"Mycobacterium massilipolynesiensis" sp. nov., a rapidly-growing mycobacterium of medical interest related to Mycobacterium phlei.

In French Polynesia, respiratory tract clinical isolate M26, displayed unusual phenotype and contradictory phylogenetic affiliations, suggesting a hitherto unidentified rapidly-growing Mycobacterium species. The phenotype of strain M26 was further characterized and its genome sequenced. Strain M26 genome consists in a 5,732,017-bp circular chromosome with a G + C% of 67.54%, comprising 5,500 protein-coding genes and 52 RNA genes (including two copies of the 16 S rRNA gene). One region coding for a putative prophage was also predicted. An intriguing characteristic of strain M26's genome is the large number of genes encoding polyketide synthases and nonribosomal peptide synthases. Phylogenomic analysis showed that strain M26's genome is closest to the Mycobacterium phlei genome with a 76.6% average nucleotide identity. Comparative genomics of 33 Mycobacterium genomes yielded 361 genes unique to M26 strain which functional annotation revealed 84.21% of unknown function and 3.88% encoding lipid transport and metabolism; while 48.87% of genes absent in M26 strain have unknown function, 9.5% are implicated in transcription and 19% are implicated in transport and metabolism. Strain M26's unique phenotypic and genomic characteristics indicate it is representative of a new species named "Mycobacterium massilipolynesiensis". Looking for mycobacteria in remote areas allows for the discovery of new Mycobacterium species.

Effect of heat killed Mycobacterium phlei on body weight gain and management of caecal coccidiosis in broiler chickens.

The objective of this study was to evaluate the immunotherapeutic potential of heat killed Mycobacterium phlei in broiler chicken against experimentally produced Eimeria tenella infection. The selected dose of E. tenella oocyst (5x10(3) sporulated oocysts per bird) was capable of producing a mild form of caecal coccidiosis as observed by significant difference in body weight gain, clinical findings and caecal lesion score. Heat killed M. phlei was fed orally at 10 mg per bird with sterile PBS vehicle at alternate day for four doses. Our study reveals that per day body weight gain was significantly (p<0.01) higher for healthy control compared to coccidia infected group. The group fed M. phlei along with coccidial challenge showed significantly (p<0.05) higher body weight gain than infected control group. Heat killed M. phlei feeding also found effective to reduce the caecal lesion score significantly (p<0.05) in comparison to E. tenella infected untreated group. IgA concentrations in serum and bile at 7-day post challenge of coccidial oocyst was also significantly (p<0.01) higher in M. phlei fed group when compared to coccidia infected and healthy control group. We concluded that use of heat killed M. phlei has a beneficial role as an immunostimulant against caecal coccidiosis in broiler chicken.

[Effect of Mycobacterium phlei F.U.36 suspended liquor on culture and proliferation of dendritic cells derived from human umbilical cord blood in vitro].

To investigate the effect of mycobacterium phlei F.U.36 suspended liquor (Utilin"s", U) on the culture and proliferation of dendritic cells (DCs) derived from human umbilical cord blood in vitro, the mononuclear cells (MNCs) were isolated from human umbilical cord blood and cultured with RPMI 1640 in the control group. Test groups consisted of Utilin"s" group (only Utilin"s"), GTI group (GM-CSF, TNF-alpha, IL-4) and GTIU group (GM-CSF, TNF-alpha, IL-4 and Utilin"s"). MNCs in all test groups were cultured with RPMI-1640. The growth of DCs was observed by the light microscopy, the phenotypes of DCs were determined by flow cytometry on the 10th day of culture, and some harvest cells were stained with Wright-Giemsa, then observed and photographed under the oil immersion objective. The results showed that the test groups all displayed some number of typical DCs; both CD1a positive cell rate and HLA-DR positive cell rate of the Utilin"s" group were higher than those of the control; HLA-DR positive cell rate of GTIU group increased most significantly and much higher than that of the GTI group. It is concluded that mycobacterium phlei F.U.36 not only promotes the proliferation of DCs derived from human umbilical cord blood in vitro, but also co-operates with rhGM-CSF, rhTNF-alpha and rhIL-4 in promoting the maturity of DCs.

Comparative analysis of phenotypic and genotypic characteristics of two desulfurizing bacterial strains, Mycobacterium phlei SM120-1 and Mycobacterium phlei GTIS10.

