[A case of Mycobacterium shimoïdei lung infection in Madagascar]. / Un cas d'infection pulmonaire à Mycobacterium shimoïdei à Madagascar.

In 1980, a 32 years-old Madagascan female developed a pulmonary tuberculosis, bacteriologically confirmed. She cured with right apical cavitary sequellae. In 1989, she presented haemoptysis again. Antituberculous treatment was adopted without bacteriological confirmation and did not improve clinical symptoms. In 1991 and 1992 cultures from sputa and bronchi aspiration yielded acid-fast bacilli identified as Mycobacterium shimoïdei. M. tuberculosis could not be detected. The patient died during treatment. This case is the fourth one in the literature. Whereas previous cases have been reported in Europe, Australia, Asia, this new case shows M. shimoïdei is also present in Africa.

Pulsed field gel electrophoresis of representatives of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains.

Using field inversion gel electrophoresis (FIGE), different Mycobacterium tuberculosis strains, such as phage prototypes, exhibit different DNA restriction patterns which are easy to compare. Virulent and avirulent variants of M. tuberculosis H37, as well as daughter strains of M. bovis BCG, display characteristic DNA profiles. BCG strains isolated from suppurative adenitis following vaccination of French patients showed patterns identical to the BCG Pasteur strain used for vaccination. These results demonstrate that FIGE of DNA restriction fragments generated by DraI represents a suitable technique for the analysis of mycobacteria at a genomic level. The DraI profiles allow the differentiation and precise identification of the BCG Pasteur, Glaxo, Russian and Japanese strains.

Mycobacterium alvei sp. nov.

A new species of rapidly growing, nonphotochromogenic mycobacteria, Mycobacterium alvei, is described. The inclusion of this organism in the genus Mycobacterium is based on its acid fastness, its mycolate pattern, and its G + C content. A study of six strains showed that they form a homogeneous group with an internal phenotypic similarity value of 97 +/- 2.22%. DNA relatedness studies showed that the six M. alvei strains which we studied form a single DNA hybridization group which is less than 49% related to 14 other species of the genus Mycobacterium; the deltaTm values determined for the strains which exhibited higher levels of DNA homology were all greater than 7.9 degrees C. A lipid analysis showed that tuberculostearic acid was present. Docosanoic and tetracosanoic acid methyl esters were detected as mycolic acid cleavage products. All six isolates which we tested contained alpha-mycolic acids and relatively large amounts of a new kind of mycolic acid containing a methoxy group of omega-1 position, a characteristic that has not been described previously in mycobacteria. Strain CR-21 is the type strain; a culture of this strain has been deposited in the Collection Nationale de Cultures de Microorganismes de l'Institut Pasteur, Paris, France, as strain CIP 103464.

Evaluation of nonradioactive DNA probes for identification of mycobacteria.

Commercial chemiluminescent DNA probes (Accuprobe; Gen-Probe, San Diego, Calif.) for the identification of Mycobacterium tuberculosis (MTB) complex, M. avium complex (MAC), M. gordonae, and M. kansasii were evaluated with 134 clinical isolates. These included 36 MTB complex, 40 MAC, 27 M. gordonae, 9 M. kansasii, and 22 Mycobacterium spp. The specificity was 100% for the four probes. The sensitivity was 100% for the MTB complex and M. gordonae probes and 95.2% for the MAC probe. Five of the nine M. kansasii isolates tested were not detected with the probe.

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.

Mycobactin analysis as an aid for the identification of Mycobacterium fortuitum and Mycobacterium chelonae subspecies.

