Morphological and biomolecular evidence for tuberculosis in 8th century AD skeletons from Bélmegyer-Csömöki domb, Hungary.

Macromorphological analysis of skeletons, from 20 selected graves of the 8th century AD Bélmegyer-Csömöki domb, revealed 19 cases of possible skeletal tuberculosis. Biomolecular analyses provided general support for such diagnoses, including the individual without pathology, but the data did not show coherent consistency over the range of biomarkers examined. Amplification of ancient DNA fragments found evidence for the Mycobacterium tuberculosis complex DNA only in five graves. In contrast, varying degrees of lipid biomarker presence were recorded in all except two of the skeletons, though most lipid components appeared to be somewhat degraded. Mycobacterial mycolic acid biomarkers were absent in five cases, but the weak, possibly degraded profiles for the remainder were smaller and inconclusive for either tuberculosis or leprosy. The most positive lipid biomarker evidence for tuberculosis was provided by mycolipenic acid, with 13 clear cases, supported by five distinct possible cases. Combinations of mycocerosic acids were present in all but three graves, but in one case a tuberculosis-leprosy co-infection was indicated. In two specimens with pathology, no lipid biomarker evidence was recorded, but one of these specimens provided M. tuberculosis complex DNA fragments.

[Mycolic acids--biological role and potential application in Mycobacterium detection and differentiation]. / Kwasy mikolowe--rola biologiczna i potencjalne zastosowanie w wykrywaniu oraz róznicowaniu rodzaju Mycobacterium.

Mycolic acids are one of the basic structural elements of the cell wall of bacteria from Corynebacterineae suborder. These compounds are long-chain α-hydroxy ß-alkyl fatty acids with two hydrocarbon chains: longer meromycolic and shorter α-chain meromycolic α-chain. The genus Mycobacterium is characterized by the presence of mycolic acids in length from 60 to 90 carbon atoms having a fully saturated α-chain with a defined length of 22, 24 or 26 carbon atoms. Current research indicates that not only the presence of mycolic acids in the cell wall of mycobacteria is essential for the virulence of mycobacteria. It is proved that the relationship between different types of mycolic acids, their length and the degree of cyclopropanation may vary depending on the stage of infection and mycobacterial culture conditions. At the same time it has been shown that some mycolic acid types are crucial for biofilm formation, antimycobacterial drug resistance or interactions with the immune system. Recent studies also indicate that analysis of mycolic acid profiles could be an alternative to conventional methods of diagnosis of diseases such as tuberculosis, leprosy or mycobacteriosis.

Mycobacterium tuberculosis complex lipid virulence factors preserved in the 17,000-year-old skeleton of an extinct bison, Bison antiquus.

Tracing the evolution of ancient diseases depends on the availability and accessibility of suitable biomarkers in archaeological specimens. DNA is potentially information-rich but it depends on a favourable environment for preservation. In the case of the major mycobacterial pathogens, Mycobacterium tuberculosis and Mycobacterium leprae, robust lipid biomarkers are established as alternatives or complements to DNA analyses. A DNA report, a decade ago, suggested that a 17,000-year-old skeleton of extinct Bison antiquus, from Natural Trap Cave, Wyoming, was the oldest known case of tuberculosis. In the current study, key mycobacterial lipid virulence factor biomarkers were detected in the same two samples from this bison. Fluorescence high-performance liquid chromatography (HPLC) indicated the presence of mycolic acids of the mycobacterial type, but they were degraded and could not be precisely correlated with tuberculosis. However, pristine profiles of C(29), C(30) and C(32) mycocerosates and C(27) mycolipenates, typical of the Mycobacterium tuberculosis complex, were recorded by negative ion chemical ionization gas chromatography mass spectrometry of pentafluorobenzyl ester derivatives. These findings were supported by the detection of C(34) and C(36) phthiocerols, which are usually esterified to the mycocerosates. The existence of Pleistocene tuberculosis in the Americas is confirmed and there are many even older animal bones with well-characterised tuberculous lesions similar to those on the analysed sample. In the absence of any evidence of tuberculosis in human skeletons older than 9,000 years BP, the hypothesis that this disease evolved as a zoonosis, before transfer to humans, is given detailed consideration and discussion.

