Predominance of Mycobacterium fortuitum-chelonae complex in Ghatampur field area, endemic for leprosy.

Non-tuberculous mycobacteria (NTM) are commonly found in the environment. As exposure to environmental mycobacteria has been reported to immunomodulatory in this study, the presence of environmental mycobacteria was investigated in soil, drinking water and drainage sample in Ghatampur, India, which is known for high endemicity for leprosy. Soil, drinking water from the hand pumps/wells and also drainage water collected in pools was collected in clean containers and cultured for environmental mycobacteria. Samples were processed according to the protocol established earlier. 69 soil, 62 drinking water and 31 drainage water samples were analysed from soil and water collected from 48 villages of this field area. After decontamination, cultures were set upon Lowenstein Jensen (LJ) medium. Mycobacteria were identified using biochemical tests and molecular techniques such as PCR-RFLP targeting hsp65 kD and rpoB region as well as 16S ribosomal sequencing in case of isolates showing variable biochemical features. NTM (non-tubercular mycobacteria) were isolated from 47.82% of soil samples, 20.69% of drinking water samples and 19.35% of the drainage water samples, overall mycobacteria could be isolated 52/162 of samples (32.09%). Among these mycobacteria, M. fortuitum-chelonae complex was predominant in this area; other species isolated were M. phlei, M. vaccae, M. terrae and M. flavescens. Relevance of exposure to these mycobacteria on endemicity needs to be studied by immunological and epidemiological parameters.

The microbial ecology of a typical Italian salami during its natural fermentation.

We have investigated the bacteria and yeast ecology of the typical Italian Ciauscolo salami that is produced in Central Italy using a polyphasic approach based on culture-dependent and -independent methods. The physico-chemical analyses showed a progressive drop in pH and water activity (aw) during ripening. The viable counts revealed a dominance of lactic acid bacteria (LAB) over coagulase negative cocci (CNC) and yeasts. From the molecular identification of the isolates, the prevalence of Lactobacillus curvatus, Lb. plantarum and Staphylococcus xylosus was shown among the bacteria, while Debaryomyces hansenii was the prevalent species among the yeasts, and it was isolated throughout the whole ripening process. Minority species, namely Rhodotorula mucillaginosa and Trichosporon brassicae, were also recovered from the meat batter. The total microbial community was profiled without cultivation by analyzing the DNA that was directly extracted from the salami samples. Moreover, the cultivable community was profiled by analyzing the DNA recovered from bulk cells that were obtained by harvesting the colonies from serial-dilution agar plates. The 16S rRNA gene V1 and V3 regions were used as targets in the denaturing gradient gel electrophoresis (DGGE) profiling of the LAB and CNC communities, respectively, while the diversity and dynamics of the yeast population were assessed by analyzing a portion of the 28S rRNA gene. Our findings suggest that the microbial diversity of fermented meat products can be successfully investigated by this polyphasic approach that is based on the assessment of both the total and the cultivable community diversity.

Use of reverse transcription polymerase chain reaction for the detection of Mycobacterium leprae in the slit-skin smears of leprosy patients.

The relevance of bacterial index (BI) for understanding the prognosis of leprosy patients on treatment has been extensively debated, as it does not give a very clear idea of the viability of the bacteria in patients under treatment. Here we used slit-skin smear samples of leprosy patients to test the suitability for studying viability of Mycobacterium leprae using reverse transcription polymerase chain reaction (RT-PCR). For this purpose, we recruited 13 multibacillary (MB) leprosy patients (8 lepromatous and 5 borderline lepromatous). Of these, 7 were relapse cases, 3 were under treatment (MB-MDT), 2 were new cases and 1 had completed treatment. We carried out extraction of RNA using Trizol reagent (Life Technologies, UK) from the slit-skin smear samples from these patients. The RNA preparation was then used for the RT-PCR using Mycobacterium leprae-specific primers for the fragment of 16s ribosomal RNA gene. Samples from both the new cases, 4 suspected relapse cases and 1 patient under treatment showed positive RT-PCR results. Other 6 patients whose smear samples did not show any amplification by RT-PCR were on MB-MDT from 8 to 30 months. The usefulness of the technique needs to be validated using mouse footpad technique and also should be more extensively explored for studying the viability of M. leprae, the efficacy of treatment and the presence of other mycobacteria in the slit-skin smear samples.

Complete DNA sequence analysis for 16S ribosomal RNA gene of the leproma-derived, cultivable and nerve-invading mycobacterium HI-75.

