• Users Online: 4865
  • Home
  • Print this page
  • Email this page


 
 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 9  |  Issue : 1  |  Page : 71-75

Targeting bedaquiline mycobacterial efflux pump to potentially enhance therapy in Mycobacterium abscessus


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 Submission20-Nov-2019
Date of Acceptance17-Dec-2019
Date of Web Publication6-Mar-2020

Correspondence Address:
Anandi Martin
Institute of Experimental and Clinical Research, Laboratory of Medical Microbiology, Université Catholique De Louvain, Avenue Hippocrate, Saint-Gilles, Brussels 54 1200
Belgium
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_181_19

Rights and Permissions
  Abstract 


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 2020 Aug 4];9:71-5. Available from: http://www.ijmyco.org/text.asp?2020/9/1/71/280139




  Introduction Top


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.[1],[2] 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).[3],[4] However, and worryingly, efflux-mediated bedaquiline resistance has been identified.[5] 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.[6] 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.[7] 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 Top


Clinical isolates

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.[8]

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[9] 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 Top


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 1: GenoType NTM-DR identification results

Click here to view
Table 2: Resistance to macrolides and aminoglycosides according to Mycobacterium abscessus subspecies

Click here to view


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

Click here to view



  Discussion Top


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[10] 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.[5],[6],[7],[8],[9],[10],[11] In 2015, Srikrishna et al.[12] 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.[13] 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.[14] 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.[15],[16] 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.[17]

It is unknown if the concentration of verapamil used inin vitro studies is attainable in patients.[18] 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.[19] 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.[20] 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.[21] Recently, Ramis et al. evaluated in silico andin vitro the tetrahydropyridine compounds as efflux inhibitors in M. abscessus subsp. abscessus.[22] 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.[23] Further studies have identified that verapamil inhibits the activity of MATE pumps.[24],[25] 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.[26] Finally, to know which EPs are involved in this activity, transcriptomic study will be a necessary.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
van Dorn A. Multidrug-resistant Mycobacterium abscessus threatens patients with cystic fibrosis. Lancet Respir Med 2017;5:15.  Back to cited text no. 1
    
2.
Nessar R, Cambau E, Reyrat JM, Murray A, Gicquel B. Mycobacterium abscessus: A new antibiotic nightmare. J Antimicrob Chemother 2012;67:810-8.  Back to cited text no. 2
    
3.
Aguilar-Ayala DA, Cnockaert M, André E, Andries K, Gonzalez-Y-Merchand JA, Vandamme P, et al.In vitro activity of bedaquiline against rapidly growing nontuberculous mycobacteria. J Med Microbiol 2017;66:1140-3.  Back to cited text no. 3
    
4.
Martin A, Godino IT, Aguilar-Ayala DA, Mathys V, Lounis N, Villalobos HR.In vitro activity of bedaquiline against slow-growing nontuberculous mycobacteria. J Med Microbiol 2019;68:1137-9.  Back to cited text no. 4
    
5.
Andries K, Villellas C, Coeck N, Thys K, Gevers T, Vranckx L, et al. Acquired resistance of Mycobacterium tuberculosis to bedaquiline. PLoS One 2014;9:e102135.  Back to cited text no. 5
    
6.
Gupta S, Cohen KA, Winglee K, Maiga M, Diarra B, Bishai WR. Efflux inhibition with verapamil potentiates bedaquiline in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2014;58:574-6.  Back to cited text no. 6
    
7.
da Silva PE, Von Groll A, Martin A, Palomino JC. Efflux as a mechanism for drug resistance in Mycobacterium tuberculosis. FEMS Immunol Med Microbiol 2011;63:1-9.  Back to cited text no. 7
    
8.
Kehrmann J, Kurt N, Rueger K, Bange FC, Buer J. Genotype NTM-DR for identifying Mycobacterium abscessus subspecies and determining molecular resistance. J Clin Microbiol 2016;54:1653-5.  Back to cited text no. 8
    
9.
Martin A, Camacho M, Portaels F, Palomino JC. Resazurin microtiter assay plate testing of Mycobacterium tuberculosis susceptibilities to second-line drugs: Rapid, simple, and inexpensive method. Antimicrob Agents Chemother 2003;47:3616-9.  Back to cited text no. 9
    
10.
Viljoen A, Raynaud C, Johansen MD, Roquet-Banères F, Herrmann JL, Daher W, et al. Verapamil improves the activity of bedaquiline against Mycobacterium abscessus in vitro and in macrophages. Antimicrob Agents Chemother 2019;63. pii: E00705-19.  Back to cited text no. 10
    
