Characterization of mycobacteria isolated from bovines by PRA-targetting hsp 65 gene region.

Bovine tuberculosis caused by the bacterium Mycobacterium bovis is a major infectious disease of animals and has zoonotic importance for humans. Even though the incidence is believed to be very low in India, human tuberculosis caused by M. bovis has been increasingly recognized in many other countries of the world. As differentiation of mycobacterial species take long time, a method for the rapid identification of mycobacteria isolated from bovine samples to the species level was used, which is based on polymerase chain reaction (PCR) of the gene encoding for the 65-kD protein followed by restriction analysis. The method involves restriction enzyme analysis of PCR products obtained with primers common to all mycobacteria and generate M. tuberculosis complex specific pattern. PRA was performed on 33 bovine isolates of which 90.9% (30/33) isolates were identified clearly as M. tuberculosis complex, M. fortuitum, M. phlei and M. smegmatis using restriction enzyme Hae III.

Comparison of the 23S ribosomal RNA genes and the spacer region between the 16S and 23S rRNA genes of the closely related Mycobacterium avium and Mycobacterium paratuberculosis and the fast-growing Mycobacterium phlei.

The 23S rDNA sequences of Mycobacterium paratuberculosis, M. avium and M. phlei and the sequences of the spacer regions between the 16S and 23S rRNA genes were determined. The overall 23S rDNA sequence identity between M. paratuberculosis and M. avium was 99.7% (nine mismatches), showing the very close relatedness of these mycobacteria. Evolutionary distances between the five known mycobacterial 23S rRNA/rDNA sequences and those of other Gram-positive G + C-rich bacteria were determined. The 23S rDNA sequences of mycobacteria showed two inserted regions compared to the other bacteria. A mycobacterial unique region contained one mismatch between M. paratuberculosis and M. avium. An Actinomycetales-specific insertion, consisting of 111 nucleotides, was completely identical for M. paratuberculosis and M. avium. The sequence of the intergenic spacer region between 16S and 23S rDNA had a length of 278 bp for M. paratuberculosis and M. avium with only two mismatches. The spacer region of the fast-growing M. phlei was 85 bp longer. No tRNA-encoding region was found in the spacer region.

DNA analysis demonstrates that mycococcus forms are not mycobacteria.

Previous experimental evidence has suggested that the mycobacteria may exist in morphological forms other than the well characterised acid-fast bacillus. However none of these studies have been able to show conclusively that these variant forms are derived from the mycobacteria and therefore much debate has centered around the exact nature of these organisms. In this study we have examined stored cultures of the mycococcus form of Mycobacterium bovis BCG and M. phlei which were prepared by Csillag in 1972 and 1969. Restriction fragment patterns of the DNA of the variant forms and the parent mycobacteria were not similar and chromosomal DNA from the parent mycobacteria did not hybridise with the DNA of the variant forms. Furthermore biochemical studies indicate that the variant forms are environmental contaminants. Although this study shows conclusively that the mycococcus is not derived from the mycobacteria, we believe that the search for variant morphological or metabolic forms of the mycobacteria should remain an active area of investigation, but that no study should be considered complete without the full use of newer molecular technology.

Mechanism of gram variability in select bacteria.

Gram stains were performed on strains of Actinomyces bovis, Actinomyces viscosus, Arthrobacter globiformis, Bacillus brevis, Butyrivibrio fibrisolvens, Clostridium tetani, Clostridium thermosaccharolyticum, Corynebacterium parvum, Mycobacterium phlei, and Propionibacterium acnes, using a modified Gram regimen that allowed the staining process to be observed by electron microscopy (J. A. Davies, G. K. Anderson, T. J. Beveridge, and H. C. Clark, J. Bacteriol. 156:837-845, 1983). Furthermore, since a platinum salt replaced the iodine mordant of the Gram stain, energy-dispersive X-ray spectroscopy could evaluate the stain intensity and location by monitoring the platinum signal. These gram-variable bacteria could be split into two groups on the basis of their staining responses. In the Actinomyces-Arthrobacter-Corynebacterium-Mycobacterium-Propionibacterium group, few cells became gram negative until the exponential growth phase; by mid-exponential phase, 10 to 30% of the cells were gram negative. The cells that became gram negative were a select population of the culture, had initiated septum formation, and were more fragile to the stress of the Gram stain at the division site. As cultures aged to stationary phase, there was a relatively slight increase toward gram negativity (now 15 to 40%) due to the increased lysis of nondividing cells by means of lesions in the side walls; these cells maintained their rod shape but stained gram negative. Those in the Bacillus-Butyrivibrio-Clostridium group also became gram negative as cultures aged but by a separate set of events. These bacteria possessed more complex walls, since they were covered by an S layer. They stained gram positive during lag and the initial exponential growth phases, but as doubling times increased, the wall fabric underlying the S layer became noticeably thinner and diffuse, and the cells became more fragile to the Gram stain. By stationary phase, these cultures were virtually gram negative.

Molecular-genetic evidence for the relationship of Mycobacterium leprae to slow-growing pathogenic mycobacteria.

