Colorimetric method for determining MICs of antimicrobial agents for Mycobacterium tuberculosis

Abstract

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.

Drug resistance mechanisms and drug susceptibility testing for tuberculosis

“…Broth microdilution methods, including using several colorimetric indicators, have been developed and used as research tools or to establish the ECOFF for new drugs. [123][124][125] A dryformat, 96-well microtitre plate assay (Sensititre MYCOTB plate; Thermo Fisher Scientific Inc., Waltham, MA, USA) has been commercially available since 2010 and early validation studies have returned promising results. [126][127][128][129][130] This assay performs MIC for 12 anti-TB drugs on the same plates.…”

Rapid drug resistance detection in Mycobacterium tuberculosis: a review of colourimetric methods

“…Colourimetric methods for detecting drug resistance in M. tuberculosis are generally based on the reduction of an oxidation-reduction indicator added to a liquid culture medium after M. tuberculosis has been exposed in vitro to different antibiotics. Resistance is detected by a change in colour of the oxidation-reduction indicator, which is directly proportional to the number of viable mycobacteria in the medium [12]. More recently, another simple format, the nitrate reductase assay (NRA), has also been proposed as a rapid method for detecting drug resistance in TB [13].…”

“…One of the first redox indicators to be used for detecting drug resistance in M. tuberculosis was Alamar blue (Trek Diagnostics, Westlake, OH, USA). This approach was first proposed by Yajko et al [12] in a study that evaluated the activity of the first-line anti-tuberculosis drugs isoniazid, rifampicin, ethambutol and streptomycin against clinical isolates of M. tuberculosis. Alamar blue is a proprietary reagent that had been used previously to study metabolism and viability in other microorganisms [14,15], and to measure toxicity in prokaryotic and eukaryotic cells [16].…”

“…It is blue in colour in the oxidised state, but turns pink when reduced as a result of bacterial metabolism, with the two colours being differentiated easily with the naked eye. The study of Yajko et al [12] was important, since it showed for the first time that MICs of isoniazid, rifampicin, streptomycin and ethambutol could be determined after incubation of M. tuberculosis isolates for only 1-2 weeks in the presence of antibiotics. When compared with results obtained using the agar proportion method, a higher level of resistance according to Alamar blue reduction was always associated with higher MICs, and the overall agreement between the two methods was 97%.…”

A protein kinase inhibitor as an antimycobacterial agent

“…Bacteria were diluted to 1U10 6 CFUs in 7H9 media as previously described [20] and were treated with varying doses of kinase inhibitors. Measurement of optical densities were done as previously described [21] Media-only controls, alamarBlue1 in media controls, BCG growth controls, and drug treatments were utilized to calculate the percent reduction of the culture [21], which is directly related to mycobacterial cell growth [20].…”