In Vitro Activity of Rifampicin and Verapamil Combination in Multidrug-Resistant Mycobacterium tuberculosis

Demitto, Fernanda de Oliveira; Amaral, Renata Claro Ribeiro do; Maltempe, Flaviane Granero; Siqueira, Vera Lúcia Dias; Scodro, Regiane Bertin de Lima; Lopes, Mariana Aparecida; Caleffi-Ferracioli, Katiany Rizzieri; Canezin, Pedro Henrique; Cardoso, Rosilene Fressatti

doi:10.1371/journal.pone.0116545

Cited by 18 publications

(13 citation statements)

References 31 publications

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“…S2). Verapamil is an efflux pump inhibitor that influences membrane potential (22) and has been previously been shown to potentiate the activity of several anti-TB drugs (23–25). In contrast, all pairwise combinations featuring sutezolid were found to be antagonistic.…”

Section: Resultsmentioning

confidence: 99%

Transcriptomic Signatures Predict Regulators of Drug Synergy and Clinical Regimen Efficacy against Tuberculosis

Jaipalli

Larkins-Ford

et al. 2019

mBio

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Multidrug combination therapy is an important strategy for treating tuberculosis, the world’s deadliest bacterial infection. Long treatment durations and growing rates of drug resistance have created an urgent need for new approaches to prioritize effective drug regimens. Hence, we developed a computational model called INDIGO-MTB that identifies synergistic drug regimens from an immense set of possible drug combinations using the pathogen response transcriptome elicited by individual drugs. Although the underlying input data for INDIGO-MTB was generated under in vitro broth culture conditions, the predictions from INDIGO-MTB correlated significantly with in vivo drug regimen efficacy from clinical trials. INDIGO-MTB also identified the transcription factor Rv1353c as a regulator of multiple drug interaction outcomes, which could be targeted for rationally enhancing drug synergy.

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Section: Resultsmentioning

confidence: 99%

Transcriptomic Signatures Predict Regulators of Drug Synergy and Clinical Regimen Efficacy against Tuberculosis

Jaipalli

Larkins-Ford

et al. 2019

mBio

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“…In particular, combinations containing the drugs chlorpromazine and verapamil were highly enriched for synergistic interactions; 77% of chlorpromazine-containing combinations and 80% of verapamil-containing combinations were found to interact synergistically (FIC < 0.9) ( Figure S2 ). Verapamil is an efflux pump inhibitor that influences membrane potential (22) and has been previously been shown to potentiate the activity of several anti-TB drugs (23-25). In contrast, all pairwise combinations featuring sutezolid were found to be antagonistic.…”

Section: Resultsmentioning

confidence: 99%

Transcriptomic signatures predict regulators of drug synergy and clinical regimen efficacy against Tuberculosis

Jaipalli

Larkins-Ford

et al. 2019

Preprint

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22 23 Classification: BIOLOGICAL SCIENCES: Microbiology; Biophysics and Computational Biology 24 25 ABSTRACT 34 35 The rapid spread of multi-drug resistant strains has created a pressing need for new drug 36 regimens to treat tuberculosis (TB), which kills 1.8 million people each year. Identifying new 37 regimens has been challenging due to the slow growth of the pathogen M. tuberculosis (MTB), 38 coupled with large number of possible drug combinations. Here we present a computational 39 model (INDIGO-MTB) that identified synergistic regimens featuring existing and emerging anti-40 TB drugs after screening in silico over 1 million potential drug combinations using MTB drug 41 transcriptomic profiles. INDIGO-MTB further predicted the gene Rv1353c as a key 42 transcriptional regulator of multiple drug interactions, and we confirmed experimentally that 43 Rv1353c up-regulation reduces the antagonism of the bedaquiline-streptomycin combination. 44 Retrospective analysis of 57 clinical trials of TB regimens using INDIGO-MTB revealed that 45 synergistic combinations were significantly more efficacious than antagonistic combinations (p-46 value = 1 x 10 -4 ) based on the percentage of patients with negative sputum cultures after 8 47 weeks of treatment. Our study establishes a framework for rapid assessment of TB drug 48 combinations and is also applicable to other bacterial pathogens. 49 50 IMPORTANCE 51 52 Multi-drug combination therapy is an important strategy for treating tuberculosis, the world's 53 deadliest bacterial infection. Long treatment durations and growing rates of drug resistance 54 have created an urgent need for new approaches to prioritize effective drug regimens. Hence, 55 we developed a computational model called INDIGO-MTB, which identifies synergistic drug 56 regimens from an immense set of possible drug combinations using pathogen response 57 transcriptome elicited by individual drugs. Although the underlying input data for INDIGO-MTB 58 was generated under in vitro broth culture conditions, the predictions from INDIGO-MTB 59 correlated significantly with in vivo drug regimen efficacy from clinical trials. INDIGO-MTB also 60identified the transcription factor Rv1353c as a regulator of multiple drug interaction outcomes, 61which could be targeted for rationally enhancing drug synergy. 62 63 64 65 66 67 68 69 70 71 131 gene associations that are correlated with synergy and antagonism. In the example above, MTB 132 upregulation of both gene 1 and gene 3 in response to the drugs measured in monotherapy is predictive 133 of antagonism when the drugs are combined. By perturbing individual genes and known targets of 134 Transcription Factors (TFs) in the model, we can infer the impact of individual gene and TF activity 135 respectively on drug interactions and subsequently engineer interaction outcomes.136 137 138

