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      Rapid, Low-Technology MIC Determination with Clinical Mycobacterium tuberculosis Isolates by Using the Microplate Alamar Blue Assay

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          ABSTRACT

          A colorimetric, microplate-based Alamar Blue assay (MABA) method was used to determine the MICs of isoniazid (INH), rifampin, streptomycin (SM), and ethambutol (EMB) for 34 Peruvian Mycobacterium tuberculosis isolates (including both pansensitive and multidrug-resistant strains) and the H 37 Rv strain by using bacterial suspensions prepared directly from solid media. Results for all isolates were available within 8 days. Discordant results were observed on initial tests for 3 of 16 INH-susceptible isolates, 5 of 31 EMB-susceptible isolates, and 2 of 4 SM-resistant isolates (by the BACTEC 460 system). The overall agreements between the MICs obtained by MABA and the results obtained with the BACTEC 460 system were 87.9% for initial results and 93.6% after retesting 12 of 17 samples with discrepant results. Interpretation of MABA endpoints improved with technical experience. The MABA is a simple, rapid, low-cost, appropriate technology which does not require expensive instrumentation and which makes use of a nontoxic, temperature-stable reagent.

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          Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium.

          L. Collins, S Franzblau (1997)
          In response to the need for rapid, inexpensive, high-throughput assays for antimycobacterial drug screening, a microplate-based assay which uses Alamar blue reagent for determination of growth was evaluated. MICs of 30 antimicrobial agents against Mycobacterium tuberculosis H37Rv, M. tuberculosis H37Ra, and Mycobacterium avium were determined in the microplate Alamar blue assay (MABA) with both visual and fluorometric readings and compared to MICs determined in the BACTEC 460 system. For all three mycobacterial strains, there was < or = 1 dilution difference between MABA and BACTEC median MICs in four replicate experiments for 25 to 27 of the 30 antimicrobics. Significant differences between MABA and BACTEC MICs were observed with 0, 2, and 5 of 30 antimicrobial agents against H37Rv, H37Ra, and M. avium, respectively. Overall, MICs determined either visually or fluorometrically in MABA were highly correlated with those determined in the BACTEC 460 system, and visual MABA and fluorometric MABA MICs were highly correlated. MICs of rifampin, rifabutin, minocycline, and clarithromycin were consistently lower for H37Ra compared to H37Rv in all assays but were similar for most other drugs. M. tuberculosis H37Ra may be a suitable surrogate for the more virulent H37Rv strain in primary screening of compounds for antituberculosis activity. MABA is sensitive, rapid, inexpensive, and nonradiometric and offers the potential for screening, with or without analytical instrumentation, large numbers of antimicrobial compounds against slow-growing mycobacteria.
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            Evaluation of bacterial survival and phagocyte function with a fluorescence-based microplate assay.

            Michael Shiloh, C Nathan Marti, J H Ruan (1997)
            To compare antibacterial function in macrophages from mice deficient in the respiratory burst oxidase or inducible nitric oxide synthase, we developed a fluorescence-based microplate assay of bacterial survival. As bacteria grow, they convert a formulation of resazurin termed AlamarBlue from its nonfluorescent oxidized state to its fluorescent reduced state. The time required to achieve a given fluorescence is inversely proportional to the number of viable bacteria present when the dye is added. This relationship allows a precise, accurate assessment of bacterial numbers with greater sensitivity and throughput and at less cost than conventional assays. The assay facilitated quantification of the killing of Escherichia coli by S-nitrosoglutathione and hydrogen peroxide and of Salmonella typhimurium by human neutrophils and mouse macrophages. Mouse macrophages lacking the 91-kDa subunit of the respiratory burst oxidase were deficient in their ability to kill S. typhimurium, while those lacking inducible nitric oxide synthase were unimpaired.
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              Colorimetric method for determining MICs of antimicrobial agents for Mycobacterium tuberculosis.

              Monika J. Madej, Benjamin A. Babst, V Cawthon … (1995)
              A colorimetric method for quantitative measurement of the susceptibility of Mycobacterium tuberculosis to antimicrobial agents is described. The method utilizes an oxidation-reduction dye, Alamar blue, as an indicator of growth. By this method, MICs of isoniazid, rifampin, streptomycin, and ethambutol were determined for 50 strains of M. tuberculosis. Colorimetric MIC results were available on the 7th, 10th, or 14th day of incubation for 29 (58%), 14 (28%), and 7 (14%) of the 50 strains, respectively. When MIC susceptibility results were compared with results obtained by the agar proportion method, increased levels of resistance detected by agar proportion were associated with higher MICs obtained by the colorimetric method. Tentative interpretive criteria for colorimetric MIC results which showed good agreement with results obtained by the agar proportion method were established. Interpretive agreement between the two methods was 98% for isoniazid, rifampin, and ethambutol and 94% for streptomycin. Overall, there was agreement between the two methods for 194 of 200 test results (97%). The colorimetric method is a rapid, quantitative, nonradiometric method for determining the antimicrobial susceptibility of M. tuberculosis.
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                Author and article information

                Journal
                Title: Journal of Clinical Microbiology
                Abbreviated Title: J Clin Microbiol
                Publisher: American Society for Microbiology
                ISSN (Print): 0095-1137
                ISSN (Electronic): 1098-660X
                Publication date Created: February 1998
                Publication date (Print): February 1998
                Volume: 36
                Issue: 2
                Pages: 362-366
                Affiliations
                [1 ]<!--label omitted: 1-->GWL Hansen’s Disease Center, Baton Rouge, Louisiana1;
                [2 ]<!--label omitted: 2-->Johns Hopkins University School of Public Health, Baltimore, Maryland2;
                [3 ]<!--label omitted: 3-->University of New Mexico Health Science Center3 and
                [4 ]<!--label omitted: 4-->Universidad Peruana Cayetano Heredia, Lima, Peru4; and
                [5 ]<!--label omitted: 5-->Scientific Laboratory Division, New Mexico State Department of Health,5 Albuquerque, New Mexico
                Article
                DOI: 10.1128/JCM.36.2.362-366.1998
                PMC ID: 104543
                PubMed ID: 9466742
                SO-VID: c586f74b-da95-429f-b2d0-f9b97c2d7a2e
                Copyright © © 1998
                License:

                https://journals.asm.org/non-commercial-tdm-license

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