New variants of polar glycopeptidolipids detected in Mycobacterium simiae, including 'habana' strains, as evidenced by electrospray ionization-ion trap-mass spectrometry.

AIMS: To determine the composition of polar glycopeptidolipids (pGPLs) of Mycobacterium simiae and, particularly, those of 'habana' strains, in a search for specific markers given the immunogenic potential of 'habana' TMC 5135 in experimental tuberculosis. METHODS AND RESULTS: pGPLs were determined in free lipid extracts using electrospray ionization-ion trap-mass spectrometry (ESI-IT-MS), working in both negative- and positive-ion mode. In the case of TMC 5135, the presence of the previously characterized GPL-II (containing 2,4-di-O-CH(3) glucuronic acid as distal sugar in the oligosaccharide antigenic moiety) and GPL-III (containing 4-O-CH(3) glucuronic acid as distal sugar) was confirmed using MS/MS and MS/MS/MS approaches. Interestingly, some 'habana' strains presented variants of GPL-II, designated GPL-II'-A and GPL-II'-B. A di-O-CH(3)-deoxy-hexose (tentatively, 2,3-di-O-CH(3)-fucose) was identified as the penultimate sugar in the oligosaccharide moiety of GPL-II'-A, whereas in GPL-II'-B the penultimate sugar was fucose (tentative identification). On the contrary, the distal sugar of the oligosaccharide chain of pGPLs of Myco. simiae ATCC 25275(T) was identified as tri-O-CH(3)-glucuronic acid (designated GPL-sim(T)-I, with two variants: GPL-sim(T)-I-A and GPL-sim(T)-I-B), O-CH(3)-glucuronic acid (designated GPL-sim(T)-II) and di-O-CH(3)-glucuronic acid (GPL-II'-A and GPL-II'-B). The penultimate sugar of the oligosaccharide chain of GPL-sim(T)-I-A and GPL-sim(T)-II was identified as di-O-CH(3)-deoxy-hexose (tentatively, 2,3-di-O-CH(3) fucose), and that of GPL-sim(T)-I-B as deoxy-hexose (tentatively, fucose). In all strains studied, each [M-H](-) and [M+Na](+) ion was revealed as a mixture of homologous compounds varying in the number of -O-CH(3) groups present in the oligosaccharide moiety and in the length of the fatty acyl linked to the peptide. CONCLUSIONS: The present work indicates that, within a similar general pattern of pGPLs, different strains of Myco. simiae present some variations, so that new compounds (GPL-II'-A, GPL-II'-B, GPL-sim(T)-I-A, GPL-sim(T)-I-B and GPL-sim(T)-II) were defined. Noteworthy was the fact that the 'habana' strains clearly differed from the type strain of Myco. simiae. SIGNIFICANCE AND IMPACT OF THE STUDY: The data obtained can be used in the delineation of the 'habana' group of Myco. simiae, including the quality control of the immunogenic strain 'habana' TMC 5135.

Lipids of 'Mycobacterium habana', a synonym of Mycobacterium simiae with vaccine potential.

'Mycobacterium habana' was proposed as a distinct species within the genus Mycobacterium; however, it is actually a synonym of Mycobacterium simiae and included in the serotype I of this species. The potential use of 'M. habana' as a vaccine in both leprosy and tuberculosis has led to the analysis of its lipid composition in an attempt to define distinctive markers that could be used in the quality control of true strains of this bacterium. Lipids of taxonomic value (fatty and mycolic acids) are similar in 'M. habana' and M. simiae; nevertheless, they clearly differ on the basis of glycopeptidolipid (GPL) composition. Thus, contrary to M. simiae, most strains of 'M. habana' can be defined by the presence of three polar compounds, designated GPL-I, GPL-II and GPL-III, easily determined by thin-layer chromatography, and characterized, respectively, by the content of l-fucose, 2,4-di-O-Me-d-glucuronic acid, and 4-O-Me-d-glucuronic acid, as epitopes.

