Characterization of Mycobacterium bohemicum isolated from human, veterinary, and environmental sources.

Chemotaxonomic and genetic properties were determined for 14 mycobacterial isolates identified as members of a newly described species Mycobacterium bohemicum. The isolates recovered from clinical, veterinary, and environmental sources were compared for lipid composition, biochemical test results, and sequencing of the 16S ribosomal DNA (rDNA) and the 16S-23S rDNA internal transcribed spacer (ITS) regions. The isolates had a lipid composition that was different from those of other known species. Though the isolates formed a distinct entity, some variations were detected in the features analyzed. Combined results of the phenotypic and genotypic analyses were used to group the isolates into three clusters. The major cluster (cluster A), very homogenous in all respects, comprised the M. bohemicum type strain, nine clinical and veterinary isolates, and two of the five environmental isolates. Three other environmental isolates displayed an insertion of 14 nucleotides in the ITS region; they also differed from cluster A in fatty alcohol composition and produced a positive result in the Tween 80 hydrolysis test. Among these three, two isolates were identical (cluster B), but one isolate (cluster C) had a unique high-performance liquid chromatography profile, and its gas liquid chromatography profile lacked 2-octadecanol, which was present in all other isolates analyzed. Thus, sequence variation in the 16S-23S ITS region was associated with interesting variations in lipid composition. Two of the isolates analyzed were regarded as potential inducers of human or veterinary infections. Each of the environmental isolates, all of which were unrelated to the cases presented, was cultured from the water of a different stream. Hence, natural waters are potential reservoirs of M. bohemicum.

Inflammatory responses in RAW264.7 macrophages caused by mycobacteria isolated from moldy houses.

Mycobacterial strains (nonpathogenic Mycobacterium terrae, potentially pathogenic Mycobacterium avium-complex and Mycobacterium scrofulaceum), isolated from a moldy building, were studied with respect to their ability to stimulate macrophages (RAW264.7) to produce inflammatory mediators, and to cause cytotoxicity. Reactive oxygen species (ROS) were measured by chemiluminescence, cytokines (TNF-alpha, IL-6, IL-1, IL-10) immunochemically, nitric oxide (NO) by Griess-method, expression of inducible NO-synthase (iNOS) with Western Blot analysis and cytotoxicity with MTT-test. All the strains induced dose- and time-dependent production of NO, IL-6 and TNF-alpha in macrophages, whereas IL-1 or IL-10 production was not detected. The production of ROS and cytotoxicity was increased with the highest doses. Interestingly, different strains had significant differences in their ability to induce these responses, M. terrae being the most potent and M. avium-complex the weakest one. These results indicate that both non- and potentially pathogenic strains of mycobacteria present in moldy buildings are capable of activating inflammatory mechanisms in macrophages.

Mycobacterium xenopi and related organisms isolated from stream waters in Finland and description of Mycobacterium botniense sp. nov.

Three scotochromogenic Mycobacterium xenopi-like organisms were isolated from stream waters in Finland. These strains grew at 36-50 degrees C but not at 30 degrees C. One of the three strains was fully compatible with the M. xenopi type strain according to GLC-MS, biochemical tests, and 16S rDNA and 16S-23S rDNA internal transcribed spacer (ITS) sequencing. Two of the strains closely resembled M. xenopi in lipid analyses and biochemical tests, but analysis by GLC-MS verified the presence of two new marker fatty acids (2,4,6,x-tetramethyl-eicosanoic acid and 2,4,6,x,x-pentamethyl-docosanoic acid). The 16S rDNA and ITS region sequences of these two strains differed from those of M. xenopi and other previously described mycobacterial sequences. Therefore, the strains are regarded as new species of slow-growing mycobacteria, for which the name Mycobacterium botniense sp. nov. is proposed. The chemical, physical and microbiological quality of the water reservoirs of M. xenopi and M. botniense are described. As far as is known, this is the first time that M. xenopi has been isolated from natural waters. The strains of M. botniense sp. nov. (E347T and E43) have been deposited in the ATCC as strains 700701T and 700702, respectively.

Separation among species of Mycobacterium terrae complex by lipid analyses: comparison with biochemical tests and 16S rRNA sequencing.

Fatty acids, alcohols, and mycolic acid cleavage products were determined for 13 ATCC strains and 24 clinical isolates, which were initially identified by biochemical and growth characteristics as the Mycobacterium terrae complex. The clinical isolates were also analyzed by partial sequencing of the 16S rRNA gene, which divided them into five genetic entities, M. triviale (three strains), M. terrae (four strains), M. nonchromogenicum sensu stricto (seven strains), Mycobacterium sp. strain MCRO 6 (seven strains), and Mycobacterium sp. strain 31958 (one strain). After acidic methanolysis, secondary alcohols were a characteristic feature in all members of the M. terrae complex but M. triviale. In addition to the prominent secondary alcohols, 2-octadecanol and 2-eicosanol, two previously unidentified alcohols, 2-(8,15-dimethyl)docosenol and 2-(8,17-dimethyl)tetracosenol, were detected in M. nonchromogenicum, Mycobacterium sp. strain MCRO 6, and Mycobacterium sp. strain 31958. Only 2-(8,17-dimethyl)tetracosenol was detected in trace amounts in M. terrae. Genetic differences were associated with differences in phenotypic characteristics, including growth at 42 degrees C and pyrazinamidase production. Based on fatty acid and alcohol composition and biochemical and genetic characteristics, M. non-chromogenicum and Mycobacterium sp. strains MCRO 6 and 31958 were found to be a closely related group, named the M. nonchromogenicum complex. Detected genetic variations associated with phenotypic characteristics may indicate further species separation of this complex. In conclusion, the results of gas-liquid chromatography fatty acid analysis, combined with those of a Tween 80 test, enable identification of the species of the M. terrae complex and their separation from other nonpigmented slowly growing mycobacteria.

Isolation of potentially pathogenic mycobacteria in the Finnish environment.

Atypical mycobacteria have become more common in clinical samples, and their reservoirs, known to be in the environment, are poorly identified. In the Finnish natural environment, mycobacteria can be cultivated from surface waters in a mean of 1500 CFU/l and from soil samples in a mean of 3.6 x 10(5) CFU/g dry weight. The majority of isolates are not pathogenic to man. Less than 10% of cultivable mycobacteria belong in species which are also found in human samples, either as infectious agents or as harmless colonizers of human epithelia. The two most important potentially pathogenic atypical mycobacteria in Finland, the Mycobacterium avium-intracellulare-scrofulaceum (MAIS) complex and M. malmoense, were detected in 40% and 4%, respectively, of the examined waters.