Capillary gas chromatographic analysis of mycolic acid cleavage products, cellular fatty acids, and alcohols of Mycobacterium xenopi.

The fatty acids, alcohols, and mycolic acids of 26 strains of Mycobacterium xenopi were studied by capillary gas chromatography and thin-layer chromatography. All strains contained alpha-, keto-, and omega-carboxymycolates. The primary mycolic acid cleavage product was hexacosanoic acid. The fatty acid patterns and, especially, the presence of 2-docosanol are characteristic markers of M. xenopi.     

Characterization of 22 Vibrio species by gas chromatography analysis of their cellular fatty acids

The cellular fatty acid compositions of 51 Vibrio strains belonging to 22 species as well as five Aeromonas strains were determined by using capillary gas-liquid chromatography (GLC). The major fatty acids were most often hexadecenoic, hexadecanoic and octadecenoic acids. Heptadecenoic acid was present in significant amounts in V. alginolyticus, V. natriegens, V. parahaemolyticus and "Vibrio navarrensis". Twenty fatty acids including branched and hydroxy acids were detected in the genus Vibrio. Quantitative results were treated by principal component analysis to display groups of strains. The first three components (accounting for 69% of the variance) showed the type strains of V. fischeri, V. ordalii, V. damsela, V. mediterranei, V. tubiashii, V. campbellii, V. pelagius, V. gazogenes, and V. nereis to be unclustered. V. alginolyticus (4 strains) and V. parahaemolyticus (4 strains) showed some overlap and the type strain of V. natriegens was in their neighborhood. V. harveyi (4 strains) formed a cluster and V. vulnificus was in its vicinity. V. cholerae (5 strains) overlapped with V. diazotrophicus (3 strains) and was close to the type strain of V. mimicus and V. anguillarum. V. metschnikovii (3 strains) clustered with the type strain of V. cincinnatiensis. A decision tree was devised for the identification of Vibrio species based on qualitative characteristics of fatty acid patterns. However, the following three groups, V. alginolyticus-V. parahaemolyticus-V. natriegens, V. metschnikovii-V. cincinnatiensis and V. cholerae-V. mimicus could not be split into such a decision tree.     

High-performance liquid chromatography patterns of Mycobacterium gordonae mycolic acids.

An important class of fatty acids contained in the cell envelopes of Mycobacterium organisms is the group of high-molecular-weight, long-chain, alpha-branched, beta-hydroxylated mycolic acids. By using standard saponification techniques and derivatization of the acids to their p-bromophenacyl esters, it is possible to differentiate them by high-performance liquid chromatography. Mycolic acid chromatograms of 63 clinical isolates of Mycobacterium gordonae were compared with conventional biochemical methods for identification. The data show two distinct pattern types for this species, only one of which has been elaborated in the literature by using this protocol. Laboratory workers who intend to use this method as a clinical tool need to be aware that these two pattern types exist.     

Gas-chromatographic analysis of mycolic acid cleavage products in mycobacteria.

Mycolic acids were detected in both reference strains and clinical isolates of mycobacteria using gas chromatography of fatty acid methyl esters prepared by acid methanolysis. The methyl esters were extracted with hexane, concentrated, and analyzed with a gas chromatograph by using two different injector temperatures. When the samples were analyzed at high injector temperatures of 300 to 350 degrees C, characteristic thermal cleavage products from mycolic acids, C22:0, C24:0, or C26:0 fatty acid methyl esters, were detected. When analyzed at injector temperatures of 235 degrees C or lower, the mycolic acids were heat stable and the characteristic methyl ester cleavage products were not observed.     

Separation of Mycobacterium bovis BCG from Mycobacterium tuberculosis and Mycobacterium bovis by using high-performance liquid chromatography of mycolic acids.

Profile analysis of mycolic acid ester patterns of Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium bovis bacillus Calmette-Gúerin (BCG) using high-performance liquid chromatography indicated that separation of BCG from M. tuberculosis and M. bovis by elution and relative retention times is possible. Mycolic acid patterns of BCG eluted from the column 0.5 min before M. tuberculosis or M. bovis, resulting in relative retention times for two peaks not seen in the pattern of M. tuberculosis or M. bovis. Identification was confirmed by phage typing, which has been the standard procedure for confirmation of BCG strains. These results showed that high-performance liquid chromatographic analysis of mycolic acid esters can be used in the mycobacterial reference laboratory for separation of BCG from M. tuberculosis and M. bovis.     

Differentiation of Mycobacterium genavense and Mycobacterium simiae by automated mycolic acid analysis with high-performance liquid chromatography.

Mycobacterium genavense, a fastidious opportunist in patients with AIDS, cannot be identified by conventional biochemical methods. Computerized mycolic acid analysis by high-performance liquid chromatography offers an alternative that distinguishes the mycolic acid profile of M. genavense from those of all other organisms in the database developed at the Centers for Disease Control and Prevention.     

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.     

