Characterization of serologic and cell-mediated reactivity of a 38-kDa antigen isolated from Mycobacterium tuberculosis

An antigen of Mycobacterium tuberculosis with an m.w. of 38,000 has been isolated by affinity chromatography using a monoclonal antibody. This antibody bound only to an antigen found in M. tuberculosis and Mycobacterium bovis BCG. The specificity of the antigen was tested in a vertical study by immunodetection on western blots reacted with hyperimmune sera against M. tuberculosis, M. bovis, and 10 other Mycobacterium species. The antigen was detected only by antisera to M. tuberculosis and M. bovis. Specificity in cell-mediated immunity was tested by skin tests in guinea pigs sensitized with M. tuberculosis, Mycobacterium intracellulare, and Mycobacterium kansasii and by lymphocyte proliferation tests. The 38-kDa antigen induced positive skin test reactions regardless of the Mycobacterium species used to sensitize the animal. The ability of the 38-kDa antigen to sensitize for cell-mediated immunity was tested by injecting mice with the 38-kDa antigen and challenging their lymphocytes in vitro with various mycobacterial antigens. Lymphocyte proliferation was observed in the presence of 38-kDa antigen, M. tuberculosis sonicate antigen, and tuberculin purified protein derivative and to M. kansasii and M. intracellulare. The 38-kDa antigen may contain a specific epitope detected by serology, but also contains epitopes that are cross-reactive for cellular immunity.     

Arylsulfatase activity of Mycobacterium avium, M. intracellulare, and M. scrofulaceum

A rapid (3-h) arylsulfatase assay for cell suspensions of mycobacteria, in which p-nitrophenyl sulfate is used as the substrate, was developed. Arylsulfatase activity was found in cell suspensions of representative strains of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum grown without the substrate in either Middlebrook 7H9 medium containing 0.2% (wt/vol) glucose and 0.05% (vol/vol) Tween 80 or Dubos broth medium, but was absent in cells grown in a low-pH, minimal medium containing 1% (vol/vol) Tween 80 as the sole carbon source. The levels of arylsulfatase activity of representatives of all three species were equal whether the activity was measured at pH 5.5, 6.5, or 7.5 and whether the cells were suspended in phosphate or Tris buffer. The addition of high levels of sulfate (present in the low-pH, Tween 80-containing medium) to Middlebrook 7H9 medium resulted in significantly lower levels of arylsulfatase activity in strains of M. scrofulaceum, but did not affect the levels in either M. avium or M. intracellulare. The levels of arylsulfatase activity were highest in M. avium, intermediate in M. intracellulare, and lowest in M. scrofulaceum strains. Polyacrylamide gel electrophoresis of crude extracts from late-log-phase cells of representatives of each species produced activity bands of unique mobility (one in M. avium, three in M. intracellulare [82, 5, and 13%], and two in M. scrofulaceum [60 and 40%]).     

Gene replacement through homologous recombination in Mycobacterium intracellulare.

Mycobacterium intracellulare is a slow-growing pathogenic mycobacterium closely related to Mycobacterium avium. In contrast to Mycobacterium tuberculosis and Mycobacterium bovis BCG, M. intracellulare has received little attention as a model species for studies of mycobacterial molecular biology and genetics. This study shows that M. intracellulare 1403 (ATCC 35761) can be transformed by electroporation with high frequencies (up to 10(6) transformants per microgram of DNA), using plasmids pYT937 and pMH94 as replicative and integrative vectors, respectively. We also describe an experimental system that we used to study DNA recombination in M. intracellulare. First, an integrative plasmid was introduced into M. intracellulare 1403. A nonreplicative, nonintegrative plasmid having homology with the integrated plasmid was then introduced, and the resultant recombinants were analyzed to distinguish between events of homologous and illegitimate recombination. No illegitimate recombination occurred; in all recombinants, a single crossover between homologous regions of the two plasmids was noted. During subsequent growth of a recombinant clone, a spontaneous deletion occurred that resulted in a gene replacement on the chromosome of M. intracellulare 1403. The ability to construct site-specific mutations in M. intracellulare will provide novel insights into the biology of slow-growing mycobacteria.     

