Mycolic acids from Rhodococcus, Gordonia, and Dietzia

The mycolic acids from 11 species of Rhodococcus, seven species of Gordonia, and one species of Dietzia were analyzed using capillary gas chromatography and mass spectrometry (GLC/MS). All strains tested in this study were divided into three groups according to the degree of double bonds and the average carbon number (Av.Nc.) of their mycolic acids. The genus Gordonia belongs to the first group possessing an Av.Nc. in the upper 50s and 60s with 0 to 5 double bonds. Some Rhodococcus species possessed Av.Nc. in the 40s with a variety of distributions of polyunsaturated fatty acids from 0 to 4. The rest of the Rhodococcus species and the genus Dietzia possessed Av.Nc. in the 30s with saturated fatty acids. We previously reported on Nocardia strains whose Av.Nc. were in the 50s. Considering the identification of mycolic acid-containing Actinomycetales at the generic level, the Av.Nc. proved to be useful as a means of differentiating the genera Rhodococcus, Gordonia and Nocardia. The genus Dietzia was found to have its own characteristic constitution of mycolic acid molecular species. The mycolic acids from D. maris 58001T were characterized by an almost equal amount of constituents of even- and odd-numbered carbon chains, whereas the major components of mycolic acids in all other strains had even-numbered carbon chains. Another characteristic of Dietzia was some even-numbered mycolic acids which contained odd-numbered straight chains with odd-numbered alpha-branches. These characteristics indicated that Dietzia might possess a novel fatty acid biosynthesis system.     

Effect of growth stage on mycolic acid structure in cell walls of Nocardia asteroides GUH-2

Mycolic acids were extracted from the cell walls of Nocardia asteroides GUH-2 during different phases of growth at 37 degrees C. These were subjected to structural analysis by combining thin-layer chromatography and gas-liquid chromatography with UV and infrared spectrophotometry and mass spectroscopy of both methyl esters and trimethyl silyl derivatives. By analyzing the fragmentation patterns of these derivatives by three different methods of mass spectroscopy combined with gas-liquid chromatographic separation, the different structural subclasses of mycolic acids were quantitated. Significant qualitative and quantitative modifications of specific mycolic acid subclasses occurred in the cell walls of N. asteroides GUH-2 that were growth stage dependent. The mycolic acids that were predominant in the log phase were polyunsaturated (greater than 2 double bonds per molecule), with long chain lengths and even carbon atom numbers (i.e., C54, C56). In contrast, those that were prominent in the stationary phase were more saturated (few or no double bonds) and of shorter overall carbon chain length (less than or equal to C52). Furthermore, stationary-phase cells had significantly increased amounts of mycolic acids with odd-numbered carbon chain lengths (i.e., C49, C51, C53).     

Mouse pathogenicity studies of Nocardia asteroides complex species and clinical correlation with human isolates

Abstract Nocardia asteroides complex organisms derived from human specimens between 1979 and 1992 were identified on the species level. Of 117 N. asteroides complex organisms, 34 (29%) were N. farcinica , 28 (24%) were N. nova , and 55 (47%) were N. asteroides sensu stricto . An analysis of the specimen sites from which the organisms were derived showed that isolates derived from blood, brain, or bone marrow were more likely to be N. farcinica than the other two species. A study of the virulence of ten strains of each species was undertaken, using a mouse model with intravenous inoculation. The 50% lethal doses (LD₅₀) for N. farcinica were significantly lower than those of the other two species. LD₅₀ values for N. nova and N. asteroides were not significantly different. The above data confirming the greater virulence of N. farcinica support the identification of species within the N. asteroides complex.     

Inactivation of kanamycin A by phosphorylation in pathogenic Nocardia.

Among the five species of pathogenic Nocardia, i.e., N. asteroides, N. brasiliensis, N. farcinica, N. nova and N. otitidiscaviarum, all strains of N. brasiliensis and N. farcinica showed resistance to an aminoglycoside antibiotic, kanamycin A, showing the MIC (minimum inhibitory concentration) values of more than 100 micrograms/ml. This species-specific difference in sensitivity was found to be explained by the production of an inactivation enzyme, aminoglycoside 3'-phosphotransferase APH(3'). Structural studies by mass and NMR spectroscopy on the inactivated substance produced by a cell-free extract of the Nocardia confirmed the conversion of kanamycin A to an inactive substance, kanamycin A 3'-phosphate. The MIC values of N. otitidiscaviarum and N. nova for kanamycin A, on the other hand, ranged from 0.78 micrograms/ml to 100 micrograms/ml, and both species were non-producers of APH(3'). Sensitivity to the antibiotic and APH(3') productivity of N. asteroides varied depending on the strain.     