11.
Xu J, Tasneen R, Peloquin CA, Almeida DV, Li SY, Barnes-Boyle K, et al. Verapamil increases the bioavailability and efficacy of bedaquiline but not clofazimine in a murine model of tuberculosis. Antimicrob Agents Chemother 2018;62. pii: e01692-17.  Back to cited text no. 11
    
12.
Srikrishna G, Gupta S, Dooley KE, Bishai WR. Can the addition of verapamil to bedaquiline-containing regimens improve tuberculosis treatment outcomes? A novel approach to optimizing TB treatment. Future Microbiol 2015;10:1257-60.  Back to cited text no. 12
    
13.
Philley JV, Wallace RJ Jr., Benwill JL, Taskar V, Brown-Elliott BA, Thakkar F, et al. Preliminary results of bedaquiline as salvage therapy for patients with nontuberculous mycobacterial lung disease. Chest 2015;148:499-506.  Back to cited text no. 13
    
14.
Vesenbeckh S, Schönfeld N, Roth A, Bettermann G, Krieger D, Bauer TT, et al. Bedaquiline as a potential agent in the treatment of Mycobacterium abscessus infections. Eur Respir J 2017;49. pii: 1700083.  Back to cited text no. 14
    
15.
Escribano I, Rodríguez JC, Llorca B, García-Pachon E, Ruiz M, Royo G. Importance of the efflux pump systems in the resistance of Mycobacterium tuberculosis to fluoroquinolones and linezolid. Chemotherapy 2007;53:397-401.  Back to cited text no. 15
    
16.
Gupta AK, Chauhan DS, Srivastava K, Das R, Batra S, Mittal M, et al. Estimation of efflux mediated multi-drug resistance and its correlation with expression levels of two major efflux pumps in mycobacteria. J Commun Dis 2006;38:246-54.  Back to cited text no. 16
    
17.
Rodrigues L, Aínsa JA, Amaral L, Viveiros M. Inhibition of drug efflux in mycobacteria with phenothiazines and other putative efflux inhibitors. Recent Pat Antiinfect Drug Discov 2011;6:118-27.  Back to cited text no. 17
    
18.
Adams KN, Szumowski JD, Ramakrishnan L. Verapamil, and its metabolite norverapamil, inhibit macrophage-induced, bacterial efflux pump-mediated tolerance to multiple anti-tubercular drugs. J Infect Dis 2014;210:456-66.  Back to cited text no. 18
    
19.
Johnston A, Burgess CD, Hamer J. Systemic availability of oral verapamil and effect on PR interval in man. Br J Clin Pharmacol 1981;12:397-400.  Back to cited text no. 19
    
20.
Gupta S, Tyagi S, Bishai WR. Verapamil increases the bactericidal activity of bedaquiline against Mycobacterium tuberculosis in a mouse model. Antimicrob Agents Chemother 2015;59:673-6.  Back to cited text no. 20
    
21.
Grossman TH, Shoen CM, Jones SM, Jones PL, Cynamon MH, Locher CP. The efflux pump inhibitor timcodar improves the potency of antimycobacterial agents. Antimicrob Agents Chemother 2015;59:1534-41.  Back to cited text no. 21
    
22.
Ramis IB, Vianna JS, Silva Junior L, von Groll A, Ramos DF, Lobo MM, et al. In silico andin vitro evaluation of tetrahydropyridine compounds as efflux inhibitors in Mycobacterium abscessus. Tuberculosis (Edinb) 2019;118:101853.  Back to cited text no. 22
    
23.
Singh M, Jadaun GP, Ramdas, Srivastava K, Chauhan V, Mishra R, et al. Effect of efflux pump inhibitors on drug susceptibility of ofloxacin resistant Mycobacterium tuberculosis isolates. Indian J Med Res 2011;133:535-40.  Back to cited text no. 23
[PUBMED]  [Full text]  
24.
Radchenko M, Symersky J, Nie R, Lu M. Structural basis for the blockade of MATE multidrug efflux pumps. Nat Commun 2015;6:7995.  Back to cited text no. 24
    
25.
Sharma A, Gupta VK, Pathania R. Efflux pump inhibitors for bacterial pathogens: From bench to bedside. Indian J Med Res 2019;149:129-45.  Back to cited text no. 25
[PUBMED]  [Full text]  
26.
Pfeifer HJ, Greenblatt DK, Koch-Wester J. Clinical toxicity of reserpine in hospitalized patients: A report from the Boston collaborative drug surveillance program. Am J Med Sci 1976;271:269-76.  Back to cited text no. 26
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Methods
Results
Discussion
References
Article Tables

 Article Access Statistics
    Viewed449    
    Printed15    
    Emailed0    
    PDF Downloaded83    
    Comments [Add]    

Recommend this journal