|Year : 2020 | Volume
| Issue : 1 | Page : 71-75
Targeting bedaquiline mycobacterial efflux pump to potentially enhance therapy in Mycobacterium abscessus
Anandi Martin1, Yasmine Bouyakoub1, Kate Soumillion1, Eléonore Ngyuvula Mantu1, Alexandre Colmant2, Hector Rodriguez-Villalobos2
1 Department of Microbiology, Institute of Experimental and Clinical Research, Laboratory of Medical Microbiology, Université Catholique De Louvain, Brussels, Belgium
2 University Hospital Saint-Luc, Brussels, Belgium
|Date of Submission||20-Nov-2019|
|Date of Acceptance||17-Dec-2019|
|Date of Web Publication||6-Mar-2020|
Institute of Experimental and Clinical Research, Laboratory of Medical Microbiology, Université Catholique De Louvain, Avenue Hippocrate, Saint-Gilles, Brussels 54 1200
Source of Support: None, Conflict of Interest: None
Background: Mycobacterium abscessus is notorious for being intrinsically resistant to most antibiotics. Antibiotic efflux is one of the mechanisms used by M. abscessus to pump out antibiotics from their cells. Inhibiting efflux pumps (EPs) can be an attractive strategy to enhance the activity of drugs. The objective of this study is to determine the activity of EP inhibitors (EPIs) to enhance the efficacy of the new drug bedaquiline against M. abscessus clinical isolates. Methods: A total of 31 phenotypically and genotypically identified M. abscessus subsp. abscessus, M. abscesss subsp. massiliense, and M. abscessus subsp. bolletii clinical isolates were studied. The contribution of EPs was determined by investigating the minimum inhibitory concentration (MIC) levels of bedaquiline reduction in the absence and presence of EPIs verapamil and reserpine using the resazurin microtiter assay. Results: The observed bedaquiline MIC reduction by verapamil was observed in 100% isolates and by reserpine in 54.8% isolates. Bedaquiline MIC was 4–32-fold using verapamil with M. abscessus subsp. bolletii showing the highest fold change and between 2- and 4-fold using reserpine. Conclusions: The results obtained in this study confirm that bedaquiline MIC decreased in the presence of EPIs verapamil and reserpine in clinical isolates of M. abscessus. Verapamil was the most effective EPI. As shown in previous studies, verapamil may have clinical potential as adjunctive therapy to enhance the effect of bedaquiline.
Keywords: Bedaquiline, efflux pump, Mycobacterium abscessus, mycobacteria
|How to cite this article:|
Martin A, Bouyakoub Y, Soumillion K, Mantu EN, Colmant A, Rodriguez-Villalobos H. Targeting bedaquiline mycobacterial efflux pump to potentially enhance therapy in Mycobacterium abscessus. Int J Mycobacteriol 2020;9:71-5
|How to cite this URL:|
Martin A, Bouyakoub Y, Soumillion K, Mantu EN, Colmant A, Rodriguez-Villalobos H. Targeting bedaquiline mycobacterial efflux pump to potentially enhance therapy in Mycobacterium abscessus. Int J Mycobacteriol [serial online] 2020 [cited 2021 May 16];9:71-5. Available from: https://www.ijmyco.org/text.asp?2020/9/1/71/280139
| Introduction|| |
Mycobacterium abscessus is a rapid-growing mycobacterium that causes severe pulmonary and skin infections and is considered as an emerging human pathogen in cystic fibrosis (CF) patients. M. abscessus remain very difficult to treat because of its innate resistance to many different classes of antimicrobial drugs, thus leading to unsatisfactory treatment outcome., Therefore, there is a significant need for search of new effective antimicrobial treatment. The introduction of new drugs could potentially improve M. abscessus treatment outcomes. Recently, two new antituberculosis (TB) drugs, bedaquiline and delamanid, have reached the market. Our group has shown that bedaquiline hasin vitro activity against nontuberculous mycobacteria (NTM)., However, and worryingly, efflux-mediated bedaquiline resistance has been identified. This mechanism of resistance decreases the intracellular drug concentration of bedaquiline, rendering the antibiotic treatment ineffective. Recent studies have explored strategies to reverse the resistance phenotype conferred by efflux pump (EP) activity by the addition of EP inhibitors (EPIs).In vitro studies shown that the EPI such as verapamil decreased the minimum inhibitory concentrations (MICs) of bedaquiline (and clofazimine) against Mycobacterium tuberculosis H37Rv by 4–16-fold. The same studies focusing on M. abscessus are, however, extremely limited. M. abscessus forms a complex of three closely related “species.” Significant differences exist between M. abscessus ssp. abscessus which is the most virulent species compared to M. abscessus ssp. bolletii and M. abscessus ssp. massiliense. Macrolides (e.g., clarithromycin) are frequently the only drug that is activein vitro against M. abscessus. However, in M. abscessus ssp. abscessus and M. abscessus ssp. bolletii, the induction of erythromycin ribosome methyltransferase gene (erm41) can lead to macrolide resistance. This inducible macrolide resistance is not found in M. abscessus ssp. massiliense due to deletions present in the erm41 gene. EPs are, thus, now largely recognized as playing an important role in induced drug resistance in mycobacteria and emerged as a major challenge in this field of bacterial resistance. However, to date, the knowledge of bedaquiline resistance mechanisms in M. abscessus is limited. EPI may have clinical potential as adjunctive treatment. The objective of this study is to determine the activity of EPI (verapamil and reserpine) for enhancing the efficacy of bedaquiline activity against M. abscessus strains isolated in our hospital, including the three subspecies.