Phylogenetic analysis and identification of different serovars of Mycobacterium intracellulare at the molecular level

Comparative 16S rRNA sequencing was used to infer the phylogenetic relationship among different serovars of the Mycobacterium avium-M. intracellulare complex as well as to define signature nucleotides characteristic for different serovars. In general, the groups defined by rRNA sequencing reflect the classification obtained with sensitin tests and pathogenicity examinations in chickens. Unique 16S rRNA sequence patterns could be defined for (1) M. avium, (2) M. intracellulare serovars 4, 5, 6, 8, 9, 10 and 11, (3) M. intracellulare serovars 12, 13, 14, 15, 17, 19 and 20, (4) M. intracellulare serovar 7 and (5) M. intracellulare serovar 18. Phylogenetically, groups 1 and 2 on one hand and groups 3, 4 and 5 on the other hand each share a common ancestor. M. paratuberculosis was indistinguishable from M. intracellulare serovars 4, 5, 6, 8, 9, 10 and 11 by this kind of analysis.     

Fluorescent acid-fast microscopy for measuring phagocytosis of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum by Tetrahymena pyriformis and their intracellular growth

Fluorescent acid-fast microscopy (FAM) was used to enumerate intracellular Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum in the ciliated phagocytic protozoan Tetrahymena pyriformis. There was a linear relationship between FAM and colony counts of M. avium cells both from cultures and within protozoa. The Ziehl-Neelsen acid-fast stain could not be used to enumerate intracellular mycobacteria because uninfected protozoa contained acid-fast, bacterium-like particles. Starved, 7-day-old cultures of T. pyriformis transferred into fresh medium readily phagocytized M. avium, M. intracellulare, and M. scrofulaceum. Phagocytosis was rapid and reached a maximum in 30 min. M. avium, M. intracellulare, and M. scrofulaceum grew within T. pyriformis, increasing by factors of 4- to 40-fold after 5 days at 30 degrees C. Intracellular M. avium numbers remained constant over a 25-day period of growth (by transfer) of T. pyriformis. Intracellular M. avium cells also survived protozoan encystment and germination. The growth and viability of T. pyriformis were not affected by mycobacterial infection. The results suggest that free-living phagocytic protozoa may be natural hosts and reservoirs for M. avium, M. intracellulare, and M. scrofulaceum.     

Occurrence of Mycobacterium Other than Mycobacterium tuberculosis in the Oral Cavity and in Sputum

Mycobacteria were cultured from 9% of 424 paired mouthwash-induced sputum specimens. The majority of the organisms were not Mycobacterium tuberculosis. Sputum cultures contributed 59% of these isolations. M. intracellulare was the species most frequently isolated. The non-M. tuberculosis mycobacteria may constitute part of the oral flora of the general population and are not more prevalent in hospitalized patients. Approximately one-third more isolations were made from the patients furnishing two or three pairs of specimens as compared to those patients providing one pair of specimens each. From the paired specimens of 4 of 113 patients, two species of Mycobacterium were isolated. M. intracellulare was isolated from two of three samples of tap water and from the fingers of 1 of 52 patients.     

