Deoxycholate metabolism in alcoholic cirrhosis

Experiences with the lipid analysis of scotochromogenic mycobacteria by means of thin-layer chromatography are described. Out of 18 strains of M. scrofulaceum 11 belong to the serotypes Lunning, Gause, and Scrofulaceum. The similarity of the lipid patterns of the M. scrofulaceum strains to the ones of the M. avium-M. intracellulare group supports the contention of some authors that these species are closely related. Lipid analysis of further species of RUNYON's group II (M. xenopi, M. gordonae, M. kansasii var. aurantiacum, M. flavescens, M. acapulcensis, M. paraffinicum, M. thermoresistibile) gives the evidence that a differentiation by means of thin-layer separation is possible in these cases, too. The answer to the question whether these lipid patterns are species specific or not can only be given when a large number of strains of every species will be investigated.     

Molecular cloning and immunologic reactivity of a novel low molecular mass antigen of Mycobacterium tuberculosis

Polypeptide Ags present in the culture filtrate of Mycobacterium tuberculosis were purified and evaluated for their ability to stimulate PBMC from purified protein derivative (PPD)-positive healthy donors. One such Ag, which elicited strong proliferation and IFN-gamma production, was further characterized. The N-terminal amino acid sequence of this polypeptide was determined and used to design oligonucleotides for screening a recombinant M. tuberculosis genomic DNA library. The gene (Mtb 8.4) corresponding to the identified polypeptide was cloned, sequenced, and expressed in Escherichia coli. The predicted m.w. of the recombinant protein without its signal peptide was 8.4 kDa. By Southern analysis, the DNA encoding this mycobacterial protein was found in the M. tuberculosis substrains H37Rv, H37Ra, Erdman, and "C" strain, as well as in certain other mycobacterial species, including Mycobacterium avium and Mycobacterium bovis BCG (bacillus Calmette-Guerin, Pasteur). The Mtb 8.4 gene appears to be absent from the environmental mycobacterial species examined thus far, including Mycobacterium smegmatis, Mycobacterium gordonae, Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium scrofulaceum. Recombinant Mtb 8.4 Ag induced significant proliferation as well as production of IFN-gamma, IL-10, and TNF-alpha, but not IL-5, from human PBMC isolated from PPD-positive healthy donors. Mtb 8.4 did not stimulate PBMC from PPD-negative donors. Furthermore, immunogenicity studies in mice indicate that Mtb 8.4 elicits a Th1 cytokine profile, which is considered important for protective immunity to tuberculosis. Collectively, these results demonstrate that Mtb 8.4 is an immunodominant T cell Ag of M. tuberculosis.     

Antibodies to mycobacterial 65-kD heat shock protein cross-react with the main mitochondrial antigens in patients with primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease associated with autoimmune disorders. The aetiology is unknown, although it has been suggested that the disease may be related to infectious agents. Previous studies revealed that sera from patients with PBC react against Mycobacterium gordonae. This specific reactivity, characterized by a recognition of two membrane polypeptides of 70-65 and 55 kD, cross-react with the two major mitochondrial autoantigens of PBC. As the most immunogenic components of mycobacteria are the heat shock proteins (hsp), which have been associated with autoimmunity, this study has been undertaken to characterize whether the reacting polypeptides in PBC are hsp from M. gordonae. Cultures of M. gordonae were incubated at 37 degrees C and 46 degrees C before sonication, protein extraction and separation by SDS-PAGE. Exposure of M. gordonae to heat shock treatment resulted in membrane protein overexpression, similar to the 70-65-kD polypeptide recognized by the sera from patients with PBC. Immunoprecipitation assays with a monoclonal antibody directed against the Hsp65 kD of mycobacteria and with sera from patients with PBC revealed similar reacting profiles characterized by the precipitation of the overexpressed 65-kD polypeptide from M. gordonae. Competitive immunoblotting showed that binding of the monoclonal antibody to the Hsp65 kD protein was prevented by preincubation with sera from patients with PBC, but not with sera from healthy subjects. Furthermore, monoclonal antibody to the Hsp65 kD protein recognized the main mitochondrial autoantigens of PBC (PDH-E2 and BCKDH-E2). These data indicate the existence of cross-reacting epitopes contained on M. gordonae Hsp65 kD and the main mitochondrial antigens in patients with PBC.     

