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PubMed1995

Direct detection of Mycobacterium tuberculosis complex in respiratory specimens by Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test and Roche Amplicor Mycobacterium Tuberculosis Test.

Two hundred and fifty-six sputum, bronchoalveolar lavage, and bronchial and tracheal aspirate specimens from 243 patients were tested for the presence of Mycobacterium tuberculosis complex by auramine fluorochrome staining, rRNA target amplification (Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test [AMTD]), and PCR (Roche Amplicor Mycobacterium Tuberculosis Test [Amplicor PCR]. The results were compared with those of conventional Löwenstein-Jensen tube culture and BACTEC radiometric liquid culture. A total of 26 specimens from 18 patients were culture positive for M. tuberculosis. In addition, seven specimens were positive by staining and by culture for other Mycobacterium species but negative by nucleic acid amplification methods and were not included in the comparison. When compared with that for culture, the sensitivities of the techniques were as follows: for staining, 80.8%; for Gen-Probe AMTD, 84.6%; and for Roche Amplicor PCR, 84.6%. The specificities were 99.1, 98.7, and 99.1%, respectively. After resolution of discrepant results by review of the patients' clinical data, 29 specimens from 21 patients were considered positive, and the overall sensitivities, specificities, and positive and negative predictive values were 89.7, 100, 100, and 98.7% for culture; 75.9, 99.5, 95.7, and 96.9% for staining; 86.2, 100, 100, and 98.2% for Gen-Probe AMTD; and 82.8, 100, 100, and 97.9% for Roche Amplicor PCR, respectively. It is concluded that both nucleic acid amplification methods are rapid, sensitive, and specific methods for the detection of M. tuberculosis in respiratory specimens.

Bacteriological Techniques↗
PubMed1988

Speciation of organisms within the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum (MAIS) complex based on restriction fragment length polymorphisms.

A DNA probe which hybridizes to all pathogenic species of slow-growing mycobacteria has been used to identify restriction-fragment-length-polymorphisms (RFLPs) in Bam Hl digests of chromosomal DNA from members of the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex. The RFLP patterns so produced were found to fall into distinct categories which were representative of each of the three species. Except for two doubtful isolates, strains of M. avium were found to fall into two related RFLP-types, one of which contained the vast majority of the strains tested. In contrast, M. intracellulare strains were found to be more heterogeneous. For these strains, we found one major RFLP-type and one subsidiary type which appears to be a sub-set of the first. We also found two further RFLP-types which contained serovars 7 and 18 respectively. We conclude from this that M. avium, M. intracellulare and M. scrofulaceum are three distinct species and that serovars belonging to the 'intermediate group' of Meissner and Anz belong to the species M. avium. Utilizing these criteria, we examined a number of isolates from the 'ambiguous' serovar 9 and found that of eight strains tested, six typed as M. avium and two typed as M. intracellulare.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Disinfection of bronchoscopes, contaminated in vitro with Mycobacterium tuberculosis, Mycobacterium avium-intracellulare and Mycobacterium chelonae in sputum, using stabilized, buffered peracetic acid solution ('Nu-Cidex').

The efficacy of 0.35% stabilized buffered peracetic acid solution ('Nu-Cidex') against clinical isolates of Mycobacterium tuberculosis, Mycobacterium avium-intracellulare and Mycobacterium chelonae in homogenized sputum was tested. An in-use method, using an automated bronchoscope washing machine, showed that over 10 cycles, at a disinfectant contact time of 5 min, M. tuberculosis and M. chelonae were effectively eradicated from the bronchoscope, even in the absence of detergent and pre-cleaning. M. avium-intracellulare was not eradicated in only one of 10 cycles with contact times of 5 and 10 min, but numbers were reduced by > 7 log10 and > 5 log10, respectively. With detergent present, M. avium-intracellulare was successfully eradicated in all cycles at a contact time of 5 min or greater. The results demonstrate that peracetic acid is an effective mycobactericidal agent for use in the disinfection of bronchoscopes.