Site-directed mutagenesis and virulence assessment of the katG gene of Mycobacterium intracellulare

Mycobacterial catalases have been suggested as acting as virulence factors by protecting intracellular mycobacteria from reactive oxidative metabolites produced by host phagocytes. Mycobacterium intracellulare , like many other mycobacteria, produces two proteins with catalase activity: a heat-stable catalase (KatE) and an inducible, heat-labile catalase peroxidase (KatG). The M. intracellulare katG gene was cloned, and a plasmid derivative with a 4 bp insertion in the katG coding sequence was constructed and used for site-directed mutagenesis of M. intracellulare 1403 (ATCC 35761). The resulting katG mutant was highly resistant to isoniazid (INH), showed an increased sensitivity to H₂O₂ and had lost peroxidase and heat-sensitive catalase activity but retained heat-stable catalase activity. The plasmid carrying the katG frameshift allele was also used for mutagenesis of the mouse virulent M. intracellulare isolate D673. After intravenous injection into BALB/c mice, D673 and the isogenic katG mutant showed the same growth kinetics in the spleen, liver and lungs of the infected mice. Our results demonstrate that the KatG catalase peroxidase mediates resistance to H₂O₂ and susceptibility to INH but is not an essential virulence factor for the survival and growth of M. intracellulare in the mouse.     

Role of stagnation and obstruction of water flow in isolation of Legionella pneumophila from hospital plumbing.

The stagnation of water in two of four hospital hot-water storage tanks found to contain Legionella pneumophila was reduced by keeping the two tanks continually on-line for 1 year. L. pneumophila colony counts in these two tanks fell quickly to low levels, whereas the organisms persisted in the two tanks that were not in use. L. pneumophila continued to be isolated from 50 to 100% of the hospital showerheads which were sampled during this period. We also examined aerators and other hospital faucet fixtures which obstruct water flow. L. pneumophila was isolated from 22 of 30 faucet aerators and 2 of 16 vacuum breakers but not from 26 nonobstructed faucets or 6 backflow preventers. Over a 7-month period, after nine faucet aerators were sterilized, 10 of 60 surveillance cultures revealed L. pneumophila, despite the inability to isolate the organism from the potable-water tanks in use. These data suggest that prevention of stagnation in hot-water tanks may be effective in reducing L. pneumophila concentrations in potable-water systems serving high-risk populations. We have also shown that faucet aerators, by providing a surface for L. pneumophila to colonize, can become secondary reservoirs for the organism in hospital plumbing.     

Characterization of mycobacterial immunoprecipitates by selective staining of enzymes

Immune precipitation patterns of Mycobacterium intracellulare, M. phlei and M. smegmatis were analysed by selective enzyme staining procedures in order to characterize individual mycobacterial antigens. Enzyme activity was shown in eight precipitinogens of M. intracellulare, seven of M. phlei, and six of M. smegmatis. The identification of mycobacterial precipitinogens as enzymes is important since only a few mycobacterial antigens have been functionally characterized.     

Heat susceptibility of aquatic mycobacteria.

An investigation was carried out to measure the heat susceptibility of opportunistic mycobacteria frequently isolated from domestic water supply systems. The study was conducted under standardized conditions designed to resemble those found in oligotrophic aquatic habitats. Strains of the following species were tested: Mycobacterium avium, M. chelonae, M. fortuitum, M. intracellulare, M. kansasii (two strains), M. marinum, M. phlei, M. scrofulaceum, and M. xenopi. Suspensions of the test strains were exposed to temperatures of 50, 55, 60, and 70 degrees C; samples were taken at defined intervals to determine the concentration of survivors. From these data, the decimal reduction times were calculated for each test strain and test temperature. The results indicate that M. kansasii is more susceptible to heat than Legionella pneumophila, whereas the heat susceptibilities of M. fortuitum, M. intracellulare, and M. marinum lie in the same order of magnitude as that of L. pneumophila. The strains of M. avium, M. chelonae, M. phlei, M. scrofulaceum, and M. xenopi were found to be more thermoresistant than L. pneumophila, with the highest resistance being found in M. xenopi. Thermal measures to control L. pneumophila may therefore not be sufficient to control the last five mycobacterial species in contaminated water systems.     

Further studies of a new pathogenic mycobacterium (M. haemophilum sp. nov.).

