Morphological changes and differentially expressed efflux pump genes in Mycobacterium tuberculosis exposed to a rifampicin and verapamil combination

Caleffi-Ferracioli, Katiany Rizzieri; Amaral, Renata Claro Ribeiro do; Demitto, Fernanda O.; Maltempe, Flaviane Granero; Canezin, Pedro Henrique; Scodro, Regiane Bertin de Lima; Nakamura, Celso V.; Leite, Clarice Queico Fujimura; Siqueira, Vera Lúcia Dias; Cardoso, Rosilene Fressatti

doi:10.1016/j.tube.2015.12.010

Cited by 36 publications

(38 citation statements)

References 26 publications

(55 reference statements)

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“…Ethidium bromide (EtBr) is considered a broad substrate of efflux pumps, most of which require the PMF ( 33 , 34 ), and has been used as a probe to monitor the activity of efflux pumps in mycobacteria ( 35 ). Verapamil has been shown to affect the intracellular accumulation of EtBr in M. tuberculosis ( 36 , 37 ), an effect generally attributed to direct inhibition of efflux pumps. However, dissipation of the PMF by verapamil could impair efflux, in turn leading to increased intracellular EtBr concentrations.…”

Section: Resultsmentioning

confidence: 99%

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Verapamil Targets Membrane Energetics in Mycobacterium tuberculosis

Chen

Gardete

Jansen

et al. 2018

Antimicrob Agents Chemother

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Mycobacterium tuberculosis kills more people than any other bacterial pathogen and is becoming increasingly untreatable due to the emergence of resistance. Verapamil, an FDA-approved calcium channel blocker, potentiates the effect of several antituberculosis (anti-TB) drugs in vitro and in vivo. This potentiation is widely attributed to inhibition of the efflux pumps of M. tuberculosis, resulting in intrabacterial drug accumulation. Here, we confirmed and quantified verapamil's synergy with several anti-TB drugs, including bedaquiline (BDQ) and clofazimine (CFZ), but found that the effect is not due to increased intrabacterial drug accumulation. We show that, consistent with its in vitro potentiating effects on anti-TB drugs that target or require oxidative phosphorylation, the cationic amphiphile verapamil disrupts membrane function and induces a membrane stress response similar to those seen with other membrane-active agents. We recapitulated these activities in vitro using inverted mycobacterial membrane vesicles, indicating a direct effect of verapamil on membrane energetics. We observed bactericidal activity against nonreplicating “persister” M. tuberculosis that was consistent with such a mechanism of action. In addition, we demonstrated a pharmacokinetic interaction whereby human-equivalent doses of verapamil caused a boost of rifampin exposure in mice, providing a potential explanation for the observed treatment-shortening effect of verapamil in mice receiving first-line drugs. Our findings thus elucidate the mechanistic basis for verapamil's potentiation of anti-TB drugs in vitro and in vivo and highlight a previously unrecognized role for the membrane of M. tuberculosis as a pharmacologic target.

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Section: Resultsmentioning

confidence: 99%

“…Verapamil has been shown to increase the intracellular accumulation of EtBr, a broad substrate of mammalian efflux pumps ( 33 , 34 ) commonly used as a probe of efflux pump activity in bacteria ( 35 ), in M. tuberculosis ( 36 ). We have reproduced those results and found EtBr and verapamil to be synergistic.…”

Section: Discussionmentioning

confidence: 99%

Verapamil Targets Membrane Energetics in Mycobacterium tuberculosis

Chen

Gardete

Jansen

et al. 2018

Antimicrob Agents Chemother

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“…Several recent studies have demonstrated the importance of the overexpression of efflux pump genes in MDR and XDR M . tuberculosis clinical strains (Calgin et al, 2013; Coelho et al, 2015; Li et al, 2015a; Yamchi et al, 2015; Kanji et al, 2016; Machado et al, 2016; Oh et al, 2017), in rifampicin monoresistant strains (Li et al, 2015b), or in the H37Rv susceptible strain after exposure to drugs (Garima et al, 2015; Caleffi-Ferracioli et al, 2016). Nevertheless, most of these studies are based on the simple assessment and evaluation of the levels of expression of M. tuberculosis efflux pump genes, and few have determined the effect of efflux inhibitors on the MICs of the antituberculosis drugs and have quantified the activity of the overexpressed efflux systems.…”