Breaking down the wall: fractionation of mycobacteria.

Mycobacterium spp. possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan-arabinogalactan complex which in turn is esterified by mycolic acids that form with other non-bound lipids an asymmetric permeability barrier and an outer layer, also called a capsule in the case of pathogenic species. In order to investigate the functional roles of the cell envelope components, especially those of the major pathogens Mycobacterium tuberculosis and Mycobacterium leprae, it is necessary to fractionate the envelope by breaking the unusual wall that covers these bacteria. To this aim we first compared the efficiency of high pressure (cell disrupter/French press) with those of pathogen-compatible breakage methods such as sonication, bead beater and lysozyme treatment using the non-pathogenic Mycobacterium smegmatis. When the distribution of various specific markers of the cell envelope compartments, which include mycolic acids, arabinose, NADH oxidase activity, cell wall and cytosolic proteins, were determined sonication combined with lysozyme treatment was found to be the best option. The protocol of subcellular fractionation was then validated for pathogenic species by applying the method to Mycobacterium bovis BCG cells, an attenuated strain of the M. tuberculosis complex.

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.

Biochemical properties and fatty acid composition of Mycobacterium haemophilum: study of 16 isolates from Australian patients.

The biochemical properties and fatty acid compositions of 16 strains of Mycobacterium haemophilum from Australian patients were studied. The strains proved to be indistinguishable from each other but could readily be differentiated from other slowly growing mycobacteria with similar cultural features. Mycolic acid analyses revealed the presence of alpha-, methoxy-, and ketomycolates. The fatty acid composition supports the validity of the fact that M. haemophilum is a distinct species. The fatty acid composition was consistent among the 16 strains, but it was unusual in that there was some resemblance to the fatty acid composition of M. leprae. The wide range of pHs (5.4 to 7.4) that supported growth of M. haemophilum on artificial medium is in keeping with suggestions that M. haemophilum exists in an environmental habitat.

Lipid composition of leprosy-derived corynebacteria, a distinct group of corynebacteria, and of a reference Corynebacterium.

Leprosy-derived corynebacteria (LDC) are diphtheroid organisms isolated from leprosy patients and previously characterized by DNA and cell wall analysis. Three groups of LDC components of taxonomic value, glycolipids, and phospholipids and cell-wall-bound lipids were analyzed in comparison with those of a reference strain C. hoffmannii (CH). The main CH glycolipid, "cord factor" (trehalose dimycolate), was missing from LDC. Among phospholipids, phosphatidylinositol and phosphatidylglycerol had lowered proportions in LDC, as compared to CH, whereas phosphatidylethanolamine and cardiolipin were absent from both microorganisms. Bound lipids in acidic extracts of delipidated LDC yielded arabinose corynomycolate in lesser quantity with respect to CH. Alkaline hydrolysis of whole cells released fatty acids and mycolic acids, which were analyzed by gas chromatography/mass spectrometry. Reference CH, grown in the absence of serum, yielded C16:0 and C18:1 (major) and C18:0 (minor) fatty acids, as well as C32, C34, and C36 corynomycolic acids. All these components, particularly mycolates, had lowered proportions when this organism was grown in the presence of serum. Dominant LDC components were, in addition to C16:0, C18:0, and CI8:u fatty acids, cholesterol from serum. Very low concentrations of corynomycolic acids with a high degree of unsaturation were found in these organisms, suggesting a dependence of lipid metabolism on growth conditions. The presence in LDC of tuberculostearic acid (C19r:0), a mycobacterial component found in some pathogenic corynebacteria, was carefully explored: Traces of C19r:0 were found in LDC 19 grown in the presence of delipidated serum, but not in LDC 15 nor in C. hoffmannii. Present data, in conjunction with previous studies on DNA and mycolic acids, disclose basic differences in the composition of LDC and conventional corynebacteria.

Location of the mycolyl ester substituents in the cell walls of mycobacteria.