Mycobacterium haemophilum is an acid-fast rod-shaped organism, originally isolated from deep subcutaneous granulomata of a patient with Hodgkin's disease. Like the other two mycobacterial skin-pathogens, M. ulcerans and M. marinum, M. haemophilum has a maximum temperature for growth below 37 degrees C. Mycobacterium haemophilum is distinguished from all other species examined by its requirement of haemin for growth and its complete lack of catalase activity. Extraneous catalase cannot replace haemin as a growth factor for this organism. Mycobacterium haemophilum can also be differentiated from other species by the patterns of electrophoresis of protein extracts and by gas-liquid chromatography of saponificated and methylated lipid extracts. A monospecific-agglutinating antiserum against M. haemophilum was obtained by adsorption of an immunoserum with M. intracellulare. A number of slow-growing mycobacterial species develop on monolayers of McCoy fibroblasts, and growth on these tissue cultures can be observed much earlier than on artificial media. Mycobacterium haemophilum is characterized by exclusively intracellular development.     

Heat susceptibility of aquatic mycobacteria.

An investigation was carried out to measure the heat susceptibility of opportunistic mycobacteria frequently isolated from domestic water supply systems. The study was conducted under standardized conditions designed to resemble those found in oligotrophic aquatic habitats. Strains of the following species were tested: Mycobacterium avium, M. chelonae, M. fortuitum, M. intracellulare, M. kansasii (two strains), M. marinum, M. phlei, M. scrofulaceum, and M. xenopi. Suspensions of the test strains were exposed to temperatures of 50, 55, 60, and 70 degrees C; samples were taken at defined intervals to determine the concentration of survivors. From these data, the decimal reduction times were calculated for each test strain and test temperature. The results indicate that M. kansasii is more susceptible to heat than Legionella pneumophila, whereas the heat susceptibilities of M. fortuitum, M. intracellulare, and M. marinum lie in the same order of magnitude as that of L. pneumophila. The strains of M. avium, M. chelonae, M. phlei, M. scrofulaceum, and M. xenopi were found to be more thermoresistant than L. pneumophila, with the highest resistance being found in M. xenopi. Thermal measures to control L. pneumophila may therefore not be sufficient to control the last five mycobacterial species in contaminated water systems.     

Comparative genomic hybridizations reveal genetic regions within the Mycobacterium avium complex that are divergent from Mycobacterium avium subsp. paratuberculosis isolates

Mycobacterium avium subsp. paratuberculosis is genetically similar to other members of the Mycobacterium avium complex (MAC), some of which are nonpathogenic and widespread in the environment. We have utilized an M. avium subsp. paratuberculosis whole-genome microarray representing over 95% of the predicted coding sequences to examine the genetic conservation among 10 M. avium subsp. paratuberculosis isolates, two isolates each of Mycobacterium avium subsp. silvaticum and Mycobacterium avium subsp. avium, and a single isolate each of both Mycobacterium intracellulare and Mycobacterium smegmatis. Genomic DNA from each isolate was competitively hybridized with DNA from M. avium subsp. paratuberculosis K10, and open reading frames (ORFs) were classified as present, divergent, or intermediate. None of the M. avium subsp. paratuberculosis isolates had ORFs classified as divergent. The two M. avium subsp. avium isolates had 210 and 135 divergent ORFs, while the two M. avium subsp. silvaticum isolates examined had 77 and 103 divergent ORFs. Similarly, 130 divergent ORFs were identified in M. intracellulare. A set of 97 ORFs were classified as divergent or intermediate in all of the nonparatuberculosis MAC isolates tested. Many of these ORFs are clustered together on the genome in regions with relatively low average GC content compared with the entire genome and contain mobile genetic elements. One of these regions of sequence divergence contained genes homologous to a mammalian cell entry (mce) operon. Our results indicate that closely related MAC mycobacteria can be distinguished from M. avium subsp. paratuberculosis by multiple clusters of divergent ORFs.     

