Comparison of Two Rapid Colorimetric Methods for Determining Resistance of Mycobacterium tuberculosis to Rifampin, Isoniazid, and Streptomycin in Liquid Medium

Abstract

The usefulness of two colorimetric methods for the determination of the susceptibility or resistance of Mycobacterium tuberculosis to rifampin, streptomycin, and isoniazid in liquid medium based on the reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was investigated. The agar proportion method was used as the reference method. Results obtained indicate that the sensitivity of the XTT reduction assay for the detection of rifampin resistance was comparable to that observed, and previously described, for the MTT assay. However, the reduction of XTT yields a water-soluble formazan that can be easily quantified without performing additional steps such as addition of lysing buffer and solubilization. Furthermore, the colorimetric assays, based on the reduction of XTT and MTT for the detection of isoniazid and streptomycin resistance in Mycobacterium tuberculosis, were standardized. The inhibition of MTT and

Rapid susceptibility testing for slowly growing nontuberculous mycobacteria using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium WST-1

“…Furthermore, it became clear that WST-1 was superior to the conventional tetrazolium salt XTT with regard to its reactive efficiency with the hydroquinones produced by the slowly growing NTM and the way in which it was influenced by the different components in the medium. The XTT colorimetric method has already been applied to the susceptibility testing of mycobacteria [21][22][23][24]. According to the previously reported method, however, XTT was added to the test solution following the incubation of the mycobacteria and the antibiotics, because of the non-cellular reduction of the XTT that can occur in the medium during the incubation period.…”

“…It is, therefore, necessary to use an electron mediator to facilitate the cellular reduction of the tetrazolium salts. In previous reports, XTT has been used in combination with an electron mediator such as 2-methyl-1,4-NQ or PMS for the susceptibility testing of mycobacteria [21][22][23][24]. The WST-1 and XTT colorimetric methods were, therefore, compared in terms of their reactivity with M. kansasii and M. avium (Fig.…”

“…The application of XTT in conjunction with an electron mediator such as 2-methyl-1,4-napthoquinone (NQ) or phenazine methosulfate (PMS) has been used in rapid colorimetric assays for the antimicrobial susceptibility testing of both bacteria and fungi [18][19][20]. The XTT colorimetric method has also been applied to the susceptibility testing of mycobacteria [21][22][23][24]. In the previously reported method, however, XTT was only added to the test solution after the mycobacteria and antibiotics had been incubated for a suitable period of time.…”

Antimycobacterial susceptibility testing methods for natural products research

“…extract dissolved in blank medium without microorganisms (52, 19). An important alternative can be the use of oxidation/reduction indicator dyes such as Alamar Blue (27, 36, 37), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (1, 22), 2,3,5-triphenyltetrazolium chloride (TTC) (44), and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazoliumhydroxide (XTT) (23), with which, the growth/inhibition can be read visually; and the reduced form of these dyes can also be quantitated colorimetrically by measuring absorbance at 570 nm, or fluorimetrically by exciting at 530 nm and detecting emission at 590 nm; the latter mode is more sensitive. These more quantitative assays are also capable of detecting partial inhibition, making it ideal for determining the relative activity of fractions using one or two concentrations (52).…”

Peroxidyme‐Amplified Radical Chain Reaction (PARCR): Visible Detection of a Catalytic Reporter

“…Chemical reactions that produce visible color changes have become indispensable to modern analytics and diagnostics, [1][2][3][4][5][6][7][8][9] as evidenced by the widespread use of enzyme immunoassays, [10][11][12] antibiotic-resistant assays, [4,6] nanoparticle-aggregation assays, [8,13,14] and many others.T ypically,t he signals produced by these assays are directly proportional to the analyte concentration. [11,15] However,l inear signal-gain systems such as these exhibit significantly worse sensitivity and dynamic range than exponential amplification systems,which are much less prevalent in the literature.…”