Microaerobic growth and anaerobic survival of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium scrofulaceum.

Representative strains of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium scrofulaceum (MAIS) grew at equal rates in laboratory medium at 21% (air) and 12% oxygen. Growth in 6% oxygen proceeded at a 1.4-1.8-fold lower rate. Colony formation was the same at 21% (air) and 6% oxygen. The MAIS strains survived rapid shifts from aerobic to anaerobic conditions as measured by two experimental approaches (Falkinham (1996) [1]). MAIS cells grown aerobically to log phase in broth were diluted, spread on agar medium, and incubated anaerobically for up to 20 days at 37°C. Although no colonies formed anaerobically, upon transfer to aerobic conditions, greater than 25% of the colony forming units (CFU) survived after 20 days of anaerobic incubation (Prince et al. (1989) [2]). MAIS cells grown in broth aerobically to log phase were sealed and vigorous agitation led to oxygen depletion (Wayne model). After 12 days anaerobic incubation, M. avium and M. scrofulaceum survival were high (>50%), while M. intracellulare survival was lower (22%). M. avium cells shifted to anaerobiosis in broth had increased levels of glycine dehydrogenase and isocitrate lyase. Growth of MAIS strains at low oxygen levels and their survival following a rapid shift to anaerobiosis is consistent with their presence in environments with fluctuating oxygen levels.

Factors influencing the chlorine susceptibility of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum.

The susceptibility of representative strains of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (the MAIS group) to chlorine was studied to identify factors related to culture conditions and growth phase that influenced susceptibility. M. avium and M. intracellulare strains were more resistant to chlorine than were strains of M. scrofulaceum. Transparent and unpigmented colony variants were more resistant to chlorine than were their isogenic opaque and pigmented variants (respectively). Depending on growth stage and growth rate, MAIS strains differed in their chlorine susceptibilities. Cells from strains of all three species growing in early log phase at the highest growth rates were more susceptible than cells in log and stationary phase. Rapidly growing cells were more susceptible to chlorine than slowly growing cells. The chlorine susceptibility of M. avium cells grown at 30 degrees C was increased when cells were exposed to chlorine at 40 degrees C compared to susceptibility after exposure at 30 degrees C. Cells of M. avium grown in 6% oxygen were significantly more chlorine susceptible than cells grown in air. Chlorine-resistant MAIS strains were more hydrophobic and resistant to Tween 80, para-nitrobenzoate, hydroxylamine, and nitrite than were the chlorine-sensitive strains.

Fluorescent acid-fast microscopy for measuring phagocytosis of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum by Tetrahymena pyriformis and their intracellular growth.

Fluorescent acid-fast microscopy (FAM) was used to enumerate intracellular Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum in the ciliated phagocytic protozoan Tetrahymena pyriformis. There was a linear relationship between FAM and colony counts of M. avium cells both from cultures and within protozoa. The Ziehl-Neelsen acid-fast stain could not be used to enumerate intracellular mycobacteria because uninfected protozoa contained acid-fast, bacterium-like particles. Starved, 7-day-old cultures of T. pyriformis transferred into fresh medium readily phagocytized M. avium, M. intracellulare, and M. scrofulaceum. Phagocytosis was rapid and reached a maximum in 30 min. M. avium, M. intracellulare, and M. scrofulaceum grew within T. pyriformis, increasing by factors of 4- to 40-fold after 5 days at 30 degrees C. Intracellular M. avium numbers remained constant over a 25-day period of growth (by transfer) of T. pyriformis. Intracellular M. avium cells also survived protozoan encystment and germination. The growth and viability of T. pyriformis were not affected by mycobacterial infection. The results suggest that free-living phagocytic protozoa may be natural hosts and reservoirs for M. avium, M. intracellulare, and M. scrofulaceum.

Water soluble palmitic acid in media for cultivation of leprosy derived psychrophilic mycobacteria from Mycobacterium leprae infected tissues

Cultivation trials for Mycobacterium leprae resulted in growth of Mycobacterium psychrophilum (L). Media were inoculated with host grown Mycobacterium leprae cells from armadillo tissues, Nu mice foot pads or human lepromata. Cultures were obtained in liquid and on semisolid multifactoria 1 media containing water soluble palmitic acid or its salts. Ammonium thioglycolate and Napalmitate served as carbon and energy sources. The water soluble palmitic acid remained in perfect solution following sterilization in the autoclave, thus easily accessible to the cells. The cyclodextrin-Fe complex served as a siderophore to grow the obtained leprosy derived psychrophilic cells. The leprosy derived cultures and subcultures grew opimally at+10 degrees Celssius but deteriorated rapidly at + 32 degrees Celsius, in the multifactorial media. No growth occurred in 7H9 media. Cultures were not identified for classification.

A simple and rapid method for the detection and identification of mycobacteria using mycobactin.

A system was developed for the identification of mycobacteria such as Mycobacterium tuberculosis and M. avium, by thin layer chromatography of 55Fe-labelled mycobactin. Approximately 2 x 10(3) mycobacteria were detected within 24 h and little operator time or skill was required. M. avium, M. intracellulare and M. scrofulaceum were found to have lower requirements for iron than other mycobacteria and this may influence their growth in host organisms.

Epidemiology of infection by nontuberculous mycobacteria. VIII. Absence of mycobacteria in chicken litter.

Overlap in the geographic distributions of (1) higher frequencies of persons reacting to antigens prepared from the Mycobacterium avium, M. intracellulare, and M. scrofulaceum (MAIS) group; (2) higher frequencies of isolation from natural waters and soils; (3) higher densities of farms producing broilers (chicken) in the southeastern United States raises the question of whether MAIS organisms occur abundantly in chicken litter (pine bark shavings containing avian fecal material) and whether litter may be a potential source of animal or human infection through its subsequent use as a fertilizer or feed supplement. We show here that potentially pathogenic mycobacteria were seldom recovered from chicken litter containing avian fecal material. Further, litter appears bactericidal to these organisms in that less than 1% of cells inoculated survived more than 6 wk, probably because of the high pH of litters.

Accumulation and transport of cadmium by tolerant and susceptible strains of Mycobacterium scrofulaceum.

Cadmium accumulation and transport were studied in two strains of Mycobacterium scrofulaceum differing in their susceptibility to Cd2+ toxicity. A 10-fold excess of either Zn2+ or Mn2+ partially antagonized inhibition of growth by Cd2+. 109Cd2+ uptake by both the tolerant and susceptible strains was temperature dependent and inhibited by a 10-fold excess of either Zn2+ or Mn2+. There were no significant differences in either the kinetics of 109Cd2+ uptake or the retention of accumulated 109Cd2+ by the tolerant and susceptible strains. Both tolerant and susceptible strains removed most of the cadmium from the culture medium, but significantly more was removed by cells of the tolerant strain. Most of the accumulated Cd2+ in the tolerant strain was in the particulate fraction, rather than in the soluble fraction. Intracellular accumulated Cd2+ was primarily in the soluble fraction of the susceptible strain. Increased Cd2+ in culture medium resulted in decreased Mn2+ and Zn2+ in cells of the susceptible strain but did not reduce the Mn2+ and Zn2+ content of cells of the tolerant strain.