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: 2.71). 08/2012; 92(6):453-88. DOI: 10.1016/
Source: PubMed


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.

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Available from: Thomas Dick, Aug 13, 2014
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    • "Redox-indicators such as tetrazolium salts (De-Logu et al., 2003) and Alamar Blue or resazurin has been found to be useful for non-fluorometric readouts (Abate, Mshana, & Miorner, 1998; Caviedes, Delgado, & Gilman, 2002; Foongladda et al., 2002 ; Martin, Camacho, Portaels, & Palomino, 2003; Martin, Portaels, & Palomino, 2007; Palomino et al., 2002). Alamar Blue reduction is the most frequently used endpoint for microplate assays, with a fluorometric (absorption at 530 nm and emission at 590 nm) and an absorbance (at 570 nm) or visual readout after 14 days of incubation (Donkeng et al., 2014; Yemoa et al., 2011; Franzblau et al., 2012). Furthermore, an Alamar Blue-based growth inhibition microplate assay has been adapted for the thermosensitive and slow-growing M. ulcerans within 8 to 10 days. "
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    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
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    • "Traditional Minimum Inhibitory Concentration (MIC) measures the activity of a compound only against AR aerobic cells, representing a non-physiological situation in low or no vascularized necrotic (caseous) granulomas.15 Thus, NR or slowly replicating Mtb assays have been developed including low oxygen (Wayne model), nutrient starvation and nitric-oxide release assays using MZ and INH as positive (growth) and negative (no growth) controls, respectively.48 The necrotic tissue has a pH of about 6.549 while the pH of active TB lesions is between 5.5 and 6.0 or lower.19 "
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    ABSTRACT: Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), which kills about 2 million people annually. Furthermore, 2 billion people worldwide are latently infected with this organism, with 10% of them reactivating to active TB due to re-growth of nonreplicating (dormant) Mtb residing in their tissues. Because of the huge reservoir of latent TB it is important to find novel drugs/drug combinations killing dormant bacilli (microaerophiles, anaerobes and drug-tolerant persisters) surviving for decades in a wide spectrum of granulomatous lesions in the lungs of TB patients. Antibiotic treatment of drug-susceptible TB requires administration of isoniazid, rifampin, pyrazinamide, ethambutol for 2 months, followed by isoniazid and rifampin for 4 months. To avoid reactivation of dormant Mtb to active pulmonary TB, up to 9 months of treatment with isoniazid is required. Therefore, a strategy to eliminate dormant bacilli needs to be developed to shorten therapy of active and latent TB and reduce the reservoir of people with latent TB. Finding drugs with high rate of penetration into the caseous granulomas and understanding the biology of dormant bacilli and in particular of persister cells, phenotypically resistant to antibiotics, will be essential to eradicate Mtb from humans. In recent years unprecedented efforts have been done in TB drug discovery, aimed at identifying novel drugs and drug combinations killing both actively replicating and nonreplicating Mtb in vitro, in animal models and in clinical trials in humans.
    Mediterranean Journal of Hematology and Infectious Diseases 11/2013; 5(1):e2013072. DOI:10.4084/MJHID.2013.072
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    • "We have used isoniazid, which is often utilized as a control in drug screening experiments,24 to provide a proof-of-concept of the feasibility of our bioluminescence–mouse model to assess drug efficacy. With this drug we have observed a similar effect on bioluminescence and cfu; however, other drugs with different mechanisms of action could affect these two readouts to different degrees, as has previously been reported for ethambutol, linezolid and moxifloxacin.14 "
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    ABSTRACT: Objectives In vivo experimentation is costly and time-consuming, and presents a major bottleneck in anti-tuberculosis drug development. Conventional methods rely on the enumeration of bacterial colonies, and it can take up to 4 weeks for Mycobacterium tuberculosis to grow on agar plates. Light produced by recombinant bacteria expressing luciferase enzymes can be used as a marker of bacterial load, and disease progression can be easily followed non-invasively in live animals by using the appropriate imaging equipment. The objective of this work was to develop a bioluminescence-based mouse model of tuberculosis to assess antibiotic efficacy against M. tuberculosis in vivo. Methods We used an M. tuberculosis strain carrying a red-shifted derivative of the firefly luciferase gene (FFlucRT) to infect mice, and monitored disease progression in living animals by bioluminescence imaging before and after treatment with the frontline anti-tuberculosis drug isoniazid. The resulting images were analysed and the bioluminescence was correlated with bacterial counts. Results Using bioluminescence imaging we detected as few as 1.7 × 103 and 7.5 × 104 reporter bacteria ex vivo and in vivo, respectively, in the lungs of mice. A good correlation was found between bioluminescence and bacterial load in both cases. Furthermore, a marked reduction in luminescence was observed in living mice given isoniazid treatment. Conclusions We have shown that an improved bioluminescent strain of M. tuberculosis can be visualized by non-invasive imaging in live mice during an acute, progressive infection and that this technique can be used to rapidly visualize and quantify the effect of antibiotic treatment. We believe that the model presented here will be of great benefit in early drug discovery as an easy and rapid way to identify active compounds in vivo.
    Journal of Antimicrobial Chemotherapy 04/2013; 68(9). DOI:10.1093/jac/dkt155
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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

    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!

    • [Show abstract] [Hide abstract]
      ABSTRACT: Mycobacteria are intracellular pathogens that survive and grow in host macrophages. Following phagocytosis, sustained intracellular bacterial growth depends on its ability to avoid destruction by macrophage-mediated host defences such as lysosomal enzymes, reactive oxygen and the reactive nitrogen intermediates.This suggests that the interaction between host cell and microbe is delicately balanced, and can be tipped in favour of either organism. The identification of Mycobacterium tuberculosis H37Rv (MTB) genes expressed within host cells would contribute greatly to the development of new strategies to fight tuberculosis. In the present study, we compared MTB gene expression in the course of intra- (human macrophages) and extracellular growth (Sauton's medium) to ascertain whether differences might occur between gene-expression patterns in the two habitats of replication. Using reverse-transcriptase polymerase chain reaction (RT-PCR) on a group of 14 MTB-Complex-specific genes, we found that MT10Sa (a small stable RNA), 35 kDa (unknown), ahpC (alkyl hydroperoxide reductase, AhpC), sigF (alternative RNA Polymerase sigma factor), and katG (catalase-peroxidase, HPI) genes are expressed in both the environments, while Ag85B, Ag85C (members of the Antigen 85 Complex), rpoV (RNA Polymerase sigma factor) and ESAT6 (early secretory antigen, 6 kDa) are expressed only in the in vitro culture; on the other hand, Ag85A (Antigen 85 Complex), rpoB (RNA Polymerase β sub-unit), pab (Protein antigen b), invA and invB genes (encoding proteins that show homologies with p60 of Listeria monocytogenes) are expressed only inside the macrophage. Positive RT-PCR products on cDNAs for these genomic regions were not obtained from approximately 1000-fold more bacteria grown in Laboratory Broth. Identification of M. tuberculosis genes expressed in response to phagocytosis by human macrophages increases our basic understanding of the host–pathogen interaction, and helps to identify bacterial factors necessary for in vivo survival and growth.
      Gene 09/2000; 253(2-253):281-291. DOI:10.1016/S0378-1119(00)00249-3

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