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PubMed1988

[Study of the minimal inhibitory concentration of rifabutine (Ansamycin LM 427) for Mycobacterium tuberculosis, Mycobacterium xenopi and Mycobacterium avium-intracellulare].

Ansamycin LM 427 (or rifabutine) is a spiropiperidyl-rifamycin which has been proposed as a treatment for tuberculosis and mycobacterial infections resistant to rifampicin. The minimal inhibitory concentration (MCI) of rifabutine and of rifampicin for 10 strains of M. tuberculosis sensitive to rifampicin, 15 strains of M. tuberculosis resistant to rifampicin, 10 strains of M. xenopi and 15 of M. avium-intracellulare have been measured in a comparative fashion on gel DIFCO 7H11 medium and also on a Loewenstein-Jensen medium. The MCI of rifampicin and of rifabutine inhibiting 90% of the strains (MCI 90) of M. tuberculosis sensitive to rifampicin have been 0.06 mg/l on 7H11 media and 1 mg/l on Loewenstein-Jensen media respectively. For strains of M. tuberculosis resistant to rifampicin they were respectively 256 and 8 mg/l on 7H11 and of 1024 and 128 Loewenstein-Jensen, for the strains of M. xenopi they were 8 and 0.5 on 7H11 and 64 and 4 mg/l on Loewenstein-Jensen respectively, and finally for strains of M. avium-intracellulare they were 64 and 2 mg on 7H11 and 256 and 16 on Loewenstein-Jensen media. With rare exceptions for proven mycobacterial infections there was a link of the order of 20:1 between the values for CMI of rifampicin and rifabutine measured on Loewenstein-Jensen medium and on 7H11; the MCIs were ten times more elevated on the first than on the second and the liaison between the MCI of rifampicin and the MCI of rifabutine tended to show that these two antibiotics have the same mode of action.(ABSTRACT TRUNCATED AT 250 WORDS)

Antitubercular Agents↗
PubMed1990

[Cross-resistance relationship between streptomycin and kanamycin resistances in Mycobacterium smegmatis (strain Jucho)--comparison of the development patterns of resistances to streptomycin and kanamycin among Mycobacterium tuberculosis, Mycobacterium avium complex, and Mycobacterium smegmatis].

The resistance development pattern of Mycobacterium smegmatis strain 17023 (Jucho) to streptomycin and kanamycin was studied. The medium used was Ogawa egg medium, and the level of resistance was determined for each clone derived from single colony by the 'actual count' method. Hence, the resistance level was estimated as the highest concentration of drugs, in which small inocula consisting of 20 to 100 colony-forming units could grow after seven days incubation. Only one type of resistance mutants resistant to more than 1,000 micrograms/ml streptomycin was isolated and these mutants were also resistant to 8 micrograms/ml kanamycin. On the other hand, only one type of kanamycin-resistant mutants resistant to 8 micrograms/ml kanamycin was isolated and these mutants were also resistant to more than 1,000 micrograms/ml streptomycin. Accordingly, there was a complete cross-resistance relationship between streptomycin and kanamycin resistances. Therefore, there existed only one phenotype, which is simultaneously resistant to streptomycin and kanamycin. The mutants occurred at a rate of about 2 x 10(-8) per viable bacterial population of the parent strain. Streptomycin-dependent mutants occurred at a rate of about 2 x 10(-9). The number of resistant phenotypes to streptomycin and kanamycin was only one in M. smegmatis, while it was five in M. tuberculosis and 2 or 3 in M. avium complex (Tsukamura, M. and Mizuno, S.: J. Gen. Microbiol. 88: 269-274, 1975; Tsukamura, M.: Kekkaku 62: 445-458, 1987). The simplicity of the resistance system of M. smegmatis suggests that this organism is evolutionally primitive in the world of mycobacteria.

Drug Resistance, Microbial↗
PubMed1990

T-cellular immune reactions (in macrophage inhibition factor assay) against Mycobacterium paratuberculosis, Mycobacterium kansasii, Mycobacterium tuberculosis, Mycobacterium avium in patients with chronic inflammatory bowel disease.

