Distribution of some mycobacterial waxes based on the phthiocerol family

Characteristic waxes, based on methoxy and keto long-chain diols, members of the phthiocerol family, have been isolated from representatives of Mycobacterium bovis, M. kansasii, M. marinum, M. microti and M. tuberculosis. M. kansasii produced essentially di-esters of the ketodiol phthiodiolone A, but the remaining species also had waxes based on the methoxy-diols phthiocerol A and phthiocerol B. Gas chromatography of derivatives of the components of the waxes showed that the phthiocerol A components from M. bovis, M. microti and M. microti and M. tuberculosis were qualitatively similar, being mainly C34 and C36, but potentially significant differences were seen in the proportions of the components from M. bovis. The phthiocerols A from M. marinum were C28 and C30 and the phthiodiolones A from M. kansasii were C25 and C27. The multimethyl-branched acids from the waxes of M. bovis were quantitatively different from those of M. microti and M. tuberculosis but all these mycocerosic acids ranged in size from C23 or C24 to C32, with C29 or C30 being the major component in most cases. M. marinum and M. kansasii strains had mainly C26 or C27 and C29 or C30 multimethyl-branched acids, respectively.     

Numerical classification of 280 strains of slowly growing mycobacteria. Proposal of Mycobacterium tuberculosis series, Mycobacterium avium series, and Mycobacterium nonchromogenicum series

Numerical classification of 280 strains of slowly growing mycobacteria was carried out by testing each strain for 76 characters. The following fourteen clusters were observed: 1. M. tuberculosis, M. bovis, M. africanum, and M. microti; 2. M. haemophilum; 3. M. ulcerans; 4. M. xenopi; 5. M. kansasii; 6. M. szulgai; 7. M. gordonae; 8. M gastri; 9. M. avium, M. intracellulare, M. scrofulaceum, and M. asiaticum; 10. M. marinum; 11. M. simiae; 12. M. nonchromogenicum, "M. novum," M. terrae, and M. triviale; 13 M. malmoense; 14. M. shimoidei. The clusters composed of M. tuberculosis, M. bovis, M. africanum, and M. microti, of M. avium, M. intracellulare, M. scrofulaceum, and M. asiaticum, and of M. nonchromogenicum, "M. novum," M. terrae, and M. triviale appeared to be reduced to a single species each. The names having priority for each species should be M. tuberculosis, M. avium, and M nonchromogenicum, respectively. However, the clusters may, in practice, be called the M. tuberculosis series (complex), the M. avium series (complex), and the M. nonchromogenicum series (complex). The type species of these series are M. tuberculosis, M. avium, and M. nonchromogenicum, respectively. These series were characterized in this study.     

The phenolic mycoside of Mycobacterium ulcerans: structure and taxonomic implications.

Mycobacterium ulcerans and some pathogenic mycobacterial species elaborate wax A consisting of related long-chain beta-diol components (phthiocerol and related compounds) esterified by multimethyl-branched fatty acids. With the exception of M. ulcerans, wax A-containing mycobacteria also synthesize glycosylated phenol phthiocerol diester and related compounds: the so-called phenolic mycosides. In a deliberate effort to characterize this latter class of compounds in M. ulcerans, 20 strains were examined. Phenolic mycosides were found in two strains. Application of chemical analyses, including one- and two-dimensional NMR spectroscopy, allowed the structural elucidation of glycolipids identified as 3-O-methyl-alpha-L-rhamnosyl phenol phthiocerol diphthioceranate investigators. As phenolic mycosides are highly species-specific molecules, this finding stresses the close phylogenetic link between M. marinum and M. ulcerans. Incidentally, a survey of the mycolate content of M. ulcerans showed that methoxymycolate could not be detected in three strains.     

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.     