Bronchoscopes↗
PubMed1990

Analysis of cellular fatty acids and proteins by capillary gas chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis to differentiate Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium scrofulaceum (MAIS) complex species.

Infections due to atypical mycobacteria have increased during the past 30 years. Species of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium scrofulaceum are among the most common non-tuberculous mycobacteria isolated from patients with AIDS or immunosuppressed. These three organisms are taxonomically closely related and identification, according to cultural characteristics and biochemical tests, is not always evident, so some of these related strains are grouped in a "MAIS" complex. Analysis of cellular constituents is an aid to identification. Gas chromatography was used to study mycolic acids and a secondary alcohol was found which is a discriminating constituent between M. scrofulaceum and the other two species. The lipidic analysis was not able to separate M. avium and M. intracellulare, so cell proteins were considered. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of proteins reflects genetic relatedness between strains; the different patterns obtained from these three species are described and it is shown that this method is very useful in classification and epidemiology.

Bacterial Proteins↗
PubMed1986

Plasmid DNA profiles as epidemiological markers for clinical and environmental isolates of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum.

Plasmid DNA was isolated, and profiles of a variety of clinical and environmental isolates of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (MAIS) were compared to determine whether plasmid DNA content would be useful as an epidemiological marker for these environmental pathogens. Since plasmids are common in clinical isolates and are stable during culture and exposure to NaOH, plasmid DNA analysis appears to be a suitable epidemiological tool. Based on the high frequency of plasmids in only clinical (56%) and aerosol (75%) isolates and low frequencies in soil (5%), dust (7%), sediment (less than 6%), and water (25%) isolates, the data suggest that MAIS-laden aerosols generated over waters of the southeastern United States are a likely source of human infection.

Aerosols↗
PubMed2000

Accumulation of rifampicin by Mycobacterium aurum, Mycobacterium smegmatis and Mycobacterium tuberculosis.

The characteristics of the accumulation of 2 mg/L [(14)C]rifampicin by wild-type strains of Mycobacterium aurum (A(+)), Mycobacterium smegmatis (mc(2)155) and Mycobacterium tuberculosis (H37Rv) were determined. After 10 min exposure M. aurum had accumulated 220 ng rifampicin/mg cells, M. smegmatis had accumulated 120 ng rifampicin/mg cells and M. tuberculosis had accumulated 154 ng rifampicin/mg cells. A steady-state concentration (SSC) of rifampicin was accumulated rapidly by M. aurum and M. tuberculosis within minutes of drug exposure, unlike M. smegmatis, which accumulated rifampicin more slowly. With an increase in the concentration of rifampicin from 0.12 mg/L to 2 mg/L there was an increase in the concentration of rifampicin accumulated by M. tuberculosis, with no detectable loss of viability over the 20 min of the accumulation experiment. With an increase in temperature there was also an increase in the concentration of rifampicin accumulated by M. tuberculosis; between 15 and 30 degrees C the increase was linear. For all three species sub-inhibitory concentrations of ethambutol increased the concentration of rifampicin accumulated. However, both growth and accumulation of rifampicin were lower in the presence of 0.05% Tween 80. Accumulation of rifampicin by M. smegmatis was unaffected by the presence of the proton motive force inhibitor, 2,4-dinitrophenol (1 mM), whether added before or after the addition of rifampicin to the mycobacterial culture. For all three species, the Gram-positive bacterial efflux inhibitor reserpine (20 mg/L) slightly increased the SSC of rifampicin, but the increase was not statistically significant. Addition of glucose to energize a putative efflux pump had little effect on the accumulation of rifampicin in the presence or absence of reserpine for M. tuberculosis; however, for M. aurum and M. smegmatis the reserpine effect was abolished by the addition of glucose. These data suggest that rifampicin may be removed from wild-type mycobacteria by efflux, but that the pump(s) is expressed at low level.

Antibiotics, Antitubercular↗
PubMed1993

Serovar determination and molecular taxonomic correlation in Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum: a cooperative study of the International Working Group on Mycobacterial Taxonomy.

