Rapid radiometric detection of mycobacterial growth from smear-positive tissue samples from pigs

Rapid demonstration of mycobacteria in slaughter pigs is important for medical, epidemiological and economic reasons. The Bactec radiometric system detected more mycobacteria in less time than conventional culture on solid medium.     

A Comparative Study of Mycobacteria from Patients' Room Dusts and from Sputa of Tuberculous Patients

The source of mycobacteria other than Mycobacterium tuberculosis occurring in sputa of tuberculous patients as casual isolates was investigated by comparing the occurrence rate of mycobacterial species between patient's room dust and patient's sputum. Almost all species of mycobacteria recovered from sputa could be found in dusts obtained from the rooms. However, the percentage of occurrence of the mycobacterial species in dusts differed from that in sputa. In dusts, Mycobacterium fortuitum (39.6%), Mycobacterium nonchromogenicum (23.5%) and Mycobacterium gordonae (16.7%) occurred in high frequencies, whereas Mycobacterium intracellulare (69.6%), M. gordonae (5.9%), Mycobacterium scrofulaceum (5.2%), and Gordona bronchialis (10.4%) were the main species found in sputa. The patterns of occurrence of mycobacterial species as illustrated above may suggest that pathogenic mycobacteria survive in the respiratory tract while nonpathogenic ones are destroyed there, thus the human body acts as a selective medium for the pathogenic mycobacterial species. Serotype studies on strains of M. intracellular as casual isolates indicated that those from sputa of tuberculous patients were different from those derived from patients with lung diseases due to this species of mycobacteria. These results led us to the conclusion that mycobacteria in patients' room dusts were the source of mycobacteria occurring in sputa as casual isolates, particularly the pathogenic mycobacteria in dusts are more likely to survive in the human respiratory tract, occasionally multiplying and causing disease under favorable circumstances.     

The isolation of Mycobacterium avium complex from soil, water, and dusts

Previously, it was difficult to isolate the Mycobacterium avium complex from soil, water, and dusts, because rapidly growing mycobacteria always overgrew slowly growing ones. We used Ogawa egg medium containing both ethambutol and ofloxacin, which inhibit the nonpathogenic slowly growing mycobacteria and most rapidly growing mycobacteria, respectively, as an aid to screen for pathogenic slowly growing mycobacteria; we could thereby isolate a number of the M. avium complex and M. scrofulaceum strains from soil, water, and dusts in this country.     

Optimization of procedures for isolation of mycobacteria from soil and water samples obtained in northern India

For isolation of environmental mycobacteria, a decontamination procedure has been standardized by which treatment with 3% sodium dodecyl sulfate plus 4% NaOH (15 and 30 min for rapid and slow growers, respectively) is followed by incubation with 2% cetrimide (5 and 15 min for fast- and slow-growing mycobacteria, respectively); this procedure was found to completely eliminate contamination with other organisms and resulted in the isolation of only mycobacteria.     

Optimization of Procedures for Isolation of Mycobacteria from Soil and Water Samples Obtained in Northern India

For isolation of environmental mycobacteria, a decontamination procedure has been standardized by which treatment with 3% sodium dodecyl sulfate plus 4% NaOH (15 and 30 min for rapid and slow growers, respectively) is followed by incubation with 2% cetrimide (5 and 15 min for fast- and slow-growing mycobacteria, respectively); this procedure was found to completely eliminate contamination with other organisms and resulted in the isolation of only mycobacteria.     

Application of the hsp65 PRA method for the rapid identification of mycobacteria isolated from clinical samples in Belgium

Biochemical identification of mycobacteria is slow and many times fail to produce correct results. We compared PCR-restriction fragment length polymorphism analysis (PRA) of hsp65 and biochemical methods for the identification of mycobacteria from human samples in Belgium. PRA was found useful in the identification of mycobacteria and simple to implement as a quick method in the laboratory.     