Thehalose mycolates from Nocardia asteroides,Nocardia farcinica,Gordona lentifragmenta and Gordona bronchialis

Isolation of glycolipids from Nocardia asteroides, N. farcinica, Gordona lentifragmenta and G. bronchialis, by column chromatography of lipid extracts on a 50% (w/w) mixture of silicic acid and silica gel H, is described. The isolated materials were partially characterized by infrared spectroscopy, optical rotation and refractive index measurements, and by identifying the products of alkaline hydrolysis. Analytical studies showed that the glycolipids released only trehalose in the aqueous phase while mycolic acids were the constituent fatty acids identified.The isolated lipids are trehalose esters in which the trehalose molecule is esterified with mycolic acids.     

Nocardia asteroides in the Soil of Kuwait

A pilot study was undertaken to determine the occurrence and distribution of pathogenic nocardiae in Kuwaiti soil. A total of 102 soil samples collected from two localities were investigated by the paraffin bait technique. Nocardia asteroides was the only species isolated from 42 (41%) soil samples. None of the isolates fulfilled the criteria required for identification of N. farcinica or N. nova. Thirty one (73.8%) isolates showed equivalent growth at 45 °C and 35 °C, 17 (40.4%) isolates utilized acetamide for carbon and nitrogen requirements and 3 (7.1%) isolates showed delayed arylsulphatase activity. Only a solitary isolate was resistant to cefamandole. Soil samples originating from the Kuwait University Campus Shuwaikh, which were rich in humus/organic matter, were more productive for N. asteroides (67%) than the samples which were devoid of it but were mixed with crude oil (39%). Sand samples that lacked organic matter and crude oil samples were least productive of N. asteroides. These preliminary findings do not suggest that massive oil contamination of soil in the Ahmadi oil field area during the Gulf war promoted the natural occurrence of N. asteroides. However, isolation of N. asteroides in as many as 41% of the soil sample is a significant observation warranting further epidemiologic studies including its possible role in the operation desert storm sickness syndrome. This is the first report on the natural occurrence of N. asteroides in Kuwait. This revised version was published online in June 2006 with corrections to the Cover Date.     

A case of lung infection caused by an unusual strain of Nocardia farcinica

A case of lung infection caused by an unusual strain of Nocardia farcinica is reported. This is the third case of the N. farcinica infection in this country. The strain failed to utilize rhamnose as sole carbon source, but could be identified by a numerical identification method. The mycolic acids contained 1-3 double bonds and the numbers of the carbon atoms of the mycolic acids were 50 to 60, average 56.     

Nocardia in soils of southeastern Spain: abundance, distribution, and chemical characterisation

Actinomycetes belonging to the genus Nocardia were isolated from the surface horizon of 15 out of 46 soil samples examined. All the nocardiae strains isolated contained mycolic acids and saturated and unsaturated straight chain fatty acids (from 12 to 18 carbon atoms) and tuberculostearic acid and were biochemically identified as members of the Nocardia asteroides complex. Nocardiae were detected in alluvial, brown, and serosem great soil groups, but not in calcic brown, solontchack, and regosol great soil groups. Numbers of nocardiae isolated varied from 5.12 X 10(2) to 1.21 X 10(4) colony-forming units per gram of dry weight, and they were statistically correlated with the carbon content (percent C) of the soil. Soil samples were, in general, very dry.     

Lipid Composition in the Classification of Nocardiae and Mycobacteria

Ninety-six strains of aerobic actinomycetes with a type IV cell wall (major amounts of meso-diaminopimelic acid, arabinose, and galactose) were analyzed for the presence of mycolic acids and nocardomycolic acids. The method used was comparatively simple and permits the separation of these organisms into two groups: the mycobacteria and the nocardiae. In general, strains received as mycobacteria contained mycolic acids, confirming the generic assignment made by other methods. On the basis of nocardomycolic acid content, Mycobacterium brevicale, M. rhodochrous, and M. thamnopheos should be placed in the genus Nocardia, and on the basis of mycolic acid content, strains recently isolated from bovine farcy should be placed in the genus Mycobacterium. Nocardia farcinica should be considered a nomen dubium and N. asteroides should be considered the type species of the genus.     

