Rapid Methods for Testing Inhibitors of Mycobacterial Growth

  • Dimitrios Evangelopoulos
  • Sanjib Bhakta
Part of the Methods in Molecular Biology book series (MIMB, volume 642)


Considering the increased concerns with controlling infectious epidemics such as tuberculosis, a global concerted effort (WHO) is now dead-lined to tackle the emergence of extensive drug resistance through identifying a novel line of therapeutics which will on the one hand shorten the course of treatment and on the other is also expected to be effective against the emerging resistant strains. Major problems with the preclinical drug screening against the uniquely slow-growing pathogen Mycobacterium tuberculosis are either found expensive, time-consuming, or require a highly complex laboratory setup. A rapid and convenient, although relatively inexpensive, method requiring very little consumption of inhibitors within a simple microbiology setup for antimycobacterial screening is thus timely. The spot-culture growth inhibition assay aims to test the biological activity of a number of newly discovered natural products and thousands of novel chemicals synthesized on the basis of basic structural and molecular biology studies. Many different classes of novel chemical entities are now independently prepared around the world by distinguished chemists on the chemical behavior of the group of molecules. To serve the purpose of antimycobacterials screening, we aim to describe a method in this chapter performed in a six-well plate format. This method can also be extended accurately to a 96-well plate format according to the necessity of the project. In addition to evaluating a range of prospective drug candidates, this method would also contribute to elucidate substrates for many putative endogenous pathways through comparing the chemical inhibition with the corresponding genetic modification.

Key words

Drug susceptibility test Mycobacteria Growth inhibition 



The spot culture growth inhibition method was initially developed in Professor Edith Sim’s laboratory at Oxford University with a Wellcome Trust Travelling fellowship (Grant Code: HBWM7) to S.B. Authors would like to thank Professor Edith Sim for her encouragement.


