Preservation of mycobacteria at -70 degrees C: survival of unfrozen suspensions in transit.

Mycobacteria stored at -70 degrees C retain 100% viability and maintain their definitive taxonomic, serologic, immunologic, and pathogenic properties. When shipped at ambient temperatures, however, suspensions of all mycobacteria lose viability in transit, with those species having a narrow temperature range for growth (Mycobacterium tuberculosis and M. bovis) being most severely affected. In spite of these losses, all strains retain their definitive taxonomic properties. If care is taken in pre-testing and post-testing the microbial populations being preserved, mycobacteria are probably best shipped in the lyophilized state, and this procedure has been successfully used for several international studies.     

Epidemiology of infection by nontuberculous mycobacteria. VII. Absence of mycobacteria in southeastern groundwaters

Using the fluorochrome auramine-O, direct microscopic counts of log phase cells of Mycobacterium avium, M. intracellulare, and M. scrofulaceum (MAIS) group showed excellent correlations with viable spread-plate counts. Accordingly, an enumeration of total acid-fast and MAIS cells by stain and culture (respectively) in groundwaters from three United States regions that differ in their incidence of human infection by MAIS was undertaken. Of 30 state-monitored, undisinfected wells, 11 were in Georgia coastal plain (high incidence), 10 were in the Virginia coastal plain (intermediate incidence), and 9 were in Montgomery County, Virginia (low incidence). Total acid-fast cells ranged widely between 280 to 5,367 per ml among the groundwaters, and with one exception showed no correlations or trends between regions of different incidence of human infection, or to total bacterial cell counts or colony-forming units. The exception was that the proportions of acid-fast cells relative to total cells were higher in the Georgia groundwaters. However, despite the relatively high auramine-O counts, few mycobacteria were recovered by culture. Of 12 wells yielding mycobacteria, 9 yielded rapidly growing and 4 slowly growing mycobacteria. Only one well in Montgomery County, Virginia (region of low incidence) yielded a MAIS isolate, albeit at low density. This research supports the conclusion that clean groundwaters are unlikely sources of MAIS infection in humans in the southeastern United States.     

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.     

Near-native structure refinement using in vacuo energy minimization

One of the greatest shortcomings of macromolecular energy minimization and molecular dynamics techniques is that they generally do not preserve the native structure of proteins as observed by x-ray crystallography. This deformation of the native structure means that these methods are not generally used to refine structures produced by homology-modeling techniques. Here, we use a database of 75 proteins to test the ability of a variety of popular molecular mechanics force fields to maintain the native structure. Minimization from the native structure is a weak test of potential energy functions: It is complemented by a much stronger test in which the same methods are compared for their ability to attract a near-native decoy protein structure toward the native structure. We use a powerfully convergent energy-minimization method and show that, of the traditional molecular mechanics potentials tested, only one showed a modest net improvement over a large data set of structurally diverse proteins. A smooth, differentiable knowledge-based pairwise atomic potential performs better on this test than traditional potential functions. This work is expected to have important implications for protein structure refinement, homology modeling, and structure prediction.     

Conformations and folding of lysozyme ions in vacuo.

Proton transfer reactivity of isolated charge states of the protein hen egg-white lysozyme shows that multiple distinct conformations of this protein are stable in the gas phase. The reactivities of the 9+ and 10+ charge state ions, formed by electrospray ionization of "native" (disulfide-intact) and "denatured" (disulfide-reduced) solutions, are consistent with values calculated for ions in their crystal structure and fully denatured conformations, respectively. Charge states below 8+ of both forms, formed by proton stripping, have similar or indistinguishable reactivities, indicating that the disulfide-reduced ions fold in the gas phase to a more compact conformation.     

Mycobacterial infections caused by nontuberculous mycobacteria

The purpose of this review is to familiarize a broad range of medical professionals with a relatively new and growing problem of infections caused by mycobacteria other than M. tuberculosis and M. leprae. There are at least 60 mycobacterial species that have been identified as causative agents of diseases in humans. They are all environmental bacteria, and they are not transmitted from person to person. The usual source of infection is water, soil, and aerosols developed from these sources. The probability of contracting such a disease depends not only on the closeness of interaction with an environment containing an enhanced concentration of bacteria but also and foremost on the sensitivity of an individual to these infections, which may depend on the state of immunity and other so-called predisposing conditions. Therefore, these infections are often referred to as opportunistic, and the group of organisms causing them are usually referred to as nontuberculous mycobacteria (NTM). This review addresses in a condensed form various aspects of these infections, including bacteriology, clinical manifestations, diagnosis, and therapy. The reader will be able to find more details on each of these topics in several reviews and some original papers cited in this article.     

Pulmonary disease caused by nontuberculous mycobacteria

Nontuberculous mycobacterial pulmonary infections have become more common in recent years. The diagnosis is often overlooked because the findings may be subtle or because the radiographic appearance may change slowly or not at all for long periods of time. As a rule, the radiographic findings of nontuberculous mycobacterial pulmonary infections are identical to those of tuberculosis in any given patient. Cavitary disease in nontuberculous mycobacterial infections is less common than in tuberculosis. The most common radiographic finding is one or more areas of clustered fibroproductive nodules that change slowly. Mycobacterium kansasii infection responds well to therapy, whereas M avium-intracellulare infection is difficult to treat. Awareness of the radiographic appearance of the nontuberculous mycobacterial pulmonary infections will facilitate their diagnosis so that appropriate therapy may be initiated before the disease is far advanced.     

Sporulation in mycobacteria

Mycobacteria owe their success as pathogens to their ability to persist for long periods within host cells in asymptomatic, latent forms before they opportunistically switch to the virulent state. The molecular mechanisms underlying the transition into dormancy and emergence from it are not clear. Here we show that old cultures of Mycobacterium marinum contained spores that, upon exposure to fresh medium, germinated into vegetative cells and reappeared again in stationary phase via endospore formation. They showed many of the usual characteristics of well-known endospores. Homologues of well-known sporulation genes of Bacillus subtilis and Streptomyces coelicolor were detected in mycobacteria genomes, some of which were verified to be transcribed during appropriate life-cycle stages. We also provide data indicating that it is likely that old Mycobacterium bovis bacillus Calmette–Guérin cultures form spores. Together, our data show sporulation as a lifestyle adapted by mycobacteria under stress and tempt us to suggest this as a possible mechanism for dormancy and/or persistent infection. If so, this might lead to new prophylactic strategies.     

Detection of mycobacteria by polymerase chain reaction]

The polymerase chain reaction (PCR) was used to detect mycobacterial DNA sequences in the cultured or the clinical specimens. Four oligonucleotide primers derived from the sequence of a gene coding 65-kilodalton antigen of Mycobacterium tuberculosis amplified DNA samples of all the 11 species of mycobacteria tested. Serial dilution of M. bovis BCG showed that DNA extracted from only 12 bacilli was enough for the detection by PCR method. However, mycobacteria in sputum were detected by PCR when more than 10(3) bacilli were present. The PCR method may become a useful tool for the rapid diagnosis of mycobacterial infections.