Veronique Levy-Frebault

RAPID DIAGNOSIS OF TUBERCULOSIS BY AMPLIFICATION OF MYCOBACTERIAL DNA IN CLINICAL SAMPLES

A method based on DNA amplification and hybridisation for the rapid detection of Mycobacterium tuberculosis was used to test 35 clinical specimens (sputum, gastric aspirate, abscess aspirate, biopsy sample) from 34 patients in whom tuberculosis was suspected. M tuberculosis was detected in 15 specimens, 2 of which were negative by standard microbiological criteria (microscopy and/or culture). 20 specimens, negative by standard methods, were also negative by the amplification method. M tuberculosis was also detected in peripheral blood samples of 2 of 4 patients with AIDS from whom the organism had been isolated.

Numerical Taxonomy of Mycobactin-Dependent Mycobacteria, Emended Description of Mycobacterium avium, and Description of Mycobacterium avium subsp. avium subsp. nov., Mycobacterium avium subsp. paratuberculosis subsp. nov., and Mycobacterium avium subsp. silvaticum subsp. nov.

We performed a numerical taxonomy analysis of 38 Mycobacterium paratuberculosis and related mycobacterial strains, including wood pigeon mycobacteria; this analysis was based on 22 tests, which were selected for their potential discriminative value from a total of 51 tests studied and produced four well-defined clusters. Cluster 1 contained the M. paratuberculosis strains, including two strains isolated from Crohns disease patients; cluster 2 contained Mycobacterium avium and Mycobacterium intracellulare reference strains; cluster 3 consisted of the wood pigeon mycobacteria; and the only strain in cluster 4 was M. paratuberculosis 316F, which is used for antigen and vaccine production. Strains in cluster 1 were mycobactin dependent even when they were subcultured, whereas strains in cluster 3 were unable to grow on egg medium and their growth was stimulated by pH 5.5. Growth stimulation by pyruvate, resistance to D-cycloserine (50 micrograms/ml), and alkaline phosphatase activity also were characteristics that were useful for discriminating between clusters 1 and 3. The results of previous DNA-DNA hybridization studies have demonstrated that M. avium Chester 1901, M. paratuberculosis Bergey et al. 1923, and the wood pigeon mycobacteria belong to a single genomic species, and we propose that the name of this species should be M. avium. On the basis of the results of previous genomic analyses based on restriction fragment length, the results of polymorphism studies, and DNA patterns determined by field inversion gel electrophoresis as well as the results of our phenotypic study, we propose that the species should be divided into subspecies which correspond to pathogenicity and host range characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)

Proposed minimal standards for the genus Mycobacterium and for description of new slowly growing Mycobacterium species.

In accordance with Recommendation 30b of the International Code of Nomenclature of Bacteria, which calls for the development of recommended minimal standards for describing new species, we propose minimal standards for describing the genus Mycobacterium and new slowly growing species of this genus. The minimal standards for assignment of a strain to the genus Mycobacterium include acid-alcohol fastness, a DNA G+C content in the range from 61 to 71 mol%, and mycolic acid detection with characterization of C22 to C26 pyrolysis esters. The recommended minimal standards for describing a new slowly growing Mycobacterium species are based on the results of phenotypic and genomic studies and include the results of the following conventional tests: growth at 25, 30, 33, 37, 42, and 45 degrees C; pigmentation; resistance to isoniazid, thiophene-2-carboxylic acid hydrazide, hydroxylamine, p-nitrobenzoic acid, sodium chloride, thiacetazone, picrate, and oleate; catalase activity; Tween hydrolysis; urease activity; niacin detection; and nitrate reductase, acid phosphatase, arylsulfatase, pyrazinamidase, and alpha-esterase activities. In addition, a mycolic acid profile should be determined, and DNA-DNA hybridization experiments in which the difference between the denaturation temperature of the homologous reaction and the denaturation temperature of the heterologous reaction is determined should be performed. This proposal has been endorsed by the members of the Subcommittee for Taxonomy of the Mycobacteria of the International Committee on Systematic Bacteriology.

Intérêt taxonomiquedes acides gras des mycobactéries: Proposition d'une méthode d'analyse

Summary A simple and quantitative method for the alkaline hydrolysis of fattyacid derivatives occurring in the lipids of mycobacteria is described. After methylation, the lipidic mixtures were chromatographed on a thin layer of silicagel. The contents in mycolates and secondary alcohols allowed the distinction of 9 groups among the 27 species studied. Such an analysis is generally insufficient to identify a species, but its discriminating power differs from that of other commonly-used methods. Complementary tests chosen according to each particular situation are necessary, including vapour phase chromatography applied to the same lipidic mixture as that used for thin-layer chromatography. Coupling of the two chromatographic methods would allow the recognition of 22 groups among the 27 species of mycobacteria studied.