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“…Multidrug efflux pumps are known to contribute to antibiotic tolerance in M. tuberculosis (38-40). It was reported that after incubation of rifampicin-exposed M. tuberculosis cells in in vitro cultures and in infected macrophages with the efflux pump inhibitor, verapamil, rifampicin levels inside the cells increased thereby enhancing susceptibility (41, 42). This study showed that verapamil-sensitive efflux pump was involved in the removal of rifampicin from the cells.…”

Section: Methodsmentioning

confidence: 99%

Reduced Permeability to Rifampicin by Capsular Thickening as a Mechanism of Antibiotic Persistence inMycobacterium tuberculosis

Sebastian

Swaminath

Ajitkumar

2019

Preprint

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17Persisters constitute a subpopulation of bacteria that can tolerate lethal concentrations 18 of antibiotics. Multiple mechanisms have been suggested for bacterial persistence 19 against antibiotics. With mycobacteria being no exception to this behaviour, we had 20 reported the de novo emergence of genetically antibiotic-resistant Mycobacterium 21 tuberculosis from persister cells upon prolonged exposure to microbicidal 22 concentrations of the anti-tuberculosis drugs, rifampicin and moxifloxacin. Here, we 23 present evidence for reduced permeability to rifampicin as a mechanism for 24 persistence of Mycobacterium tuberculosis in vitro. We observed that rifampicin 25 persistent M. tuberculosis cells developed a thick outer layer (TOL) capsule. The TOL 26 restricted the entry of fluorochrome-conjugated rifampicin, 5-carboxyfluorescein-27 rifampicin (5-FAM-rifampicin), which retained only 2.5% of its original bactericidal 28 activity, but high levels of permeability, on actively growing mid-log phase cells. Gentle 29 mechanical removal of TOL significantly enhanced 5-FAM-rifampicin entry into the 30 persister cells. The level of 5-FAM-rifampicin in the persister cells was not affected by 31 the pre-incubation of the cells with verapamil, a drug efflux pump inhibitor, ruling out 32 the involvement of efflux pumps in the reduced intracellular concentration of 5-FAM-33 rifampicin. GC-MS analysis of TOL showed the presence of ~7-fold, ~5-fold and ~2-34 fold higher levels of α-D-glucopyranoside, 1,2,5-linked-mannitol, and 3,4-linked 35 mannose, respectively, among ~2-fold higher levels of derivatives of several other 36 types of sugars such as arabinose and galactose. Taken together, the present study 37 reveals that rifampicin-persistent M. tuberculosis cells develop TOL that enables the 38 bacilli to restrict entry of rifampicin and thereby remain tolerant to the antibiotic in vitro.39 40 65 4unknown strategy to ensure sub-lethal concentration of antibiotics inside persister cells 66 even though they remain exposed to lethal concentrations of the antibiotics. In order 67 to verify this possibility, we examined whether the permeability of rifampicin into M. 68 tuberculosis persister cells was affected by any ultrastructural changes. The present 69 study reports the ultrastructural changes of rifampicin persistent M. tuberculosis cells 70 and their contribution to the restricted permeability of rifampicin, in comparison with 71 the entry of rifampicin into actively growing mid-log phase cells. 72 73 MATERIALS AND METHODS 74 75 Rifampicin treatment of Mycobacterium tuberculosis 76 Mycobacterium tuberculosis H37Ra (National JALMA Institute of Leprosy and Other 77 Mycobacterial Diseases, Agra, India) was used in all the experiments. Bacteria were 78 grown in Middlebrook 7H9 broth (BD) supplemented with 0.2% glycerol, 0.05% 79 Tween-80 and 10% ADS (albumin, dextrose, sodium chloride) until mid-log phase 80 (MLP, 0.6 at OD600 nm) at 37ºC under shaking at 170 rpm. Cultures were then exposed 81 to 1 µg/ml (10x MBC; 12) of ...

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In Vitro Activity of Rifampicin and Verapamil Combination in Multidrug-Resistant Mycobacterium tuberculosis

Cited by 18 publications

References 31 publications

Transcriptomic Signatures Predict Regulators of Drug Synergy and Clinical Regimen Efficacy against Tuberculosis

Transcriptomic Signatures Predict Regulators of Drug Synergy and Clinical Regimen Efficacy against Tuberculosis

Transcriptomic signatures predict regulators of drug synergy and clinical regimen efficacy against Tuberculosis

Reduced Permeability to Rifampicin by Capsular Thickening as a Mechanism of Antibiotic Persistence inMycobacterium tuberculosis

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