Characteristic new members of the phthiocerol and phenolphthiocerol families from Mycobacterium ulcerans

Diacyl phthiodiolone A and phenolphthiodiolone A lipids were isolated from two strains of Mycobacterium ulcerans. The diol units of the phthiodiolone A and phenolphthiodiolone A components were shown to have erythro stereochemistry by infrared spectroscopy and proton nuclear magnetic resonance of an acetal derivative. This stereochemistry is shared only by related diols from M. marinum, the diols from M. bovis, M. kansasii, M. leprae and M. tuberculosis having threo stereochemistry.     

Genotyping Mycobacterium ulcerans and Mycobacterium marinum by using mycobacterial interspersed repetitive units

A novel category of variable tandem repeats (VNTR) called mycobacterial interspersed repetitive units (MIRUs) has been identified for Mycobacterium ulcerans (n = 39), M. marinum (n = 27), and one related organism. Fifteen MIRU loci were identified in the genome of M. marinum and were used to genotype M. ulcerans, M. marinum, and an M. marinum-like organism that is considered a possible missing link between M. marinum and M. ulcerans. Seven MIRU loci were polymorphic, and locus-specific PCRs for four of these loci differentiated seven M. ulcerans genotypes, four M. marinum genotypes, and a unique genotype for the missing link organism. The seven M. ulcerans genotypes were related to six different geographic origins of isolates. All isolates from West and Central Africa, including old and recent isolates, belonged to the same genotype, emphasizing the great spatiotemporal homogeneity among African isolates. Unlike the M. ulcerans genotypes, the four M. marinum genotypes could not be clearly related to the geographic origins of the isolates. According to MIRU-VNTR typing, all M. ulcerans and M. marinum isolates of American origin were closely related, suggesting a common American ancestor for these two pathogenic species on the American continents. MIRU typing has significant potential value for discriminating between reoccurrence and reinfection for M. ulcerans disease.     

PCR with subsequent sequencing of the 16S gene rRNA in species identification of mycobacteria of the non-tuberculosis complex

One hundred of mycobacterium cultures were assayed by the method of PCR with subsequent sequencing of the 16S rRNA region. The below mycobacterium species were identified: M. tuberculosis complex (n = 55), M. avium (n = 17), M. intracellulare (n = 4), M. scrofaleceum (n = 2), M. kansasii - M. gastri (n = 3), M. gordonae (n = 3), M. ulcerans - M. marinum (n = 1), M. smegmatis (m = 2), M. fortuitum (n = 11), M. peregrinum (n = 1) and M. chelonae - M. abscessus (n = 1). The method enabled the differentiation of species M. avium from M. intracellulare and M. peregrinum from M. fortuitum, which could not be differentiated by using the classic biochemical and bacteriological methods. Genetic heterogeneity of the mycobacterium strains of M. avium, M. fortuitum and M. gordonae was also established by PCR plus sequencing of the 16S rRNA region.     

Mycoside G, a Specific Glycolipid in Mycobacterium marinum (Balnei).

Navalkar, R. G. (University of Wisconsin, Madison), E. Wiegeshaus, E. Kondo, H. K. Kim, and D. W. Smith. Mycoside G, a specific glycolipid in Mycobacterium marinum (Balnei). J. Bacteriol. 90:262-265. 1965.-A new specific glycolipid in extracts prepared from strains designated Mycobacterium marinum and M. balnei has been demonstrated by use of the techniques of column chromatography and infrared spectroscopy. Since there is now agreement among many workers that M. marinum and M. balnei are identical, the demonstration of the same specific glycolipid in both species is not surprising. This substance, which we have designated mycoside G, is chemically similar to mycosides A and B, and apparently differs only in the sugar moiety. In addition, the lipids extracted from these cultures contain phthiocerol dimycocerosate, a wax component found also in M. tuberculosis and M. bovis.     