Differentiation of Peptococcus and Peptostreptococcus by gas-liquid chromatography of cellular fatty acids and metabolic products

Gas-liquid chromatographic (GLC) profiles of cellular fatty acids and metabolic products were useful in identifying strains of Peptococcus saccharolyticus, Peptococcus asaccharolyticus, Peptostreptococcus anaerobius, Peptostreptococcus micros, and Streptococcus intermedius. The GLC results supported the recent taxonomic decision to transfer aerotolerant Peptostreptococcus species to the genus Streptococcus. Because inconsistencies in the results prevented our differentiating Peptococcus prevotii. Peptococcus magnus, and Peptococcus variabilis by GLC, additional strains will have to been examined. These GLC techniques are amenable to routine use; however, for interlaboratory results to be meaningful, the classification and nomenclature of the anaerobic gram-positive cocci should be standardized.     

Identification of Achromobacter species by cellular fatty acids and by production of keto acids.

The cellular fatty acid composition and metabolic products of 12 reference strains of Achromobacter sp. and A. xylosoxidans were determined by gas-liquid chromatography (GLC). Results showed that the two Achromobacter groups are strikingly different and can be readily distinguished on the basis of cellular fatty acids and the short-chain acids produced by Achromobacter sp. The major cellular fatty acids of Achromobacter sp. were octadecenoic (18:1) and a 19-carbon cyclopropanoic (19:0 delta) acid, whereas hexadecanoic (16:0) and a 17-carbon cyclopropanoic (17:0 delta) acid were principal components of the lipids of A. xylosoxidans. Hydroxy acids were not found in strains of Achromobacter sp. but comprised approximately 20% of the cellular fatty acids of A. xylosoxidans. In addition, Achromobacter sp. produced relatively large amounts of 2-ketoisocaproic acid, which was detected in only trace amounts from strains of A. xylosoxidans. The data show that GLC tests provide additional criteria for differentiating groups which are very closely related when evaluated with conventional tests. The GLC tests can be readily adapted in the clinical laboratory because they are rapid, highly reproducible, relatively inexpensive, and simple to perform.     

Analysis of fatty acids of the genus Rochalimaea by electron capture gas chromatography: detection of nonanoic acid.

The fatty acid compositions of Rochalimaea quintana, strains Fuller and Guadalupe, and R. vinsonii, the Canadian vole agent, were determined in an effort to further characterize these bacteria. The cells were saponified with 5% NaOH in 50% methanol and acidified to pH 2. The methanolysates were extracted with chloroform, derivatized with 2,2,2-trichloroethanol, and analyzed using a Hewlett-Packard gas chromatograph equipped with a frequency pulse-modulated electron capture detector and a 3% OV-101 packed-glass column. The fatty acid profiles of the three Rochalimaea strains were similar, with octadecenoic acid (C18:1) the most abundant, followed by octadecanoic (C18:0) and hexadecanoic (C16:0) acids. Moderate to trace amounts of other acids were also present. Unexpectedly, well-defined peaks of nonanoic acid (C9) were found consistently. A portion of this acid, but not all, was extractable with chloroform. Since C9 is not reported as a usual component of bacteria and most analyses do not include a search for this fatty acid, this study was extended to three strains of Legionella and one of Campylobacter. Comparable results were obtained. Since these bacteria were grown in complex media which contain some C9, it is possible that the medium is the source of bacterial C9. Whether this compound can be synthesized by the bacteria remains to be investigated.     

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.     

High-performance liquid chromatography patterns of mycolic acids as criteria for identification of Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium smegmatis.

Rapidly growing mycobacteria of clinical significance were identified by mycolic acids detected with high-performance liquid chromatography. Mycolic acids from whole cells were extracted, derivatized, and detected by a modified high-performance liquid chromatography procedure in less than 3 h. Use of an internal standard allowed differentiation of Mycobacterium chelonae and Mycobacterium fortuitum by comparison of relative retention times. Peak height ratios were used for subidentification of M. chelonae strains; however, M. fortuitum and Mycobacterium smegmatis could not be separated by this system.     

Structure and stereochemistry of mycolic acids of Mycobacterium marinum and Mycobacterium ulcerans

Mycobacterium marinum and M. ulcerans were previously shown to synthesize lipid compounds which are stereochemically different from the corresponding molecules isolated from M. tuberculosis and other species. Stereochemical and biogenetic studies of mycolic acids isolated from M. marinum showed that the absolute configurations of the chiral centres occurring in the mycolates are identical to those of the other mycobacterial species examined so far. Furthermore, all the methyl branches were found to come from methionine whatever the configuration of the centre. The structures of the mycolates synthesized by M. marinum and M. ulcerans were found to be identical, consisting of dicyclopropyl and monocyclopropyl monoenoic mycolates, monoenoic keto- and methoxymycolates, thus reinforcing the taxonomical relationship between the two species.     

Gas-liquid chromatography of cellular fatty acids for identification of staphylococci.