Immunodiffusion studies of some Nocardia strains

Forty-three strains of Nocardia, one of Actinomadura and two of Nocardiopsis were studied using the comparative immunodiffusion technique. Three reference precipitation systems were employed: one represented Nocardia asteroides N10, one N. asteroides ATCC 19247, and one N. otitidis-caviarum ATCC 14629. One tight cluster was formed by the N. otitidis-caviarum strains and another tight cluster was formed by some of the N. asteroides strains studied. However, other strains of N. asteroides were distinct from the latter cluster. Furthermore, N. asteroides ATCC 19247, which is the type strain, differed from most ot the N. asteroides strains tested. Strains of the species N. asteroides, N. brasiliensis, N. farcinica and N. otitidis-caviarum were found to be closely related, while N. amarae strains differed slightly from this group. The strains referred to Actinomadura and Nocardiopsis were clearly distinct from the three Nocardia reference strains; nevertheless, three antigens common to these genera were revealed.     

Isonicotinic acid hydrazide induced changes and inhibition in mycolic acid synthesis in Nocardia and related taxa

The mycolic acid compositions of Nocardia rubra and related bacteria grown in media containing different concentrations of antituberculous isonicotinic acid hydrazide (INH) were determined in detail by gas chromatography-mass spectrometry. On the basis of molecular species composition, average carbon numbers of mycolic acids were calculated. In Nocardia rubra, N. lutea and Rhodococcus rhodochrous IFO-13161, the ratio of mycolic to non-mycolic fatty acids and the average carbon numbers of mycolic acids were decreased at the INH concentrations of higher than 1 g/ml, paralleling with the significant inhibition of growth. In above three species the synthesis of longer chain mycolic acids (longer than C₄₄ or C₄₆) was inhibited more significantly than shorter homologues such as C₃₈ or C₄₀. In contrast, neither growth inhibition nor change in corynomycolic acid composition was observed in Corynebacteria xerosis and Rhodococcus rhodochrous IFO-13165 at the concentration region of INH up to 100 g/ml. The direct mass fragmentographic analysis of the trimethylsilylated (TMS) derivatives of mycolic acid methyl esters, monitoring [M-15] ions of individual molecular species, revealed that the chain shortening of total mycolic acid molecule by INH occurred more greatly in more highly unsaturated subclasses than in less unsaturated subclasses. Furthermore, mass fragmentographic analysis, monitoring fragment ions (A) and (B), due to straight chain and branched chain alkyl units, respectively, demonstrated the inhibition of mycolic acids was not attributed to the shortening of -alkyl chain, but to the inhibition of chain elongation of C₂₈ to C₃₂ straight chain meromycolic acids. It was also indicated the amounts of trehalose mono- and di-mycolate (cord factor) decreased significantly with the addition of INH (1 to 20 g/ml) in the above strains. From the results obtained above, INH appeared to inhibit the synthesis of mycolic acids longer than C₄₄ or C₄₆ specifically by inhibiting chain elongation or desaturation of precursor long chain fatty acids longer than C₂₈ or C₃₀.     

Effects of culture conditions on the mycolic acid composition of isolates of Rhodococcus spp. from activated sludgefoams

The influence of two different carbon sources and three incubation temperatures on the mycolic acid compositions of three Rhodococcus isolates from activated sludge was examined using Selective Ion Monitoring (SIM) gas chromatography-mass spectrometry (GC-MS). Considerable qualitative and quantitative differences were detected in the mycolic acid compositions of the three very closely related isolates grown under the same conditions. Culture age also affected both the chain lengths and proportions of saturated mycolic acids detected in cell extracts, but not in the same manner for each isolate. Mycolic acids generally were of shorter chain lengths in cells grown with Tween 80 compared to glucose grown cells in strain 11R but the opposite situation occurred with strains A7 and D5. In all three, the proportion of unsaturated mycolic acids decreased with increasing growth temperatures. The taxonomic relevance of these observations and possible explanations for the observed changes in mycolic acid composition under various culture conditions are discussed.     

Free mycolic acids as criteria in the classification of Nocardia and the 'rhodochrous' complex.

The methyl esters of free mycolic acids from representative strains of Nocardia asteroides, N. brasiliensis, N. caviae and the 'rhodochrous' complex were subjected to detailed mass spectral analysis. The anhydromycolic esters of the Nocardia strains consisted of homologous series containing from zero to three double bonds, with the main components of the parent mycolic acids centred on C52 to C54 (range C46 to C58). The anhydromycolates from one rhodochrous strain, Nocardia opaca, had a molecular weight range similar to the nocardiae (C46 to C57) but the remaining rhodochrous strains gave an homologous series of anhydromycolates containing from zero to two double bonds, with the main components of the parent mycolic acids centred on C38, C42, C44 or C46 (total range from C34 to C50). The mycolic acids from the rhodochrous strains with chain lengths centred around C40 form a group intermediate in size between corynomycolic acids (centred around C32) and nocardomycolic acids (centred around C50). These data weaken the case for retaining the 'rhodochrous' complex in the genus Mycobacterium, and also show that many rhodochrous strains can be distinguished from true nocardiae and corynebacteria. These results confirm the value of lipid characters in the classification of these organisms.     