A cooperative study was conducted by the International Working Group on Mycobacterial Taxonomy to correlate the agglutination serovar designations of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum strains with the species ascriptions of these organisms according to molecular criteria and cultural properties and to assess the reproducibility of serovar determinations for a set of 63 reference strains of these species. Among the molecular criteria, the level of agreement between results obtained with nucleic acid probes and T-catalase serology results was 94% for strains of M. avium and M. intracellulare. Nucleic acid probes were not available for M. scrofulaceum, but none of the 10 strains ascribed to this species on the basis of catalase serology data reacted with a nucleic acid probe for M. avium or M. intracellulare. Ascription to a species on the basis of mycolic acid high-performance liquid chromatography patterns was in agreement with catalase serology results in 86% of the cases examined. Most strains belonging to serovars 1 through 6 and 8 through 11 were identified by molecular criteria as M. avium, most strains belonging to serovars 7, 12 through 20, 23, and 25 were identified as M. intracellulare, and most strains belonging to serovars 41 through 43 were identified as M. scrofulaceum, in agreement with common current practice. Evidence for assigning serovar 27 to M. scrofulaceum was obtained. However, two strains of a given serovar may, on occasion, be placed in different species. The dominant species assignments for strains belonging to serovars 21, 24, 26, and 28 remain unresolved.(ABSTRACT TRUNCATED AT 250 WORDS)

Agglutination Tests↗
PubMed1996

Organization of the origins of replication of the chromosomes of Mycobacterium smegmatis, Mycobacterium leprae and Mycobacterium tuberculosis and isolation of a functional origin from M. smegmatis.

The genus Mycobacterium is composed of species with widely differing growth rates ranging from approximately three hours in Mycobacterium smegmatis to two weeks in Mycobacterium leprae. As DNA replication is coupled to cell duplication, it may be regulated by common mechanisms. The chromosomal regions surrounding the origins of DNA replication from M. smegmatis, M. tuberculosis, and M. leprae have been sequenced, and show very few differences. The gene order, rnpA-rpmH-dnaA-dnaN-recF-orf-gyrB-gyrA, is the same as in other Gram-positive organisms. Although the general organization in M. smegmatis is very similar to that of Streptomyces spp., a closely related genus, M. tuberculosis and M. leprae differ as they lack an open reading frame, between dnaN and recF, which is similar to the gnd gene of Escherichia coli. Within the three mycobacterial species, there is extensive sequence conservation in the intergenic regions flanking dnaA, but more variation from the consensus DnaA box sequence was seen than in other bacteria. By means of subcloning experiments, the putative chromosomal origin of replication of M. smegmatis, containing the dnaA-dnaN region, was shown to promote autonomous replication in M. smegmatis, unlike the corresponding regions from M. tuberculosis or M. leprae.

Bacterial Proteins↗
PubMed2003

Factors influencing the chlorine susceptibility of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum.

The susceptibility of representative strains of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (the MAIS group) to chlorine was studied to identify factors related to culture conditions and growth phase that influenced susceptibility. M. avium and M. intracellulare strains were more resistant to chlorine than were strains of M. scrofulaceum. Transparent and unpigmented colony variants were more resistant to chlorine than were their isogenic opaque and pigmented variants (respectively). Depending on growth stage and growth rate, MAIS strains differed in their chlorine susceptibilities. Cells from strains of all three species growing in early log phase at the highest growth rates were more susceptible than cells in log and stationary phase. Rapidly growing cells were more susceptible to chlorine than slowly growing cells. The chlorine susceptibility of M. avium cells grown at 30 degrees C was increased when cells were exposed to chlorine at 40 degrees C compared to susceptibility after exposure at 30 degrees C. Cells of M. avium grown in 6% oxygen were significantly more chlorine susceptible than cells grown in air. Chlorine-resistant MAIS strains were more hydrophobic and resistant to Tween 80, para-nitrobenzoate, hydroxylamine, and nitrite than were the chlorine-sensitive strains.