Abnormal prostate development in C3(1)-bcl-2 transgenic mice

Two bacillus Calmette-Guérin (BCG)-susceptible mouse strains, BALB/c and C57BL/6, were infected intravenously with Mycobacterium intracellulare, M. avium or M. scrofulaceum and monitored during 3 months for mycobacterial replication and antibody and Th1-type cytokine production in response to cytoplasmic and secreted antigens from M. bovis BCG. Whereas initial colony-forming unit (CFU) counts of M. intracellulare and M. avium were higher in lungs than in spleen, the opposite was observed for M. scrofulaceum. Mycobacterium intracellulare was the most virulent species and its replication could not be controlled in either mouse strain. It also induced the strongest antibody response. Mycobacterium avium was eliminated in both mouse strains and M. scrofulaceum finally was eliminated in C57BL/6 but multiplied in spleen from BALB/c mice. Significant sustained interleukin-2 and interferon-gamma production towards BCG antigens was only found in M. scrofulaceum infection. As in BCG-vaccination, M. scrofulaceum-infected C57BL/6 mice demonstrated a higher response towards whole BCG culture filtrate, BCG extract and purified antigen 85 complex (Ag85) from BCG than did BALB/c mice. The data suggest that the presence of M. scrofulaceum in the environment may possibly interfere in genetically predisposed subjects with BCG vaccine and its protective efficacy against M. tuberculosis.     

Genetic diversity among Mycobacterium avium complex strains recovered from children with and without human immunodeficiency virus infection

The genetic diversity and molecular epidemiology of Mycobacterium avium complex (MAC) infections in children with and without human immunodeficiency virus (HIV) infection were evaluated. Isolates recovered from 136 children were subtyped by sequence analysis of a 360-bp region of the gene (hsp65) encoding a 65-kDa heat-shock protein. Twenty-one distinct hsp65 alleles were identified. On the basis of hsp65 genotype, 6 isolates were not MAC organisms. Of the remaining 130 samples, 61% were M. avium, 37% were Mycobacterium intracellulare, and 2% were species nonspecific MAC. Eighty-eight percent of the isolates obtained from HIV-infected children were M. avium. In contrast, only 38% of the isolates obtained from children without HIV infection were M. avium (chi2 test, P < .001). M. avium isolates were further subtyped by Southern blot analysis with insertion element IS1245. Taken together, no evidence for a single clonal M. avium strain causing infection was detected.     

Isolation of a novel mycobacterium from an adolescent with cervical lymphadenitis

A slow-growing mycobacterium was isolated from a cervical lymph node of an adolescent male. This isolate produced small, smooth, scotochromogenic colonies after 6 weeks of incubation at 25 degrees C and 30 degrees C (but not at 37 degrees C or 43 degrees C). The results of 16S-rRNA gene sequencing and high-performance liquid chromatography suggest that this isolate belongs to a hitherto unrecognised pathogenic species.     

Clinicopathological study of cases with Mycobacterium szulgai infection

Pulmonary mycobacteriosis is usually caused by Mycobacterium tuberculosis, Mycobacterium avium complex, or Mycobacterium kansasii. There are, however, other slow-growing mycobacteria which can cause pulmonary infection. Mycobacterium szulgai, first reported in 1972, is a scotochromogenic species which can affect human lungs, although human-to-human spread of infection is thought to be unlikely. We have recently treated three cases of middle-aged to elderly persons (45-87 year-old), two of them had underlying diseases (one with intrapulmonary and the other with extrapulmonary). All patients had constitutional symptoms (cough, sputum, dyspnea), and chest roentgenograms demonstrated either cavitation with scattered nodules or peripheral infiltrates predominantly in upper lobes, resembling pulmonary tuberculosis. In two cases, M. szulgai was identified by using DNA-DNA hybridization method. The in vitro susceptibility of M. szulgai to antimycobacterial drugs was better than that of M. avium complex, and it was resistant only to paraaminosalicylate, cycloserine, and partially to isoniazid. Pulmonary disease of three patients were successfully treated with a combination of multiple antimycobacterial agents including rifampin, ethambutol, isoniazid, or streptomycin.     