Isolation and automated ribotyping of Mycobacterium lentiflavum from drinking water distribution system and clinical specimens

Automated ribotyping as a tool for identifying of nontuberculous mycobacteria was evaluated. We created a database comprising of riboprints of 60 strains, representing 32 species of nontuberculous mycobacteria. It was shown that combined ribopatterns generated after digestion with EcoRI and PvuII were distinguishable between species of both slow-growing and rapid-growing mycobacteria. The findings were in good agreement with the 16S rRNA gene sequencing results, allowing correct identification of Mycobacterium lentiflavum isolated from clinical specimens and from biofilms growing in public water distribution system. The automated ribotyping was powerful in discriminating between M. lentiflavum and closely related species M. simiae and M. palustre. Mycobacterium lentiflavum strains from drinking water biofilms were resistant to two to four antimycobacterial drugs. The drinking water distribution system may, thus, be a source of nontuberculous mycobacteria resistant to multiple drugs.     

Mycobacteria from leprous tissue of an armadillo cultivated on a hyaluronic acid based medium.

Mycobacteria were isolated from pooled leprous tissues of an armadillo. The suspensions of acid fast bacilli obtained were inoculated into a culture medium composed of umbilical cord extract, supplemented with yeast extract powder and glycerol with sheep serum added. Incubation temperature was 34 degrees C. An abundant growth of mycobacteria was observed in the primo culture in four weeks. The culture was easily sub-cultured on the homologous media. The primo culture did not grow on Lo?wenstein medium. The identity of the cultures of mycobacteria obtained is not yet established. The same strain of mycobacteria was cultured in media inoculated with suspensions of M. leprae decontaminated with sodium hydroxide-citrate solution. We confirm the findings of Skinsnes et al. (1975) that mycobacteria from human and animal leprous tissue can be cultured repeatedly on a hyaluronic based medium.     

Comparison of NaOH-N-acetyl cysteine and sulfuric acid decontamination methods for recovery of mycobacteria from clinical specimens

We compared the NaOH-N-acetyl cysteine (NaOH-NALC) and the sulfuric acid decontamination procedure in the detection of mycobacteria using the Mycobacteria Growth Indicator Tube (MGIT). In total 219 sputum specimens were collected from 142 Zambian patients and subjected to mycobacterial culture. One half of the specimen was decontaminated with NaOH-NALC and the other half was decontaminated with sulfuric acid. From the 438 samples a total of 261 (60%) cultures yielded growth of mycobacteria, consisting of 22 different species. The sulfuric acid method was more successful than the NaOH-NALC method in recovering mycobacteria in MGITs (146 versus 115 respectively, p = 0.001). Of the 146 positive mycobacterial cultures recovered after sulfuric acid decontamination 28 were Mycobacterium tuberculosis, 84 nontuberculous mycobacteria (NTM) and 34 acid fast bacterial isolates which could not be identified to the species level. The 115 mycobacteria recovered by the NaOH-NALC method consisted of 34 M. tuberculosis strains, 55 NTM and 26 acid fast bacteria that could not be identified. The most frequently isolated NTM were Mycobacterium lentiflavum and Mycobacterium intracellulare. Comparing the two decontamination methods the recovery of NTM in the sulfuric acid group was significant higher than in the NaOH-NALC group (p = 0.001). In contrast, no significant difference was found for the recovery of M. tuberculosis. These results show that the decontamination method used affects the recovery of nontuberculous mycobacteria in particular.     

Characterisation of porin genes from Mycobacterium fortuitum and their impact on growth

Background  

Highly pathogenic mycobacteria like Mycobacterium tuberculosis are characterised by their slow growth and their ability to reside and multiply in the very hostile phagosomal environment and a correlation between the growth rate of mycobacteria and their pathogenicity has been hypothesised. Here, porin genes from M. fortuitum were cloned and characterised to address their impact on the growth rate of fast-growing and pathogenic mycobacteria.     

The PCR amplification of non-tuberculous mycobacterial 16S rRNA sequences from soil

Non-tuberculous mycobacteria are free living saprophytic organisms commonly found in soil and water. Some are major causes of opportunistic infection, particularly in immuno-compromised patients, and may influence the efficacy of bacille Calmette-Guérin vaccinations. Many of these organisms are not amenable to culture, so information about their distribution is limited. PCR primers designed to amplify part of the mycobacterial 16S rRNA gene were applied to DNA extracted from cultured organisms and soil. The PCR products from soil contained sequences with similarity to slow growing mycobacteria similar to Mycobacterium lentiflavum, and to fast growing mycobacteria such as the xenobiotic degraders PYR-I and RJGII.     