Deoxyribonucleic Acid Relatedness Study of the Mycobacterium fortuitum-Mycobacterium chelonae Complex

A deoxyribonucleic acid relatedness study of 13 Mycobacterium chelonae strains indicated that M. chelonae subsp. chelonae and M. chelonae subsp. abscessus are two different genomic species, both distinct from Mycobacterium fortuitum and “Mycobacterium peregrinum.” One strain of M. chelonae constitutes a third genomic species. The type strain of M. fortuitum and the reference strain of “M. peregrinum” (strain ATCC 14467) belong to two different genomic groups.

The phenolic mycoside of Mycobacterium ulcerans: structure and taxonomic implications.

Mycobacterium ulcerans and some pathogenic mycobacterial species elaborate wax A consisting of related long-chain beta-diol components (phthiocerol and related compounds) esterified by multimethyl-branched fatty acids. With the exception of M. ulcerans, wax A-containing mycobacteria also synthesize glycosylated phenol phthiocerol diester and related compounds: the so-called phenolic mycosides. In a deliberate effort to characterize this latter class of compounds in M. ulcerans, 20 strains were examined. Phenolic mycosides were found in two strains. Application of chemical analyses, including one- and two-dimensional NMR spectroscopy, allowed the structural elucidation of glycolipids identified as 3-O-methyl-alpha-L-rhamnosyl phenol phthiocerol diphthioceranate investigators. As phenolic mycosides are highly species-specific molecules, this finding stresses the close phylogenetic link between M. marinum and M. ulcerans. Incidentally, a survey of the mycolate content of M. ulcerans showed that methoxymycolate could not be detected in three strains.

Lack of correlation between colony morphology and lipooligosaccharide content in the Mycobacterium tuberculosis complex

Rough and smooth colony variants of the Mycobacterium tuberculosis complex were compared with respect to their composition in trehalose-containing glycolipid antigens in view of the results of a recent investigation suggesting that the chemical basis of rough and smooth colony morphology in mycobacteria may reside in the occurrence of lipooligosaccharides. A careful chemical characterization of the individual glycolipids of the selected strains allowed the identification of the major glycolipids. The comparative study of the glycolipid content of the smooth Canetti strain, its spontaneous rough variant, and 16 additional strains of M. tuberculosis, M. bovis and M. africanum showed that the presence of lipooligosaccharides was not related to the morphology of the colonies.

Mycobacterium fallax sp. nov.

A new species, Mycobacterium fallax, is described. A study of 22 strains showed that they form a homogeneous group with an internal phenotypic similarity value of 94.6 ± 4.1%. The colony morphology resembled that of Mycobacterium tuberculosis, with cord formation on solid medium. The characteristic features of these strains were that they were nonchromogenic and grew rapidly at 30° but slowly at 37°. Most formed a thermolabile catalase and produced nitrate reductase. A lipid analysis showed that tuberculostearic acid was present and that only α-mycolic acids were formed. These α-mycolic acids were di- and tri-unsaturated acids, a feature that has not been described previously in the mycobacteria. The type strain has been deposited in the Collection Nationale de Cultures de Microorganismes, Paris, France, as strain CIP 8139.

New kinds of unsaturated mycolic acids from Mycobacterium fallax sp. nov.

Abstract Mycobacterium fallax was found to contain only α-mycolic acids which were separated by argentation chromatography into four fractions called M1 to M4 according to their decreasing Rf. The structures of these products were determined by chemical oxidation followed by the analysis of the oxidative fragments by GC and GC/MS, or alternatively by a direct mass spectrometric study of the TMS ethers of the α-diols formed by dihydroxylation of the double bonds. The diunsaturated mycolates M1 contained an allylic methyl branch on one double bond. Two different classes were thus detected, depending on the position of the methyl branch. The M1A mycolates possessed an allylic branch near the remote double bond towards the methyl end, whereas in M1B mycolates, it was located near the first double bond towards the carbonyl end. The M2 mycolates were diunsaturated without any methyl branch. The M3 and M4 mycolates were tri and tetraunsaturated species, respectively. It seems that this is the first description of M1A type mycolates, and also of tri and tetraunsaturated ‘true’ mycolic acids, with both a total carbon number from 70 to 80 and a carbon number of the fatty acids from pyrolysis equal to 22 and 24.

Mutations Affecting Pigment Synthesis in Mycobacterium aurum

Pigmentation mutants of Mycobacterium aurum were isolated after chemical mutagenesis. Examination of the pigments extracted from these mutants indicated that at least 15 carotenoids were formed. beta-Carotene was not detected and the major carotene of M. aurum appeared to be leprotene. The possible biosynthetic pathway is discussed on the basis of these results.