  1. 1.
    Sutter VL, Finegold SM (1971) Antibiotic disc susceptibility tests for rapid presumptive identification of Gram-negative anaerobic bacilli. Appl Microbiol 21(1):13–20PubMedGoogle Scholar
  2. 2.
    Barker LP, Lien BA, Brun OS, Schaak DD, McDonough KA, Chang LC (2007) A Mycobacterium marinum zone of inhibition assay as a method for screening potential antimycobacterial compounds from marine extracts. Planta Med 73(6):559–563CrossRefPubMedGoogle Scholar
  3. 3.
    Casal MJ, Rodriguez FC (1981) Simple, new test for rapid differentiation of the Mycobacterium fortuitum complex. J Clin Microbiol 13(5):989–990PubMedGoogle Scholar
  4. 4.
    Kim SJ (2005) Drug-susceptibility testing in tuberculosis: methods and reliability of results. Eur Respir J 25(3):564–569CrossRefPubMedGoogle Scholar
  5. 5.
    Isenberg HD (2004) Clinical microbiology procedures handbook, 2nd edn. ASM, Washington, DCGoogle Scholar
  6. 6.
    Canetti G, Fox W, Khomenko A, Mahler HT, Menon NK, Mitchison DA et al (1969) Advances in techniques of testing mycobacterial drug sensitivity, and the use of sensitivity tests in tuberculosis control programmes. Bull World Health Organ 41(1):21–43PubMedGoogle Scholar
  7. 7.
    Siddiqi SH, Libonati JP, Middlebrook G (1981) Evaluation of rapid radiometric method for drug susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol 13(5):908–912PubMedGoogle Scholar
  8. 8.
    DeCoster DJ, Vena RM, Callister SM, Schell RF (2005) Susceptibility testing of Mycobacterium tuberculosis: comparison of the BACTEC TB-460 method and flow cytometric assay with the proportion method. Clin Microbiol Infect 11(5):372–378CrossRefPubMedGoogle Scholar
  9. 9.
    Coban AY, Bilgin K, Uzun M, Akgunes A, Yusof A, Durupinar B (2008) Comparative study for determination of Mycobacterium tuberculosis susceptibility to first- and second-line antituberculosis drugs by the Etest using 7H11, blood, and chocolate agar. J Clin Microbiol 46(12):4095–4098CrossRefPubMedGoogle Scholar
  10. 10.
    Palomino JC, Traore H, Fissette K, Portaels F (1999) Evaluation of Mycobacteria Growth Indicator Tube (MGIT) for drug susceptibility testing of Mycobacterium tuberculosis. Int J Tuberc Lung Dis 3(4):344–348PubMedGoogle Scholar
  11. 11.
    Martin A, von Groll A, Fissette K, Palomino JC, Varaine F, Portaels F (2008) Rapid detection of Mycobacterium tuberculosis resistance to second-line drugs by use of the manual mycobacterium growth indicator tube system. J Clin Microbiol 46(12):3952–3956CrossRefPubMedGoogle Scholar
  12. 12.
    Tortoli E, Benedetti M, Fontanelli A, Simonetti MT (2002) Evaluation of automated BACTEC MGIT 960 system for testing susceptibility of Mycobacterium tuberculosis to four major antituberculous drugs: comparison with the radiometric BACTEC 460TB method and the agar plate method of proportion. J Clin Microbiol 40(2):607–610CrossRefPubMedGoogle Scholar
  13. 13.
    Bemer P, Palicova F, Rusch-Gerdes S, Drugeon HB, Pfyffer GE (2002) Multicenter evaluation of fully automated BACTEC Mycobacteria Growth Indicator Tube 960 system for susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol 40(1):150–154CrossRefPubMedGoogle Scholar
  14. 14.
    Huang TS, Tu HZ, Lee SS, Huang WK, Liu YC (2002) Antimicrobial susceptibility testing of Mycobacterium tuberculosis to first-line drugs: comparisons of the MGIT 960 and BACTEC 460 systems. Ann Clin Lab Sci 32(2):142–147CrossRefPubMedGoogle Scholar
  15. 15.
    Scarparo C, Ricordi P, Ruggiero G, Piccoli P (2004) Evaluation of the fully automated BACTEC MGIT 960 system for testing susceptibility of Mycobacterium tuberculosis to pyrazinamide, streptomycin, isoniazid, rifampin, and ethambutol and comparison with the radiometric BACTEC 460TB method. J Clin Microbiol 42(3):1109–1114CrossRefPubMedGoogle Scholar
  16. 16.
    Abate G, Mshana RN, Miorner H (1998) Evaluation of a colorimetric assay based on 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) for rapid detection of rifampicin resistance in Mycobacterium tuberculosis. Int J Tuberc Lung Dis 2(12):1011–1016PubMedGoogle Scholar
  17. 17.
    Franzblau SG, Witzig RS, McLaughlin JC, Torres P, Madico G, Hernandez A et al (1998) Rapid, low-technology MIC determination with clinical Mycobacterium tuberculosis isolates by using the microplate alamar blue assay. J Clin Microbiol 36:362–366PubMedGoogle Scholar
  18. 18.
    Martin A, Camacho M, Portaels F, Palomino JC (2003) Resazurin microtiter assay plate testing of Mycobacterium tuberculosis susceptibilities to second-line drugs: rapid, simple, and inexpensive method. Antimicrob Agents Chemother 47(11):3616–3619CrossRefPubMedGoogle Scholar
  19. 19.
