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

Abstract

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...

TheMycobacterium tuberculosis phoPROperon Is Positively Autoregulated in the Virulent Strain H37Rv

“…It was clearly demonstrated that this point mutation is responsible for the absence of SL, DAT, and PAT in H37Ra (10). This finding may explain some striking similarities between H37Ra and M. tuberculosis phoP mutants, as many of the phenotypes of the H37Ra strain, including virulence attenuation (9, 37), formation of smaller colonies on agar plates, loss of acid fastness (44), lack of reactivity with neutral red (13,29,41), and cording defects (17,30), are correlated with the phenotypes of phoP mutants constructed from virulent M. tuberculosis strains (19,32,49).…”

Role of the Mmr Efflux Pump in Drug Resistance in Mycobacterium tuberculosis

“…Neutral red staining was used to ensure that the constructed strains keep free lipids of the cell wall, such as the virulence factor phthiocerol dimycocerosates (PDIMs). Neutral red staining was performed in a test tube, as described previously (26). Briefly, mycobacterial strains were grown at 37°C on Middlebrook 7H10 medium supplemented with 10% ADC and kanamycin (20 g/ml) or hygromycin B (50 g/ml) when required.…”

A Point Mutation in the Two-Component Regulator PhoP-PhoR Accounts for the Absence of Polyketide-Derived Acyltrehaloses but Not That of Phthiocerol Dimycocerosates in Mycobacterium tuberculosis H37Ra

“…Radiolabeling of whole M. tuberculosis cells with [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]propionate (specific activity, 56.7 Ci mol Ϫ1 , MP Biomedicals Inc.), extraction and analyses of lipids were performed as described (16). Neutral red staining of tubercle bacilli was performed as described by Soto and collaborators (36). For the analysis of cording properties, a drop of M. tuberculosis culture grown in Sauton's medium was smeared onto a glass slide, stained by the standard Kinyoun procedure and observed under a light microscope at ϫ1,000 magnification.…”