Cellular fatty acids of Alcaligenes and Pseudomonas species isolated from clinical specimens.

The cellular fatty acid composition of 25 clinical isolates of Alcaligenes and Pseudomonas was determined by gas-liquid chromatography (GLC). The GLC fatty acid profiles of three species of Pseudomonas were markedly different from those of Alcaligenes. The most significant differences were the presence and relative amounts of hydroxy, branched-chain, and cyclopropane fatty acids. One of the major fatty acids in A. faecalis was a 17-carbon cyclopropane (17 delta) acid, whereas a 15-carbon branched-chain acid (13-methyl tetradecanoate) characterized isolates of P. putrefaciens. The determination of these fatty acids by GLC provides a rapid and specific means of distinguishing clinical isolates of Pseudomonas and Alcaligenes.     

Application of gas-liquid chromatography to the routine identification of nonfermenting gram-negative bacteria in clinical specimens

A total of 430 strains of glucose-nonfermenting gram-negative bacteria representing 35 species were analyzed for their cellular fatty acid composition by gas-liquid chromatography (GLC). On the basis of qualitative differences in their cellular fatty acid composition, these bacteria could be divided into 19 distinct chromatographic groups. Eight Pseudomonas species, Achromobacter xylosoxidans, group Vd, and Agrobacterium radiobacter were identified from their fatty acid compositions alone. The other glucose-nonfermenting gram-negative bacterial species studied here, classified within nine distinct GLC groups, were easily recognized by using the GLC fatty acid analysis supplemented with a limited number of conventional biochemical tests. The results support the hypothesis that bacterial fatty acid composition is rather specific and that qualitative GLC fatty acid analysis can be adapted in the clinical laboratory either to provide additional criteria for differentiation of closely related groups or to serve as a rapid and highly reproducible method for their routine identification.     

Identification of strains of Alcaligenes and Agrobacterium by a polyphasic approach

The number of stable discriminant biochemical characters is limited in the genera Alcaligenes and Agrobacterium, whose species are consequently difficult to distinguish from one another by conventional tests. Moreover, genomic studies have recently drastically modified the nomenclature of these genera; for example, Alcaligenes xylosoxidans was transferred to the genus Achromobacter in 1998. Twenty-five strains of Achromobacter xylosoxidans, three strains of an Agrobacterium sp., five strains of an Alcaligenes sp., and four unnamed strains belonging to the Centers for Disease Control and Prevention group IVc-2 were examined. These strains were characterized by conventional tests, including biochemical tests. The assimilation of 99 carbohydrates, organic acids, and amino acids was studied by using Biotype-100 strips, and rRNA gene restriction patterns were obtained with the automated Riboprinter microbial characterization system after cleavage of total DNA with EcoRI or PstI restriction endonuclease. This polyphasic approach allowed the two subspecies of A. xylosoxidans to be clearly separated. Relationships between five strains and the Ralstonia paucula type strain were demonstrated. Likewise, three strains were found to be related to the Ochrobactrum anthropi type strain. We showed that substrate assimilation tests and automated ribotyping provide a simple, rapid, and reliable means of identifying A. xylosoxidans subspecies and that these two methods can be used as alternative methods to characterize unidentified strains rapidly when discriminant biochemical characters are missing.     

Cellular fatty acids and metabolic products of Pseudomonas species obtained from clinical specimens.

The cellular fatty acid composition of 112 reference strains and clinical isolates of Pseudomonas species was determined by gas-liquid chromatography (GLC). The presence and relative amounts of cyclopropane, hydroxy, and branched-chain fatty acids were distinguishing features of these strains. Determination of short-chain fatty acids extracted from spent growth media provided an additional means for identifying some strains. Our results show that clinical isolates of pseudomonads can be divided into eight distinct GLC groups. The procedures were especially useful for distinguishing glucose-nonoxidizing pseudomonads, which are difficult to identify by conventional criteria. Since the GLC procedures are simple, rapid, and highly reproducible, they are useful in diagnostic laboratories that process large numbers of cultures. Coupled with selected conventional tests, the analysis of short-chain and cellular fatty acids can be very useful for rapid screening of clinical isolates of Pseudomonas species.     

Cellular fatty acids of Brucella canis and Brucella suis.

The cellular fatty acid composition of Brucella canis and Brucella suis was determined by gas-liquid chromatography. The presence of relatively large amounts of a 19-carbon cyclopropane fatty acid in B. suis was a major distinguishing feature between these organisms. The gas-liquid chromatography test for cellular fatty acids provides an additional criterion for the distinction of antigenically rough strains of B. suis which cannot be differentiated from B. canis by conventional procedures.     