Mycobactin patterns from 65 Mycobacterium fortuitum and Mycobacterium chelonae strains have been determined by thin-layer chromatography. By use of a rich liquid medium containing an iron chelator (ethylenediamine-di-o-hydroxyphenylacetic acid [EDDA]) to ensure iron starvation, all strains were able to form mycobactins. The method developed here allows sensitive detection of mycobactin by thin-layer chromatography from as little as 5 ml of culture after a 2-week incubation. Within M. fortuitum two mycobactin patterns were identified, whereas within M. chelonae four were recognized. Comparisons with the subspecific identification performed by using biochemical tests showed that 73% of the M. fortuitum subsp. fortuitum strains shared the same mycobactin pattern (designated F), whereas 75% of the M. fortuitum subsp. peregrinum strains shared the other mycobactin pattern (designated P). Within the M. fortuitum strains that cannot be assigned to a subspecies on the basis of their biochemical features, only F and P patterns were determined. Similarly, 93% of the M. chelonae subsp. chelonae strains produced the so-called C1 and C2 patterns and 86% of the M. chelonae subsp. abscessus strains produced A1 and A2 patterns. C2 and A2 were the patterns most frequently encountered; they were represented by 65 and 50% of the M. chelonae subsp. chelonae and M. chelonae subsp. abscessus strains, respectively. Within the biochemically M. chelonae strains that did not fit any subspecies on the basis of biochemical test results, C1, C2, and A1 patterns were found. Whereas about 30% of both M. fortuitum and M. chelonae strains cannot be characterized to the subspecies level on the basis of biochemical tests, 100% of the strains of both species can be characterized on the basis of mycobactin patterns.

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.

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.

Fourth report of the cooperative, open-ended study of slowly growing mycobacteria by the International Working Group on Mycobacterial Taxonomy.

The open-ended study of the International Working Group on Mycobacterial Taxonomy is an ongoing project to characterize slowly growing strains of mycobacteria that do not belong to well-established or thoroughly characterized species. In this fourth report we describe two numerical taxonomic clusters that represent subspecies or biovars of Mycobacterium simiae, one cluster that encompasses the erstwhile type strain of the presently invalid species "Mycobacterium paraffinicum," one cluster that is phenotypically very similar to Mycobacterium avium and Mycobacterium intracellulare but may be a separate genospecies, one cluster that appears to be phenotypically distinct from M. avium but reacts with a nucleic acid probe specific for M. avium, and three tentatively defined clusters in proximity to a cluster that encompasses the type strain of Mycobacterium malmoense. Of special practical interest is the fact that one of the latter three clusters is composed of clinically significant scotochromogenic bacteria that can be misidentified as the nonpathogenic organism Mycobacterium gordonae if insufficient biochemical tests are performed.

The genes coding for the antigen 85 complexes of Mycobacterium tuberculosis and Mycobacterium bovis BCG are members of a gene family: cloning, sequence determination, and genomic organization of the gene coding for antigen 85-C of M. tuberculosis.

A gene encoding the 33-kDa secreted protein of Mycobacterium tuberculosis (antigen 85-C) was isolated and sequenced. The corresponding DNA sequence contains a 1,020-bp coding region. The deduced amino acid sequence corresponds to a 340-residue protein consisting of a 46-amino-acid signal peptide and a 294-amino-acid mature protein. Comparison with previously described genes for the 30-kDa antigen (the alpha antigen of M. bovis BCG, also called antigen 85-B) and the 32-kDa antigens from M. bovis BCG and M. tuberculosis (antigens 85-A) indicates that the three genes share considerable sequence homology (70.8 to 77.5%) but may also code for distinctive epitopes. Strong differences among the three sequences are clearly visible upstream and downstream from the region coding for the mature proteins. The three genes have been detected in the genome of M. bovis BCG by Southern blot hybridization with three type-specific probes. Furthermore, hybridization of large DNA fragments (100 to 1,000 kbp) from M. tuberculosis separated by pulsed-field gel electrophoresis showed that the three genes coding for the antigen 85 complex are not clustered within the bacterial genome.

Restriction fragment length polymorphism analysis using IS6110 as an epidemiological marker in tuberculosis.

The mycobacterial insertion sequence IS6110 has been shown to be present in multiple copies in the chromosome of Mycobacterium tuberculosis. IS6110 restriction fragment length polymorphism analysis of strains isolated from patients who developed tuberculosis showed identical patterns over a 2- to 3-year period. In contrast, a high degree of polymorphism was observed between strains of the M. tuberculosis complex isolated from different patients. This study demonstrates that the presence of IS6110 does not induce in vivo major genomic rearrangements over a 2- to 3-year period and confirms its use as a valuable epidemiological marker in tuberculosis.

Characterization of a Mycobacterium tuberculosis insertion sequence, IS6110, and its application in diagnosis.