| Methods|| |
A total of 31 clinical isolates of M. abscessus obtained from sputum samples from CF and non-CF patients from the University Hospital Saint-Luc, Brussels, Belgium, were included in the present study.
Efflux pump inhibitors and drug
Verapamil and reserpine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solution of verapamil was dissolved in distilled water, whereas reserpine was prepared in dimethyl sulfoxide (DMSO). Bedaquiline was kindly provided by Janssen Pharmaceutica (Beerse, Belgium) and was dissolved in DMSO.
Identification and resistance profile of isolates
Isolates were identified as M. abscessus by MALDI-TOF and then subtyped by the GenoType NTM-DR line probe assay version 1.0 (Hain Lifescience, Nehren, Germany) for the identification and resistance profile determination of the three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii according to the manufacturer's recommendations and as previously described.
Effect of efflux pump inhibitors on the minimum inhibitory concentration levels of bedaquiline
MIC levels for bedaquiline were determined using the resazurin microtiter assay (REMA) as previously described in the presence or absence of EPI (verapamil and reserpine). Final concentrations used in REMA for verapamil were 40 mg/l and for reserpine 12 mg/l. A total of 100 μl volume of Middlebrook 7H9 broth supplemented with 10% oleic acid, albumin, dextrose, and catalase and 0.5% glycerol was dispensed in the wells of a 96-well cell culture plate. Bedaquiline concentrations used ranged from 2, 1, 0.5, 0.25, 0.125, and 0.062 mg/l. M. abscessus freshly grown on 7H10 agar plate was taken to prepare a bacterial suspension of 0.5 McFarland standard and diluted to 1:10 in 7H9 broth. This diluted suspension (100 μl) was used to inoculate each well of the plate. Plates were sealed and incubated at 37°C for 2–3 days. After that, the resazurin dye (Sigma, USA) (0.01%, 30 μl) was added to each well and the plates were re-incubated for two more days. A change in color from blue to pink indicated the growth of bacteria, and the MIC was read as the lowest bedaquiline concentration that prevented the color change in resazurin dye.
| Results|| |
Identification of isolates
GenoType NTM-DR V.1.0 line probe assay enables M. abscessus subspecies identification and the simultaneous determination of antibiotic resistance to macrolides and aminoglycosides of mutations at position 28 in erm (41), position 2058/2059 in rrl, and position 1408 in rrs. Of 31 M. abscessus tested, the GenoType NTM-DR identified 8 M. abscessus subsp. abscessus erm (41) genotype t28, 3 M. abscessus subsp. abscessus erm (41) genotype c28, 10 M. abscessus subsp. Bolletii, and 10 M. abscessus subsp. massiliense [Table 1]. [Table 2] shows the genotype resistance patterns to macrolides and aminoglycosides in detail according to each M. abscessus subsp. tested.
|Table 2: Resistance to macrolides and aminoglycosides according to Mycobacterium abscessus subspecies|
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Minimum inhibitory concentrations of bedaquiline with and without efflux pump inhibitors verapamil and reserpine
MICs of bedaquiline determined in the absence of efflux inhibitors (verapamil or reserpine) were compared with those determined in the presence of verapamil or reserpine. Two-fold or more reduction in MIC levels was considered as an indication of the presence of EP activity in bedaquiline M. abscessus isolates. It was observed a presence of EP activity in all clinical isolates (100%) tested in the presence of verapamil and in 17 of 31 (54.8%) isolates in the presence of reserpine. MICs of bedaquiline and fold change in bedaquiline MIC in the presence of verapamil and reserpine are shown in [Table 3]. For the control, MICs of bedaquiline and efflux inhibitors alone have been tested. For verapamil, for the majority of M. abscessus subsp. abscessus isolates, 9 of 11 (81%) show 4-fold change reduction in bedaquiline MIC and 2 isolates show 8-fold change reduction. For M. abscessus subsp. bolletii, 4 of 10 isolates (40%) showed a decrease in bedaquiline MIC with 16–32-fold, 4 isolates (40%) 8-fold change reduction, and 2 isolates (20%) a 4-fold change. For M. abscessus subsp. massiliense half of the isolates, 5 of 10 (50%) show an 8-fold change and 50% a 4-fold change. For reserpine, the 17 of 31 (54.8%) isolates that show EP activity had a 4-fold change reduction in bedaquiline MIC.