The question of the precise location of mycolic acids, the single most distinctive cell wall entity of members of the Mycobacterium genus, has now been addressed. The free hydroxyl functions of the arabinogalactan component of the mycobacterial cell wall were O-methylated under conditions in which the mycolyl esters were not cleaved. Subsequent replacement of the mycolyl functions with O-ethyl groups resulted in an acid- and base-stable differentially O-alkylated surrogate polysaccharide, more amenable to analysis. Complete hydrolysis, reduction, acetylation, and gas chromatography/mass spectrometry revealed the unexpected finding that the mycolyl substituents were selectively and equally distributed on the 5-hydroxyl functions of terminal- and 2-linked arabinofuranosyl (Araf) residues. Further analysis of the O-alkylated cell wall through partial acid hydrolysis, NaB[2H]4 reduction, pentadeuterioethylation, and gas chromatography/mass spectrometry demonstrated that the mycolyl units are clustered in groups of four on the previously recognized nonreducing terminal pentaarabinosyl unit [beta-Araf-(1----2)-alpha-Araf)2-3, 5-alpha-Araf. However, only about two-thirds of the available pentasaccharide units are so substituted. Thus, the antigenicity of the arabinan component of mycobacterial cell walls may be explained by the fact that about one-third of the pentaarabinosyl units are not mycolyated and are available for interaction with the immune system. On the other hand, the extreme hydrophobicity and impenetrability of the mycobacterial cell may be explained by the same motif also acting as the fulerum for massive esterified paraffin residues. New fundamental information on the structure of mycobacterial cell walls will aid in our comprehension of its impenetrability to antibiotics and role in immunopathogenesis and persistence of disease.

Appearance of a methoxy mycolate-like component by the acid methanolysis of Mycobacterium leprae.

It has been reported that Mycobacterium leprae contains two types of mycolic acid, namely, alpha- and keto-mycolic acids, thus it is taxonomically similar to M. bovis BCG. However, there was some controversy about the presence of methoxy mycolic acid which was observed in small amounts only in the case of experimentally infected (W45) armadillo-derived M. leprae. To investigate this fact, mycolic acids were extracted from the cell-wall structure of M. leprae and characterized using chromatographic techniques. The results showed the appearance of a methoxy mycolate-like component for both purified bacilli and infected human skin tissue materials. However, this appearance occurred only when the acid methanolysis procedure was followed for the release of mycolic acids from these bacilli. No such component appeared on the chromatogram when the alkaline methanolysis procedure was followed. Nevertheless, the consistent presence of this methoxy mycolate-like component by acid methanolysis is an important finding which has to be kept in mind while identifying this pathogen when using chromatographic techniques.

Biochemical characteristics and fatty acid compositions of some armadillo-derived mycobacteria and their relation to Mycobacterium gordonae.

The long-chain components of 75 strains of mycobacteria, cultivated from Mycobacterium leprae-infected or non-infected armadillos, and of eight clinical and 15 environmental isolates of M. gordonae, were compared. Four major groups could be distinguished based on the presence of 10-methyloctadecanoic (tuberculostearic) and 2-methyl 3-hydroxyeicosanoic acids and secondary alcohols (2-octadecanol and 2-eicosanol). Some heterogeneity was found in strains assigned to M. gordonae: the characteristic absence of tuberculostearic acid and secondary alcohols and the presence of the branched C14 and the hydroxylated C20 acids were seen in only 34 of the 49 strains studied. Three strains were identified as M. malmoense, one as M. kansasii, ten as belonging to the M. avium-M. intracellulare-M. scrofulaceum complex and eight as belonging to new groups of armadillo-derived mycobacteria (ADM 1, ADM 2 and ADM 3) by conventional bacteriological tests and fatty acid compositions, though M. malmoense was heterogeneous in its fatty acids composition. Four strains, identified as M. avium by conventional tests, differed from this species by their fatty acid compositions. Thirteen strains showed some similarity to M. simiae and ten strains differed from all other known mycobacteria.

Pyrolysis gas chromatography-mass spectrometry of mycobacterial mycolic acid methyl esters and its application to the identification of Mycobacterium leprae.