Structure of the major triglycosyl phenol-phthiocerol of Mycobacterium tuberculosis (strain Canetti)

Phenol-phthiocerol glycolipids have been found previously in Mycobacterium leprae, M. kansasii, M. bovis and M. marinum, but not in M. tuberculosis. A search for glycolipids in this latter species showed that the Canetti strains of M. tuberculosis synthesize a major triglycosyl phenol-phthiocerol, accompanied by minor amounts of other glycolipids with a similar aglycone moiety. The triglycoside moiety has the following structure: 2,3,4-tri-O-methyl L-fucopyranosyl(alpha 1----3)L-rhamnopyranosyl(alpha 1----3)2-O-methyl L-rhamnopyranosyl(alpha 1-. The aglycone moiety consists in phenol-phthiocerol (two homologs). Its two secondary alcohol functions are esterified by mycocerosic acids (homologs with 26-32 carbon atoms and with 2-4 methyl branches). The proposed structure differs on several points from the M. leprae glycolipids, but presents some analogy with the major glycolipid of M. kansasii. A minor monoglycosyl phenol-phthiocerol was also studied. Its overall structure is very similar to that of M. bovis, with 2-O-methyl rhamnose as sugar moiety.     

The free lipids of Mycobacterium leprae harvested from experimentally infected nine-banded armadillos

The free lipids of a sample of Mycobacterium leprae were extracted by a procedure designed to produce separate non-polar and polar fractions. The composition of these lipids was analysed semi-quantitatively by five special thin-layer chromatographic systems covering the total range of mycobacterial lipid polarities. In order of increasing polarity, the major lipids were dimycocerosates of phthiocerol A, phthiocerol B and phthiodiolone A, glycosyl phenolphthiocerol dimycocerosates and phospholipids, including monoacylphosphatidylinositol di- and pentamannosides. The diacylated forms of these latter lipids, found in most mycobacteria, were not present. The composition of the free lipids of the leprosy bacillus, surveyed over the total polarity range for the first time, showed that the patterns were particularly related to those of Mycobacterium bovis, Mycobacterium kansasii and Mycobacterium marinum.     

Differentiation Between Mycobacterium kansasii and Mycobacterium marinum by Gas-Liquid Chromatographic Analysis of Cellular Fatty Acids

Comparison of the cellular fatty acids of 10 strains of Mycobacterium marinum and 35 strains of Mycobacterium kansasii revealed similarities within each species but differences between these two photochromogenic mycobacteria. A branched-chain fatty acid characteristic of M. kansasii was found in trace amounts in 2 of the 10 strains of M. marinum.     

Subtractive hybridization reveals a type I polyketide synthase locus specific to Mycobacterium ulcerans

Mycobacterium ulcerans causes Buruli ulcer, the third most prevalent mycobacterial infection of immunocompetent humans after tuberculosis and leprosy. Recent work has shown that the production by M. ulcerans of mycolactone, a novel polyketide, may partly explain the pathogenesis of Buruli ulcer. To search for the genetic basis of virulence in M. ulcerans, we took advantage of the close genetic relationship between M. ulcerans and Mycobacterium marinum by performing genomic suppressive subtractive hybridization of M. ulcerans with M. marinum. We identified several DNA fragments specific to M. ulcerans, in particular, a type I polyketide synthase locus with a highly repetitive modular arrangement. We postulate that this locus is responsible for the synthesis of mycolactone in M. ulcerans.     

Gas chromatographic assay of cellular fatty acids and alcohols for the identification of Mycobacterium species.

Ten mycobacterial species obtained from 141 cultures isolated from clinical specimens were studied. The cultures were grown on solid medium and then analysed-after saponification, methylation, extraction with organic solvent and washing of the organic phase--by capillary gas-liquid chromatography for fatty acid and secondary alcohol composition. The absence of secondary alcohols was characteristic of M. genavense, M. tuberculosis and the following Mycobacterium species with specific branched-chain fatty acids allowing their direct identification: M. gordonae, M. kansasii and M. marinum. The presence of secondary alcohols was characteristic of M. avium, M. phlei, M. scrofulaceum, M. terrae and M. xenopi. In the case of M. xenopi direct identification was made possible by the presence of a specific alcohol.     