Bactericidal activity of the membrane fraction isolated from phagocytes of mice and its stimulation by melittin

The membrane fraction prepared from mouse peritoneal exudate cells was incubated with mycobacteria, staphylococci, or E. coli in acetate buffer of pH 5.6 to follow the fate of viable bacilli. The membrane fraction exhibited bactericidal effect on mycobacteria and staphylococci, but not on E. coli. The activity to kill mycobacteria, as well as the endogenous phospholipase A2 activity, of the membrane fraction was markedly enhanced by melittin, a basic peptide from bee venom, and inhibited by indomethacin and EDTA. The role of the enzyme activity in the bactericidal activity was discussed.     

Relationships between Mycobacterium isolates from patients with pulmonary mycobacterial infection and potting soils

High numbers of mycobacteria, including known pathogenic species such as Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium chelonae, were recovered from aerosols produced by pouring commercial potting soil products and potting soil samples provided by patients with pulmonary mycobacterial infections. The dominant mycobacteria in the soil samples corresponded to the dominant species implicated clinically. Profiles of large restriction fragments obtained by pulsed-field gel electrophoresis demonstrated a closely related pair of M. avium isolates recovered from a patient and from that patient's own potting soil. Thus, potting soils are potential sources of infection by environmental mycobacteria. Use of dust-excluding masks should be considered during potting or other activities that generate aerosol with soil.     

The diverse family of MmpL transporters in mycobacteria: from regulation to antimicrobial developments

Mycobacterial genomes contain large sets of loci encoding membrane proteins that belong to a family of multidrug resistance pumps designated Resistance‐Nodulation‐Cell Division (RND) permeases. Mycobacterial membrane protein Large (MmpL) transporters represent a subclass of RND transporters known to participate in the export of lipid components across the cell envelope. These surface‐exposed lipids with unusual structures play key roles in the physiology of mycobacteria and/or can act as virulence factors and immunomodulators. Defining the substrate specificity of MmpLs and their mechanisms of regulation helps understanding how mycobacteria elaborate their complex cell wall. This review describes the diversity of MmpL proteins in mycobacteria, emphasising their high abundance in a few opportunistic rapid‐growing mycobacteria. It reports the conservation of mmpL loci between Mycobacterium tuberculosis and non‐tuberculous mycobacteria, useful in predicting the role of MmpLs with unknown functions. Paradoxically, whereas MmpLs participate in drug resistance mechanisms, they represent also attractive pharmacological targets, opening the way for exciting translational applications. The most recent advances regarding structural/functional information are also provided to explain the molecular basis underlying the proton‐motive force driven lipid transport. Overall, this review emphasises the Janus‐face nature of MmpLs at the crossroads between antibiotic resistance mechanisms and exquisite vulnerability to drugs.     

A method to extract intact and pure RNA from mycobacteria

We describe a high-yielding, simple, and aerosol-free protocol for the isolation of RNA from mycobacteria that does not require sophisticated instruments. The method yielded 50 μg of RNA from 10⁷ cells, 50 times more than a recently reported method. Our method can extract total RNA from aerobically grown bacteria and from in vitro hypoxia-induced dormant bacilli and mycobacteria residing within infected macrophages.     

Lipoarabinomannans: from structure to biosynthesis

Mycobacterium tuberculosis, the causative agent of tuberculosis, is one of the most effective human pathogens and the molecular basis of its virulence remains poorly understood. Here, we review our current knowledge about the structure and biosynthesis of the mycobacterial cell-wall lipoglycans, lipoarabinomannans (LAM). LAM are ubiquitous of mycobacteria and appear as the most potent non-peptidic molecules to modulate the host immune response. Nevertheless, LAM structure differs according to the mycobacterial species and three types of LAM have been described: mannose-capped LAM (ManLAM), phospho-myo-inositol-capped LAM (PILAM) and non-capped LAM (AraLAM). The type of capping is a major structural feature determining the ability of LAM to modulate the immune response. ManLAM, found in slow-growing mycobacteria, such as M. tuberculosis, have been demonstrated to be powerful anti-inflammatory molecules and emerge as key virulence factors that may be relevant drug targets. LAM-like molecules are not only confined to mycobacteria but are also present in actinomycetes (including the genera Rhodococcus, Corynebacterium or Gordonia). This offers the possibility of comparative studies that should help in deciphering the structure-function relationships and biosynthesis of these complex molecules in the future.     

