Comparison of mycobacteria-induced cytotoxicity and inflammatory responses in human and mouse cell lines.

Environmental mycobacteria, which are ubiquitous in nature, are also detected in moisture-damaged buildings. Their potential role inducing the adverse health effects associated with living in moisture damaged buildings requires clarification. To establish a model for these studies, we evaluated inflammatory responsiveness in different cell lines exposed to environmental mycobacterial species. Four mycobacterial isolates belonging to Mycobacterium avium complex and Mycobacterium terrae, recovered from the indoor air sampled when a moldy building was being demolished, were studied for their cytotoxicity and ability to stimulate the production of inflammatory mediators in mouse RAW264.7 and human 28SC macrophage cell lines, and human A549 lung epithelial cell line. Lipopolysaccharide (LPS) was used as a positive control. Production of cytokines (tumor necrosis factor alpha, TNF-alpha; interleukin 6, IL-6; and interleukin beta, IL-1beta) was analyzed immunochemically, nitric oxide (NO) by the Griess method, expression of inducible NO synthase with Western blot analysis, and cytotoxicity with the MTT test. Both human and mouse cells produced NO and IL-6 after mycobacterial exposure. Mouse macrophages also showed production of TNF-alpha induced by both mycobacteria and LPS, whereas the human cell lines failed to produce TNF-alpha after mycobacterial exposure and the human epithelial cell line also failed to respond to LPS. Similarly, only mouse macrophages produced IL-1beta. Mycobacterial exposure was not cytotoxic to human cells and was only slightly cytotoxic to mouse macrophages. The results indicate that environmental mycobacterial isolates from moldy buildings are capable of activating inflammatory mechanisms in both human and murine cells. The human and mouse cell lines, however, differ significantly in the grade and type of the responses.

PCR-based typing of Mycobacterium avium isolates in an epidemic among farmed lesser white-fronted geese (Anser erythropus).

Mycobacterium avium is an important veterinary pathogen causing avian tuberculosis in birds. The aim of the study was to evaluate the genetic relatedness in M. avium isolates from deep tissues of farmed lesser white-fronted geese with avian tuberculosis and in samples from the farm environment. The strains were analyzed by two PCR-based typing methods, inverted repeat (IR) typing and random amplified polymorphic DNA (RAPD) analysis. The primers for the inverted repeats of the insertion sequences IS1245 and IS1311 were used in IR typing, and the RAPD analysis was performed with six primers. Seven of the nine avian strains yielded an identical pattern in the IR typing, but they could be divided into two groups in the RAPD analysis. The remaining two bird isolates had an identical IR pattern (IR cluster II) which they shared with two environmental isolates. However, the RAPD analysis revealed that these environmental isolates had a RAPD pattern (RAPD cluster VI) distinct and different from either of the bird isolates (RAPD clusters II and IV). In all, four M. avium strains were verified as being inducers of avian tuberculosis in birds, and all were distinct from the three environmental strains identified. Thus, the results did not confirm the preliminary idea that a single strain had caused the epidemic. The polymorphism among M. avium strains highlighted the great biodiversity among an M. avium population even in a limited environmental setting during a short time span, and indicated the high susceptibility to avian tuberculosis of lesser white-fronted geese.

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.

Copper and silver ions more effective against legionellae than against mycobacteria in a hospital warm water system.

We studied the influence of electrolytically released copper and silver ions on the microbiological quality in a warm water system of a hospital. The concentration of nontuberculous mycobacteria was followed for three, and that of legionellae and other heterotrophic bacteria in the water for four years. The highest concentrations of copper and silver ions were 220 and 68 microg/l, respectively. Silver ion concentration of about 3 microg/l was sufficient to control the growth of legionellae in circulating warm water. The results showed that it is more difficult to eradicate legionellae from taps and showers: these points were colonized by a small number of legionellae after the metal ion concentrations were increased in the circulating water. A regular use of water eradicated legionellae from the shower. One tap was still used irregularly, and this may be a reason why it still contained small concentrations of legionellae also in the last years of the study. Mycobacteria were occasionally isolated from the circulating water and repeatedly from the shower, even when the metal concentrations were high. To control legionella bacteria in warm water systems, silver concentrations of only 3 microg/l are needed if all taps and showers of the system are regularly used. Such low copper and silver concentrations, however, are not efficient against nontuberculous mycobacteria or other heterotrophic bacteria.

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.

Isolation of mycobacteria from indoor swimming pools in Finland.