[Implication of ermX genes in macrolide- and telithromycin-resistance in Corynebacterium jeikeium and Corynebacterium amycolatum]. / Implicacion de los genes ermX en la resistencia a los macrolidos y la telitromicina de Corynebacterium jeikeium y Corynebacterium amycolatum.

The activity of seven macrolides, clindamycin and telithromycin against clinical isolates of Corynebacterium spp. was studied. Of these, 36 isolates were identified as C. jeikeium and 57 as C. amycolatum. The frequency of resistance to erythromycin and other macrolides as well as clindamycin was high, with CMI(90) >256 microg/ml. Telithromycin showed the best activity, with 52.3% of C. amycolatum and 70% of C. jeikeium erythromycin-resistant strains susceptible to this ketolide. All strains had the MLSb constitutive phenotype. The ermX gene was present in all erythromycin-resistant strains, and in C. amycolatum was 100% homologous with that of C. striatum and C. diphtheriae.

Biochemical, antimicrobial susceptibility and genotyping studies on Corynebacterium urealyticum isolates from diverse sources.

Thirty-two isolates of Corynebacterium urealyticum, isolated between 1991 and 1995, were studied by biochemical tests, phospholipid content, analysis of fatty and mycolic acids, ribotyping, whole-cell protein patterns and antimicrobial susceptibility to six antibiotics. Nineteen isolates were from human and human-related sources (HHRS); the remainder were from animal and animal-related sources (AARS). Most C. urealyticum isolates were similar in their biochemical and whole-cell protein profiles, although most HHRS isolates were alkaline phosphatase-positive (84%) and produced almost identical protein patterns, whereas AARS isolates were quite diverse. The qualitative composition of cellular fatty acids was identical for all isolates examined. Twelve different ribotypes were obtained with HindIII producing four-to-seven bands. Ribotypes 8, 9 and 10 were predominant in isolates from HHRS, whereas in isolates from AARS, ribotypes 5 and 6 predominated. AARS isolates were significantly less antibiotic-resistant, in comparison with HHRS isolates. Ribotyping appeared to be the most useful tool for strain characterisation.

Detection of mycoloylglycerol by thin-layer chromatography as a tool for the rapid inclusion of corynebacteria of clinical origin in the genus Corynebacterium.

A chemotaxonomic study of some corynebacteria isolated from clinical samples revealed characteristic thin-layer chromatographic patterns for meso-diaminopimelic acid containing species included in the genera Corynebacterium, Dermabacter and Brevibacterium. Notably, a specific compound was consistently detected in mycolic acid containing species of the genus Corynebacterium. This compound was composed by glycerol and mycolic acids and structural analyses carried out by fast atom bombardment mass spectrometry in C. minutissimum confirmed its identification as mycoloylglycerol. The chain length of mycoloyl groups in this molecule ranged from 28 to 34 carbon atoms, being mono-, di- or triunsaturated. Detection of mycoloylglycerol by thin-layer chromatography may be thus useful for the rapid inclusion of a great variety of corynebacteria of clinical origin in the genus Corynebacterium in laboratories employing chromatographic techniques as an adjunct for the identification of these microorganisms.

Microbiological characterization and clinical significance of Corynebacterium amycolatum strains.

The laboratory records of patients with bacillus isolates identified as Corynebacterium xerosis were reviewed in an attempt to establish the clinical significance of the isolates, and the isolated strains were reidentified. Of the 22 strains available for reidentification, four were identified as Corynebacterium striatum and 18 as Corynebacterium amycolatum. Forty-one patients were considered to have Corynebacterium amycolatum isolates, and in 13 (31.7%) of these patients a genuine clinical infection occurred, comprising catheter-related infection in seven cases, surgical wound infection in five cases, and pilonidal cyst infection in one case. Most patients were treated with antimicrobial agents (vancomycin in seven cases and amoxicillin/clavulanic acid in four cases). All patients were cured. Corynebacterium amycolatum can cause genuine infection, usually hospital-acquired, and the clinical significance of isolates must be determined to ensure proper management of patients.

Acyl phosphatidylglycerol: a major phospholipid of Corynebacterium amycolatum.

