Analysis of cellular fatty acids and proteins by capillary gas chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis to differentiate Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium scrofulaceum (MAIS) complex species.

Infections due to atypical mycobacteria have increased during the past 30 years. Species of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium scrofulaceum are among the most common non-tuberculous mycobacteria isolated from patients with AIDS or immunosuppressed. These three organisms are taxonomically closely related and identification, according to cultural characteristics and biochemical tests, is not always evident, so some of these related strains are grouped in a "MAIS" complex. Analysis of cellular constituents is an aid to identification. Gas chromatography was used to study mycolic acids and a secondary alcohol was found which is a discriminating constituent between M. scrofulaceum and the other two species. The lipidic analysis was not able to separate M. avium and M. intracellulare, so cell proteins were considered. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of proteins reflects genetic relatedness between strains; the different patterns obtained from these three species are described and it is shown that this method is very useful in classification and epidemiology.

Gas chromatographic characterization of the relationship between some Mycobacterium avium and Mycobacterium paratuberculosis strains.

Mycobacterium avium strain P-55 and M. avium strain DENT differ from M. avium strain 16909-338 on the basis of their fatty acid spectra (C14:0, C18:0 and tuberculostearic [TBS] acids) studied by multivariate statistical analyses. Strains P-55 and DENT are closer to M. paratuberculosis strain 5889 than to M. avium strain 16909-338, a finding which is in harmony with earlier immunological observations. The recently isolated M. paratuberculosis strain 385 has proved different from M. paratuberculosis strain 5889.

Radiolabelling of Mycobacterium avium oligosaccharide determinant and use in macrophage studies.

Internal radiolabelling procedures were used to radiolabel the oligosaccharide determinant of the glycopeptidolipids (GPL) from serovars 4 and 20 of the Mycobacterium avium complex. Mycobacteria were cultured in the presence of [6-3H]fucose, [2-3H]mannose or [methyl-3H]methionine, after which radiolabelled native lipid was extracted and distribution of radioactivity in native and deacetylated lipid was determined by thin-layer chromatographic methods. Incorporation of radiolabel was confirmed by examining acid hydrolysates of purified GPL for 3H-labelled sugars on cellulose thin-layer plates. Least incorporation of radiolabel into GPL was observed with [6-3H]fucose, whereas better incorporation was obtained with [2-3H]mannose and [methyl-3H]methionine. Use of [methyl-3H]methionine resulted in the radiolabelling of the methylated sugars in both the oligosaccharide determinant and the 3,4-di-O-methylrhamnose located at the terminus of the peptide core. Use of [2-3H]mannose resulted in the incorporation of radioactivity into the oligosaccharide determinant as 2-O-methylfucose, found in the GPL of both serovars 4 and 20. GPL radiolabelled with [2-3H]mannose were subsequently examined in macrophage cultures and found to be relatively inert to degradation by those phagocytic cells. These results substantiate earlier findings with the GPL of serovar 20 and indicate that these mycobacterial components may play a role in pathogenesis.

Characterization of distinct layers of the Mycobacterium avium envelope in respect of their composition by fatty acids, proteins, oligosaccharides and antigens.

The distribution of fatty acids, proteins, polysaccharides and antigens in subcellular fractions of Mycobacterium avium is described. Significant qualitative differences in the chemical composition of the various fractions have been used to further characterize the tripartite structure of the cell wall. In the outer dense layer (POL), in addition to previously described complex amphiphatic lipids, new oligosaccharides (lipooligosaccharides?) and a major glycoprotein were located; and it was found that tuberculostearic acid (TSA) esterified the phospholipids of this outerlayer. Judging from the data, it was proposed that the phospholipids formed a basic matrix monolayer in which other compounds of the POL intercalated. It was suggested that in an aqueous environment the hydrophobic ends of the phospholipids oriented to face the mycolic acid residues of the cell wall skeletons (or CWS) to form the 12 nm thick electron transparent layer. The purified CWS contained alpha-, keto-, and dicarboxylic mycolic acids; alanine, glutamic acid and diaminopimelic acid; and arabinose and galactose. Two additional nonidentified amino acids and an unidentified sugar were found in the CWS. Also, in the CWS the fatty acids: palmitic acid (21.8%), oleic acid (4.3%), stearic acid (9.2%) and TSA (4.3%), were detected. The main fatty acids detected in the cytoplasmic membrane (CM) were palmitic (20%), oleic (14.5%) and stearic (8.6%) acids. Mycolic acids and TSA were absent in the CM phospholipids. The major proteins of the CM (86, 40, and 26 Kd proteins) were distinct from the major proteins detected in the cytosol (CYT) fraction (43, 36, and 19 Kd proteins). A 58 Kd protein was present in both the CM and the CYT. The CYT and CM antigens were found absent in surface antigens extracted using sodium dodecyl sulphate (SDS).

