Use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide for rapid detection of rifampin-resistant Mycobacterium tuberculosis

Abstract

We describe a test which uses the ability of viable cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to detect resistance to a bactericidal drug, rifampin, in in vitro-cultured Mycobacterium tuberculosis. The assay shows a linear relationship between the number of viable bacteria and the ability to reduce MTT. Dead mycobacteria were unable to reduce MTT. Rifampin-sensitive M. bovis (BCG) and M. tuberculosis exposed to rifampin showed a rifampin concentration-dependent inhibition of the ability to reduce MTT, while the resistant strains were unaffected. The inhibition of MTT reduction after treatment with rifampin paralleled the reduction in the number of CFU. By using mixing experiments in which the population percentages of rifampin-sensitive and -resistant strains were varied, the assay could detect the presence of rifampin resistance in the mixture when at least 1% of the bacterial population was composed of drug-resistant strains. The assay is cheap, can be visually read, and requires less than 3 days to obtain susceptibility results. The total time required to obtain results, from the time sputum is received in the laboratory, is, in most cases, less than 4 to 5 weeks, which is the time required for primary culture of the bacteria. The MTT assay could, in combination with a test to detect resistance to isoniazid, be a cheap and rapid screening method for multidrug-resistant M. tuberculosis that is affordable even by low-income countries where tuberculosis is a major public health problem.

FIG. 1

Reduction of MTT by M. bovis BCG in vitro is dependent on the concentration of the bacilli. Bacilli killed by treatment with 5% sodium hypochlorite for 15 min cannot reduce MTT. Heat killing (immersion in a boiling water bath at 91°C) was less efficient, especially at high bacterial concentrations.

FIG. 2

Effect of rifampin on the ability of M. bovis BCG to reduce MTT. BCG treated with various concentrations of rifampin for 48 or 72 h showed a drug dose-dependent inhibition of the ability to reduce MTT (A). This reduction in the ability of rifampin-treated bacilli to reduce MTT paralleled a reduction in the number of CFU when these bacilli were recultured (B). In both experiments, 5 × 10⁷ bacilli were set up in culture, treated for 48 or 72 h with rifampin, and processed for the MTT or CFU assay as described in Materials and Methods.

FIG. 3

Standard rifampin-resistant (•; H₃₇Rv [ATCC 35838]) and rifampicin-sensitive (▪; H₃₇Ra [ATCC 35836]) M. tuberculosis strains were set up in culture and treated with rifampin (1 μg/ml) for 48 h before being processed for the MTT reduction assay. Rifampin at 1 μg/ml was able to completely inhibit MTT reduction by the rifampin-sensitive M. tuberculosis strain, while it did not affect the rifampin-resistant strain.

FIG. 4

Different MTT reduction patterns of rifampin-resistant (A) and -sensitive (B) M. tuberculosis isolates obtained after treatment with rifampin for 48 h. Bacilli (5 × 10⁷) were set up in cultures containing rifampin and processed for MTT reduction. Drug-resistant isolates were not inhibited in the ability to reduce MTT, while drug-sensitive isolates showed a drug dose-dependent reduction in the ability to reduce MTT after treatment with rifampin. The sensitivity of the clinical isolates had previously been determined by using the BACTEC system. Strains 35838 and 35836 are rifampin-resistant and rifampin-sensitive reference strains, respectively.

FIG. 5

Detection of a rifampin-resistant subpopulation. Cultures containing various proportions of rifampin-resistant (H₃₇Rv [ATCC 35838]) and rifampin-sensitive (H₃₇Ra [ATCC 35836]) bacteria were set up in culture, treated with rifampin at 1 μg/ml, and processed for the MTT reduction assay. Bacterial populations containing a drug-resistant subpopulation of more than 1% could be detected in that their ability to reduce MTT was not inhibited by rifampin at 1 μg/ml. Further, the ability to continue to reduce MTT in the presence of rifampin increased with the proportion of the drug-resistant subpopulation.