Cellular fatty acid composition of Pseudomonas marginata and closely associated bacteria.

The cellular fatty acid compositions of the lectotype strain and four clinical isolates of Pseudomonas marginata were determined by gas-liquid chromatography and compared with 11 strains of the Centers for Disease Control Pseudomonas-like group 2, which are similar to P. marginata in a number of conventional biochemical tests. Isolates of P. marginata were readily distinguished from Pseudomonas-like group 2 by the presence of a C17:0 cyclopropane acid and hydroxy acids 3-OH-C14:0, 2-OH-C16:0, 3-OH-C16:0, and 2-OH-C18:1, whereas strains of Pseudomonas-like group 2 contained C16:1 delta 9 as a major acid with small amounts of 3-OH-C12:0 and 2-OH-C14:0 acids. Our data show that cellular fatty acid composition provides useful additional information that can be combined with selected conventional tests to provide a more reliable and rapid identification of P. marginata and related bacteria.     

Cellular fatty acids of Capnocytophaga species.

The cellular fatty acid composition of 18 clinical isolates and 4 reference strains of Capnocytophaga species (Capnocytophaga ochracea, Capnocytophaga gingivalis, and Capnocytophaga sputigena) was determined by gas-liquid chromatography. The fatty acid profiles of the 22 cultures were essentially identical and were characterized by major amounts (60% or greater) of a saturated, iso-branched-chain, 15-carbon acid (13-methyl-tetradecanoate) and the presence of two relatively uncommon saturated, iso-branched, 3-hydroxy acids (13-methyl-3-hydroxy-tetradecanoate and 15-methyl-3-hydroxy-hexadecanoate). The presence and relative amounts of these acids distinguish Capnocytophaga spp. from other gliding bacteria.     

Unusual fatty acid substitution in lipids and lipopolysaccharides of Helicobacter pylori.

Cellular fatty acids, phospholipid fatty acids, and lipopolysaccharide fatty acids of four strains of Helicobacter pylori were analyzed by gas-liquid chromatography. The presence of myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, 19-carbon cyclopropane fatty acid, beta-hydroxypalmitic acid, and beta-hydroxystearic acid was confirmed. In phospholipids, myristic acid and 19-carbon cyclopropane fatty acid were the major fatty acids. Hydroxy fatty acids and unsaturated fatty acids were not detected or occurred only in small amounts. The major fatty acids of lipopolysaccharides were stearic acid, beta-hydroxypalmitic acid, and beta-hydroxystearic acid. Unsaturated fatty acids and 19-carbon cyclopropane fatty acid were not found. The unusual compositions of H. pylori phospholipid and lipopolysaccharide fatty acids may have important implications for the taxonomy, physicochemical membrane properties, and biological activity of lipopolysaccharides.     

Cellular fatty acid composition of Campylobacter fetus.

The cellular fatty acid composition of 29 human and animal isolates of Campylobacter fetus was determined with gas-liquid chromatography. All strains contained small amounts of 3-hydroxytetradecanoic acid, suggesting the presence of lipid A. Each of 13 different blood or stool isolates of C. fetus subsp. jejuni obtained from humans or fowl contained a 19-carbon cyclopropane acid which was not present in C. fetus subsp. fetus (6 strains from cattle) or C. fetus subsp. intestinalis (10 strains from humans and cattle). These findings indicate that C. fetus subsp. jejuni can be rapidly differentiated from other C. fetus by gas-liquid chromatography analysis of cellular fatty acids.     

Cellular fatty acid composition of Campylobacter pylori from primates and ferrets compared with those of other campylobacters.