Identification of major slowly growing pathogenic mycobacteria and Mycobacterium gordonae by high-performance liquid chromatography of their mycolic acids.

A rapid, reverse-phase high-performance liquid chromatography method was used to detect rho-bromophenacyl mycolic acid ester patterns for strains of four major pathogenic Mycobacterium species and for the most commonly encountered saprophytic species, Mycobacterium gordonae. Mycobacteria in low numbers (2.5 X 10(6) CFU) were detected and identified to the species level. Standard chromatographic patterns characteristic of each species were established. Simple pattern recognition enabled rapid identification of M. tuberculosis, M. kansasii, M. avium, M. intracellulare, and M. gordonae.

Structure of the cell envelope of Mycobacterium avium.

In this report the cell wall of Mycobacterium avium is shown as a triple-layered structure where the outermost layer was stained by the ruthenium red staining for polysaccharides. The outermost layer hindered the diffusion of chemotherapeutic agents across the wall thus causing multiple drug-resistance by exclusion. The concerted electron microscopy and chemical analysis of chloroform-methanol and Triton X-100 extracts indicated that the outer layer was made of diverse amphiphil glycolipids (mycosides C, glycolipids, peptidolipids, phospholipids) that formed a matrix in which proteins were embedded. The examination of a spontaneous rough mutant indicated that mutations blocking the synthesis of one or several of the amphiphils must leave unsubstituted mycolic acid residues, thus causing surface hydrophobicity and roughness. Judging from our data, a model describing the overall cell envelope of M. avium was proposed. From the comparative analysis of M. avium, its spontaneous rough mutant, and its spheroplasts, some of the functions of the outermost layer were disclosed.

Heating cells in acid methanol for 30 min without freeze-drying provides adequate yields of fatty acids and alcohols for gas chromatographic characterization of mycobacteria.

We studied the release of mycobacterial fatty acids (as methyl esters) and secondary alcohols after heating both wet and freeze-dried cells in methanolic hydrogen chloride for different time periods. A 30-min heating of the mycobacteria without prior freeze-drying was found adequate in a routine gas chromatographic procedure for strain and species characterization.

Mycolic acid analysis for clinical identification of Mycobacterium avium and related mycobacteria.

We examined the mycolic acid composition of 133 strains belonging to MAIS complex (Mycobacterium avium-M. intracellulare-M. scrofulaceum) and MAIS intermediate strains and the related species M. asiaticum, M. malmoense, M. shimoidei, and M. simiae. The analysis revealed that about 10% of the strains identified as M. avium-M. intracellulare complex by conventional cultural and biochemical tests were in fact M. simiae strains according to their mycolate composition. Of 25 strains previously studied by the International Working Group on Mycobacterial Taxonomy, 2 (MAIS intermediate and M. asiaticum) presented patterns incompatible with the clusters to which they had been assigned. M. malmoense and both M. simiae serovars shared the same pattern with alpha-, alpha'-, and ketomycolates. We describe here the method used to identify the mycolic acid profiles in detail. We found it to be highly reproducible and convenient for use in mycobacterial reference laboratories.

Analysis of mycolic acid cleavage products and cellular fatty acids of Mycobacterium species by capillary gas chromatography.

After growth and experimental conditions were established, the mycolic acid cleavage products, constituent fatty acids, and alcohols of representative strains of Mycobacterium tuberculosis, M. smegmatis, M. fortuitum complex, M. kansasii, M. gordonae, and M. avium complex were determined by capillary gas chromatography. Reproducible cleavage of mycolic acid methyl esters to tetracosanoic (24:0) or hexacosanoic (26:0) acid methyl esters was achieved by heating the sample in a high-temperature muffle furnace. The major constituent fatty acids in all species were hexadecanoic (16:0) and octadecenoic (18:1 omega 9-c, oleic) acids. With the exception of M. gordonae, 10-methyloctadecanoic acid was found in all species; moreover, M. gordonae was the only species tested which contained 2-methyltetradecanoic acid. M. kansasii was characterized by the presence of 2,4-dimethyltetradecanoic acid, M. avium complex by 2-eicosanol, and M. tuberculosis by 26:0 mycolic acid cleavage product. The mycolic acid cleavage product in the other five species tested was 24:0. Although a limited number of strains and species were tested, preliminary results indicate that this gas chromatographic method can be used to characterize mycobacterial cultures by their mycolic acid cleavage products and constituent fatty acid and alcohol content.