A commercially available, computer-assisted microbial identification system (MIS) employs gas-liquid chromatographic analyses of bacterial fatty acids. The MIS was used to identify 470 isolates of Staphylococcus species. The accuracy of the MIS was compared with the accuracies of conventional methods. There was a complete agreement between the MIS and conventional methods in the identification of 413 (87.8%) strains. For 36 of 45 misidentified strains, the correct identification was listed by the MIS as a choice but not as the first choice. Twelve strains could not be matched. All strains of Staphylococcus cohnii, S. epidermidis, S. intermedius, S. lugdunensis, S. schleiferi, S. sciuri, S. simulans, and S. xylosus were correctly identified. Two species, S. hominis and S. saprophyticus, accounted for 52.6% (30 of 57) of the misidentifications. Seventy-eight organisms were retested. Identification of 73 organisms remained unchanged, and for five organisms, the second choice became first and vice versa. The overall performance of the MIS is acceptable, and the system can be used as an alternate identification method for staphylococci.     

Discriminant analysis of cellular fatty acids of Candida species, Torulopsis glabrata, and Cryptococcus neoformans determined by gas-liquid chromatography.

We used discriminant analysis of cellular fatty acid compositions determined by gas-liquid chromatography to differentiate yeastlike fungi (a total of 190 strains; including 37 Candida albicans strains, 21 Candida krusei strains, 13 Candida guilliermondii strains, 37 Candida tropicalis strains, 10 Candida pseudotropicalis strains, 24 Candida parapsilosis strains, 32 Torulopsis glabrata strains, and 16 Cryptococcus neoformans strains). Previous results with a standard strain of C. albicans indicated that reproducible fatty acid chromatograms can be obtained with cells grown in a medium of 2% Sabouraud glucose agar at 35 degrees C for between 48 and 72 h. These conditions were also maintained in cultures of the other organisms that we studied. The cellular fatty acid compositions of the organisms were determined quantitatively by gas-liquid chromatography and analyzed by discriminant analysis. The total correct identification expressed as relative peak percent was 95.8% (89.2% for C. albicans to 100% for C. krusei, C. guilliermondii, C. pseudotropicalis, T. glabrata, and C. neoformans). The total correct identification expressed as the common peak (palmitic acid) ratio was 94.7% (87.5% for C. parapsilosis to 100% for C. pseudotropicalis, T. glabrata, and C. neoformans). Both results suggest that cellular fatty acid compositions can be differentiated by this method.     

Gas-liquid chromatography of bacterial fatty acids with a fused-silica capillary column.

The use of flexible, fused-silica capillary column for gas-liquid chromatographic analysis of bacterial fatty acids is illustrated with Propionibacterium acnes, Propionibacterium shermanii, and a standard methyl ester mixture.     

Differentiation of Bordetella avium and related species by cellular fatty acid analysis.

The fatty acids of 18 strains of Bordetella avium, 3 strains of Alcaligenes faecalis, 5 strains of Bordetella bronchiseptica, and 12 strains of a B. avium-like organism were examined by gas chromatography-mass spectrometry. The presence of a significant amount of the acid 2-OH C14:0 characterized B. avium and the B. avium-like organism. B. avium and the B. avium-like organism differed in their relative concentrations of C16:1 and 3-OH C14:0 acids. B. bronchiseptica and A. faecalis were distinguishable by comparison of the relative concentrations of C18:0 and C18:1 acids.     

High-performance liquid chromatography of mycolic acids as a tool in the identification of Corynebacterium, Nocardia, Rhodococcus, and Mycobacterium species

High-performance liquid chromatography of bromophenacyl esters of mycolic acid was used as an aid to assign a particular organism to one of four mycolic acid-containing genera. A gradient elution system, with methanol and chloroform, was used to distinguish representative mycolic acid patterns for the genera Corynebacterium, Rhodococcus, Nocardia, and Mycobacterium.     

Determination of cellular fatty acid compositions of various yeasts by gas-liquid chromatography.

The cellular fatty acid composition of 51 cultures of various species of yeasts was determined by gas-liquid chromatography. Analysis was done with a fused-silica gas-liquid chromatography capillary column, with resolution of all components including mono-, di-, and tri-unsaturated 18-carbon acids. The cultures were placed into one of four distinct gas-liquid chromatography groups on the basis of large quantitative differences in fatty acids. Group I contained only Saccharomyces species and group II only Torulopsis glabrata. Most Candida species were placed into group III, and group IV contained only basidiomycetous yeasts.     

Analysis by gas liquid chromatography of production of volatile fatty acids by anaerobic bacteria grown on solid medium.

Volatile fatty acids produced in Robertson's cooked meat medium by a range of clinically relevant anaerobes were compared by gas liquid chromatography with those produced in blood agar. The same volatile fatty acid profiles were obtained in both media, although the concentration of acids was lower in blood agar. We conclude that detection of volatile fatty acids from a pure culture of an organism on solid medium is practicable and offers advantages over the conventional technique.