AIM: To compare few phenotypic and genotypic characteristics of two desulfurizing bacterial strains, Mycobacterium phlei SM120-1 and Mycobacterium phlei GTIS10. METHODS AND RESULTS: In the present study, dibenzothiophene (DBT) desulfurizing activity, composition of fatty acids of cell membranes, DBT sulfone monoxygenase gene (bdsA) and the selection pressure applied during the growth and enrichment of the bacterial strains M. phlei SM120-1 and M. phlei GTIS10 were compared in our laboratory. The DBT desulfurization activity of M. phlei SM120-1 was found to be 0.17 +/- 0.02 micromol 2-HBP min(-1) (gram dry cell weight)(-1) and that of the bacterial strain M. phlei GTIS10 was 1.09 +/- 0.05 micromol 2-HBP min(-1) (gram dry cell weight)(-1). Fatty acid methyl ester analysis of cell membranes of these two bacterial strains in the presence of light gas oil showed that both the strains had different fatty acid profiles in their cell membranes. Comparison of the full gene sequences of the desulfurization gene bdsA in the two bacterial strains showed significant difference in the bdsA gene sequences. There was a significant difference observed in the selection pressure applied during the growth and enrichment of the two bacterial strains. CONCLUSIONS: The results of the comparative study of the bacterial strains, M. phlei SM120-1 and M. phlei GTIS10 showed that there were considerable differences in the phenotypic and genotypic characteristics of these two strains. SIGNIFICANCE AND IMPACT OF STUDY: The present study would broaden the understanding of biodesulfurization trait at intra-species level.

Estimation of efflux mediated multi-drug resistance and its correlation with expression levels of two major efflux pumps in mycobacteria.

Multidrug resistance has been posing an increasing problem in the treatment of tuberculosis. Mutations in the genomic targets of drugs have been identified as the major mechanism behind this resistance. However, high degree of resistance in some isolates towards major drugs like rifampicin, isoniazid, ethambutol and streptomycin can not be explained solely on the basis of mutations. Besides this, certain other mechanisms like efflux pumps have also been considered as alternative mechanisms in the drug resistant isolates where there is no mutation and these mechanisms are specially important for drug resistance in non-tuberculous mycobacteria (NTM). In this study, we have estimated efflux pump mediated drug resistance in different mycobacterial species with the help of efflux pump inhibitors. All major anti-tuberculous drugs have been shown to be extruded by efflux pumps and the degree to which these drugs are extruded, vary in different mycobacterial species and isolates. The correlation of this resistance with functional activity of two major efflux pump genes pstB and Rv1258c was also assessed by reverse transcription PCR. Besides the significant role of these pumps observed, other efflux pumps, present in mycobacteria, may also be involved in drug resistance and need to be investigated.

Study of the properties of a channel-forming protein of the cell wall of the gram-positive bacterium Mycobacterium phlei.

The gram-positive bacterium Mycobacterium phlei was treated with detergents. Reconstitution experiments using lipid bilayers suggested that the detergent extracts contain a channel forming protein. The protein was purified to homogeneity by preparative SDS-PAGE and identified as a protein with an apparent molecular mass of about 135 kDa. The channel-forming unit dissociated into subunits with a molecular mass of about 22 kDa when it was boiled in 80% dimethylsulfoxid (DMSO). The channel has on average a single channel conductance of 4.5 nS in 1 m KCl and is highly voltage-dependent in an asymmetric fashion when the protein is added to only one side of the membrane. Zero-current membrane potential measurements with different salts implied that the channel is highly cation-selective because of negative point charges in or near the channel mouth. Analysis of the single-channel conductance as a function of the hydrated cation radii using the Renkin correction factor and the effect of the negative point charges on the single-channel conductance suggest that the diameter of the cell wall channel is about 1.8 to 2.0 nm. The channel properties were compared with those of other members of the mycolata and suggest that these channels share common features. Southern blots demonstrated that the chromosome of M. phlei and other mycolata tested contain homologous sequences to mspA (gene of the cell wall porin of Mycobacterium smegmatis).

Modulation of interleukin-12 synthesis by DNA lacking the CpG motif and present in a mycobacterial cell wall complex.

