A Study of the Neutral Red Reaction for Determining the Virulence of Mycobacteria

Morse, Warren C.; Dail, Martha C.; Olitzky, I.

doi:10.2105/ajph.43.1.36

Cited by 7 publications

(3 citation statements)

References 2 publications

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“…The neutral red assay has been used extensively to distinguish between avirulent and virulent strains of MTb because of its specificity for labeling cells producing SL-1 (15,32,34,35). The capacity to bind this molecule has been interpreted as indicating a surface-accessible location of the sulfatides, whose strongly acidic sulfate interacts ionically with the cationic dye (36).…”

Section: Disruption Of the Mtb Mmpl8mentioning

confidence: 99%

The Role of MmpL8 in Sulfatide Biogenesis and Virulence of Mycobacterium tuberculosis

Domenech

Reed

Dowd

et al. 2004

Journal of Biological Chemistry

147

142

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To study the role of MmpL8-mediated lipid transport in sulfatide biogenesis, we insertionally inactivated the mmpL8 gene in Mycobacterium tuberculosis. Characterization of this strain showed that the synthesis of mature sulfolipid SL-1 was interrupted and that a more polar sulfated molecule, termed SL-N, accumulated within the cell. Purification of SL-N and structural analysis identified this molecule as a family of 2,3-diacyl-␣,␣-D-trehalose-2-sulfates. This structure suggests that transport and biogenesis of SL-1 are coupled and that the final step in sulfatide biosynthesis may be the extracellular esterification of two trehalose 6-positions with hydroxyphthioceranic acids. To assess the effect of the loss of this anionic surface lipid on virulence, we infected mice via aerosol with the MmpL8 mutant and found that, although initial replication rates and containment levels were identical, compared with the wild type, a significant attenuation of the MmpL8 mutant strain in time-to-death was observed. Early in infection, differential expression of cytokines and cytokine receptors revealed that the mutant strain less efficiently suppresses key indicators of a Th1-type immune response, suggesting an immunomodulatory role for sulfatides in the pathogenesis of tuberculosis.

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Section: Disruption Of the Mtb Mmpl8mentioning

confidence: 99%

The Role of MmpL8 in Sulfatide Biogenesis and Virulence of Mycobacterium tuberculosis

Domenech

Reed

Dowd

et al. 2004

Journal of Biological Chemistry

147

142

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“…Dubos and Middlebrook reported that H37Rv, together with 22 M. tuberculosis and Mycobacterium bovis isolates, fixed neutral red in an alkaline aqueous environment and became red in color, whereas H37Ra cells did not (8). Two subsequent studies in which larger numbers of M. tuberculosis clinical isolates were used (about 200 strains in each) corroborated these findings (11,15). At the same time, Desbordes and coworkers concluded that, unlike attenuated and avirulent strains, the virulent strain H37Rv strongly fixed basic dyes, such as Nile blue and neutral red, in their anionic forms (red and blue, respectively) in very alkaline environments.…”

mentioning

confidence: 75%

Simple and Rapid Differentiation ofMycobacterium tuberculosisH37Ra fromM. tuberculosisClinical Isolates through Two Cytochemical Tests Using Neutral Red and Nile Blue Stains

et al. 2002

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The attenuated Mycobacterium tuberculosis strain H37Ra is one of the most commonly used controls for M. tuberculosis identification in the clinical laboratory and is a source of false-positive results for M. tuberculosis as a consequence of cross-contamination. Therefore, the ability to discriminate between H37Ra and real clinical isolates has important public health implications. To date, differentiation of H37Ra from M. tuberculosis clinical isolates is possible only by IS6110 genotyping and spoligotyping. In the 1950s, some authors reported that the virulent strain H37Rv and M. tuberculosis clinical isolates were able to fix basic dyes in their anionic forms, while H37Ra was not. We have studied the different techniques described for M. tuberculosis cytochemical staining and have chosen the best of these, introducing certain modifications in order to increase their discriminative power and reproducibility. We describe cytochemical staining of M. tuberculosis cells with neutral red and Nile blue, which differentiates H37Ra from virulent strains. This method could be used as an easy laboratory tool for distinguishing between H37Ra and real M. tuberculosis clinical isolates.At present, Mycobacterium tuberculosis identification is normally carried out in clinical laboratories by using conventional tests such as niacin, nitrate reduction, pyrazinamidase, thiopen-2-carbonic acid hydrazide (TCH), or catalase (13,21,22). In addition, the M. tuberculosis complex can be distinguished from other mycobacteria by commercialized nucleic acid probes (20) and high-performance liquid chromatography analysis of mycolic acid (4). However, it is not possible to distinguish the attenuated strain H37Ra from M. tuberculosis clinical isolates by use of these techniques. Nevertheless, H37Ra is used as a control in identification tests, and false diagnosis of tuberculosis due to cross-contamination of laboratory specimens with H37Ra is both likely and frequent (3,16). To date, reliable differentiation of H37Ra from virulent strains has been possible only through spoligotyping and IS6110 genotyping (1), laborious and time-consuming procedures.Cytochemical reactions in tubercle bacilli were studied in great depth in the 1940s and 1950s in order to find phenotypic markers correlated to virulence. The action of many basic dyes, such as Nile blue, methylene blue, neutral red, and methyl violet, on different Mycobacterium strains has been studied (5,6,7,8). The most successful results were obtained with neutral red and Nile blue (6, 8). Dubos and Middlebrook reported that H37Rv, together with 22 M. tuberculosis and Mycobacterium bovis isolates, fixed neutral red in an alkaline aqueous environment and became red in color, whereas H37Ra cells did not (8). Two subsequent studies in which larger numbers of M. tuberculosis clinical isolates were used (about 200 strains in each) corroborated these findings (11,15). At the same time, Desbordes and coworkers concluded that, unlike attenuated and avirulent strains, the virulent strain H37Rv strongly...

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“…The specific phenomenon observed in this test is elicited by a chemically induced alteration of the dye color, rather than by the physical washing out of unfixed dye. Its usefulness has been limited to recognition of virulent mammalian tubercle bacilli, which have been grown by routine methods (Richmond and Cummings, 1950;Morse et al, 1953). Modification of the technique to make it applicable to the staining of colonies has yielded a differential tool for visualization of tubercle bacillus colonies grown on molecular filter membranes.…”

Section: Methodsmentioning

confidence: 99%

Cultivation and Visualization of Mycobacteria on Molecular Filter Membranes

Wayne

1955

J Bacteriol

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A Study of the Neutral Red Reaction for Determining the Virulence of Mycobacteria

Cited by 7 publications

References 2 publications

The Role of MmpL8 in Sulfatide Biogenesis and Virulence of Mycobacterium tuberculosis

The Role of MmpL8 in Sulfatide Biogenesis and Virulence of Mycobacterium tuberculosis

Simple and Rapid Differentiation ofMycobacterium tuberculosisH37Ra fromM. tuberculosisClinical Isolates through Two Cytochemical Tests Using Neutral Red and Nile Blue Stains

Cultivation and Visualization of Mycobacteria on Molecular Filter Membranes

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