A total of 1170 nucleotides of the 16S rRNA from Mycobacterium leprae were compared to the homologous regions of M. tuberculosis, M. bovis Vallée, M. avium, M. scrofulaceum, M. phlei, M. fortuitum and one representative each of the genera Corynebacterium, Nocardia, and Rhodococcus. Homology values were calculated and a phylogenetic tree was constructed from the evolutionary distance values. Despite differences in DNA G + C content and genome size, M. leprae is a true member of the slow-growing pathogenic mycobacteria, branching off intermediate to the other members of this subgroup. Slow- and fast-growing mycobacteria are phylogenetically well separated but constitute an individual branch of the actinomycetes proper. Significant structural variation of certain regions of the 16S rRNA may allow construction of M. leprae-specific probes used for rapid identification.

Determination of genome size and DNA homology between an unclassified Mycobacterium species isolated from patients with Crohn's disease and other mycobacteria.

The genome size of an unclassified Mycobacterium species, isolated from a patient with Crohn's disease, was determined by measurement of DNA renaturation kinetics as 3.1 X 10(9) Da. The percentage of DNA homology of this organism to DNA of related mycobacteria was evaluated by measurement of DNA renaturation with heterologous DNA. These studies supported the classification of this organism as Mycobacterium paratuberculosis but failed to distinguish between M. paratuberculosis and organisms of the Mycobacterium avium-intracellulare groups.

Influence of culture medium and incubation time on the fatty acid compositions of some rapid-growing mycobacteria as analysed by packed and capillary column gas chromatography.

Cellular fatty acids of four rapid-growing mycobacterial species (Mycobacterium chelonei, M. fortuitum, M. phlei, and M. smegmatis) were analysed by packed and capillary column gas chromatography after one, three, four, six, eight, and twelve days of incubation on Löwenstein-Jensen and Sauton agar media. Variations in incubation time did not affect the chromatograms except in the case of twelve-day incubated M. smegmatis. Mycobacteria cultivated on Sauton agar medium contained more tuberculostearic and less oleic acid compared with Löwenstein-Jensen. For informative and reproducible analytical results, we recommend using a chemically defined culture medium and splitless injection on a capillary column capable of separating not only the methyl esters of the cellular fatty acids but also the diagnostically important secondary alcohols containing 18 and 20 carbon atoms.

[Characteristics of the protein complexes of clinical strains of mycobacteria]. / Kharakteristika belkovykh kompleksov klinicheskikh shtammov mikobakterii.

The protein composition of Mycobacterium tuberculosis and the molecular weight of proteins contained in these organisms have been determined by the method of electrophoresis in the porosity gradient of polyacrylamide gel. Close similarity between the electrophoregrams of M. tuberculosis clinical and laboratory strains has been revealed. The study of 18 strains of different groups of mycobacteria has shown that M. tuberculosis are essentially different from opportunistic mycobacteria and acid-resistant saprophytes. These data may be important for the identification and taxonomy of mycobacteria.

[Bacteriophage occurrence in "Mycobacterium phlei" (author's transl)]. / Présence d'une bactériophage chez Mycobacterium phlei.

Surface growth of synchronized bacteria was obtained by means of a suspension of Mycobacterium phlei cells in pentane, the dispersion of which resulted from passage through glass (Ballotini) column. By using standardized conditions, a series of identical cultures were obtained, suitable for studying their evolution as a function of time. By counting colonies every twenty minutes, during ten hours, two doublings were observed, with a generation time of five hours. At the end of a plateau, just before the next doubling, the curve exhibited a marked decrease. Bacteriophages were found in culture medium at the time corresponding to this decrease. In thin sections of the pellicles collected at this time, condensations resembling DNA from phage heads could be noticed within the bacterial cells, as well as free phages in th close neighbourhood of burst cells. The relations between phage and bacteria, and the possible relation between the presence of the phage and the synthesis of phleates has not been determined.

Reclassification of Mycobacterium phlei PN-bb as Rhodococcus bronchialis PN-bb.

Mycobacterium phlei PN-bb, induced in the parental strain PN by gamma-radiation and ethyl methanesulfonate, should be reclassified as Rhodococcus bronchialis PN-bb. The reclassification is based on the results of lipid analysis, DNA-DNA hybridization and several biochemical tests, performed in comparison with the parental strain PN, earlier reclassified as R. bronchialis, and R, bronchialis (N654).

Comparative studies of the strains PA and PN of Mycobacterium phlei leading to their reclassification: examination of lipids and DNA, biochemical tests and phage typing.

Study of lipid and DNA, biochemical tests and phage typing performed on the strain PA previously labelled Mycobacterium phlei, lead to the conclusion that this strain belongs to the species M. smegmatis. Parallel studies performed on strain PN, isolated from a culture of strain PA, as well as DNA homology percentage of the two strains, do not support the assumption that strain PN could have resulted from a mutation of strain PA Strain PN produces mycolic acids similar to those found in Rhodococcus bronchialis; the few biological tests applied quite agree with such a classification.