Chlorine↗
PubMed1980

Immunotherapy of experimental cancer by intralesional injection of emulsified nonliving mycobacteria: comparison of Mycobacterium bovis (BCG), Mycobacterium phlei, and Mycobacterium smegmatis.

Mycobacterium bovis (BCG), Mycobacterium phlei, and Mycobacterium smegmatis were each tested in emulsified form for their potency to cause regression of transplants of a syngeneic murine fibrosarcoma and of a syngeneic guinea pig hepatoma. On a weight basis, M. phlei and M. smegmatis were as effective as BCG in causing tumor regression. M. phlei and M. smegmatis were comparable to BCG in provoking delayed cutaneous hypersensitivity reactions in guinea pigs sensitized to M. phlei or M. smegmatis. In BCG-sensitized guinea pigs, M. phlei and M. smegmatis provoked weaker delayed cutaneous hypersensitivity reactions than did BCG. Purified protein derivative of M. tuberculosis was more active in eliciting delayed cutaneous hypersensitivity in BCG-sensitized guinea pigs than in animals sensitized with M. phlei or M. smegmatis.

Animals↗
PubMed1989

Homology of the 70-kilodalton antigens from Mycobacterium leprae and Mycobacterium bovis with the Mycobacterium tuberculosis 71-kilodalton antigen and with the conserved heat shock protein 70 of eucaryotes.

Two lambda gt11 recombinant clones, JKL2 and JKL15, each containing an insert coding for part of the highly immunogenic 70-kilodalton (kDa) protein antigen, were isolated from a Mycobacterium leprae genomic library by immunoscreening with the monoclonal antibody L7. Clone JKL2 contained the largest insert, 2.3 kilobase pairs. Nonoverlapping fragments of this insert were used as probes and showed strong hybridization to a number of Mycobacterium tuberculosis-lambda gt11 recombinants producing proteins recognized by an anti-M. tuberculosis 71-kDa monoclonal antibody, IT11. One clone from a recombinant Mycobacterium bovis library was also characterized by using L7, and the insert from this clone, B5bt, hybridized strongly to the M. leprae probes as well. The nucleotide sequence of the 1,037-base-pair coding region of the JKL2 M. leprae clone which encodes the carboxy-terminal half of the 70-kDa protein had extensive homology with genes from a number of species. In all cases, these genes, including the recently described Ag63 and Ag361 of Plasmodium falciparum, were found to be members of the heat shock protein 70 (hsp 70) family of genes. At the amino acid level, homology was maximal between amino acids 83 through 107 and 159 through 184, which showed extreme conservation (92 and 85% identity) with Escherichia coli DnaK amino acids 386 through 409 and 460 through 485, respectively, and was 51% homologous over the entire coding region (amino acids 1 through 344 of JKL2). In contrast, amino acids 129 through 158 had maximal homology with the phylogenetically more distant Xenopus laevis hsp70. Homology declined substantially in the carboxy-terminal 34 amino acids. The predicted ATP-binding functional activity of the 70-kDa antigen from M. bovis was confirmed with affinity purification of the antigen by binding to ATP-agarose and elution with ATP. In view of the conservation of sequences between these mycobacterial antigens and mammalian endogenous cellular enzymes, further evaluation of these molecules in vivo may aid in understanding tolerance to self-antigens as well as provide potentially useful immunodiagnostic reagents.

Adenosine Triphosphate↗
PubMed1990

Identification of various serovar strains of Mycobacterium avium complex by using DNA probes specific for Mycobacterium avium and Mycobacterium intracellulare.