Purification and structure analysis of mycolic acids in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum is widely used for producing amino acids. Mycolic acids, the major components in the cell wall of C. glutamicum might be closely related to the secretion of amino acids. In this study, mycolic acids were extracted from 5 strains of C. glutamicum, including ATCC 13032, ATCC 13869, ATCC 14067, L-isoleucine producing strain IWJ-1, and L-valine producing strain VWJ-1. Structures of these mycolic acids were analyzed using thin layer chromatography and electrospray ionization mass spectrometry. More than twenty molecular species of mycolic acid were observed in all 5 strains. They differ in the length (20–40 carbons) and saturation (0–3 double bonds) of their constituent fatty acids. The dominant species of mycolic acid in every strain was different, but their two hydrocarbon chains were similar in length (14–18 carbons), and the meromycolate chain usually contained double bonds. As the growth temperature of cells increased from 30°C to 34°C, the proportion of mycolic acid species containing unsaturated and shorter hydrocarbon chains increased. These results provide new information on mycolic acids in C. glutamicum, and could be useful for modifying the cell wall to increase the production of amino acids.     

Immunochemical characterization of Nocardia asteroides antigens: support for a single species concept

Nocardia asteroides strains are highly heterogeneous. They show morphological, physiological, and immunological differences. In a previous study, we delineated seven immunotypes of N. asteroides. In the present study, we compared the culture filtrate antigens of these immunotypes by antigen-antibody crossed-immunoelectrophoresis and by rocket electrophoresis. We have also compared the antigen preparations by two-dimensional electrophoresis. While unique components constitute the major portion of the components, the results indicate that similar components are present in the culture filtrates of all strains. This finding supports the view of retaining all the immunotypes in the species Nocardia asteroides rather than designating different species such as N. farcinica and N. sebivorans.     

Cell wall modification resulting from in vitro induction of L-phase variants of Nocardia asteroides.

The chemical composition of the cell walls of several L-form revertants derived from Nocardia asteroides 10905 was determined at different stages of growth. It was observed that each L-form revertant had a cell well that differed from that of the parental strain when grown under identical conditions. In some strains the peptidolipid and mycolic acid components were affected the most, whereas in other strains the fatty acid, sugar, and mycolic acid moieties were altered. Shifts in mycolic acid size were prominent, whereas the basic peptidoglycan structure appeared to be affected the least. Both the method used to induce the L-form of N. asteroides 10905 and the length of time these organisms were maintained in the wall-less state affected the degree of cell wall modification during the reversion process. Thus, removal of the cell wall appeared to potentiate and select for mutational alterations within the cell envelope of N. asteroides, and these changes resulted in altered cellular and colonial morphology.     

Trafficking pathways of mycolic acids: structures, origin, mechanism of formation, and storage form of mycobacteric acids

Mycolic acids, the hallmark of mycobacteria and related bacteria, are major and specific components of their cell envelope and essential for the mycobacterial survival. Mycobacteria contain structurally related long-chain lipids, but the metabolic relationships between these various classes of compounds remain obscure. To address this question a series of C(35) to C(54) nonhydroxylated fatty acids (mycobacteric acids), ketones, and alcohols structurally related to the C(70-80) dicyclopropanated or diethylenic mycolic acids were characterized in three mycobacterial strains and their structures compared. The relationships between these long-chain acids and mycolic acids were established by following the in vivo traffic of (14)C labeled alpha-mycolic acids purified from the same mycobacterial species. The labeling was exclusively found in mycobacteric acids. The mechanism of this degradation was established by incorporation of (18)O(2) into long-chain lipids and shown to consist in the rupture of mycolic acids between carbon 3 and 4 by a Baeyer-Villiger-like reaction. We also demonstrated that mycobacteric acids occur exclusively in the triacylglycerol (TAG) fraction where one molecule of these acids esterifies one of the three hydroxyl groups of glycerol. Altogether, these data suggest that these compounds represent a pathway of metabolic energy that would be used by mycobacteria in particular circumstances.     