Characterization of a Mycobacterium bovis BCG insertion sequence related to the IS21 family

The structure and distribution of a Mycobacterium bovis BCG insertion element of the IS21 family were investigated. Several IS21-like elements found in mycobacterial genomes were separated in four types, following their nucleic acid similarities. The M. bovis BCG IS21 element is highly similar to IS1533 (class I), 70% similar to IS1534 (class II), 52% similar to IS1532 (class III) of Mycobacterium tuberculosis, and 54% similar to both an Mycobacterium avium serovar 2 and an M. avium silvaticum IS (class IV). The M. bovis BCG IS21 element of the class I appears to be present in a single copy in the genome of M. bovis BCG, M. bovis, M. tuberculosis and Mycobacterium africanum and to be absent from all other tested species of the Corynebacteria-Mycobacteria-Nocardia group.     

Identification of Mycobacterium species by comparative analysis of the dnaA gene

For the establishment of a diagnostic tool for mycobacterial species, a part of the dnaA gene was amplified and sequenced from clinically relevant 27 mycobacterial species as well as 49 clinical isolates. Sequence variability in the amplified segment of the dnaA gene allowed the differentiation of all species except for Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacterium microti, which had identical sequences. Partial sequences of dnaA from clinical isolates belonging to three frequently isolated species revealed a very high intraspecies similarity, with a range of 96.0-100%. Based on the dnaA sequences, a species-specific primer set for Mycobacterium kansasii and Mycobacterium gastri was successfully designed for a simple loop-mediated isothermal amplification method. These results demonstrate that the variable sequences in the dnaA gene were species specific and were sufficient for the development of an accurate and rapid diagnosis of Mycobacterium species.     

Taxonomic studies on the Mycobacterium tuberculosis series

Numerical classification of slowly growing mycobacteria, including 159 strains received as Mycobacterium tuberculosis, M. bovis, M. africanum, and M. microti, was carried out using 88 characters, and the following results were obtained. 1) All 159 strains received as M. tuberculosis, M. bovis, M. africanum, and M. microti formed one cluster, and no clear-cut differentiation among four species was achieved. These four species should be reduced to one species, M. tuberculosis. 2) Within the cluster, two subclusters appeared, with a number of strains located outside the subclusters. One subcluster was composed, except for two strains, of only M. tuberculosis strains, and another of M. bovis and M. africanum strains only. The subclusters were regarded as subspecies tuberculosis and subspecies bovis, respectively. M. africanum was regarded as a synonym of M. bovis, i.e., a niacin-positive variety of M. bovis (subsp. bovis). 3) An intermediate subcluster was observed outside the M. tuberculosis and M. bovis subclusters. This may be regarded as intermediate between the two subspecies, tuberculosis and bovis. 4) In the last two decades, the characteristics of M. tuberculosis strains isolated from patients seemed to be approaching those of M. bovis strains. The recently isolated strains showed stronger arylsulfatase activity and lower resistance to thiophene-2-carboxylic acid hydrazide than the strains isolated 16 to 19 years ago.     

Amplified ribosomal DNA restriction analysis in the differentiation of related species of mycobacteria

This study explores the potential of the amplified ribosomal DNA restriction analysis (ARDRA) for intra- and interspecies identification of the genus Mycobacteria. A set of primers was used to amplify part of the 16S and 23S rDNA as well as the 16S-23S rDNA spacer from 121 isolates belonging to 13 different mycobacterial species. Restriction analysis was carried out with five different restriction enzymes, namely CfoI, HaeIII, RsaI, MspI and TaqI. Restriction digestion of the PCR product using CfoI enabled differentiation between 9 of the 13 mycobacterial species, whereas the remaining four enzymes differentiated between 7 of these 13 species. None of the five enzymes distinguished between different isolates of Mycobacterium tuberculosis or between species within the M. tuberculosis complex i.e., M. tuberculosis, M. bovis, M. bovis BCG and M. africanum. Although ARDRA analysis of the 16S-23S rDNA does not seem to have a potential for intraspecies differentiation, it has proven to be a rapid and technically relatively simple method to recognise strains belonging to the M. tuberculosis complex as well as to identify mycobacterial species outside this complex.     