Mycobacterium avium and Mycobacterium intracellulare chromatotypes defined by curvilinear gradient HPLC of mycolic acids

Seventy-nine strains of Mycobacterium avium complex bacteria (MAC), previously characterized by genetic probe analysis, were assayed using two methods of reverse phase high-performance liquid chromatography (HPLC) that employed curvilinear gradients. Although different in column length and cycle time, the methods produced equivalent results, yielding seven distinct chromatographic patterns (chromatotypes) of M. avium and M. intracellulare based on the ratio of mycolate concentrations in the late vs. the middle of three peak clusters (L:M ratio). The M. avium strains (n = 36) were assigned to chromatotypes 1 through 4 (L:M ratios less than 3), and the M. intracellulare strains (n = 25) to chromatotypes 5 through 7 (L:M ratios greater than 4). Of 18 Mycobacterium 'X' strains, seven resembled M. avium, seven others resembled M. intracellulare, and four were intermediate between M. avium and M. intracellulare.     

Influence of pork consumption on human infection with Mycobacterium avian-intracellulare.

The hypothesis that eating pork from animals infected with organisms of the Mycobacterium avium-intracellular complex is a significant cause of human mycobacteriosis was examined by skin testing students with purified protein derivative from M. intracellulare to determine their sensitivity to M. avium-intracellulare. No significant differences in sensitivity could be demonstrated between those individuals who had never eaten pork and those who ate pork routinely. It was concluded that sensitivity to purified protein derivative from M. intracellulare in this population did not involve eating pork.     

Identification of cytoplasmic membrane protein antigens of Mycobacterium avium, M. intracellulare, and M. scrofulaceum

The cytoplasmic membrane isolated from representative strains of the Mycobacterium avium, M. intracellulare, and M. scrofulaceum (MAIS) group contained approximately 20 proteins, as identified by SDS - polyacrylamide gel electrophoresis. One membrane protein predominated, comprising up to 50% of the total membrane protein. This major cytoplasmic membrane protein (MCMP) had a molecular weight of 31,000 and was surface accessible based on its susceptibility to proteinase digestion. The composition of the culture medium strongly influenced the amount of MCMP in the membrane fraction. Western blot analysis revealed that the MCMP and several other membrane proteins reacted with serum samples from patients infected with M. avium-intracellulare, M. tuberculosis, or other mycobacteria.     

A weakly antimalarial biflavanone from Rhus retinorrhoea

The biflavanone (2S,2"S)-7,7"-di-O-methyltetrahydroamentoflavone and five known flavonoids, 7-O-methylnaringenin, 7,3'-O-dimethylquercetin, 7-O-methylapigenin, 7-O-methylluteolin, and eriodictyol were isolated from the leaves of Rhus retinorrhoea Steud, Ex Olive. The biflavanone exhibited moderate antimalarial activity with IC50 0.98 microg/ml against Plasmodium falciparum (W2 Clone) and weak activity against P. falciparum (D6 Clone) with IC50 2.8 microg/ml. Nevertheless, it did not display any cytotoxicity. 7-O-Methylnaringenin showed weak antimicrobial activity against Candida albicans, C. krusei, Staphylococcus aureus, Mycobacterium smegmatis, M. intracellulare, and M. xenopi with MIC approximately 100 microg/ml. Characterization of each compound was based on spectral analysis and comparison with reported data.     

Sequence-based differentiation of strains in the Mycobacterium avium complex.