    Palomino JC, Martin A, Camacho M, Guerra H, Swings J, Portaels F (2002) Resazurin microtiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 46(8):2720–2722CrossRefPubMedGoogle Scholar
  20. 20.
    Leonard B, Coronel J, Siedner M, Grandjean L, Caviedes L, Navarro P et al (2008) Inter- and intra-assay reproducibility of microplate Alamar blue assay results for isoniazid, rifampicin, ethambutol, streptomycin, ciprofloxacin, and capreomycin drug susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol 46(10):3526–3529CrossRefPubMedGoogle Scholar
  21. 21.
    Martin A, Morcillo N, Lemus D, Montoro E, Telles MA, Simboli N et al (2005) Multicenter study of MTT and resazurin assays for testing susceptibility to first-line anti-tuberculosis drugs. Int J Tuberc Lung Dis 9(8):901–906PubMedGoogle Scholar
  22. 22.
    Martin A, Portaels F, Palomino JC (2007) Colorimetric redox-indicator methods for the rapid detection of multidrug resistance in Mycobacterium tuberculosis: a systematic review and meta-analysis. J Antimicrob Chemother 59(2):175–183CrossRefPubMedGoogle Scholar
  23. 23.
    Akcali S, Cicek C, Surucuoglu S, Ozbakkaloglu B (2005) E-test: an alternative method for susceptibility testing of Mycobacterium tuberculosis. Med Princ Pract 14(4):264–267CrossRefPubMedGoogle Scholar
  24. 24.
    Hazbon MH, del Socorro Orozco M, Labrada LA, Tovar R, Weigle KA, Wanger A (2000) Evaluation of Etest for susceptibility testing of multidrug-resistant isolates of Mycobacterium tuberculosis. J Clin Microbiol 38(12):4599–4603PubMedGoogle Scholar
  25. 25.
    Wanger A, Mills K (1996) Testing of Mycobacterium tuberculosis susceptibility to ethambutol, isoniazid, rifampin, and streptomycin by using Etest. J Clin Microbiol 34(7):1672–1676PubMedGoogle Scholar
  26. 26.
    Pina-Vaz C, Costa-de-Oliveira S, Rodrigues AG (2005) Safe susceptibility testing of Mycobacterium tuberculosis by flow cytometry with the fluorescent nucleic acid stain SYTO 16. J Med Microbiol 54(Pt 1):77–81CrossRefPubMedGoogle Scholar
  27. 27.
    Gali N, Dominguez J, Blanco S, Prat C, Quesada MD, Matas L et al (2003) Utility of an in-house mycobacteriophage-based assay for rapid detection of rifampin resistance in Mycobacterium tuberculosis clinical isolates. J Clin Microbiol 41(6):2647–2649CrossRefPubMedGoogle Scholar
  28. 28.
    Riska PF, Su Y, Bardarov S, Freundlich L, Sarkis G, Hatfull G et al (1999) Rapid film-based determination of antibiotic susceptibilities of Mycobacterium tuberculosis strains by using a luciferase reporter phage and the Bronx Box. J Clin Microbiol 37(4):1144–1149PubMedGoogle Scholar
  29. 29.
    Anderton MC, Bhakta S, Besra GS, Jeavons P, Eltis LD, Sim E (2006) Characterization of the putative operon containing arylamine N-acetyltransferase (nat) in Mycobacterium bovis BCG. Mol Microbiol 59(1):181–192CrossRefPubMedGoogle Scholar
  30. 30.
    Madikane VE, Bhakta S, Russell AJ, Campbell WE, Claridge TD, Elisha BG et al (2007) Inhibition of mycobacterial arylamine N-acetyltransferase contributes to anti-mycobacterial activity of Warburgia salutaris. Bioorg Med Chem 15(10):3579–3586CrossRefPubMedGoogle Scholar
  31. 31.
    Smellie IA, Bhakta S, Sim E, Fairbanks AJ (2007) Synthesis of putative chain terminators of mycobacterial arabinan biosynthesis. Org Biomol Chem 5(14):2257–2266CrossRefPubMedGoogle Scholar
  32. 32.
    O’Donnell G, Poeschl R, Zimhony O, Gunaratnam M, Moreira JB, Neidle S, et al (2009) Bioactive pyridine-N-oxide disulfides from Allium stipitatum. J Nat Prod 72(3):360–365CrossRefPubMedGoogle Scholar
  33. 33.
    Allen BW (1998) Mycobacteria: general culture methodology and safety considerations. In: Parish TaS NG (ed) Mycobacteria protocols. Humana Press, Totowa, New Jersey, pp 15–30CrossRefGoogle Scholar
  34. 34.
    Ramon-Garcia S, Martin C, Ainsa JA, De Rossi E (2006) Characterization of tetracycline resistance mediated by the efflux pump Tap from Mycobacterium fortuitum. J Antimicrob Chemother 57(2):252–259CrossRefPubMedGoogle Scholar
  35. 35.
    Bhusal Y, Shiohira CM, Yamane N (2005) Determination of in vitro synergy when three antimicrobial agents are combined against Mycobacterium tuberculosis. Int J Antimicrob Agents 26(4):292–297CrossRefPubMedGoogle Scholar
  36. 36.
    Odds FC (2003) Synergy, antagonism, and what the chequerboard puts between them. J Antimicrob Chemother 52(1):1CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Dimitrios Evangelopoulos
    • 1
  • Sanjib Bhakta
    • 1
  1. 1.Faculty of Science/ Institute of Structural and Molecular Biology, Birkbeck College, School of Biological and Chemical SciencesUniversity of LondonLondonUK

Personalised recommendations