Article

Comprehensive analysis of methods used for the evaluation of compounds against Mycobacterium tuberculosis

Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, 833 S. Wood Street, Chicago, IL 60621-7231, USA.
Tuberculosis (Edinburgh, Scotland) (Impact Factor: 3.5). 08/2012; 92(6):453-88. DOI: 10.1016/j.tube.2012.07.003
Source: PubMed

ABSTRACT In drug development, there are typically a series of preclinical studies that must be completed with new compounds or regimens before use in humans. A sequence of in vitro assays followed by in vivo testing in validated animal models to assess the activity against Mycobacterium tuberculosis, pharmacology and toxicity is generally used for advancing compounds against tuberculosis in a preclinical stage. A plethora of different assay systems and conditions are used to study the effect of drug candidates on the growth of M. tuberculosis, making it difficult to compare data from one laboratory to another. The Bill and Melinda Gates Foundation recognized the scientific gap to delineate the spectrum of variables in experimental protocols, identify which of these are biologically significant, and converge towards a rationally derived standard set of optimized assays for evaluating compounds. The goals of this document are to recommend protocols and hence accelerate the process of TB drug discovery and testing. Data gathered from preclinical in vitro and in vivo assays during personal visits to laboratories and an electronic survey of methodologies sent to investigators is reported. Comments, opinions, experiences as well as final recommendations from those currently engaged in such preclinical studies for TB drug testing are being presented. Certain in vitro assays and mouse efficacy models were re-evaluated in the laboratory as head-to-head experiments and a summary is provided on the results obtained. It is our hope that this information will be a valuable resource for investigators in the field to move forward in an efficient way and that key variables of assays are included to ensure accuracy of results which can then be used for designing human clinical trials. This document then concludes with remaining questions and critical gaps that are in need of further validation and experimentation.

Download full-text

Full-text

Available from: Thomas Dick, Aug 13, 2014
2 Followers
 · 
162 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Buruli ulcer (BU) caused by Mycobacterium ulcerans is the third most common chronic mycobacterial infection in humans. Approximately 5000 cases are reported annually from at least 33 countries around the globe, especially in rural African communities. Even though anti-mycobacterial therapy is often effective for early nodular or ulcerative lesions, surgery is sometimes employed for aiding wound healing and correction of deformities. The usefulness of the antibiotherapy nonetheless is challenged by huge restrictive factors such as high cost, surgical scars and loss of income due to loss of man-hours, and in some instances employment. For these reasons, more effective and safer drugs are urgently needed, and research programs into alternative therapeutics including investigation of natural products should be encouraged. There is the need for appropriate susceptibility testing methods for the evaluation of potency. A number of biological assay methodologies are in current use, ranging from the classical agar and broth dilution assay formats, to radiorespirometric, dye-based, and fluorescent/luminescence reporter assays. Mice, rats, armadillo, guinea pigs, monkeys, grass cutters and lizards have been suggested as animal models for Buruli ulcer. This review presents an overview of in vitro and in vivo susceptibility testing methods developed for the determination of anti-Buruli ulcer activity of natural products and derivatives. Copyright © 2015. Published by Elsevier Inc.
    Journal of pharmacological and toxicological methods 03/2015; 73. DOI:10.1016/j.vascn.2015.03.001 · 2.15 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Mycobacterium tuberculosis is exposed to hypoxia and acidity within granulomatous lesions. In this study, an acidic culture model of M. tuberculosis was used to test drug activity against aerobic, 5-day-old (A5) and hypoxic, 5-, 12-, 19-day-old (H5, H12, H19, respectively) bacilli after 7, 14, 21 days of exposure. In A cultures, CFUs and pH rapidly increased while in H cultures growth stopped and pH increased slightly. Ten drugs were tested: rifampin (R), isoniazid (I), pyrazinamide (Z), ethambutol (E), moxifloxacin (MX), amikacin (AK), metronidazole (MZ), nitazoxanide (NZ), niclosamide (NC), PA-824 (PA). Rifampin was the most active against A5, H5, H12, H19 bacilli. Moxifloxacin and AK efficiently killed A5 and H5 cells, I was active mostly against A5 cells, Z against H12 and H19 cells, E was low active. Among nitrocompounds, NZ, NC and PA were effective against A5, H5, H12, H19 cells while MZ was active against H12 and H19 cells. To kill all A and H cells, A5- and H5-active agents R, MX and AK were used in combination with MZ, NZ, NC or PA, in comparison with R-I-Z-E, currently used for human therapy. Mycobacterial viability was determined by CFUs and a sensitive test in broth (day-to-positivity, MGIT 960 system). As shown by lack of regrowth in MGIT, the most potent combination was R-MX-AK-PA, which killed all A5, H5, H12, H19 cells in 14 days. These observations demonstrate the sterilizing effect of drug combinations against different M. tuberculosis stages grown in aerobic and hypoxic acidic conditions.
    Antimicrobial Agents and Chemotherapy 01/2013; DOI:10.1128/AAC.02154-12 · 4.45 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Tuberculosis is a serious global health problem caused by the bacterium Mycobacterium tuberculosis. There is an urgent need for discovery and development of new treatments, but this can only be accomplished through rapid and reproducible M. tuberculosis assays designed to identify potent inhibitors. We developed an automated 96-well assay utilizing a recombinant strain of M. tuberculosis expressing a far-red fluorescent reporter to determine the activity of novel compounds; this allowed us to measure growth by monitoring both optical density and fluorescence. We determined that optical density and fluorescence were correlated with cell number during logarithmic phase growth. Fluorescence was stably maintained without antibiotic selection over 5 days, during which time cells remained actively growing. We optimized parameters for the assay, with the final format being 5 days' growth in 96-well plates in the presence of 2% w/v DMSO. We confirmed reproducibility using rifampicin and other antibiotics. The dual detection method allows for a reproducible calculation of the minimum inhibitory concentration (MIC), at the same time detecting artefacts such as fluorescence quenching or compound precipitation. We used our assay to confirm anti-tubercular activity and establish the structure activity relationship (SAR) around the imidazo[1,2-a]pyridine-3-carboxamides, a promising series of M. tuberculosis inhibitors.
    PLoS ONE 04/2013; 8(4):e60531. DOI:10.1371/journal.pone.0060531 · 3.53 Impact Factor