Pyrolysis gas chromatography-mass spectrometry of methyl mycolates from 32 species of mycobacteria, including Mycobacterium leprae, was carried out. The mycobacteria could be classified into four groups in respect of the fatty acid ester patterns detected within the range C20 to C26. The applicability of this pyrolysis-gas chromatographic method for identifying M. leprae is discussed.

Results from cation and mass fingerprint analysis of single cells and from ATP measurements of M. leprae for drug sensitivity testing: a comparison.

The physiologic states of Mycobacterium leprae isolated from patient biopsies were studied using single cell mass spectrometry by laser microprobe mass analysis (LAM-MA) and ATP bioluminescence assay. The changes in the physiologic state of M. leprae after the patients had been treated with dapsone (DDS) monotherapy were also studied. The shift of the low intracellular Na+, K+-ratio of untreated M. leprae cells to higher values under DDS therapy, as measured from a limited number of single bacteria, correlates with a decrease in the ATP content. Further information on the influence of the drug could be drawn from the multivariate analysis of mass fingerprints of the organic matrix of the cells. Evidence is provided that the combination of the measurement of the intracellular cation ratios and of the mass fingerprint analysis could give fast answers to the question of drug resistance and to the persister hypothesis. The ATP bioluminescence assay and the single cell mass analysis should be alternatives to the mouse foot pad test.

Quantitative comparison of the mycolic and fatty acid compositions of Mycobacterium leprae and Mycobacterium gordonae.

The mycolic and fatty acids of three samples each of Mycobacterium leprae and Mycobacterium gordonae were compared. Acids released by whole-organism alkaline hydrolysis were converted to 4-nitrobenzyl esters and mycolic acids were further derivatized to t-butyldimethylsilyl ethers. Thin-layer chromatography of the derivatized long-chain extracts showed that all three M. leprae preparations contained so-called alpha-mycolates and ketomycolates but that the M. gordonae samples had a methoxymycolate in addition to the above types. Silica gel normal-phase high-performance liquid chromatography of the total mycolic acid derivatives confirmed the lack of detectable amounts of methoxymycolates in M. leprae and reverse-phase chromatography of the individual mycolate types demonstrated the homogeneity of the chain lengths of the mycolic acids in each species. Non-hydroxylated fatty acid 4-nitrobenzyl esters were transformed to methyl esters and examined by gas chromatography. Tuberculostearic (10-methyloctadecanoic) acid was a major component of the lipids of all three M. leprae preparations but it was absent in one M. gordonae strain and a very minor component in the other representatives of this latter species. On the basis of fatty and mycolic acid compositions, therefore, a previously suggested close relationship between M. leprae and M. gordonae was not supported.

Comparative study on the thin-layer chromatographic pattern of methyl mycolates of M. leprae and related species.

The thin-layer chromatographic (TLC) pattern of methyl mycolates of Mycobacterium leprae has been compared with the lipids extracted from M. leprae-infected tissue material and also with those of other cultivable mycobacterial species. It has been found that M. leprae-derived methyl mycolates give two spots, namely, alpha- and keto-derivatives, after charring the TLC plates with K2Cr2O7/H2SO4 spray. Lipids extracted from infected tissue material also show spots with the same Rf values when compared with those of armadillo-derived M. leprae. Normal skin tissue extracts, however, do not show any presence of mycolates. This TLC pattern of M. leprae-derived methyl mycolates is characteristic and is not shown by any other mycobacterial species isolated from leprosy lesions. The strain and species which have been earlier found to resemble M. leprae antigenically or biochemically could readily be distinguished with the help of the TLC pattern of their methyl mycolates.

Characterization of Mycobacterium leprae by lipid analysis.

The lipid composition of the leprosy bacillus, harvested from experimentally infected nine-banded armadillos, strongly supports it status as a distinct species of the genus Mycobacterium. Phthiocerol dimycocerosate waxes and glycosylated phenophthiocerol dimycocerosates are distinct from those characterised from a number of other mycobacteria. The polar lipids of a single isolate lack diacylated forms of phosphatidylinositol di- and pentamannosides, lipids usually found in most mycobacteria. A simple mycolic acid pattern composed of alpha-mycolates and ketomycolates is characteristic of most preparations of M. leprae.