The cellular fatty acid profiles of newly described campylobacters were determined on a polar, capillary column. Six isolates of the gastric spiral organism, Campylobacter pylori subsp. mustelae, from ferrets from Australia, England, and the United States were all found to have a similar fatty acid profile which was different from that of C. pylori from humans; C. pylori subsp. mustelae did not have 3-hydroxyoctadecanoic acid (3-OH C18:0) and had much less tetradecanoic acid (C14:0) and much more hexadecanoic acid (C16:0). Inasmuch as Lambert et al. (M.A. Lambert, C.M. Patton, T.J. Barrett, and C.W. Moss, J. Clin. Microbiol. 25:706-713, 1987) have proposed that campylobacters can be grouped by cellular fatty acid composition, we propose this organism should be in a new gas-liquid chromatography (GLC) group, group J. Seven isolates of gastric spiral organisms from macaque monkeys and baboons, including three from Macaca nemestrina, and one isolate from a pig were found to have fatty acid profiles very similar to that of C. pylori; but a second type of organism (type B) from M. nemestrina had a unique profile without 19-carbon cyclopropane fatty acid (C19:0 cyc) but with 3-hydroxy tetradecanoic acid (OH C14:0), which is not present in other gastric spiral bacteria. We propose that this organism (nemestrina type B) should be in a new GLC group, group K. The cellular fatty acid profile of seven isolates of C. jejuni subsp. doylei was found to be similar to that for C. jejuni, but with possibly significant differences in that the former did not have 3-OH C14:0 but did have 3-hydroxyhexadecanoic acid (3-OH C16:0) and had more C14:0 than did C. jejuni. Two strains of urease-positive thermophilic campylobacters were found to have a profile similar to that of "C. cinaedi" and thus should be included with them in GLC group D. We confirm that C. sputorum has a unique cellular fatty acid composition and suggest that it should be in a new group, group H.     

Differentiation of Campylobacter and Campylobacter-like organisms by cellular fatty acid composition.

The cellular fatty acid compositions of 368 strains of Campylobacter species or Campylobacter-like organisms were determined by gas-liquid chromatography. Most of the strains (339) were placed in one of three groups based on differences in fatty acid profiles. Group A contained Campylobacter jejuni (97%) and most C. coli (83%) strains and was characterized by the presence of a 19-carbon cyclopropane fatty acid (19:0 cyc) and 3-hydroxytetradecanoic acid (3-OH-14:0). Group B included all C. laridis and some C. coli (17%) strains; its profile was similar to that of group A, except that 19:0 cyc was absent. Group C contained C. fetus subsp. fetus and C. fetus subsp. veneralis and was characterized by the presence of 3-OH-14:0 and 3-hydroxyhexadecanoic acid (3-OH-16:0) and the absence of 19:0 cyc. Twenty-nine isolates were placed in four additional groups. Group D included the type strain of "C. cinaedi" and 14 other isolates, which were differentiated by the presence of dodecanoic acid (12:0), 3-hydroxydodecanoic acid (3-OH-12:0), and 3-OH-16:0 and the absence of hexadecenoic acid (16:1) and 3-OH-14:0. Group E contained the type strain of "C. fennelliae" and two additional isolates, which were differentiated by the presence of a 16-carbon aldehyde and a 16-carbon dimethylacetyl and the absence of 16:1. Group F included the type strain and one reference strain of C. cryaerophila and six human isolates whose phenotypic characteristics were similar to those of this species; this group was distinguished by the presence of two isomers of 16:1, tetradecenoic acid (14:1), and 3-OH-14:0. Group G included three stains of C. pyloridis and was characterized by the presence of 19:0 cyc, 3-OH-16:0, and 3-hydroxyoctadecanoic acid (3-OH-18:0) and by the absence of 16:1 and 3-OH-14:0.     

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.     

Differentiation of Brucella ovis from Brucella abortus by gas-liquid chromatographic analysis of cellular fatty acids

The cellular fatty acid composition of Brucella ovis and Brucella abortus strains was determined by gas-liquid chromatography. Both species were characterized by the presence of fatty acids 16:0, 17:0, 17:0 cyclopropane, 18:0, 18:1, and 19:0 cyclopropane; B. ovis also contained some 15:0. There were differences in the relative proportions of the fatty acids present, and it was possible to differentiate B. ovis from B. abortus on the basis of the absence of 15:0, lower concentrations of 17:0 and 18:1, and higher concentrations of 19:0 cyclopropane in B. abortus. The data indicate that analysis of cellular fatty acid composition by gas-liquid chromatography can be used for the identification of B. ovis and its differentiation from B. abortus.     

Cultural and chemical characterization of CDC groups EO-2, M-5, and M-6, Moraxella (Moraxella) species, Oligella urethralis, Acinetobacter species, and Psychrobacter immobilis.

