Elsevier

Tuberculosis

Volume 107, December 2017, Pages 111-118
Tuberculosis

Drug Discovery and Resistance
Ionophore A23187 shows anti-tuberculosis activity and synergy with tebipenem

https://doi.org/10.1016/j.tube.2017.09.001Get rights and content

Abstract

The objective of this study was to find molecules with anti-mycobacterial activity from a natural compounds library, investigate their mechanisms of resistance, and assess their synergy with antibiotics. We screened a library of 2582 natural compounds with Mycobacterium aurum with the aim of identifying molecules with anti-mycobacterial activity. The hits with the lowest MICs in M. aurum were also tested for their antimicrobial activity in other mycobacterial species including M. tuberculosis complex strains. The chequerboard titration assay was chosen for determining drug interactions in vitro. Spontaneous resistant mutants were isolated and their whole genome sequences compared to wild type and resistant mutants to identify resistance mechanisms. We found that ionophores show anti-mycobacterial activity in vitro. Resistance mechanism to ionophores is mediated by the MmpL5-MmpS5 transporter overexpression. Ionophore A23187 enhanced beta-lactam activity in M. tuberculosis infected macrophage. It will help in the investigation of new drug combinations against bacterial infections including tuberculosis.

Introduction

Among infectious diseases, tuberculosis (TB) remains a major public health problem with increasing cases of multidrug-resistant (MDR) TB [1]. According to the WHO, MDR-TB is defined as those cases are resistant to isoniazid and rifampin, with or without resistance to other first-line drugs. The treatment of MDR cases makes use of 2nd line drugs that are less effective and more toxic than those used for 1st line treatment, which have been shown to be effective in a six months treatment for non- MDR tuberculosis cases. The search for new molecules with anti-TB activity using cell-based systems and chemical libraries of synthetic molecules or antibiotic repurposing has led to the discovery of several candidates. Several of them are in clinical trials, such as pretomanid and SQ109 [2]. Others like bedaquiline and delamanid can be used for the treatment of severe MDR-TB cases although severe side effects were observed for bedaquiline [3]. In consequence, more anti-TB drugs need to be identified.

We therefore decided to screen a library of purified natural compounds, a number of them synthesized by soil microorganisms, and approved for human use. This would lower safety concerns and the extent of necessary in vivo analysis before using such molecules as new antimicrobials. Recently, examples of microbial products that prevent the growth of other bacterial species, even killing them have been reported [4].

The utilization of a combination of non-pathogenic fast-growing mycobacterial species facilitates genetic approaches to decipher antimicrobial activity and resistance mechanisms before ultimate testing with the slow growing M. tuberculosis pathogenic species that requires BSL3 safety containments [5]. For this, we used the fast-growing non-pathogenic species M. aurum, which grows without forming aggregates to screen the chemical library more easily. M. aurum grows rapidly on simple media such as LB broth and is sensitive to many anti-TB drugs.

Our screening of a natural product library led to discover the anti-mycobacterial activity of a group of compounds including nigericin, the antibiotic A23187, and salinomycin that have been shown previously to exhibit antimicrobial activity probably due to their property as ionophores at the bacterial level [6], [7], [8]. We describe here the activity of these compounds against mycobacterial species, the mechanisms of resistance to these compounds found in mycobacteria, and their synergy with beta-lactam antibiotics, which constitute a promising way for repurposing this family of antibiotics for treating mycobacterial infections.

Section snippets

Drug and reagents preparation

The following antibiotics were used: rifampin, kanamycin, ofloxacin, amikacin, bedaquiline, clavulanate, meropenem, faropenem and tebipenem. Antibiotic solutions were prepared at a concentration of 1 μg/mL in distilled water, filter sterilized, and frozen until use. All compounds to be screened were dissolved in 100% dimethylsulfoxide (DMSO) and stored as frozen stocks at a concentration of 2 mg/mL. Resazurin sodium salt powder was prepared at 0.01% (w/v) in distilled water filter sterilized,

Screening of chemical library

We identified 19 hits active against M. aurum from among 2582 compounds. The activity of these compounds was evaluated by determining their MICs in BCG. Three compounds (nigericin, A23187 and salinomycin) had lower MICs in M. aurum (0.16, 0.08, and 0.04 μg/mL respectively) and two of them had lower MICs in BCG (nigericin 0.625 μg/mL and A23187 1.25 μg/mL respectively). The three compounds that showed antibacterial activity on M. aurum are ionophores, known to promote ion transport through cell

Discussion

The phenotypic screening of natural product library for molecules with anti-mycobacterial activity allowed the identification of several hits. Among them, ionophores (nigericin, A23187 and salinomycin) were previously described with antimicrobial activity [6], [7], [8]. Another study described salinomycin and other ionophores as a new class of antimalarial drugs [23]. However, the mechanisms of resistance to these molecules have not been described to date.

Nigericin and salinomycin are

Conclusions

We identified ionophores as a new class of antibacterials against mycobacteria in vitro. A23187 could enhance the activity of beta-lactams against intracellular bacteria. This opens the way to use beta-lactam antibiotics combined with clavulanate or other inhibitors of betalactamase, cephalosporinase, or carbapenemase activity, together with ionophores, such as A23187, for the treatment of MDR-TB and other infectious bacterial diseases by oral administration.

Funding

This work was supported by European Union's Seventh Framework Program for research, technological development (604237), Gabonese Republic, Chinese Academy of Science; Institut Pasteur of Shanghai; Institut Pasteur of Paris.

Competing interests

None declared.

Ethical approval

In conformity with French regulations, the biobank has been declared to and recorded by both the French Ministry of Research and a French Ethics Committee under the reference DC-2008-68 collection 2.

References (35)

  • L.L. Ling et al.

    A new antibiotic kills pathogens without detectable resistance

    Nature

    (2015)
  • A. Gupta et al.

    Fast- growing, non-infectious and intracellularly surviving drug-resistant Mycobacterium aurum: a model for high-throughput antituberculosis drug screening

    J Antimicrob Chemother

    (2009)
  • B.C. Pressman

    Biological applications of ionophores

    Annu Rev Biochem

    (1976)
  • A. Martin et al.

    Multicenter study of MTT and resazurin assays for testing susceptibility to first- line anti-tuberculosis drugs

    Int J Tuberc Lung Dis

    (2005)
  • J.C. Palomino et al.

    Resazurin microtiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis

    Antimicrob Agents Chemother

    (2002)
  • P. Chen et al.

    Synergistic interactions of SQ109, a new ethylene diamine, with front-line antitubercular drugs in vitro

    J Antimicrob Chemother

    (2006)
  • F.C. Odds

    Synergy, antagonism, and what the chequerboard puts between them

    J Antimicrob Chemother

    (2003)
  • Cited by (0)

    1

    Wei Huang and Julien Briffotaux contributed equally to this article.

    View full text