The type strain and several clinical isolates of Corynebacterium amycolatum were examined for lipid composition as a chemotaxonomic character for routine identification. The phospholipid profile was composed of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides, together with various unidentified compounds. One of them, accounting for 20-29% of total phospholipids, was purified and characterized as acyl phosphatidylglycerol by chromatographic and spectrometric techniques. The acyl substituents on the phosphatidyl moiety were characterized as tetradecanoyl, pentadecanoyl, hexadecenoyl, hexadecanoyl, heptadecenoyl, heptadecanoyl, octadecenoyl (the major one), and octadecanoyl. The acyl group on the polar head (glycerol) was only octadecenoyl. Phospholipid analysis by thin-layer chromatography of a collection of Corynebacterium strains proved that this compound is widely distributed, although it only represents a minor (2-9%) component among mycolic acid-containing species. Acyl phosphatidylglycerol can be considered as a useful chemical marker for the identification of C. amycolatum in addition to the absence of mycolic acids.

[Evaluation of Mycobacterium fortuitum acyltrehaloses in the serologic diagnosis of pulmonary tuberculosis]. / Evaluación de aciltrehalosas de Mycobacterium fortuitum en el diagnóstico serológico de tuberculosis pulmonar.

BACKGROUND: Several glycolipids of Mycobacterium tuberculosis are antigenic and their use in the serodiagnosis of pulmonary tuberculosis has been postulated. Acyltrehaloses (SL-IV) are among the strongest antigenic glycolipids of M. tuberculosis; similar compounds have been found in M. fortuitum. The aim of the present study was to evaluate the usefulness of the acyltrehaloses of M. fortuitum in the serodiagnosis of pulmonary tuberculosis. METHODS: Two glycolipids, identified as triacyl- (TAT-MF) and diacyl- (DAT-MF) trehaloses, were studied by an ELISA method. Two independent analyses were carried out. In the first one, IgG and IgM were determined in sera from patients with pulmonary tuberculosis (84-24 bacteriologically not confirmed--), healthy individuals (46) and patients with respiratory pathologies other than pulmonary tuberculosis (38), using TAT-MF. In the second one, IgG was determined in sera from pulmonary tuberculosis patients (34) and from healthy individuals (20), using DAT-MF. RESULTS: The sensitivity of the IgG ELISA using TAT-MF varied, according to the cut-off point, between 79.8 and 39.3%; the specificity values ranged between 83.3 and 98.8%. In sera from bacteriologically not confirmed pulmonary tuberculosis patients the sensitivity was 87.5-45.8%. The sensitivity for IgM was very low using TAM-MF (10.7-2.3%), with specificity values ranging from 77.4 to 100%. The sensitivity and specificity values of IgG using DAT-MF were, respectively, 34.3-9.3% and 90-100%. CONCLUSIONS: The IgG ELISA using TAT-MF has similar values of sensitivity and specificity to those reported for the acyltrehaloses of M. tuberculosis, although this antigen, by itself, can not be used in the serodiagnosis of pulmonary tuberculosis. DAT-MF has no value in the serodiagnosis of pulmonary tuberculosis.

A family of diacyltrehaloses isolated from Mycobacterium fortuitum.

A trehalose-containing glycolipid was detected in several strains of Mycobacterium fortuitum and characterized as 2,3-di-O-acyltrehalose (DAT) by combined NMR spectroscopy, IR spectroscopy, GLC and GLC-MS. Lipid constituents of the molecule were identified as a mixture of straight-chain (14-18 carbon atoms) and methyl-branched-chain (17-21 carbon atoms) fatty acyl groups. DAT was further fractionated by reverse phase TLC into four fractions that were designated DAT-I-DAT-IV. DAT-I contained 70-75% straight-chain acyl substituents (hexadecanoyl and octadecanoyl predominating) and 25-30% 2-methyl branched substituents (mainly 2-methyl octadecadienoyl). DAT-II was composed of a mixture in which the acyl groups were almost exclusively 2-methyl branched, with 2-methyl octadecadienoyl and 2-methyl octadecen-2-oyl predominating. DAT-III, which was the major isolated fraction, consisted of compounds in which the ratio linear to branched acyl groups varied between 0.8 to 0.9, 2-methyl octadecen-2-oyl, hexadecanoyl and octadecanoyl being the most abundant. Finally, DAT-IV comprised a mixture of DAT molecules containing mostly 2-methyl octadecadienoyl, 2-methyl octadecen-2-oyl, 2-methyl eicosadienoyl and 2-methyl eicosen-2-oyl groups.