Hydrogenase and ribulose diphosphate carboxylase during autotrophic, heterotrophic, and mixotrophic growth of scotochromogenic mycobacteria

Two key autotrophic enzyme systems, hydrogenase and ribulose diphosphate carboxylase, were examined in Mycobacterium gordonae and two other chemolithotrophic, scotochromogenic mycobacteria under different cultural conditions. In all three organisms both enzymes were inducible and were produced in significant levels only in the presence of the specific substrate, hydrogen or carbon dioxide. M. gordonae exhibited increased growth rates and yields, indicating mixotrophic growth, in the presence of a number of single organic substrates, including acetate, pyruvate, glucose, fructose, and glycerol. In contrast to other aerobic hydrogen autotrophs, the presence of either acetate or pyruvate did not repress ribulose diphosphate carboxylase, and mixotrophic growth was rapid with these substrates. In the absence of carbon dioxide, growth in glycerol medium under an atmosphere of hydrogen and oxygen was severely inhibited, even with cells preadapted to heterotrophic growth on glycerol. Cyclic adenosine monophosphate was not effective in inducing hydrogenase or carboxylase in heterotrophic, mixotrophic, or hydrogen-inhibited cultures.     

Bacteriology of mycobacteria: taxonomic and morphological characteristics

Bacteriological characteristics of organisms belonging to Genus Mycobacterium which involves more than 60 species are described. Mycobacterial organisms can be divided into the following groups having differential characteristics, on the basis of the results of biological, biochemical, and genetic investigations, including lipid analysis, DNA probe test, and comparative 16S ribosomal RNA sequencing. First, Mycobacterium tuberculosis complex (M. tuberculosis, M. bovis, M. africanum, etc.). Second, cultivable but slowly growing nontuberculous mycobacteria, including photochromogens (Runyon Group I) such as M. kansasii, M. marinum, M. simiae, M. intermedium, and M. asiaticum, scotochromogens (Runyon Group II) such as M. scrofulaceum, M. szulgai, M. injectum, M. lentiflavum, and M. gordonae, nonphotochromogenens (Runyon Group III) such as M. avium, M. intracellulare, M. xenopi, M. malmoense, M. genavense, M. celatum, and M. gastri. Third, cultivable rapidly growing nontuberculous mycobacteria (Runyon Group IV) including M. fortuitum, M. chenolae, M. abscessus, M. phlei, and M. smegmatis. Fourth, noncultivable mycobacteria including M. leprae. About 30 species of Mycobacterium cause pulmonary, dermal, lymphatic, and disseminated infections in human beings. This paper mainly deals with the taxonomic, morphological, and other biological characteristics of these mycobacterial organisms.     

Quantitative research for improving respiratory muscle contraction by breathing exercise

Mycobacterium gordonae is an atypical mycobacterium of very low pathogenic potential. It is widely distributed in soil and water and often detected on the mucous membranes of healthy persons. In recent years, there have been numerous reports of infections by M. gordonae in immunocompromised patients. In contrast, only four cases of skin infections by M. gordonae in immunocompetent patients have been published. We report on another patient without evidence of immunodeficiency who developed an atypical mycobacteriosis after a thorn injury during gardening. M. gordonae was isolated by tissue culture. The skin lesion cleared completely after treatment with doxycycline for three months.     

Cloning and expression of portions of the 34-kilodalton-protein gene of Mycobacterium paratuberculosis: its application to serological analysis of Johne's disease

Paratuberculosis (Johne's disease), an endemic mycobacteriosis of cattle that is caused by Mycobacterium paratuberculosis, is characterized by incoercible diarrhea and fecal shedding of bacteria. The present work aimed at developing a specific serological test for this disease. We have recently shown that a 34-kDa protein belonging to the major antigen complex A36 of M. paratuberculosis is immunodominant and contains epitopes specific with respect to all mycobacteria tested, including Mycobacterium bovis and the closely related species Mycobacterium avium. From a lambda gt11 genomic library of M. paratuberculosis, three portions of the gene coding for this 34-kDa protein have been isolated. Two of them expressed cross-reacting mycobacterial epitopes. One portion (in clone a362) expressed a polypeptide which cross-reacted with all tested M. paratuberculosis strains but not with 20 other bacteria tested, including many strains of the M. avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum group. The occurrence at the M. paratuberculosis surface of epitopes corresponding to the a362 polypeptide was shown by immune electron microscopy. The recombinant a362 polypeptide was used as reagent for an enzyme-linked immunoassay for paratuberculosis. This assay correctly diagnosed all the tested blood samples from infected cattle at all stages of the disease.     