A mycobacterial cell wall complex prepared from the non-pathogenic microorganism Mycobacterium phlei, where mycobacterial DNA is preserved and complexed to cell wall fragments, possesses anticancer and immunomodulatory activity. DNA from a number of prokaryotes has been found to modulate the immune system and to induce cytokine synthesis. We have therefore determined whether the DNA associated with this complex has the ability to induce the synthesis of interleukin-12 (IL-12), a potent anticancer cytokine. Mycobacterial DNA complexed with cell wall fragments or DNA purified from M. phlei induced IL-12 synthesis by murine and human monocytes and macrophages in vitro, and was capable of inducing IL-12 synthesis in vivo in mice following i.p. administration. Neutralization of DNA with cationic liposomes or digestion with DNase I significantly decreased the ability of the cell wall complex to induce IL-12. CpG methylation of DNA extracted from these cell walls or from M. phlei did not affect the induction of IL-12 synthesis by monocytes and macrophages. In contrast, CpG methylation of DNA from Escherichia coli abolished its ability to induce IL-12 synthesis. These results demonstrate that unmethylated CpG motifs present in M. phlei DNA are not a prerequisite for the induction of IL-12 synthesis. The size of the mycobacterial DNA, in the range of 5 bp to genomic DNA, did not influence its capacity to induce IL-12. Our results emphasize that M. phlei DNA associated with the cell wall complex makes a significant contribution to the overall immunomodulatory and anticancer activity of this mycobacterial cell wall preparation and that these activities are not correlated with the presence of CpG motifs.

Mycobacterium lepraemurium, a well-adapted parasite of macrophages: I. Oxygen metabolites.

We measured the release of reactive oxygen intermediaries [ROI (hydrogen peroxide and superoxide anion)] by murine peritoneal macrophages challenged in vitro with Mycobacterium lepraemurium (MLM), complement-opsonized yeast, M. bovis BCG, M. phlei, or phorbol myristate acetate (PMA). We found that except for MLM, all of the other materials provoked the release of significant amounts of hydrogen peroxide and superoxide. MLM entered the macrophages without triggering their oxidative metabolism. Pre-infection of macrophages with MLM did not alter these cells' capacity to release the normal amounts of ROI in response to other microorganisms or PMA. Killing of MLM did not revert the macrophages' failure to release ROI upon ingestion of the microorganism, nor were macrophages able to produce these toxic metabolites when pre-incubated in the presence of murine gamma interferon (IFN-gamma). MLM has several attributes that allow it to survive within macrophages: a) it is a nontoxigenic microorganism (it does not harm its host), b) it resists the harsh conditions of the intraphagolysosomal milieu (a property perhaps dependent on its thick lipidic envelope), and c) it penetrates the macrophages without triggering their oxidative response (thus avoiding the generation of the toxic intermediaries of oxygen). For these attributes (and others discussed in this paper), we recognize MLM as a highly evolved, well-adapted parasite of macrophages. In addition, the results of the present study prompted the analysis of the biochemical pathways used by MLM and M. bovis BCG to penetrate into their cellular hosts, a subject now under investigation in our laboratory.

Use of a dot enzyme-linked immunosorbent assay on absorbed sera for the diagnosis of bovine paratuberculosis.

This study describes the response of cattle to a dot enzyme-linked immunosorbent assay (ELISA) using sera absorbed with Mycobacterium phlei. Results obtained by visual observation are compared with those obtained using a densitometer. Infection status of cattle was determined by faecal culture. Cattle of different levels of exposure and disease manifestation were examined. A significantly higher dot ELISA response was observed (using both absorbed and non-absorbed sera) in animals with heavy shedding of M. paratuberculosis than in animals which tested negative by faecal culture or shed M. paratuberculosis at lower levels (P < 0.05). Paratuberculosis was diagnosed by visual determination of dot ELISA results using non-absorbed sera in 29 of 44 (65.9%) clinically-suspect animals giving positive results by faecal culture, and 85 of 93 (91.4%) cattle testing negative by faecal culture. With absorbed sera, the sensitivity of visual determination decreased to 15 of 44 (34.1%), while specificity increased to 91 of 93 (97.8%). Approximately 75% of cattle yielding positive results by dot ELISA were heavy bacterial shedders (> 1,500 colonies/g of faeces) at the time of serological testing. Comparison of the dot ELISA results determined visually with results obtained by objective densitometric measurement showed compatible specificity. Sensitivity of the dot ELISA was 65.9% for non-absorbed sera using visual evaluation and 87.5% using densitometric evaluation at a cut-off optical density value of 0.2. For absorbed sera, the values were 34.1% and 82.5%, respectively.