|Table 3: Effect of efflux pump inhibitors on bedaquiline minimum inhibitory concentrations in Mycobacterium abscessus isolates|
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| Discussion|| |
In this study, we explored the impact of EP activity on bedaquiline in M. abscessus. We have demonstrated verapamil and reserpine activities through a simplein vitro phenotypic screening test that measures the changes in the MICs of the bedaquiline in the absence and presence of EPIs. This study adds more data and confirms the previous finding on existing EPIs effective against M. abscessus. Moreover, this is the first observation regarding the effect of EPIs on the activity of bedaquiline against clinical isolates of M. abscessus, including the three subspecies isolated in our hospital, showing its potential clinical significance in the treatment. Our results show that the MICs of bedaquiline in M. abscessus clinical isolates were affected in the presence of verapamil and reserpine suggesting a role of EP activity in bedaquiline efficacy. Results of this study are concordant with the recently published study in which verapamil improved the activity of bedaquiline against M. abscessus with a 4- and 8-fold reduction of the bedaquiline MIC. That study, however, did not investigate the effect of reserpine. Furthermore, in M. tuberculosis, previousin vitro studies have shown that verapamil decreases the MIC of bedaquiline by 8–16-fold.,,,,,, In 2015, Srikrishna et al. showed in a preclinical study that verapamil potentiated the activity of bedaquiline against M. tuberculosis reducing the dose required for cure. The study of Philley et al. demonstrates the potential clinical and microbiologic activity of bedaquiline in patients with Mycobacterium avium and M. abscessus lung disease. Furthermore, it has been shown that bedaquiline could be an alternative in multidrug treatment regimens for severe or relapsing disease, potentially including patients with underlying CF. Other studies have also shown a decrease in the MIC of isoniazid, rifampicin, streptomycin, ciprofloxacin, ofloxacin, and linezolid against M. tuberculosis and NTM in the presence of EPIs., In other mycobacteria, Rodrigues et al. found a significant reduction of resistance to clarithromycin and erythromycin in M. avium ATCC 25291 in the presence of verapamil.
It is unknown if the concentration of verapamil used inin vitro studies is attainable in patients. Verapamil is a small molecule that acts as an ion channel blocker and is used in the treatment of hypertension. Verapamil is also known to have negative cardiac side effects. It is, therefore, vital to further investigate EPIs as a viable treatment option by improving current EPIs.In vivo use of EPIs as an adjuvant to treatment regimens in M. abscessus has only recently been explored, and Gupta group has demonstrated the treatment shortening by inclusion of verapamil into standard treatment regimens in a mouse model of infection and its adjunctive use could help preserve bedaquiline activity on standard TB treatment. However, there is a limited amount of information on model organisms for which the efficacy of EPIs was evaluated in animal models. In another mouse model of infection study, timcodar treatment resulted in 1.0 and 0.4 log10 reduction in bacterial burden when used in combination with rifampicin and isoniazid, respectively. This suggests its promise as an adjuvant treatment. Recently, Ramis et al. evaluated in silico andin vitro the tetrahydropyridine compounds as efflux inhibitors in M. abscessus subsp. abscessus. Based on their analysis, this compound can be a potential pharmacophore candidate for the development of a therapeutic adjuvant for M. abscessus infections. Studies in M. tuberculosis have shown that verapamil potentiates the activity of bedaquiline and ofloxacin. Further studies have identified that verapamil inhibits the activity of MATE pumps., It has a low amount of toxicity toward bacterial cells not expressing MATE EPs, suggesting specificity toward bacteria expressing these pumps and a competitive mode of inhibition. Reserpine, an antipsychotic drug extracted from the roots of Rauwolfia serpentina, is a promising EPI that targets EPs of the MFS and RND. The clinical application of reserpine with clinically used antibiotics, however, has not yet been achieved due to its nephrotoxic nature. Finally, to know which EPs are involved in this activity, transcriptomic study will be a necessary.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]