Delineation of molecular species of a family of diacyltrehaloses from Mycobacterium fortuitum by mass spectrometry.

A family of 2,3-di-O-acyl trehaloses (DAT), previously identified in Mycobacterium fortuitum, was studied by fast-atom bombardment mass spectrometry to establish the combinations of fatty acyl substituents and, hence, to delineate the molecular species there comprised. The mass spectra indicated the possible existence of 41 molecular species, with a total of 35-40 carbon atoms and 0-4 double bonds in the lipid moiety. The principal components were situated a m/z (M+ +23) 919 (formulated as 2-methyloctadec-2-enoyl,2-methyloctadecadienoyl trehalose and as 2-methylhexadec-2-enoyl,2-methyleicosadienoyl trehalose) and m/z (M+ +23) 921 (formulated as di-2-methyloctadec-2-enoyl trehalose and as 2-methylhexadec-2-enoyl,2-methyleicos-2-enoyl trehalose). The data obtained revealed that DAT was composed of three types of combinations of fatty acyl groups; (i) linear plus linear; (ii) linear plus 2-methyl branched; and (iii) 2-methyl branched plus 2-methyl branched.

Ethambutol inhibition of glucose metabolism in mycobacteria: a possible target of the drug.

The addition of D-arabinose, D-galactose, D-glucosamine, or D-mannose to the growth medium of Mycobacterium smegmatis suppressed the inhibitory effects of ethambutol both on acetate labeling of cell wall-linked mycolic acids and on the increase in the delipidated cell dry weight. The addition of D-glucose or D-fructose had no effect. It is proposed that ethambutol inhibits an early step of glucose conversion into the monosaccharides used for the biosynthesis of structurally and biologically important cell wall polysaccharides: arabinogalactan, arabinomannan, and peptidoglycan.

Numerical analysis of fatty and mycolic acid profiles of Corynebacterium urealyticum and other related corynebacteria.

The fatty and mycolic acid profiles of 52 strains of clinical origin belonging to Corynebacterium urealyticum were subjected to numerical analysis along with those of representative members of Corynebacterium ammoniagenes, Corynebacterium bovis, Corynebacterium glutamicum, Corynebacterium jèikeium, Corynebacterium minutissimum, Corynebacterium pseudodiphtheriticum, Corynebacterium pseudotuberculosis, Corynebacterium xerosis, Corynebacterium renale, Corynebacterium cystitidis, "Corynebacterium ulcerans" and one strain of the Corynebacterium F1 group. Strains were divided into eight clusters at an amalgamation distance of 7.4. Corynebacterium urealyticum appeared as an homogeneous cluster clearly distant from others, that included several members of the genus Corynebacterium, and it was characterized by its content on unsaturated mycolic acids of mainly 28 (28:1) and 30 (30:3) carbon atoms. On the basis of these results the taxonomic "status" of Corynebacterium urealyticum, a new species within the genus Corynebacterium "sensu stricto", is further justified.

Classification of coryneform bacteria associated with human urinary tract infection (group D2) as Corynebacterium urealyticum sp. nov.

Urealytic strains of coryneform bacteria that are designated Corynebacterium group D2 and are isolated from human urine are a cause of urinary tract infections. Cell wall and lipid analyses confirmed that these organisms are members of the genus Corynebacterium but can be separated from other species in the genus on the basis of DNA base composition and DNA-DNA hybridization values. Biochemically, strains in this taxon can be distinguished from other Corynebacterium spp. by their failure to produce acid from carbohydrates, by their failure to reduce nitrates, and by their ability to hydrolyze urea. We regard these bacteria as a new species of the genus Corynebacterium and propose the name Corynebacterium urealyticum. The type strain is strain NCTC 12011 (= ATCC 43042).

