to mutations in Rv0678, a transcriptional repressor of the genes encoding the MmpS5-MmpL5 efflux pump. Efflux-based resistance was identified in paired isolates from patients treated with BDQ, as well as in mice, in which it was confirmed to decrease bactericidal efficacy. The efflux inhibitors verapamil and reserpine decreased the minimum inhibitory concentrations of BDQ and CFZ in vitro, but verapamil failed to increase the bactericidal effect of BDQ in mice and was unable to reverse efflux-based resistance in vivo. Cross-resistance between BDQ and CFZ may have important clinical implications.
Subject(s)
Diarylquinolines/pharmacology , Drug Resistance, Bacterial/drug effects , Mycobacterium tuberculosis/drug effects , Animals , Antitubercular Agents/pharmacology , Antitubercular Agents/therapeutic use , Bacterial Proteins/metabolism , Base Sequence , Clofazimine/pharmacology , Diarylquinolines/therapeutic use , Genes, Bacterial , Genetic Fitness , Humans , Mice , Microbial Sensitivity Tests , Models, Biological , Molecular Sequence Data , Mutation/genetics , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/isolation & purification , Reserpine/pharmacology , Tuberculosis/drug therapy , Tuberculosis/microbiology , Up-Regulation/drug effects , Verapamil/pharmacologyABSTRACT
Although the classical antibiotic spectinomycin is a potent bacterial protein synthesis inhibitor, poor antimycobacterial activity limits its clinical application for treating tuberculosis. Using structure-based design, we generated a new semisynthetic series of spectinomycin analogs with selective ribosomal inhibition and excellent narrow-spectrum antitubercular activity. In multiple murine infection models, these spectinamides were well tolerated, significantly reduced lung mycobacterial burden and increased survival. In vitro studies demonstrated a lack of cross resistance with existing tuberculosis therapeutics, activity against multidrug-resistant (MDR) and extensively drug-resistant tuberculosis and an excellent pharmacological profile. Key to their potent antitubercular properties was their structural modification to evade the Rv1258c efflux pump, which is upregulated in MDR strains and is implicated in macrophage-induced drug tolerance. The antitubercular efficacy of spectinamides demonstrates that synthetic modifications to classical antibiotics can overcome the challenge of intrinsic efflux pump-mediated resistance and expands opportunities for target-based tuberculosis drug discovery.