Isolation and restriction endonuclease analysis of mycobacterial DNA.

A method for the isolation of DNA from mycobacteria propagated in vitro is described that utilizes organic solvents to extract lipoidal components from the outer membrane, and digestion with a protease (nagarse) and lysozyme to penetrate the cell wall. The mycobacterial cells were lysed by the addition of detergent and the DNA was purified by digestion with pronase, sequential phenol and chloroform extractions, and digestion with RNAase A. The isolated DNA, which was obtained in good yields, was of a relatively high Mr and could be readily digested by restriction endonucleases. By this method, the genomes of Mycobacterium avium, M. intracellulare, M. lepraemurium, 'M. lufu', M. marinum, M. phlei, M. scrofulaceum, M. smegmatis and M. tuberculosis were isolated and the restriction endonuclease digestion patterns analysed. Each species could be distinguished by the digestion patterns, indicating that this approach can be used for identifying mycobacterial species. This approach is also sufficiently sensitive to differentiate strains since we were able to distinguish two independently isolated strains of M. tuberculosis, H37 and H4. In addition, no evidence was obtained for the presence of methylcytosine residues in the sequences 5'.CCGG.3',5'.CCCGGG.3',5'.CC(A/T) GG.3' or for methyladenine at 5'.GATC.3' in the DNA of the nine mycobacterial species examined using pairs of restriction enzymes that recognize and cleave at the same nucleotide sequence but differ in their sensitivity to 5-methylcytosine or 6N-methyladenine.

Use of gas chromatography to differentiate Mycobacterium leprae from cultivable armadillo-derived mycobacteria, M. avium/intracellulare, and M. lepraemurium by analysis of secondary alcohols.

Two long-chain secondary alcohols, 2-octadecanol and 2-eicosanol, were demonstrated by gas chromatography in hydrolysates of Mycobacterium avium/intracellulare, in cultivable, armadillo-derived mycobacteria, and in M. lepraemurium grown in vivo, but they were not found in purified suspensions of M. leprae isolated from experimentally infected armadillos. Gas chromatographic analysis of these alcohols constitutes a method for rapid detection and quantification of contaminating mycobacteria in preparations of M. leprae intended, for example, for vaccine use. The technique may also be of value for critical evaluation of cultures of "in vitro-grown" M. leprae.

Cytochemical characterization of mycobacterial outer surfaces.

A cytochemical study of mycobacterial outer surfaces was carried out on both pathogenic (M. leprae, M. avium) and non pathogenic (M. aurum) strains. Different cytochemical markers were used: Ruthenium Red, Concanavalin A, Wheat Germ Agglutinin, Colloidal Iron and Cationized Ferritin. The cytochemical staining pattern varied according to the species studied. The relationship between outer surface properties of mycobacteria and their capacity of adhesion to or ingestion by bone marrow macrophages was also considered.

Free fatty acid and triglyceride content of Mycobacterium avium cultured under different growth conditions.

Previous investigations demonstrated that Mycobacterium avium has a requirement for fatty acid, which can be fulfilled by palmitic (C16:0) or oleic (C18:1) acids, and that it incorporates the fatty acid into triglycerides that are later utilized. Mycobacterium avium was grown in continuous culture or batch-cultured in medium that contained palmitic acid as the fatty acid source, but lacked albumin. Cells were extracted and free fatty acids and triglycerides were obtained by preparative thin-layer chromatography. The triglycerides were further purified by column chromatography. The free fatty acids were methylated and analyzed by gas chromatography. Oleic acid represented 40 to 64% of the total free fatty acids, except for cells batch-cultured and limited for nitrogen. The latter cells contained about 27% oleic acid, and 24% of the free fatty acids were of sizes greater than C24:0. The amount of palmitic acid varied considerably, but it and oleic acid together usually accounted for 70% of the total free fatty acids. The overall fatty acid content of the triglycerides was similar to that of the free fatty acids. However, two size classes of triglycerides were found that approximated the sizes of tristearin (C54) and tricaprylin (C24), molecular weights of 892 and 471 daltons, respectively. It is concluded that these types of studies may eventually lead to more accurate identification of pathogenic mycobacteria by means of gas chromatographic analyses.