The complete 16S-23S rDNA internal transcribed spacer (ITS) was sequenced in 35 reference strains of the Mycobacterium avium complex. Twelve distinct ITS sequences were obtained, each of which defined a "sequevar"; a sequevar consists of the strain or strains which have a particular sequence. ITS sequences were identified which corresponded to M. avium (16 strains, four ITS sequevars) and Mycobacterium intracellulare (12 strains, one ITS sequevars). The other seven M. avium complex strains had ITS sequences which varied greatly from those of M. avium and M. intracellulare and from each other. The 16S-23S rDNA ITS was much more variable than 16S rDNA, which is widely used for genus and species identification. Phylogenetic trees based on the ITS were compatible with those based on 16S rDNA but were more detailed and had longer branches. The results of ITS sequencing were consistent with the results of hybridization with M. avium and M. intracellulare probes (Gen-Probe) for 30 of 31 strains tested. Serologic testing correlated poorly with ITS sequencing. Strains with the same sequence were different serovars, and those of the same serovar had different sequences. Sequencing of the 16S-23S rDNA ITS should be useful for species and strain differentiation for a wide variety of bacteria and should be applicable to studies of epidemiology, diagnosis, virulence, and taxonomy.     

坑酸分枝杆菌和相关菌全细胞枝菌酸甲基酯的薄层分析

Mycolic acid methanolysates of whole-cell in Mycobacterium and related bacteria were analysed by thin-layer chromatography. The experimental results show that five of twenty-two species, M. tuberculosis, M. bovis, M. kansasii, M. marinum and M. gastri have similar pattern of mycolates, composed of alpha-mycolates, methoxymycolates, ketomycolates and two unknown components. M. gilvum, M. phleri, M. avium, M. intracellulare, M. xenopi and M. nonchromogenicum contain alpha-mycolates, ketomycolates and wax-ester. The patterns of TLC for other tested species were different from each other. Nocardia, Rhodococcus and Corynebacterium show a relatively simple pattern which principally contain alpha-mycolates. The four genus can be differentiated. Spots of mycolic acids of nine strains Mycobacterium sp. isolated from patients in this hospital were similar to M. tuberculosis. These strains were also identified to the same result as above by traditional methods. The method is of value in the classification and identification of Mycobacterium.     

Structural analysis and biosynthesis gene cluster of an antigenic glycopeptidolipid from Mycobacterium intracellulare

Mycobacterium avium-Mycobacterium intracellulare complex (MAC) is the most common isolate of nontuberculous mycobacteria and causes pulmonary and extrapulmonary diseases. MAC species can be grouped into 31 serotypes by the epitopic oligosaccharide structure of the species-specific glycopeptidolipid (GPL) antigen. The GPL consists of a serotype-common fatty acyl peptide core with 3,4-di-O-methyl-rhamnose at the terminal alaninol and a 6-deoxy-talose at the allo-threonine and serotype-specific oligosaccharides extending from the 6-deoxy-talose. Although the complete structures of 15 serotype-specific GPLs have been defined, the serotype 16-specific GPL structure has not yet been elucidated. In this study, the chemical structure of the serotype 16 GPL derived from M. intracellulare was determined by using chromatography, mass spectrometry, and nuclear magnetic resonance analyses. The result indicates that the terminal carbohydrate epitope of the oligosaccharide is a novel N-acyl-dideoxy-hexose. By the combined linkage analysis, the oligosaccharide structure of serotype 16 GPL was determined to be 3-2'-methyl-3'-hydroxy-4'-methoxy-pentanoyl-amido-3,6-dideoxy-beta-hexose-(1-->3)-4-O-methyl-alpha-L-rhamnose-(1-->3)-alpha-L-rhamnose-(1-->3)-alpha-L-rhamnose-(1-->2)-6-deoxy-alpha-L-talose. Next, the 22.9-kb serotype 16-specific gene cluster involved in the glycosylation of oligosaccharide was isolated and sequenced. The cluster contained 17 open reading frames (ORFs). Based on the similarity of the deduced amino acid sequences, it was assumed that the ORF functions include encoding three glycosyltransferases, an acyltransferase, an aminotransferase, and a methyltransferase. An M. avium serotype 1 strain was transformed with cosmid clone no. 253 containing gtfB-drrC of M. intracellulare serotype 16, and the transformant produced serotype 16 GPL. Together, the ORFs of this serotype 16-specific gene cluster are responsible for the biosynthesis of serotype 16 GPL.