A rapid, low pH, nutrient stress, assay to determine the bactericidal activity of compounds against non-replicating Mycobacterium tuberculosis

Autoři: Julie V. Early aff001;  Steven Mullen aff001;  Tanya Parish aff001
Působiště autorů: TB Discovery Research, Infectious Disease Research Institute, Seattle, Washington, United States of America aff001
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0222970


There is an urgent need for new anti-tubercular agents which can lead to a shortened treatment time by targeting persistent or non-replicating bacilli. In order to assess compound activity against non-replicating Mycobacterium tuberculosis, we developed a method to detect the bactericidal activity of novel compounds within 7 days. Our method uses incubation at low pH in order to induce a non-replicating state. We used a strain of M. tuberculosis expressing luciferase; we first confirmed the linear relationship between luminescence and viable bacteria (determined by colony forming units) under our assay conditions. We optimized the assay parameters in 96-well plates in order to achieve a reproducible assay. Our final assay used M. tuberculosis in phosphate-citrate buffer, pH 4.5 exposed to compounds for 7 days; viable bacteria were determined by luminescence. We recorded the minimum bactericidal concentration at pH 4.5 (MBC4.5) representing >2 logs of kill. We confirmed the utility of the assay with control compounds. The ionophores monensin, niclosamide, and carbonyl cyanide 3-chlorophenylhydrazone and the anti-tubercular drugs pretomanid and rifampicin were active, while several other drugs such as isoniazid, ethambutol, and linezolid were not.

Klíčová slova:

Antibiotics – Fluorescence – Luciferase – Luminescence – Oxygen – Ionophores – Outgrowth assay


1. WHO. Global Tuberculosis Report 2018 2018. Available from: http://www.who.int/tb/publications/global_report/en/.

2. WHO. Guidelines for treatment of tuberculosis 2010. Available from: http://www.who.int/tb/publications/2010/9789241547833/en/.

3. Gordon SV, Parish T. Microbe Profile: Mycobacterium tuberculosis: Humanity's deadly microbial foe. Microbiology (Reading, England). 2018;164(4):437–9. Epub 2018/02/22. doi: 10.1099/mic.0.000601 29465344.

4. Gomez JE, McKinney JD. M. tuberculosis persistence, latency, and drug tolerance. Tuberculosis (Edinb). 2004;84(1–2):29–44. 14670344.

5. McDermott W, Tompsett R. Activation of pyrazinamide and nicotinamide in acidic environments in vitro. American review of tuberculosis. 1954;70(4):748–54. 13197751.

6. Wayne LG, Hayes LG. An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through two stages of nonreplicating persistence. Infect Immun. 1996;64(6):2062–9. 8675308; PubMed Central PMCID: PMC174037.

7. Cho SH, Warit S, Wan B, Hwang CH, Pauli GF, Franzblau SG. Low-oxygen-recovery assay for high-throughput screening of compounds against nonreplicating Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2007;51(4):1380–5. doi: 10.1128/AAC.00055-06 17210775; PubMed Central PMCID: PMC1855511.

8. Gold B, Warrier T, Nathan C. A multi-stress model for high throughput screening against non-replicating Mycobacterium tuberculosis. Methods in molecular biology (Clifton, NJ). 2015;1285:293–315. Epub 2015/03/18. doi: 10.1007/978-1-4939-2450-9_18 25779324.

9. Deb C, Lee CM, Dubey VS, Daniel J, Abomoelak B, Sirakova TD, et al. A novel in vitro multiple-stress dormancy model for Mycobacterium tuberculosis generates a lipid-loaded, drug-tolerant, dormant pathogen. PLoS One. 2009;4(6):e6077. doi: 10.1371/journal.pone.0006077 19562030; PubMed Central PMCID: PMC2698117.

10. Zhang M, Sala C, Hartkoorn RC, Dhar N, Mendoza-Losana A, Cole ST. Streptomycin-starved Mycobacterium tuberculosis 18b, a drug discovery tool for latent tuberculosis. Antimicrob Agents Chemother. 2012;56(11):5782–9. doi: 10.1128/AAC.01125-12 22926567; PubMed Central PMCID: PMC3486556.