Acidic methanolysis v. alkaline saponification in gas chromatographic characterization of mycobacteria: differentiation between Mycobacterium avium-intracellulare and Mycobacterium gastri.

Mycobacterium avium-intracellulare and M.gastri were analyzed with capillary gas chromatography after each strain had been subjected to acidic methanolysis or to alkaline saponification followed by methylation. Prominent peaks of myristic, palmitoleic, palmitic, oleic, stearic and tuberculostearic acids were found in the chromatograms of both species, whereas 2-octadecanol and 2-eicosanol were detected only in M. avium-intracellulare. In initial runs, both of the derivatization principles yielded virtually identical chromatograms for a given strain. After repeated injections of extracts from alkaline saponification, however, the alcohol peaks showed pronounced tailing and finally almost disappeared from the chromatograms. This disadvantage, which was not observed when only acid methanolysis was used, could be overcome with trifluoroacetylation. Restored peak shape of the underivatized alcohols could be achieved by washing the cross-linked stationary phase in the capillary tubing with organic solvents. The study demonstrated the importance of conditions which enable separation of 2-octadecanol and 2-eicosanol when gas chromatography is used for species identification of mycobacteria.

Gaschromatography of constitutive fatty acids in Mycobacterium leprae.

A constitutive saturated and monounsaturated fatty acid pattern of Mycobacterium leprae, isolated from the liver of a nine-banded armadillo with experimental leprosy, was analyzed gaschromatographically and compared with that of cultured M. lepraemurium, M. avium, M. bovis, strain BCG and M. smegmatis. In comparing the fatty acid pattern thus obtained and the known structure of mycolic acids in these mycobacteria, an experiential rule that each species of mycobacteria has a relatively high content of normal (straight-chained) saturated fatty acid having two more carbons than those of the alpha-branch in this species' mycolic acids, coincided well for all mycobacteria tested. In particular, M. leprae was found to contain a relatively high content of behenic acid (n-C22:0) and the carbon-number of the alpha-branch in this species' mycolic acids is 20 as we previously reported. These data suggested the possibility of simple detection of M. leprae by gaschromatography, and results sustaining this possibility were obtained.

Fatty acid and polar lipid analysis as tools in the identification of Mycobacterium leprae and some related slow-growing mycobacterial species.

Some species of slow-growing Mycobacteria, including Mycobacterium leprae (1 strain), M. lepraemurium (2 strains), M. paratuberculosis (12 strains) and a group of 12 M. avium-like strains (isolates from wild animals) were examined by gas chromatography (GC) for cellular fatty acids and by thin-layer chromatography (TLC) for polar lipids. All the GC patterns, including that of M. leprae, contained high levels of tuberculostearic-, stearic-, octadecenoic- and palmitic acid. Tetradecanoic-, pentadecanoic-, hexadecenoic- and heptadecanoic acid were also generally present but in lower concentrations. In addition to these acids shared by all strains, each bacterial species or group was found to exhibit compounds which were not detected (or detected in considerably lower quantities) in the other taxa examined. Thus each bacterial species or group could be distinguished by their GC profiles. The corresponding TLC patterns were also rather complex. A total of 39 different spots were distinguished. A few of these were shared by all strains, some were characteristic of certain species or groups, whereas others were strain-specific. Both M. leprae and M. lepraemurium shared several features with the other strains but could be distinguished from each other and from the others by their patterns of slow-moving (polar) spots. The 12 M. avium-like strains were divided into two main groups, one with only a few slow-moving spots (rough stains), and one with several slow-moving spots (smooth strains) which included the M. avium reference strains.

Technique for extracting niacin from Mycobacterium tuberculosis cultured on 7H-10 and 7H-11 agars.

Niacin extractions from M. tuberculosis growing on 7H-10 or 7H-11 agar base medium yield consistent results when performed at 37 degrees C for 2 h. The technique does not require any modification of the media formulation.