Taxonomic implications of the chemical analysis of the D2 group of corynebacteria.

Whole cell acid methanolysates from corynebacteria of the D2 group were found to contain meso-diaminopimelic acid, arabinose and galactose. Among lipids of taxonomic value, saturated and unsaturated straight chain fatty acids (14 to 18 carbon atoms), tuberculostearic acid (10-methyl octadecanoic acid) and mycolic acids were present. The last compounds ranged from 26 to 36 carbon atoms, the predominant types being 28.2, 28.1, 30.3, 30.2, 32.3 and 32.2. By reverse phase thin-layer chromatography the major menaquinone was identified as MK-9(H2)-containing nine isoprene units with two additional hydrogens. Moreover, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides were detected among the phospholipids of these bacteria. Thus, on these bases, the D2 group appears to be closely related to the true corynebacteria.

Establishment of 2-docosanol as a cellular marker compound in the identification of Mycobacterium xenopi.

Sixteen strains of Mycobacterium xenopi were studied by gas chromatography and thin-layer chromatography. Data on the cellular fatty acids, fatty alcohols, mycolic acids, and glycolipids indicated that this bacterium possesses a specific lipid composition. 2-Docosanol, detected in all studied strains, was found to constitute a useful chemical marker in the identification of M. xenopi.

Tetraenoic and pentaenoic mycolic acids from Mycobacterium thamnopheos. Structure, taxonomic and biosynthetic implications.

On the basis of the analysis of mycolates, the type strain of Mycobacterium thamnopheos has been considered as a member of the genus Nocardia. In a comparative study conducted on mycobacterial species we found that M. thamnopheos synthesized two types of mycolate having the same mobilities on thin-layer chromatography as those of mycobacteria, but different from nocardomycolates. Mass spectrometry analyzes showed that the major series of both types consisted of polyunsaturated mycolic acids, ranging from C72 to C78 with four or five double bonds. On pyrolytic mass spectrometry or gas chromatography, the least polar mycolates released mainly monounsaturated C22 esters whereas the other type yielded saturated C20 and C22 esters. These results suggested that M. thamnopheos might be more related to the Aurantiaca taxon than to mycobacteria and Nocardia. The permanganate-periodate oxidation products of esters obtained by pyrolysis of the least polar mycolates showed that they contained docosen-4-oic and docosen-6-oic acids. Both types of mycolate esters yielded the same set of long-chain meroaldehydes on pyrolysis. These meroaldehydes were significantly distinct from those of mycobacterial mycolates in the location of the double bonds. After hydrogenation of the double bond located in the alkyl-branched chain, the two types of mycolates had the same mobility on thin-layer chromatography, indicating that the difference of migration was due to the additional double bond found in the least polar mycolates. Based on stereochemical data, the relative configuration of both mycolates was found to be threo, like that established for all mycolates studied so far.

Fatty and mycolic acids of Mycobacterium malmoense.

The fatty acids and mycolic acids of 16 clinical isolates of Mycobacterium malmoense were studied by gas chromatography and thin-layer chromatography. All strains contained 2-methyleicosanoic and 2,4,6-trimethyltetracosanoic acids and alpha-, alpha'-, and keto-mycolic acids. The reported findings suggest that lipid analysis is a very useful approach in the species identification of M. malmoense.

A thin-layer chromatographic method for separating methyl esters of mycobacterial mycolic acids.

A two-dimensional thin-layer chromatographic method was developed which allowed separation of the different mycobacterial mycolic acids as methyl esters. Dichloromethane (once) and petroleum ether:acetone (95:5, v/v twice or three times) were used as solvents. Alkaline saponification of freeze-dried cells followed by methylation of the mycolic acids using iodomethane gave satisfactory results, whereas methylation using boron trichloride-methanol complex or trans-esterification through direct acid methanolysis was found to degrade epoxy-mycolates. The chromatographic method developed here is rapid and informative, and should prove valuable in routine mycobacterial differentiation.