Immunogenicity of Cell Walls from Various Mycobacteria Against Airborne Tuberculosis in Mice

Protective potency of oil-treated cell walls of various mycobacteria against airborne infection of mice with a few cells of Mycobacterium tuberculosis H37Rv was compared with that of viable BCG. Although less potent than BCG cell walls, the cell walls of atypical mycobacteria of Runyon's groups I to IV protected against challenge by aerosol to some degree. Protection afforded by cell walls of H37Rv and of the avirulent mutants H37Ra and Washington II was comparable to that provided by BCG cell walls. However, cell walls of a highly virulent strain of M. bovis (Bovinus I) provided the best protection yet achieved. Present evidence suggests that protective substances are shared by all mycobacteria but in differing amounts; the relationship between virulence and immunogenicity has yet to be clarified.     

Isolation of mycobacteria from acidic forest soil samples: comparison of culture methods

To evaluate which combination of decontamination method and medium is most reliable when examining acidic, organic forest soils for mycobacteria, three decontamination methods and five media supplemented with cycloheximide were compared. Before decontamination, the samples were incubated at 37°C for 5 h to allow germination of microbial spores. The recovery of mycobacteria was significantly influenced both by the method and by medium. Decontamination with NaOH or H₂SO₄ both combined with malachite green and cycloheximide yielded higher viable counts of mycobacteria than decontamination with NaOH followed by oxalic acid. Egg media at pH 5·5 resulted in lower mycobacterial counts than egg media at pH 6·5 or Mycobacteria 7H11 agar. The numbers of slopes totally free of contaminants revealed Mycobacteria 7H11 agar medium to be more prone to contamination than the four egg media tested. The highest counts of mycobacteria and a low rate of contamination were obtained when decontamination with NaOH-malachite green–cycloheximide was combined with culture on glycerol and cycloheximide supplemented egg medium at pH 6·5.     

A cooperative taxonomic study of mycobacteria isolated from armadillos infected with Mycobacterium leprae

Seventeen strains of mycobacteria, recovered from six armadillos experimentally infected with Mycobacterium leprae, were examined in ten different laboratories. This collaborative study included use of conventional bacteriological tests, lipid analyses, determination of mycobactins and peptidoglycans, characterization by Py-MS, and immunological, metabolic, pathological and DNA studies. These armadillo-derived mycobacteria (ADM) formed five homogeneous groups (numbered ADM 1 to 5) on the basis of phenetic analyses. However, DNA studies revealed only four homogeneous groups since group ADM 1 and one of the two strains in group ADM 3 showed a high level of DNA relatedness. The phenetic and DNA studies confirmed that the ADM strains differed from all other known mycobacteria. Cultural, biochemical, metabolic and pathogenic properties as well as DNA-DNA hybridizations clearly differentiated these ADM from M. leprae.     

Relationships between Mycobacterium Isolates from Patients with Pulmonary Mycobacterial Infection and Potting Soils

High numbers of mycobacteria, including known pathogenic species such as Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium chelonae, were recovered from aerosols produced by pouring commercial potting soil products and potting soil samples provided by patients with pulmonary mycobacterial infections. The dominant mycobacteria in the soil samples corresponded to the dominant species implicated clinically. Profiles of large restriction fragments obtained by pulsed-field gel electrophoresis demonstrated a closely related pair of M. avium isolates recovered from a patient and from that patient's own potting soil. Thus, potting soils are potential sources of infection by environmental mycobacteria. Use of dust-excluding masks should be considered during potting or other activities that generate aerosol with soil.