Section: Discussionmentioning

confidence: 99%

Interplay between Mutations and Efflux in Drug Resistant Clinical Isolates of Mycobacterium tuberculosis

et al. 2017

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Numerous studies show efflux as a universal bacterial mechanism contributing to antibiotic resistance and also that the activity of the antibiotics subject to efflux can be enhanced by the combined use of efflux inhibitors. Nevertheless, the contribution of efflux to the overall drug resistance levels of clinical isolates of Mycobacterium tuberculosis is poorly understood and still is ignored by many. Here, we evaluated the contribution of drug efflux plus target-gene mutations to the drug resistance levels in clinical isolates of M. tuberculosis. A panel of 17 M. tuberculosis clinical strains were characterized for drug resistance associated mutations and antibiotic profiles in the presence and absence of efflux inhibitors. The correlation between the effect of the efflux inhibitors and the resistance levels was assessed by quantitative drug susceptibility testing. The bacterial growth/survival vs. growth inhibition was analyzed through the comparison between the time of growth in the presence and absence of an inhibitor. For the same mutation conferring antibiotic resistance, different MICs were observed and the different resistance levels found could be reduced by efflux inhibitors. Although susceptibility was not restored, the results demonstrate the existence of a broad-spectrum synergistic interaction between antibiotics and efflux inhibitors. The existence of efflux activity was confirmed by real-time fluorometry. Moreover, the efflux pump genes mmr, mmpL7, Rv1258c, p55, and efpA were shown to be overexpressed in the presence of antibiotics, demonstrating the contribution of these efflux pumps to the overall resistance phenotype of the M. tuberculosis clinical isolates studied, independently of the genotype of the strains. These results showed that the drug resistance levels of multi- and extensively-drug resistant M. tuberculosis clinical strains are a combination between drug efflux and the presence of target-gene mutations, a reality that is often disregarded by the tuberculosis specialists in favor of the almost undisputed importance of antibiotic target-gene mutations for the resistance in M. tuberculosis.

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“…Along with cell wall permeability, active efflux systems also provide resistance by expelling the drug molecules that enter the cell. Several mycobacterial drug efflux pumps have been identified and can be grouped into five different structural families: the major facilitator superfamily (MFS) (Siddiqi et al, 2004; Balganesh et al, 2012; Xu et al, 2015), the small multidrug resistance (SMR) family (De Rossi et al, 2002; Garima et al, 2015), the resistance-nodulation-cell division (RND) superfamily (De Rossi et al, 2006; Murakami et al, 2006), the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily (Bhatt et al, 2000; Choudhuri et al, 2002; Pasca et al, 2004; Lubelski et al, 2007; Caleffi-Ferracioli et al, 2016) and the multidrug and toxic compound extrusion (MATE) family (Table 2) (Mishra and Daniels, 2013). …”

Section: Drug Resistance Mechanisms In Mycobacteriamentioning

confidence: 99%

New Insights in to the Intrinsic and Acquired Drug Resistance Mechanisms in Mycobacteria

et al. 2017

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Infectious diseases caused by clinically important Mycobacteria continue to be an important public health problem worldwide primarily due to emergence of drug resistance crisis. In recent years, the control of tuberculosis (TB), the disease caused by Mycobacterium tuberculosis (MTB), is hampered by the emergence of multidrug resistance (MDR), defined as resistance to at least isoniazid (INH) and rifampicin (RIF), two key drugs in the treatment of the disease. Despite the availability of curative anti-TB therapy, inappropriate and inadequate treatment has allowed MTB to acquire resistance to the most important anti-TB drugs. Likewise, for most mycobacteria other than MTB, the outcome of drug treatment is poor and is likely related to the high levels of antibiotic resistance. Thus, a better knowledge of the underlying mechanisms of drug resistance in mycobacteria could aid not only to select the best therapeutic options but also to develop novel drugs that can overwhelm the existing resistance mechanisms. In this article, we review the distinctive mechanisms of antibiotic resistance in mycobacteria.

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Morphological changes and differentially expressed efflux pump genes in Mycobacterium tuberculosis exposed to a rifampicin and verapamil combination

Cited by 36 publications

References 26 publications

Verapamil Targets Membrane Energetics in Mycobacterium tuberculosis

Verapamil Targets Membrane Energetics in Mycobacterium tuberculosis

Interplay between Mutations and Efflux in Drug Resistant Clinical Isolates of Mycobacterium tuberculosis

New Insights in to the Intrinsic and Acquired Drug Resistance Mechanisms in Mycobacteria

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