An insertion sequence-like element, IS6110, was isolated from a Mycobacterium tuberculosis cosmid library as a repetitive sequence. IS6110 shows similarities with elements of the IS3 family. This insertion sequence was found to be specific to mycobacteria belonging to the M. tuberculosis complex. For detection and identification of M. tuberculosis bacilli in uncultured specimens, oligonucleotides derived from the IS6110 sequence were used as primers and probes in polymerase chain reaction studies. The results obtained were consistent with results of classical identification procedures, bacteriological data, and clinical criteria.

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 Crohn's 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)

DNA polymorphism in Mycobacterium paratuberculosis, "wood pigeon mycobacteria," and related mycobacteria analyzed by field inversion gel electrophoresis.

Mycobacterium paratuberculosis strains, mycobacteria from patients suffering from Crohn's disease, "wood pigeon mycobacteria," and representatives of Mycobacterium avium-Mycobacterium intracellulare were compared by restriction endonuclease DraI digestion and field inversion gel electrophoresis. Characteristic profiles were seen for M. paratuberculosis, including isolates from patients suffering from Crohn's disease, for wood pigeon mycobacteria, and for M. avium-M. intracellulare serotypes 2, 16, 18, and 19. Two M. paratuberculosis strains used for vaccine production (St 18 and 316 F) presented patterns different from those of the other M. paratuberculosis strains. Strains St 18 yielded a pattern identical to that of the M. avium type strain serotype 2, whereas 316 F gave a unique pattern. The method developed in this study represents a useful taxonomic tool for the identification and classification of mycobacteria.

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.

Detection and identification of mycobacteria by amplification of mycobacterial DNA.

A 383bp segment of the gene coding for the 65kD mycobacterial antigens from Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium paratuberculosis, and Mycobacterium fortuitum was amplified using Taq polymerase and synthetic oligonucleotide primers and the amplified DNAs from four of these species were compared by nucleotide sequencing. Although the gene segments from these species showed considerable similarity, oligonucleotide probes which could distinguish M. tuberculosis/M. bovis, M. avium/M. paratuberculosis and M. fortuitum could be identified. Samples containing 10(6) human cells and serial dilutions of a suspension of intact mycobacteria were prepared, DNA was extracted, the segment of the mycobacterial DNA sequence amplified, and the amplified DNA hybridized with oligonucleotide probes. In two independent experiments, this procedure permitted the detection and identification of less than 100 mycobacteria in the original sample. These results suggest that this approach may prove useful in the early diagnosis of mycobacterial infection.

Ability of smooth and rough variants of Mycobacterium avium and M. intracellulare to multiply and survive intracellularly: role of C-mycosides.

A spontaneous rough (Rg) mutant of M. avium ATCC 15769 was mutagenized using N-methyl-N-nitro-nitrosoguanidine (MNNG). Out of 54 clones initially chosen on the basis of their morphological appearance on the 7H11 agar, seven Rg clones were selected on the basis of their response to current biochemical tests, and were later characterized for their cell wall amphiphilic contents (analysis of loosely-bound surface lipids for mycosides, peptidolipids, phospholipids, and mycolic acids by thin layer chromatography, and fatty acids by gas chromatography), and ability to grow intracellularly inside J-774 macrophages. A parallel study was also performed on a previously reported Rg mutant of M. intracellulare serovar 20 (W.W. Barrow and P.J. Brennan, J. Bact. 150 (1982) 381-384) which lacked the ability to synthesize mycosides C (MYC-). The results obtained were compared to parental smooth (Sm) colony-types of the respective M. avium-intracellulare strains. Our results showed that neither the ninhydrin-reacting lipids (probably peptidolipids) nor the glycopeptidolipids (mycosides C) were primary factors responsible for the intracellular survival and multiplication of these bacteria. Ultrastructural studies showed that although the polysaccharide-rich outer wall layer (POL) in case of MYC- Rg strain was not uniformly distributed and contained blebs, these bacteria formed the characteristic electron-transparent zone (ETZ) upon phagocytes by macrophages. Furthermore, the multiplication of MYC- Rg strain inside macrophages did not result in intracellular selection of MYC+ bacteria, nor in Rg to Sm transition.