Identification and strain differentiation of Mycobacterium species on the basis of DNA 16S-23S spacer region polymorphism

Amplification of the region separating the genes coding for the two rRNA species 16S and 23S was performed to identify 56 mycobacterial strains, belonging to eleven species: Mycobacterium tuberculosis, M. avium, M. kansasii, M. gordonae, M. abscessus, M. fortuitum, M. xenopi, M. bovis, M. bovis/BCG, M. africanum and M. intracellulare. Reproducible amplification patterns were obtained with most species with the exception of M. kansasii which showed heterogeneity, confirming the existence of a genetically distinct subspecies within this species. In addition, we used the amplified products as target DNA for restriction endonuclease digestion and RAPD (randomly amplified polymorphic DNA) analysis to compare strains of M. abscessus, M. tuberculosis and M. avium. The discriminatory power of these two typing methods was higher than when whole genomic DNA is used as target. Our results demonstrate that the two-step approach to identification and typing on the basis of the hypervariability of 16S-23S spacer region is reliable, rapid and simple, and consequently could be an epidemiological tool in clinical laboratories.     

Relationship between streptomycin susceptibility and rpsL mutations of Mycobacterium tuberculosis strains

The relationship between streptomycin (SM) susceptibility and rpsL mutations of Mycobacterium tuberculosis strains was studied. Of 18 clinically isolated SM-resistant M.tuberculosis strains, mutation was suspected in 9 strains (50%) with SM MICs of > or = 256 micrograms/ml by PCR-single strand conformation polymorphism targeting rpsL gene. On the other hand, using PCR-direct sequence method, amino acid substitution caused by single nucleotide point mutation in rpsL gene was demonstrated in 11 out of 18 strains (61%). The same amino acid substitution at codon 43 (Lys-->Arg) was observed in all 11 strains with SM MICs of > or = 256 micrograms/ml. In addition, PCR products obtained from these 11 strains could not be cut by a restriction enzyme, Mbo II, while H37Rv strain and the other 32 strains with SM MICs of < 256 micrograms/ml were cut into 2 fragments. In conclusion, our results suggest that highly SM-resistant M.tuberculosis strains with MICs of > or = 256 micrograms/ml could be rapidly and easily detected by the restriction enzymatic method.     

Mycobacterium novocastrense sp. nov., a rapidly growing photochromogenic mycobacterium

A strain isolated from a biopsy sample taken from a slowly spreading skin granulation on a child's hand was found to have properties consistent with its classification in the genus Mycobacterium. An almost complete gene sequence of the 16S rRNA of the strain was determined following the cloning and sequencing of the amplified gene. The sequence was aligned with those available for mycobacteria, and phylogenetic trees were inferred with four tree-making algorithms. The organism, which formed a distinct phyletic line within the evolutionary radiation occupied by rapidly growing mycobacteria, was readily distinguished from members of validly described species of rapidly growing mycobacteria on the basis of its mycolic acid pattern and a number of other phenotypic features, notably its ability to form yellow pigmented colonies when incubated in the light. The name proposed for this new species is Mycobacterium novocastrense. The type strain is DSM 44203.     

The identification of Mycobacterium marinum genes differentially expressed in macrophage phagosomes using promoter fusions to green fluorescent protein

Mycobacterium marinum, like Mycobacterium tuberculosis, is a slow-growing pathogenic mycobacteria that is able to survive and replicate in macrophages. Using the promoter-capture vector pFPV27, we have constructed a library of 200-1000 bp fragments of M. marinum genomic DNA inserted upstream of a promoterless green fluorescent protein (GFP) gene. Only those plasmids that contain an active promoter will express GFP. Macrophages were infected with this fusion library, and phagosomes containing fluorescent bacteria were isolated. Promoter constructs that were more active intracellularly were isolated with a fluorescence-activated cell sorter, and inserts were partially sequenced. The promoter fusions expressed intracellularly exhibited homology to mycobacterial genes encoding, among others, membrane proteins and biosynthetic enzymes. Intracellular expression of GFP was 2-20 times that of the same clones grown in media. Several promoter constructs were transformed into Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis. These constructs were positive for GFP expression in all mycobacterial strains tested. Sorting fluorescent bacteria in phagosomes circumvents the problem of isolating a single clone from macrophages, which may contain a mixed bacterial population. This method has enabled us to isolate 12 M. marinum clones that contain promoter constructs differentially expressed in the macrophage.     