The presence of mycobacteria in seven indoor pools in Finland was evaluated by multiple culture methods. Replicate samples, with and without inactivation of chlorine by sodium thiosulfate, were cultured in two laboratories. Laboratory I used two methods: (A) no decontamination and (B) cetylpyridinium chloride (0.005%, 20 min); and Laboratory II two methods: (C) cetylpyridinium chloride (0.005%, 18 h) and (D) oxalic acid (5%, 15 min). Samples processed by methods (A) and (B) were cultured on different egg media of pH 6.3 or 5.8; by method (C) on Middlebrook and Cohn 7H10 (+OADC) agar of pH 5.5; and by method (D) on Middlebrook and Cohn 7H10 agar (+OADC) with cycloheximide (500 microg/ml). Mycobacteria were recovered from five (71%) of seven pools. Detection of mycobacteria depended on the method used. High isolation rates (36-46% of the samples) were obtained by methods (A), (B) and (D). Contamination was a problem only with method (A). Inactivation of chlorine had a variable impact on mycobacterial detection. Isolates included M. kansasii, M. gordonae, M. fortuitum complex, M. sphagni, and M. vaccae, as well as M. simiae-like and M. chubuense-like organisms. In addition, a group of slowly growing and a group of rapidly growing isolates with previously unknown fatty acid and alcohol composition were isolated. No M. avium was detected. Mycobacterial counts were highest in a small pool with high temperature, low pH, and low content of free available chlorine.

Environmental factors affecting the occurrence of mycobacteria in brook sediments.

The occurrence of mycobacteria was studied in aerobic brook sediments from 39 drainage areas in Finland. The culturable counts of mycobacteria were related to climatic conditions, characteristics of the drainage area, chemical characteristics of the sediment and water, culturable counts of other heterotrophic bacteria, and microbial respiration rate in the sediment. The counts of mycobacteria varied from 1.1 x 10(2) to 1.5 x 10(4) cfu g-1 dry weight of sediment. They correlated positively with the proportion of the drainage area consisting of peatland, total content of C, content of Pb, microbial respiration rate in the sediment, and chemical oxygen demand of the water. The correlations of the mycobacterial counts with pH of sediment and alkalinity of water were negative. The results of the present sediment study and of the forest soil study published earlier strongly suggest that an increase in acidity increases the counts of mycobacteria and decreases the counts and activity of other heterotrophic bacteria. Mycobacterial counts were more than 100 times higher (per dry weight) in forest soils with pH 3.5-4.3 than in sediments with pH 4.5-6.3.

Comparison of some decontamination methods and growth media for isolation of mycobacteria from northern brook waters.

Two decontamination methods and five media were compared for the isolation of mycobacteria from brook waters of different physical, chemical and bacteriological characteristics. The decontaminants used were: 0.7 mol l-1 NaOH followed by 50 g l-1 oxalic acid and 0.9 mol l-1 H2SO4 combined with 0.5 g l-1 cycloheximide. The media compared were: Mycobacteria 7H11 agar with OADC enrichment (pH 6.6), glycerol egg (pH 6.5 and 5.5), and pyruvate egg (pH 6.5 and 5.5). All media contained cycloheximide, 0.5 g l-1. The NaOH-oxalic acid method generally resulted in lower contamination and higher isolation of mycobacteria than the H2SO4-cycloheximide method. With the NaOH-oxalic acid method, all five media were equal in positivity rates but contamination was a problem on Mycobacteria 7H11 agar. Of the four egg media tested, the highest positivity rate (92% of the samples) was obtained on the pyruvate modification (pH 6.5), and the highest mean colony count of mycobacteria (900 cfu l-1) on the glycerol modification (pH 6.5). Characteristics of water and sampling site had similar effects on the isolation frequencies of mycobacteria obtained by different combinations.

Effect of freezing of water samples on viable counts of environmental mycobacteria.

The effect of prolonged storage on mycobacteria and other heterotrophic bacteria in brook water samples was examined by determination of viable counts from fresh samples and again after water concentrates had been stored in nutrient broth at -75 degrees C for 15 months. The counts of mycobacteria were on average three times higher after storage (range of ratio 0.9-10.4). In contrast, the viable counts of other heterotrophic bacteria were reduced by 69%. The increase in mycobacterial counts was probably due to break-up of microcolonies or release of attached bacteria from particles. The possibility of cultivating mycobacteria from frozen samples is of practical help in large-scale field surveys.

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.

Environmental factors affecting the occurrence of mycobacteria in brook waters.

To evaluate the impact of environmental factors on the occurrence of environmental mycobacteria, viable counts of mycobacteria were measured in samples of brook water collected from 53 drainage areas located in a linear belt crossing Finland at 63 degrees north latitude. The numbers of mycobacteria were correlated with characteristics of the drainage area, climatic parameters, chemical and physical characteristics of the water, and counts of other heterotrophic bacteria in the water. The numbers of mycobacteria in the water ranged from 10 to 2,200 CFU/liter. The counts correlated positively (P < 0.001) with the presence of peatlands, precipitation data, chemical oxygen demand, water color, and concentrations of Fe, Al, Cu, Co, and Cr. The mycobacterial counts correlated negatively (P < 0.001) with water pH, whereas other heterotrophic bacterial counts lacked any correlation with pH. A linear regression model with four independent variables (i.e., peatlands in the drainage area, chemical oxygen demand, concentration of potassium, and pH) explained 83% of the variation in mycobacterial counts in brook waters. Our results suggest that acidification may enhance the growth of environmental mycobacteria.