Questions & Answers about this publication

  • Kathryn Sydney Doornbos added an answer in MDR-TB:
    What could explain the decrease in CFU/ml for my control during a time-kill analysis?

    I am working MDR-TB and performing a time-kill assay. I use 1 x 10^5 CFU/ml cells from 7H10 OADC agar (~14 days incubation) and inoculate them into liquid culture (Sauton media). I then monitor CFU/ml on treatment days of 0,1,2,3 and 6 using 7H10 OADC. I noticed that my control MDR-TB have a gradual decrease over the days i.e, with regards to log10 CFU/ml, 5.09, 5.27, 5.20, 5.05 and 4.64. Comparing day 0 to day 6, there is difference of 0.45. Statistically, there is no difference between the control and RIF+INH treatment, but a difference for the peptide I am testing. 

    I would like to know if I can still use these results and if the decrease of my MDR-TB control is something natural.

    Before I got to the time-kill assay, I was using the microtiter Alamar Blue assay and by 6, the MDR-TB control was sufficiently changing Alamar Blue from blue to pink indicating sufficient growth.

    I also performed the assay using H37Ra and there was an increase in growth over the six days.

    I really hope someone can shed light on this issue?

    All experiments were conducted in triplicate and independent of each other.

    Kathryn Sydney Doornbos · University of Alabama at Birmingham

    Can you clarify why you are going from 7H10/OADC to Sauton's to 710/OADC? What detergents are you using in each medium?

    The decrease you are seeing may be due to the fact that Sauton's is a defined medium that is very, very minimal compared to 7H9/OADC. In our hands, cultures grown in Sauton's grow more slowly, seem to aggregate more and do not reach comparable ODs when compared to 7H9/OADC. You should also be aware that growth in Sauton's changes the transcriptome of Mtb, can alter its susceptibility to anti-mycobacterial compounds and its susceptibility to other metabolites. If you don't need a defined medium at this step, which you may since you are testing peptides and not conventional compounds, I'd suggest using 7H9/OADC and determining if you still observe this effect.

    If there is a bona fide reason that a defined medium must be used, you have a few options: you could pre-culture your strains in 7H9/OADC to mid-log, wash and re-innoculate into Sauton's, and then allow this culture to reach mid log before restarting new cultures for your time-course assay. This acclimates your bacteria to the minimal media and would ensure that the you aren't seeing 'growing pains' in your time-kill assay. Alternately, you could try HdB, another minimal media, and go about acclimating it as above. Some strains grow better in HdB vs Sauton's or vice-versa. Just be sure to use tyloxapol, not tween-80, as your detergent in any minimal media as we have observed that tween without BSA is toxic to the culture. Good luck!