Identification of mycobacteria infecting fish to the species level using polymerase chain reaction and restriction enzyme analysis

An assay is described utilizing PCR technology for a rapid diagnostic test to identify fish infection with Mycobacterium marinum, M. fortuitum and M. chelonae. A 924 bp DNA fragment from a highly conserved area of the mycobacterial 16S rRNA gene was amplified using mycobacteria genus-specific primers and digested with restriction enzymes (BanI and ApaI). This examination yielded unique restriction patterns for each mycobacterial specie enabling identification of mycobacteria infecting fish to the species level. The protocol can be applied to purified DNA, a simple colony preparation or infected fish tissue. This protocol can be completed in 1-2 days.     

A novel pathogenic taxon of the Mycobacterium tuberculosis complex, Canetti: characterization of an exceptional isolate from Africa

In an attempt to characterize an unusual mycobacterial strain isolated from a 2-year-old Somali patient with lymphadenitis, we applied various molecular methods not previously used for the taxonomic classification of mycobacteria. This isolate, designated So93, did not differ from Mycobacterium tuberculosis in the biochemical tests and in its 16S rRNA sequence, but produced smooth and glossy colonies, which is highly exceptional for this species. This smooth phenotype was unstable and switched nonreversibly to a rough colony morphology with a low frequency. The two colony types were equally virulent for the guinea pig, exhibiting characteristic tuberculous disease. Both morphotypes had shorter generation times than the M. tuberculosis reference laboratory strain H37Rv and clinical isolates of M. tuberculosis and Mycobacterium bovis. Furthermore, the So93 isolate differed from all M. tuberculosis complex strains described thus far by having only a single copy of insertion sequence IS1081, an unusual composition of the direct repeat cluster, and a characteristic phenolic glycolipid and lipooligosaccharide. This glycolipid had previously been observed only in a smooth isolate of M. tuberculosis obtained in 1969 by Canetti in France. Analysis of the Canetti strain showed that it shared virtually all genetic properties characteristic of So93, distinguishing these two strains from the known M. tuberculosis complex taxa, M. tuberculosis, Mycobacterium africanum, M. bovis, and Mycobacterium microti. The natural reservoir, host range, and mode of transmission of the group of bacteria described in this paper are presently unknown. This study, partly based on not previously used molecular criteria, supports the idea that the established members within the M. tuberculosis complex and the newly described Canetti grouping should be regarded as a single species, which likely will be designated "M. tuberculosis".     

Etiological agents of mycobacterioses in pet birds between 1986 and 1995

Between May 1986 and June 1995, mycobacteriosis was diagnosed by histology and microscopy in 204 (3.8%) of 5,345 necropsied pet birds. The predominant macroscopic changes were enlargement of the liver and spleen and thickening of intestinal walls. Attempts to cultivate mycobacteria were made in 110 cases. Acid-fact bacilli grew in 66 specimens (60%) only. In 18 cases we failed to obtain subcultures. Therefore, species identification could be performed for only 48 isolates. Identification was carried out by conventional biochemical tests as well as by PCR-mediated sequencing of the 16S rRNA gene. The majority of the isolates were Mycobacterium genavense (34 isolates), followed by M. avium complex (8), M. fortuitum (2), M. tuberculosis (2), M. gordonae (1), and M. nonchromogenicum (1). The significance of M. genavense as a zoonotic agent remains to be determined.     

Differentiation between Mycobacterium tuberculosis and Mycobacterium avium by amplification of the 16S-23S ribosomal DNA spacer

Differentiation between Mycobacterium tuberculosis and M. avium is helpful for the treatment of disseminated mycobacterial infection in AIDS patients. This can traditionally be done by time-consuming biochemical tests or with Accuprobe. Previously, PCR restriction enzyme analysis (PCR-REA) of the 16S-23S rRNA gene spacer was shown to be able to identify a limited number of strains of Mycobacterium. In this study the method was improved by using more specific primers and was tested with 50 clinical isolates of M. tuberculosis and 65 clinical isolates of M. avium complex. Probes specific to the spacers of M. tuberculosis and M. avium were also tested. Both M. tuberculosis and M. avium could be reliably identified either by PCR-REA or by PCR-hybridization, with the results completely agreeing with those obtained by biochemical tests and with the Accuprobe, respectively. The method may therefore be useful as an alternative in-house method for identification of the bacteria.