Identification of phthiodiolone ketoreductase, an enzyme required for production of mycobacterial diacyl phthiocerol virulence factors

Diacyl phthiocerol esters and their congeners are mycobacterial virulence factors. The biosynthesis of these complex lipids remains poorly understood. Insight into their biosynthesis will aid the development of rationally designed drugs that inhibit their production. In this study, we investigate a biosynthetic step required for diacyl (phenol)phthiocerol ester production, i.e., the reduction of the keto group of (phenol)phthiodiolones. We utilized comparative genomics to identify phthiodiolone ketoreductase gene candidates and provide a genetic analysis demonstrating gene function for two of these candidates. Moreover, we present data confirming the existence of a diacyl phthiotriol intermediate in diacyl phthiocerol biosynthesis. We also elucidate the mechanism underlying diacyl phthiocerol deficiency in some mycobacteria, such as Mycobacterium ulcerans and Mycobacterium kansasii. Overall, our findings shed additional light on the biosynthesis of an important group of mycobacterial lipids involved in virulence.     

Use of selected ion monitoring for detection of tuberculostearic and C32 mycocerosic acid in mycobacteria and in five-day-old cultures of sputum specimens from patients with pulmonary tuberculosis

Gas chromatography/mass spectrometry and selected ion monitoring (SIM), employing both electron (EI) and chemical ionization (CI), was used to detect 10-methyloctadecanoic (tuberculostearic) and 2, 4, 8, 8-tetramethyloctacosanoic (C32 mycocerosic) acids in bacteria of 14 species of Mycobacterium and 3 species of Nocardia. Tuberculostearic acid was found in all species studied, while C32 mycocerosic acid was demonstrated only in M. africanum, M bovis, M. bovis strain BCG, M. kansasii and M. tuberculosis. The relative amounts of these acids in the organisms of these five species varied, thereby constituting a presumptive diagnostic technique. The lowest detectable amount of C32 mycocerosic acid was approximately 5 pg when using EI-SIM, monitoring at m/zz 88 and m/z 101. When using CI, employing isobutane as reactant gas, and focusing at m/z 495, 2 pg could be detected, and when ammonia was the reactant gas, the corresponding figure was 1 pg, monitoring at m/z 512. Tuberculostearic acid was demonstrated in 5-day incubated sputum specimens from 6 patients with pulmonary tuberculosis, including 5 patients infected with M tuberculosis and 1 patient infected with M. avium. C32 mycocerosic acid was detected in 4 of the 5 patients with M. tuberculosis infection. None of the acids was found in a further 8 patients who had viral or bacterial (non-mycobacterial) pneumonia. Tuberculostearic acid could be demonstrated in 10 of another 12 sputum specimens from patients with tuberculosis, when the samples were analyzed directly, viz prior to culturing. The possibility of using SIM for the rapid diagnosis of pulmonary tuberculosis is thus worth consideration.     

The identification of antigenic determinants on Mycobacterium bovis using monoclonal antibodies

Murine hybridomas secreting monoclonal antibodies to Mycobacterium bovis were produced and three soluble antigens were identified using radioimmunoassays and immunoblotting from polyacrylamide electrophoresis gels. Antibody MB3 (IgM, k chain) reacted with 20-100 kDal antigens produced by all mycobacterial strains examined while antibody MB5 (IgG2a, k chain) identified a 29.8 kDal antigen detected in field isolates of M. bovis and M. bovis strains Vallée and AN5. There was insignificant binding to M. bovis BCG, M. tuberculosis, M. microti, M. africanum, M. avium or M. paratuberculosis. Monoclonal antibody MB17 (IgA, k chain) reacted with a 17.4 kDal antigen present in M. bovis, M. tuberculosis and M. microti. Absorption of monoclonal antibodies with antigens from different species of Mycobacterium confirmed the specificities of MB3 and MB5 but the binding of MB17 was inhibited to some extent by all the extracts examined. The antigen identified by MB3 was present in purified protein derivative (PPD) from M. bovis, M. paratuberculosis and M. avium but antigens identified by MB5 and MB17 were not detected in these reagents.