The complete 1493 nucleotide sequence of the 16SrRNA gene of the leproma-derived and cultivable mycobacterium HI-75 strain was analyzed to elucidate the taxonomic characteristics by direct sequencing of the polymerase chain reaction (PCR) products. The results revealed that the sequence of mycobacterium HI-75 was mostly similar to that of Mycobacterium scrofulaceum with 5 bases differences in the sequenced 1493 bases (0.35%) of the 16SrRNA gene. M. leprae differed from the strain with 47 bases (3.3%). Sasaki and Hamit reported the nerve-invasive activity of the inoculated mycobacterium HI-75 in nude mice or the 131I-treated immunocompromised Swiss mice. The results indicate that mycobacterium HI-75 could be a mutant of M. scrofulaceum possessing the ability to invade the peripheral nerve in addition to developing leproma-like lesions.

Nucleotide sequence and secondary structures of precursor 16S rRNA of slow-growing mycobacteria.

Slow-growing mycobacteria have a single ribosomal RNA (rrn) operon, with the genes for 16S, 23S and 5s rRNA being present in that order. The transcription start site of the rrn operon of Mycobacterium tuberculosis was identified in Escherichia coli. PCR methodology was used to amplify parts of the rrn operon, namely the leader region and the spacer-1 region separating the 16S rRNA and 23S rRNA genes of Mycobacterium avium, Mycobacterium paratuberculosis, Mycobacterium intracellulare, 'Mycobacterium lufu', Mycobacterium simiae and Mycobacterium marinum. The amplified DNA was sequenced. The sequence data, together with those obtained previously for Mycobacterium leprae and M. tuberculosis, were used to identify putative antitermination signals and RNase III processing sites within the leader region. Notable features include a highly conserved Box B element and a sequence of 31 nucleotides which is common to all eight slow-growers which were scrutinized. A secondary structure for mycobacterial precursor-16S rRNA was devised, based on sequence homologies and homologous nucleotide substitutions. The 18 nucleotides at the 5'-end of spacer-1 have the capacity of binding sequences close to the 5'- and 3'-ends of mature 16S rRNA, suggesting that secondary structure is important to the maturation process. All the slow-growers, including M. leprae, conform to the same scheme of secondary structure. The scheme proposed for M. tuberculosis is a variant of the main theme. The leader and spacer sequences may prove a useful supplement to 16S rRNA sequences in establishing phylogenetic relationships between very closely related species. 'M. lufu' appears to be a close relative of M. intracellulare.

Polymerase chain reaction amplifying DNA coding for species-specific rRNA of Mycobacterium leprae.

The sensitivity of the polymerase chain reaction (PCR) on the DNA coding for the species-specific fragment of 16S rRNA of Mycobacterium leprae studied on mouse foot pad harvests and human skin biopsies varies widely between 1 and 3 x 10(4) organisms. This is probably the result of variations in the proportions of organisms with sufficiently intact DNA suitable for PCR. Preserving human skin biopsies for 3 weeks at an ambient temperature even after boiling for 6 minutes gives rise to a 10-fold decrease in sensitivity. Fixation of tissues in formol 10% or Lowy fixative or preserving in Dubos OAA broth is very harmful to the PCR, mainly due to the enhancement of an inhibitory effect on the PCR reaction. For preservation, the best choice at the moment seems to be alcohol 70%. Sample preparation of five cycles of freeze-boiling is simple and generally more efficient than proteinase K treatment and DNA extraction.

The 16S ribosomal RNA of Mycobacterium leprae contains a unique sequence which can be used for identification by the polymerase chain reaction.

Nucleotide sequence data for bacterial 16S ribosomal RNA was used to identify oligodeoxyribonucleotide primers suitable for probing the rRNA gene of mycobacteria and related organisms, with the polymerase chain reaction. The method enabled us to distinguish mycobacteria from other closely related genera, and to differentiate between slow- and fast-growing mycobacteria. Mycobacterium leprae fell within the slow-growing group of mycobacteria but there are significant differences between the sequence of the M. leprae 16S rRNA gene and that of other slow-growing mycobacteria. These differences were used to devise a rapid, non-radioactive method for detecting M. leprae in infected tissue.

The 16S rRNA nucleotide sequence of Mycobacterium leprae: phylogenetic position and development of DNA probes.

The almost complete 16S rRNA sequence from Mycobacterium leprae was determined by direct sequencing of the chromosomal gene amplified by the polymerase chain reaction. The primary sequence revealed an insertion of 12 nucleotides at the 5' end of the 16S rRNA gene, which consists of an A-T stretch and appears to be unique for M. leprae. Within the mycobacteria M. leprae branches off with a group of slow-growing species comprising M. scrofulaceum, M. kansasii, M. szulgai, M. malmoense, M. intracellulare and M. avium. A systematic comparison of the nucleotide sequence resulted in the characterization of oligonucleotide probes which are highly specific for M. leprae. The probes hybridized exclusively to 16S rRNA nucleic acids from M. leprae, but not to nucleic acids from 20 cultivable fast- and slow-growing mycobacteria.