Tuberculosis remains a major global cause of morbidity and mortality. There is an urgent need for improved bacteriologic diagnosis of Mycobacterium tuberculosis infection. Three methods for rapid identification of M. tuberculosis in sputum samples (direct microscopy, gas chromatography-mass spectrometry [GC-MS], and polymerase chain reaction [PCR]), were compared with culture on Lowenstein-Jensen medium. Growth of M. tuberculosis was observed in 38 of 145 sputum samples. Detection of acid-fast bacilli by direct microscopy gave a sensitivity of 66% and a specificity of 100%. Detection of tuberculostearic acid by GC-MS gave a sensitivity of 55% and a specificity of 87%. Amplification by PCR of a fragment of the insertion sequence IS6110 gave a sensitivity of 95% and a specificity of 93% compared with culture and a corrected specificity of 99% compared with both culture and clinical data. This study indicates that PCR can be adapted for clinical use and is the method of choice for rapid diagnosis of pulmonary tuberculosis.
Examination of the lipid components of microbes in recent sediments has provided a convenient, quantitative, and comprehensive method to define the viable biomass, community composition, and nutritional/physiological activities of the biological communities in the sediments. The lipid extraction provides both a concentration and purification of the lipids from the soils and sediments. The subsequent fractionation, purification, and derivatization, sets up the definitive separation and structural identification by capillary gas chromatography with mass spectral identification of each component. As a part of this signature lipid biomarker (SLB) analysis, the lipid extraction also lyses the cells and allows for recovery of purified nucleic acids for subsequent gene probing with and without enzymatic amplification. This polyphasic analysis adds powerful specificity to the analysis of community microbial ecology. Since the SLB analysis involves detection by mass spectrometry, rates of incorporation of non -radioactive 13 C and 15 N mass-labeled precursors into signature biomarkers can be utilized to gain insight into specific metabolic activities. Application of electrospray and other external ionization sources to ion-trap mass spectrometry will greatly increase the specificity and sensitivity of the SLB analysis.The assessment of the microbes and their in situ interactions in various environments has proven to be a major problem as it has become increasingly apparent that communities of microbes act differently in geochemical cycles than the sum of the isolated individuals. This has required the application of non-traditional methodology. Classical microbiological methods, that were so successful with infectious disease, have severe limitations for the analysis of environmental samples. Pure-culture isolation, 22
Twenty-seven strains belonging to 12 Mycobacterium species were studied for 3-hydroxy fatty acid composition. Mycobacterial cells were subjected to both mild and strong acid methanolysis, after which the liberated hydroxy fatty acids were purified and analyzed by gas chromatography-mass spectrometry as methyl ester trimethylsilyl ether derivatives. Altogether, 21 3-hydroxy fatty acids containing 14 to 28 carbon atoms were detected; 10 were straight chain, 6 were 2-methyl branched chain, and 5 were 2,4,6-trimethyl branched chain. The mycobacterial strains were classified in groups according to 3-hydroxy fatty acid patterns.Mycobacterial cell walls have been shown to contain typespecific antigenic glycolipids, such as phenolic glycolipids, glycopeptidolipids (GPL), and trehalose-containing lipooligosaccharides (LOS) (4, 5). Long-chain 3-hydroxy fatty acids (3-OH-FAs) have been identified in several glycolipids, for example, in GPL from Mycobacterium peregrinum and M. smegmatis (7,12), in LOS from M. szulgai and M. tuberculosis Canetti (9, 10), and in acyltrehaloses from M. tuberculosis Canetti and H37Rv (3,6). Furthermore, 3-OH-FAs have been detected in other bioactive mycobacterial structures, i.e., in ornithine-amide lipids and in lipopeptides in rough mutants of M. avium (2, 11). The present work was undertaken to systematically study the 3-OH-FA composition of several clinically important mycobacteria.MATERIALS AND METHODS Chemicals. N,O-bis(trimethylsilyl)trifluoroacetamide (BST-FA) was purchased from Fluka Chemica (Buchs, Switzerland), diethyl ether and dichloromethane were from Janssen Chemica (Beerse, Belgium), pyridine and acetylchloride were from Merck (Darmstadt, Germany), 3-hydroxytridecanoic acid (3-OH C13:0) was from Larodan Lipids (Malmo, Sweden), and the solvents n-hexane and methanol were from Lab-Scan (Dublin, Ireland).Mycobacteria. Altogether, 27 strains belonging to 12 Mycobacterium species were analyzed. Twenty-five of the strains were grown at 37 or 33°C (M. marinum) in Sauton medium, and two (M. peregrinum and M. chelonae) were grown in Dubos medium (Table 1). The cultures were autoclaved, harvested by centrifugation, washed twice with sterile distilled water, and freeze-dried.Methanolysis and derivatization. Portions (8 mg) of dried mycobacterial cells were divided into two equal parts; one was subjected to mild methanolysis, i.e., heating in 2 M methanolic HCl (60°C, 2 h), and the other was subjected to strong methanolysis, i.e., heating in 4 M methanolic HCl (100°C, 18 h). After cooling, 50 ng of 3-OH C13:0 methyl ester was added as an internal standard, and the reaction mixture was extracted with 1 ml of hexane-water (1.5:1, vol/vol). The hexane phase was collected, and the lower phase was subjected to a second * Corresponding author. hexane extraction. The two hexane phases were then combined, dried, and redissolved in 1 ml of hexane-dichloromethane (1:1, vol/vol). Hydroxylated and nonhydroxylated fatty acid methyl esters were separated by using a disposable silica gel column (1 ml; Bon...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.