A mycobacterial aetiology has been suggested for Crohn's disease. A slow growing mycobacterium, biochemically and genetically identical to M paratuberculosis, the causative agent of enteritis in ruminants (Johne's disease), has been isolated from gut specimens of patients affected by Crohn's disease. If M paratuberculosis or other mycobacteria play a role in the pathogenesis of Crohn's disease, then patients may have been sensitised to these mycobacteria or show an anergy immune reaction. We therefore investigated the T-cell mediated immune response to sonicates of M paratuberculosis, M kansasii, M avium, and M tuberculosis in 35 patients with Crohn's disease, 28 with ulcerative colitis, and 25 controls using a macrophage inhibition factor assay on peripheral blood lymphocytes. Two types of reaction patterns were identified--that is, 'responders' (subjects with a macrophage inhibition factor assay in which a dose response relation was present and a percentage of inhibition exceeding 20%), and 'non-responders'. There was no significant difference in the prevalence of responders (59%-80%) and non-responders (20%-41%) to these mycobacteria between the group of Crohn's disease, ulcerative colitis, and control group. We found also that a large proportion of controls showed T-cell immunisation to the mycobacteria which supports the contention that the antigens are practically commensal. Our results do not support the proposed involvement of mycobacteria in the pathogenesis of Crohn's disease.

Adult↗
PubMed1995

A multiplex polymerase chain reaction for distinguishing Mycobacterium tuberculosis from Mycobacterium tuberculosis complex.

A multiplex polymerase chain reaction has been developed which is able to distinguish Mycobacterium tuberculosis from other members of the M. tuberculosis complex. The assay is based on the simultaneous amplification of two different targets: a 396bp region from the mtp40 species-specific gene sequence of M. tuberculosis and a 245bp fragment from the M. tuberculosis complex insertion sequence IS986. Results have been obtained for 54 mycobacterial strains including five non-M. tuberculosis complex isolates. All 49 strains of the M. tuberculosis complex were positive for IS986 but only the 27 M. tuberculosis isolates were positive for both IS986 and mtp40.

Base Sequence↗
PubMed1999

Early identification of Mycobacterium tuberculosis and Mycobacterium avium using the polymerase chain reaction on samples positive by a rapid commercial culture system.

A combination of two methods -- a rapid culture method [Mycobacteria Growth Indicator Tube (MGIT); Becton-Dickinson, USA] and a double polymerase chain reaction (PCR) assay -- was assessed for the detection and identification of Mycobacterium tuberculosis and Mycobacterium avium from clinical samples. The aim of the study was to evaluate the ability of the system to offer rapid and accurate diagnosis of mycobacterial infections. After decontamination, clinical samples (n = 554) were stained and cultured in parallel on solid media and in MGITs following standard procedures. The performance of the two culture systems was compared. Positive MGITs were tested for the presence of Mycobacterium tuberculosis and Mycobacterium avium by PCR of IS6110 (Mycobacterium tuberculosis) and the 16S rRNA gene (Mycobacterium avium). A total of 41 mycobacteria -- 27 Mycobacterium tuberculosis isolates, eight Mycobacterium avium isolates, and six other species of mycobacteria -- were isolated by one or both culture media. The MGIT system recovered 36 (87.8%) mycobacteria and the solid media 33 (80.4%). The mean time to detection by the two culture systems did not differ overall, but the mean time to detection of Mycobacterium avium from smear-positive specimens was shorter in MGITs than in solid media (5.25 days vs. 16.25 days, P < 0.05). The double PCR assay performed on the 36 positive MGITs correctly identified all 24 Mycobacterium tuberculosis-positive MGITs and all six Mycobacterium avium-positive vials. Therefore, application of the PCR assay to positive MGITs may mean that Mycobacterium tuberculosis and Mycobacterium avium can be identified at an earlier stage than with current methods.

Bacteriological Techniques↗
PubMed2003

Polymorphic nucleotide within the promoter of nitrate reductase (NarGHJI) is specific for Mycobacterium tuberculosis.