Mycolic acid metabolic filiation and location in Mycobacterium aurum and Mycobacterium phlei

Synchronous cultures of Mycobacterium aurum were used to prove a close relationship between cellular division and active synthesis of mycolic acids (characteristic long-chain 3-hydroxyacids, branched at position 2), confirming previous proposals. Mycolic acid biosynthesis was studied in two species (Mycobacterium phlei and M. aurum) each producing three types of mycolic acids: di-unsatured mycolates, oxomycolates and wax-ester mycolates (ester of dicarboxymycolic acid and 2-icosanol or 2-octadecanol). It was shown that unsaturated mycolates and oxomycolic acids were not directly related, whereas a metabolic filiation was confirmed between oxomycolate and wax ester mycolate: the latter derived from the former by a Baeyer-Villiger oxidation step, as has been proposed on the basis of structural considerations. By observing the labelling of the different mycolate pools in the cell, i.e. the organic-solvent-extractable fraction (essentially containing esters of trehalose and of glycerol) and the cell residue (assumed to be the cell-wall polymers), it was clear that oxomycolates and unsaturated mycolates appeared first in the extractable lipids, then in the wall-linked mycolates while wax-ester mycolates appeared first as wall-linked derivatives. Thus, it is proposed that mycolates could follow separate routes involving differently located enzymes to reach their complex forms either in extractable lipids or in the wall-linked arabino-galactan.     

Differentiation of nocardia species by PCR-randomly amplified polymorphic DNA fingerprinting

Representatives of fifteen validly described and three non-validly described species of Nocardia were assigned to nineteen groups based on an optimised PCR-randomly amplified polymorphic DNA fingerprinting technique. Species specific banding patterns were recognised for the representatives of N. brasiliensis, N. crassostreae, N. farcinica, N. otitidiscaviarum and N. seriola. Unique banding patterns were also seen for the type strains of N. brevicatena, N. carnea, N. salmonicida, N. uniformis and N. vaccinii, and for the single representatives of "N. fusca", "N. pseudosporangifera", and "N. violaceofusca". More than one banding pattern was detected for the N. asteroides, N. flavorosea, N. nova, N. pseudobrasiliensis and N. transvalensis strains though in the case of the representative strains of N. nova and N. transvalensis the patterns were similar for each of these species. The results are in line with current trends in nocardial systematics thereby indicating that PCR-randomly amplified polymorphic DNA fingerprinting provides valuable data for the classification and identification of pathogenic nocardiae to the species level.     

Endogenous phospholipids of swine liver: effect of fat deprivation on molecular species of phosphatidylcholine and phosphatidylethanolamine

Three 1-yr-old swine and two 2.5-wk-old swine were fed a fat-free diet for 1 month and 5 months, respectively. The hepatic phosphatidylcholine and phosphatidylethanolamine were fractionated by silver ion thin-layer chromatography. A distinctive feature of the chromatographic procedure was the development of the chromatograms at low temperatures: -10 degrees C for phosphatidylcholine and 4 degrees C for phosphatidylethanolamine. The chromatographic fractions were hydrolyzed with phospholipase A(2), and the fatty acids were characterized. Significant concentrations of odd-chain saturated and unsaturated fatty acids were found in the swine deprived of fat for 5 months. The major molecular species of phosphatidylcholine in both groups contained monoenoic fatty acids: 16:0/18:1(n - 9), 18:0/18:1(n - 9), and 18:1(n - 9)/18:1(n - 9). Their concentrations changed only slightly with the diet. The molecular species of phosphatidylethanolamine were more sensitive to dietary changes. In the swine deprived of fat for 1 month, about 50% of the molecular species of phosphatidylethanolamine contained tetraenoic fatty acids: 16:0/20:4(n - 6), 18:0/20:4(n - 6), and 18:1(n - 9)/20:4(n - 6). The phosphatidylethanolamine of animals deprived of fat for 5 months contained only 3% molecular species with tetraenoic acids, 18:0/20:4(n - 6), but 36% molecular species with trienoic acids: 18:0/20:3(n - 9), 18:1(n - 9)/20:3(n - 9), 18:0/19:3(n - 8), 16:0/20:3(n - 9), and 17:0/20:3(n - 9). Doubly unsaturated species, such as 18:1(n - 9)/18:1(n - 9), 18:1(n - 9)/20:3(n - 9), and 18:1(n - 9)/20:4(n - 6), were found in both groups of swine, although their total concentrations were higher in the group deprived of fat for a longer period.