Reference strains of the Mycobacterium avium complex (MAC) belonging to serovars 1 to 28 were examined with DNA probes (Gen Probe Rapid Diagnostic System for the MAC; Gen Probe Inc., San Diego, Calif.) specific for either M. avium or Mycobacterium intracellulare. This study revealed that the earlier designations of the MAC serovars, in which serovars 1 to 3 and 4 to 28 were regarded as M. avium and M. intracellular, respectively, should be revised as follows. First M. avium includes serovars, 1 to 6, 8 to 11, and 21. Second, M. intracellulare includes serovars 7, 12 to 20, and 25. However, other serovars, such as serovars 22 to 24 and 26 to 28, involve M. intracellulare, Mycobacterium scrofulaceum, and MAC that are lacking in the reactivity with either DNA probe and that are too disordered to enable a conclusive description here, particularly concerning their taxonomic positions in the MAC.

Antigens, Bacterial↗
PubMed1999

Multisite reproducibility of results obtained by the broth microdilution method for susceptibility testing of Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium fortuitum.

A multicenter study was conducted to assess the interlaboratory reproducibility of broth microdilution testing of the more common rapidly growing pathogenic mycobacteria. Ten isolates (four Mycobacterium fortuitum group, three Mycobacterium abscessus, and three Mycobacterium chelonae isolates) were tested against amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, sulfamethoxazole, and tobramycin (M. chelonae only) in four laboratories. At each site, isolates were tested three times on each of three separate days (nine testing events per isolate) with a common lot of microdilution trays. Agreement among MICs (i.e., mode +/- 1 twofold dilution) varied considerably for the different drug-isolate combinations and overall was best for cefoxitin (91.7 and 97.2% for one isolate each and 100% for all others), followed by doxycycline, amikacin, and ciprofloxacin. Agreement based on the interpretive category, using currently suggested breakpoints, also varied and overall was best for doxycycline (97.2% for one isolate and 100% for the rest), followed by ciprofloxacin and clarithromycin. Reproducibility among MICs and agreement by interpretive category was most variable for imipenem. Based on results reported from the individual sites, it appears that inexperience contributed significantly to the wide range of MICs of several drugs, especially clarithromycin, ciprofloxacin, and sulfamethoxazole. New interpretive guidelines are presented for the testing of M. fortuitum against clarithromycin; M. abscessus and M. chelonae against the aminoglycosides; and all three species against cefoxitin, doxycycline, and imipenem.

Amikacin↗
PubMed2000

Multisite reproducibility of Etest for susceptibility testing of Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium fortuitum.

A multicenter study was conducted to assess the inter- and intralaboratory reproducibility of the Etest for susceptibility testing of the rapidly growing mycobacteria. The accuracy also was evaluated by comparing Etest results to those obtained by broth microdilution. Ten isolates (four of the Mycobacterium fortuitum group, three of Mycobacterium abscessus, and three of Mycobacterium chelonae) were tested against amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, and trimethoprim-sulfamethoxazole in each of four laboratories. At each site, isolates were tested three times on each of three separate days (nine testing events per isolate) using common lots of media and Etest strips. Interlaboratory agreement among MICs (i.e., mode +/- 1 twofold dilution) varied for the different drug-isolate combinations and overall was best for trimethoprim-sulfamethoxazole (75% for one isolate and 100% for all others), followed by doxycycline and ciprofloxacin. Interlaboratory agreement based on interpretive category also varied and overall was best for doxycycline (100% for all isolates), followed by trimethoprim-sulfamethoxazole and ciprofloxacin. Interlaboratory reproducibility among MICs was most variable for imipenem, and agreement by interpretive category was lowest for imipenem and amikacin. Modal Etest MICs agreed with those by broth microdilution only for doxycycline and the sulfonamides. For all other drugs, the modal MICs by the two methods differed by more than +/- 1 twofold dilution for one or more isolates. In all cases, the Etest MIC was higher and would have caused reports of false resistance. In summary, the Etest in this evaluation did not perform as well as broth microdilution for susceptibility testing of the rapidly growing mycobacteria. It was problematic for most species and drugs, primarily because of a trailing endpoint and/or high MICs compared to broth. Its use will necessitate further investigation, including determination of the optimal medium and incubation conditions and clarification of endpoint interpretation.

Anti-Bacterial Agents↗
PubMed1992

Epidemiology of infection by nontuberculous mycobacteria. Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum in acid, brown-water swamps of the southeastern United States and their association with environmental variables.

Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (MAIS) organisms were isolated and identified from waters, soils, aerosols, and droplets ejected from water collected from four geographically separate aquatic environments (Okefenokee Swamp, GA; Dismal Swamp, VA; Claytor Lake, VA; and Cranberry Glades, WV) during several seasons. Recovery of MAIS was significantly higher from waters, soils, and aerosols collected from the two acid, brown-water swamps located in the southeastern coastal plain. High MAIS numbers correlated with warmer temperature, low pH, low dissolved oxygen, high soluble zinc, high humic acid, and high fulvic acid. This research, in relation to previous findings for the geographic distribution and physiologic ecology of MAIS, supports the conclusion that waters, soils, and aerosols of the acid, brown-water swamps of the southeastern United States coastal plain represent major environmental sources likely connected with the higher incidence of human infection in this region.

Georgia↗
PubMed2006

Exposure to Mycobacterium avium can modulate established immunity against Mycobacterium tuberculosis infection generated by Mycobacterium bovis BCG vaccination.

Mycobacterium bovis bacille Calmette Guerin (BCG), the current vaccine against infection with Mycobacterium tuberculosis, offers a variable, protective efficacy in man. It has been suggested that exposure to environmental mycobacteria can interfere with the generation of BCG-specific immunity. We hypothesized that exposure to environmental mycobacteria following BCG vaccination would interfere with established BCG immunity and reduce protective efficacy, thus modeling the guidelines for BCG vaccination within the first year of life. Mice were vaccinated with BCG and subsequently given repeated oral doses of live Mycobacterium avium to model exposure to environmental mycobacteria. The protective efficacy of BCG with and without subsequent exposure to M. avium was determined following an aerogenic challenge with M. tuberculosis. Exposure of BCG-vaccinated mice to M. avium led to a persistent increase in the number of activated T cells within the brachial lymph nodes but similar T cell activation profiles in the lungs following infection with M. tuberculosis. The capacity of BCG-vaccinated mice to reduce the bacterial load following infection with M. tuberculosis was impaired in mice that had been exposed to M. avium. Our data suggest that exposure to environmental mycobacteria can negatively impact the protection afforded by BCG. These findings are relevant for the development of a vaccine administered in regions with elevated levels of environmental mycobacteria.

Animals↗
PubMed2005

Elemental analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cell's endosomal system.

Mycobacterium avium and Mycobacterium tuberculosis are human pathogens that infect and replicate within macrophages. Both organisms live in phagosomes that fail to fuse with lysosomes and have adapted their lifestyle to accommodate the changing environment within the endosomal system. Among the many environmental factors that could influence expression of bacterial genes are the concentrations of single elements within the phagosomes. We used a novel hard x-ray microprobe with suboptical spatial resolution to analyze characteristic x-ray fluorescence of 10 single elements inside phagosomes of macrophages infected with M. tuberculosis and M. avium or with avirulent M. smegmatis. The iron concentration decreased over time in phagosomes of macrophages infected with Mycobacterium smegmatis but increased in those infected with pathogenic mycobacteria. Autoradiography of infected macrophages incubated with (59)Fe-loaded transferrin demonstrated that the bacteria could acquire iron delivered via the endocytic route, confirming the results obtained in the x-ray microscopy. In addition, the concentrations of chlorine, calcium, potassium, manganese, copper, and zinc were shown to differ between the vacuole of pathogenic mycobacteria and M. smegmatis. Differences in the concentration of several elements between M. avium and M. tuberculosis vacuoles were also observed. Activation of macrophages with recombinant IFN-gamma or TNF-alpha before infection altered the concentrations of elements in the phagosome, which was not observed in cells activated following infection. Siderophore knockout M. tuberculosis vacuoles exhibited retarded acquisition of iron compared with phagosomes with wild-type M. tuberculosis. This is a unique approach to define the environmental conditions within the pathogen-containing compartment.

Animals↗