Mycobacterium tuberculosis rapidly reduces nitrate, leading to the accumulation of nitrite. This characteristic served for the past 40 years to differentiate M. tuberculosis from other members of the Mycobacterium tuberculosis complex (MTBC), such as Mycobacterium bovis (non-BCG [referred to here as simply "M. bovis"]), Mycobacterium bovis BCG, Mycobacterium africanum, or Mycobacterium microti. Here, a narG deletion in M. tuberculosis showed that rapid nitrite accumulation of M. tuberculosis is mediated by narGHJI. Analysis of narG mutants of M. bovis and M. bovis BCG showed that, as in M. tuberculosis, nitrite accumulation was mediated by narGHJI, and no other nitrate reductase was involved. However, in contrast to M. tuberculosis, accumulation was delayed for several days. Comparison of the narGHJI promoter revealed that, at nucleotide -215 prior to the start codon of narG, M. tuberculosis carried a thymine residue, whereas the bovine mycobacteria carried a cytosine residue. Using LightCycler technology we examined 62 strains of M. tuberculosis, M. bovis, M. bovis BCG, M. microti, and M. africanum and demonstrated that this single nucleotide polymorphism was specific for M. tuberculosis. For further differentiation within the MTBC, we included, by using LightCycler technology, the previously described analysis of oxyR polymorphism, which is specific for the bovine mycobacteria, and the RD1 polymorphism, which is specific for M. bovis BCG. Based on these results, we suggest a LightCycler format for rapid and unambiguous diagnosis of M. tuberculosis, M. bovis, and M. bovis BCG.

Anaerobiosis↗
PubMed2002

The methyl-branched fortifications of Mycobacterium tuberculosis.

Mycobacterium tuberculosis continues to be the predominant global infectious agent, annually killing over three million people. Recommended drug regimens have the potential to control tuberculosis, but lack of adherence to such regimens has resulted in the emergence of resistant strains. Mycobacterium tuberculosis has an unusual cell envelope, rich in unique long-chain lipids, that provides a very hydrophobic barrier to antibiotic access. Such lipids, however, can be drug targets, as exemplified by the action of the front-line drug isoniazid on mycolic acid biosynthesis. A number of these lipids are potential key virulence factors and their structures are based on very characteristic methyl-branched long-chain acids and alcohols. This review details the history, structure, and genetic aspects of the biosynthesis of these methyl-branched components, good examples of which are the phthiocerols and the mycocerosic and mycolipenic acids.

Cell Membrane↗
PubMed2000

Comparison of phenotypic and genotypic methods for pyrazinamide susceptibility testing with Mycobacterium tuberculosis.

Mycobacterium tuberculosis converts pyrazinamide to its active form by using the enzyme pyrazinamidase. This enzyme is coded for on the pncA gene, and mutations in the pncA gene result in absence of active enzyme, conferring resistance to the drug pyrazinamide. We investigated 27 strains of Mycobacterium tuberculosis suspected of being multidrug resistant. Each isolate was tested for susceptibility to pyrazinamide by the BACTEC 460TB method, and 19 were pyrazinamide resistant. The presence of active pyrazinamidase enzyme was sought by using the Wayne assay, which was positive in all of the sensitive isolates and four of the resistant isolates. The pncA gene was amplified by PCR, and mutations were sought by single-strand conformation polymorphism (SSCP) analysis. We identified four isolates which were phenotypically resistant to pyrazinamide, but which had active pyrazinamide enzyme on the Wayne assay and normal pncA gene SSCP. MICs measured by BACTEC 460TB and susceptibility testing at a lower pH of 5.5 confirmed genuine resistance. The pncA gene was sequenced in these four isolates and found not to have any mutations. This implies that an alternative mechanism of resistance exists in these strains. We conclude that genotypic assessment of pyrazinamide resistance is unreliable, because it depends on the identification of a single resistance mechanism. Phenotypic methods such as the BACTEC 460TB technique remain the best methods for pyrazinamide susceptibility testing.

Amidohydrolases↗
PubMed1987

Whole chromosomal DNA probes for rapid identification of Mycobacterium tuberculosis and Mycobacterium avium complex.

Whole chromosomal DNA probes were used to identify clinical isolates of Mycobacterium tuberculosis, Mycobacterium avium complex, and Mycobacterium gordonae. The probe for M. tuberculosis was prepared from Mycobacterium bovis BCG, which has been shown to be closely related to M. tuberculosis. A probe for the M. avium complex was prepared from three strains representing each of the three DNA homology groups in the M. avium complex. The probes were used in dot blot assays to identify clinical isolates of mycobacteria. The dot blot test correctly identified 57 of the 61 (93%) cultures grown on solid media, and 100% of antibiotic-treated broth-grown cells were correctly identified. Identification by dot blot required a maximum of 48 h. When the probes were tested against 63 positive BACTEC (Johnston Laboratories, Inc., Towson, Md.) cultures of clinical specimens, 59% were correctly identified. However, of the 14 BACTEC cultures that had been treated with antibiotics before being lysed, 13 (93%) were correctly identified.