11. Grant SS, Kawate T, Nag PP, Silvis MR, Gordon K, Stanley SA, et al. Identification of novel inhibitors of nonreplicating Mycobacterium tuberculosis using a carbon starvation model. ACS Chem Biol. 2013;8(10):2224–34. doi: 10.1021/cb4004817 23898841; PubMed Central PMCID: PMC3864639.

12. Andreu N, Fletcher T, Krishnan N, Wiles S, Robertson BD. Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence. The Journal of antimicrobial chemotherapy. 2012;67(2):404–14. doi: 10.1093/jac/dkr472 22101217; PubMed Central PMCID: PMC3254196.

13. Andreu N, Zelmer A, Fletcher T, Elkington PT, Ward TH, Ripoll J, et al. Optimisation of bioluminescent reporters for use with mycobacteria. PLoS One. 2010;5(5):e10777. doi: 10.1371/journal.pone.0010777 20520722; PubMed Central PMCID: PMC2875389.

14. Zhang JH, Chung TD, Oldenburg KR. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. Journal of biomolecular screening. 1999;4(2):67–73. doi: 10.1177/108705719900400206 10838414.

15. Yates RM, Hermetter A, Taylor GA, Russell DG. Macrophage activation downregulates the degradative capacity of the phagosome. Traffic. 2007;8(3):241–50. doi: 10.1111/j.1600-0854.2006.00528.x 17319801.

16. Early J, Alling T. Determination of compound kill kinetics against Mycobacterium tuberculosis. Methods in molecular biology (Clifton, NJ). 2015;1285:269–79. doi: 10.1007/978-1-4939-2450-9_16 25779322.

17. Early J, Ollinger J, Darby C, Alling T, Mullen S, Casey A, et al. Identification of Compounds with pH-Dependent Bactericidal Activity against Mycobacterium tuberculosis. ACS infectious diseases. 2019;5(2):272–80. Epub 2018/12/07. doi: 10.1021/acsinfecdis.8b00256 30501173; PubMed Central PMCID: PMC6371205.

18. Betts JC, Lukey PT, Robb LC, McAdam RA, Duncan K. Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling. Molecular Microbiology. 2002;43(3):717–31. doi: 10.1046/j.1365-2958.2002.02779.x 11929527

19. Carroll P, Muwanguzi-Karugaba J, Parish T. Codon-optimized DsRed fluorescent protein for use in Mycobacterium tuberculosis. BMC research notes. 2018;11(1):685. Epub 2018/10/05. doi: 10.1186/s13104-018-3798-3 30285840; PubMed Central PMCID: PMC6167837.

20. Iversen PW, Beck B, Chen YF, Dere W, Devanarayan V, Eastwood BJ, et al. HTS Assay Validation. In: Sittampalam GS, Coussens NP, Brimacombe K, Grossman A, Arkin M, Auld D, et al., editors. Assay Guidance Manual. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004.

21. Stover CK, Warrener P, VanDevanter DR, Sherman DR, Arain TM, Langhorne MH, et al. A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis. Nature. 2000;405(6789):962–6. Epub 2000/07/06. doi: 10.1038/35016103 10879539.

22. Iacobino A, Piccaro G, Giannoni F, Mustazzolu A, Fattorini L. Fighting tuberculosis by drugs targeting nonreplicating Mycobacterium tuberculosis bacilli. Int J Mycobacteriol. 2017;6(3):213–21. doi: 10.4103/ijmy.ijmy_85_17_85_17 28776518.

23. Salfinger M, Heifets LB. Determination of pyrazinamide MICs for Mycobacterium tuberculosis at different pHs by the radiometric method. Antimicrobial agents and chemotherapy. 1988;32(7):1002–4. doi: 10.1128/aac.32.7.1002 3142340.

24. Piccaro G, Poce G, Biava M, Giannoni F, Fattorini L. Activity of lipophilic and hydrophilic drugs against dormant and replicating Mycobacterium tuberculosis. J Antibiot (Tokyo). 2015;68(11):711–4. doi: 10.1038/ja.2015.52 25944535.

25. (CLSI) CaLSI. Methods for Determining Bactericidal Activity of Antimicrobial Agents; Approved Guideline. Wayne, Pennsylvania: Clinical and Laboratory Standards Institute; 1999.

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2019 Číslo 10
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