Use of an enzyme-linked immunosorbent assay for serodiagnosis of clinical paratuberculosis in goats. Study by western blotting of false-positive reactions.

An enzyme-linked immunosorbent assay (ELISA) was performed for diagnosis of paratuberculosis in goats, using as antigen a protoplasmic extract (PPA-3). The test was developed on the basis of the results obtained with two serum reference pools, positive and negative respectively. To avoid day-to-day variations, dilutions of the positive serum pool were included in each plate to obtain an arbitrary system, transforming absorbance into immunoglobulin (Ig) G anti-Mycobacterium paratuberculosis units. The ELISA was used on sera of two reference groups of animals. One group consisted of 35 goats suspected of being infected with paratuberculosis, which was confirmed by histological findings and isolation of M. paratuberculosis. The negative group consisted of 61 healthy goats from a farm free of paratuberculosis. The test showed a sensitivity of 100% and a specificity of 91.8%. Absorption of sera with a Mycobacterium phlei suspension did not modify either the sensitivity or the specificity of the test. Sera from the negative group were analysed by Western blotting, and four of them recognized two fractions with a molecular weight of 17.3 and 28.1 kDa.

Phenotypic changes in mycobacteria grown in oxygen-limited conditions.

Laboratory strains of Mycobacterium phlei, M. smegmatis, M. fortuitum, M. gordonae, M. kansasi, M. bovis, M. tuberculosis and M. intracellulare were adapted to grow in an anaerobic environment. Concomitant with the transition to anaerobic growth was loss of acid-fastness, loss or modification of colonial pigmentation, and loss of ability to grow on a malachite green-containing medium. The mycobacteria grown anaerobically produced acid from a greater range of carbohydrates than aerobically grown cultures, lost iron-uptake activity, and showed a reduction of urease, catalase and nitratase activity. Back adaption of mycobacteria from an anaerobic to an aerobic environment resulted in the acquisition of acid-fastness, pigmentation, and other characteristics used in the taxonomy of mycobacteria. These results suggest that mycobacterial cultures, if grown in an anaerobic environment, may be erroneously identified in clinical laboratories.

In vitro interaction of Mycobacterium avium with intestinal epithelial cells.

Human intestinal epithelial cell monolayers were inoculated with cultures of Mycobacterium avium serotype 2, 8, or 10 that were viable, autoclaved, Formalin killed, exposed to UV light, or suspended in anti-M. avium serotype 2 serum. The effects of four reagents known to block phagocytosis or endocytosis (cytochalasin B, dibutyryl cyclic adenosine monophosphate, iodoacetate, and 2,4-dinitrophenol) on the bacteria-cell interaction were also studied. The maximum uptake of pathogenic M. avium by human intestinal epithelial cells occurred after 2 to 3 h of incubation. Serotype 2 was taken up in greater quantity than serotype 8 or 10. Saprophytic mycobacteria did not attach to or penetrate the host cells. The data showed that viable mycobacteria are ingested by host cells, whereas dead organisms are not. Components of the bacterial cells are partially, but not solely, responsible for the phagocytosis of M. avium serotype 2 by human intestinal epithelial cells. Furthermore, uptake of M. avium by human intestinal epithelial cells was suppressed by reagents which inhibit uptake by known phagocytic cells, suggesting that the mechanism of uptake is an endocytic process induced by virulent mycobacteria.

Mutagenic effect of n-methyl-N-nitrosourea on Mycobacterium phlei.

N-Methyl-N-nitrosourea was used to induce auxotrophic, scotochromogenic and isonicotinic acid hydrazide resistant mutants in Mycobacterium phlei and its effect was compared with that of nitrosoguanidine. Seventeen auxotrophic mutants requiring amino acids or vitamins and 52 scotochromogenic mutants with orange colonies were induced. The frequency of isonicotinic acid hydrazide-resistant mutants increased by two orders of magnitude.