Chromosomes, Bacterial↗
PubMed1998

Use of microscopic morphology in smears prepared from radiometric cultures for presumptive identification of Mycobacterium tuberculosis complex, Mycobacterium avium complex, Mycobacterium kansasii, and Mycobacterium xenopi.

The aim of this study was to assess the feasibility of a method for presumptive identification of mycobacteria, based on the morphology in smears prepared from radiometric Bactec-positive cultures (Becton Dickinson, USA) and to select the appropriate DNA probe (AccuProbe; Gen Probe, USA). The smear morphology of acid-fast bacilli was evaluated in 468 positive cultures from clinical samples: 313 Mycobacterium tuberculosis complex, 67 Mycobacterium avium complex, 32 Mycobacterium kansasii, 49 Mycobacterium xenopi, and seven Mycobacterium gordonae. The sensitivity and specificity for various morphological patterns were as follows: cord formation for Mycobacterium tuberculosis complex 90% and 100%, respectively; striped bacilli for Mycobacterium kansasii, 66% and 99%; sea urchin for Mycobacterium xenopi, 96% and 99%; short bacilli for Mycobacterium avium complex, 61% and 99%; fine-striped bacilli associated with Mycobacterium avium complex from blood samples, 33% and 98%. This criterion was applied in the selection of a suitable DNA probe for the identification of 178 cultures. The correct probe was selected in 98%, 97%, and 72% of cultures, respectively, for Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Mycobacterium kansasii. The observation of acid-fast bacilli morphology in radiometric cultures is a rapid and cost-efficient method for presumptive identification of common clinical isolates of mycobacteria.

Bacteriological Techniques↗
PubMed1992

Separation of Mycobacterium bovis BCG from Mycobacterium tuberculosis and Mycobacterium bovis by using high-performance liquid chromatography of mycolic acids.

Profile analysis of mycolic acid ester patterns of Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium bovis bacillus Calmette-Gúerin (BCG) using high-performance liquid chromatography indicated that separation of BCG from M. tuberculosis and M. bovis by elution and relative retention times is possible. Mycolic acid patterns of BCG eluted from the column 0.5 min before M. tuberculosis or M. bovis, resulting in relative retention times for two peaks not seen in the pattern of M. tuberculosis or M. bovis. Identification was confirmed by phage typing, which has been the standard procedure for confirmation of BCG strains. These results showed that high-performance liquid chromatographic analysis of mycolic acid esters can be used in the mycobacterial reference laboratory for separation of BCG from M. tuberculosis and M. bovis.

Bacteriophage Typing↗
PubMed2002

A family of autocrine growth factors in Mycobacterium tuberculosis.

Mycobacterium tuberculosis and its close relative, Mycobacterium bovis (BCG) contain five genes whose predicted products resemble Rpf from Micrococcus luteus. Rpf is a secreted growth factor, active at picomolar concentrations, which is required for the growth of vegetative cells in minimal media at very low inoculum densities, as well as the resuscitation of dormant cells. We show here that the five cognate proteins from M. tuberculosis have very similar characteristics and properties to those of Rpf. They too stimulate bacterial growth at picomolar (and in some cases, subpicomolar) concentrations. Several lines of evidence indicate that they exert their activity from an extra-cytoplasmic location, suggesting that they are also involved in intercellular signalling. The five M. tuberculosis proteins show cross-species activity against M. luteus, Mycobacterium smegmatis and M. bovis (BCG). Actively growing cells of M. bovis (BCG) do not respond to these proteins, whereas bacteria exposed to a prolonged stationary phase do. Affinity-purified antibodies inhibit bacterial growth in vitro, suggesting that sequestration of these proteins at the cell surface might provide a means to limit or even prevent bacterial multiplication in vivo. The Rpf family of bacterial growth factors may therefore provide novel opportunities for preventing and controlling mycobacterial infections.

Bacterial Proteins↗
PubMed2005

[The in-vitro interferon-gamma release assay for the diagnosis of tuberculosis and Mycobacterium tuberculosis infections].

OBJECTIVE: To investigate the in-vitro interferon-gamma (IFN-gamma) release assay based on three different Mycobacterium tuberculosis antigens in the diagnosis of tuberculosis and Mycobacterium tuberculosis infections. METHODS: The peripheral blood mononuclear cells (PBMC) were collected from the patients with tuberculosis (tuberculosis group, n = 57), patients with lung cancer (lung cancer group, n = 29), and healthy controls (healthy control group 2, n = 27). The PBMCs were co-cultured for 5 days with different antigens: purified protein derivatives (PPD) of tuberculin, early secretary antigenic target 6,000 protein (ESAT6) and 38,000 antigen. The protein levels of IFN-gamma were detected by ELISA, and the results were compared to those with the tuberculin skin test (TST). RESULTS: (1) For healthy controls, the TST was positively related to the history of BCG vaccination and the closeness of contact with sputum-positive tuberculosis patients (P = 0.047, P = 0.041 respectively). The ESAT6 based IFN-gamma release assay was only significantly related to the closeness of contact with sputum-positive tuberculosis patients (P = 0.005), but not to the history of BCG vaccination. (2) There was no significant difference of the TST results among the three groups (P > 0.05). (3) The receiver operating characteristic (ROC) curve analysis indicated that the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of the IFN-gamma release assay based on 38,000 antigen were 64.9%, 89.3%, 77.0%, 86.0%, and 71.0% respectively for the diagnosis of tuberculosis. CONCLUSIONS: IFN-gamma release assay based on ESAT6 appears to be better than TST in the diagnosis of infection of Mycobacterium tuberculosis, while IFN-gamma release assay based on 38,000 may be helpful for the diagnosis of tuberculosis.

Adult↗
PubMed1970

Complexing of fatty acids by Triton WR1339 in relation to growth of Mycobacterium tuberculosis.

Mycobacterium tuberculosis grew in the presence of toxic concentrations of free fatty acids when an adequate amount of triton WR1339 was added to the medium. Triton produced a solubilizing effect on turbid suspensions of fatty acids, indicating the formation of a detergent-lipid complex. Oleic acid complexed with triton served as a source of carbon for growth of M. tuberculosis.

Fatty Acids↗
PubMed2002

Purification, biochemical characterization and immunogenicity of SA5K, a secretion antigen of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB) secretory proteins are generally considered important antigens for immune protection against tuberculosis (TB). An 8.3-kDa secretory antigen of MTB and Mycobacterium bovis bacillus Calmette-Guérin (BCG), called SA5K, was recently identified and cloned in our laboratory. In this report, recombinant SA5K containing a histidine hexamer was expressed in Escherichia coli and purified to investigate its biochemical structure and to establish whether it was immunogenic for healthy sensitized and nonsensitized human donors and for patients infected with MTB. The protein nucleotide sequence was shown to be identical in BCG and in MTB. SA5K revealed an abnormal electrophoretic mobility in SDS-PAGE that made it look lighter than it is in Western blotting. While recombinant SA5K was poorly recognized by T lymphocytes from patients with pulmonary TB, it elicited proliferation of CD4+ T lymphocytes in the vast majority of healthy individuals sensitized to mycobacterial antigens by BCG vaccination. At a serum dilution of 1 : 80, antibodies reacting against recombinant SA5K were found in 67% of sera from TB patients and in 73% of sera from healthy subjects. The percentage of positive subjects dropped at higher serum dilutions, but no significant difference in the recognition rate was observed between TB patients and healthy donors and between healthy vaccinated and nonvaccinated subjects. Owing to the high percentage of sera from healthy subjects who recognized SA5K in Western blotting, the antigen seems to exhibit, at least in the present form, a poor specificity for an employment for a serodiagnosis of TB.

Antigens, Bacterial↗
PubMed2005

Insights into mechanisms of induction and ligands recognition in the transcriptional repressor EthR from Mycobacterium tuberculosis.

Mycobacterium tuberculosis EthR is a repressor of ethA, a gene encoding a mono-oxygenase required for the activation of the prodrug ethionamide. Two EthR crystal structures have been reported recently, either in a ligand-bound (Frenois F, Engohang-Ndong J, Locht C, Baulard AR, Villeret V. Mol Cell 2004; 16: 301-7) or in a presumed apo conformation (Dover LG, Corsino PE, Daniels IR, Cocklin SL, Tatituri V, Besra GS, Futterer K. J Mol Biol 2004; 340: 1095-105). In order to infer the EthR induction mechanism, we have compared these structures. It appears that the two structures are in a conformation incompatible with repressor function, due to the presence in both proteins of fortuitous and structurally unrelated ligands. This observation paves the way to the design of specific drugs that could increase the sensitivity of M. tuberculosis to ethionamide.

Antitubercular Agents↗