We determined phenotypic characteristics, cellular fatty acid composition, and isoprenoid quinone content of representative strains of CDC groups EO-2, M-5, and M-6, Moraxella (Moraxella) species, Oligella urethralis, Acinetobacter species, and Psychrobacter immobilis. All organisms contained ubiquinone with eight isoprene units as the major isoprenolog, but distinct differences were observed in fatty acid composition. Twenty-eight of the original collection of CDC group EO-2 strains were further identified as P. immobilis, EO-2, or EO-3 by distinctive cellular fatty acid profiles, cellular morphology, and pigment production. The cellular fatty acid compositions of M-5 and M-6 were similar but were clearly different from those of other organisms. The genus Acinetobacter was differentiated from other organisms in the study by small amounts of 2-hydroxydodecanoic acid (2-OH-12:0), and P. immobilis was differentiated by small amounts of decanoic acid (10:0) and a branched-chain 17-carbon acid (i-17:0). All Moraxella species were distinguished by small amounts of decanoic acid (10:0) and the absence of i-17:0. M. bovis, M. nonliquefaciens, and some strains of M. lacunata formed a single fatty acid group, while M. osloensis, M. phenylpyruvica, M. atlantae, and other strains of M. lacunata (M. lacunata II) had species-specific fatty acid profiles. O. urethralis differed from Moraxella species by the presence of large amounts (49%) of cis-vaccenic acid (18:1 omega 7c), small amounts (1%) of 3-hydroxyhexadecanoate (3-OH-16:0), and the absence of 10:0 and 3-hydroxydodecanoate (3-OH-12:0). The combined use of chemical data and a small number of conventional tests permitted rapid identification and differentiation of these organisms from each other and from related organisms.     

Chemical characterization of clinical isolates which are similar to CDC group DF-3 bacteria.

Six clinical isolates, taken from blood or wounds, that had biochemical characteristics most similar to Centers for Disease Control group DF-3 bacteria were examined for cellular fatty acid composition and isoprenoid quinone content to evaluate their chemical relatedness to known bacterial species and groups. The fatty acids were liberated from whole cells by base hydrolysis, methylated, and analyzed by capillary gas-liquid chromatography. The isoprenoid quinones were extracted from lyophilized whole cells and analyzed by reverse-phase high-performance liquid chromatography. All six strains, which were designated group DF-3-like, possessed a distinct fatty acid profile that was characterized by large amounts (greater than 20%) of 13-methyltetradecanoate (i-C15:0) and 12-methyltetradecanoate (a-C15:0), moderate amounts of saturated branched-chain 13-carbon acids (i-C13:0 and a-C13:0) and hexadecanoate (n-C16:0), and small to moderate amounts of both branched- and straight-chain hydroxy acids (i-3-OH-C15:0, 3-OH-C16:0, i-3-OH-C17:0, and 2-OH-C17:0). This fatty acid profile was unique compared with the profiles of group DF-3 and other bacteria we have previously tested and is useful for the rapid identification of group DF-3-like isolates. The isoprenoid quinone content of four group DF-3-like strains was similar, with ubiquinone-9 (Q-9) and Q-10 as their major quinones, while the other two group DF-3-like strains contained Q-7 as their major quinones, with smaller amounts of Q-8 and Q-9.     

Biochemical and chemical characterization of pink-pigmented oxidative bacteria.

The biochemical and chemical characteristics were determined for 156 clinical isolates of pink-pigmented bacteria that are similar to but distinct from Methylobacterium extorquens (synonymous with Pseudomonas mesophilica). These isolates were gram-negative, nonfermentative, usually nonvacuolated, coccoid rods; all grew at 35 degrees C and were catalase and urease positive; the majority grew on MacConkey agar and were variable for oxidase production and motility. On the basis of oxidation of xylose and mannitol and hydrolysis of esculin, these 156 strains were subdivided into four groups that were designated "pink coccoid" groups I, II, III, and IV. Groups I, II, and III are similar to an unnamed taxon described by Gilardi and Faur in 1984; only strains of group IV hydrolyze esculin. The cellular fatty acid compositions of strains of groups I, II, and III were essentially identical and differed from strains of group IV by the absence of 3-OH-C14:0 and the presence of C19:0 delta and 2-OH-C19:0 delta. The fatty acid composition of group IV strains was most similar to that of M. extorquens but differed by the presence of small amounts of two C17:1 acids, 3-OH-C16:0, and 2-OH-C18:1.     

Characterization of CDC group DF-3 by cellular fatty acid analysis.

Fourteen strains of Centers for Disease Control group DF-3 bacteria were examined for cellular fatty acid composition to evaluate their chemical relatedness to known bacterial species and groups. The fatty acids were liberated from whole cells by base hydrolysis, methylated, and analyzed by capillary gas-liquid chromatography. All group DF-3 strains possessed a distinct fatty acid profile which was characterized by large amounts (24%) of 12-methyltetradecanoate (a-C15:0), moderate amounts of saturated iso-branched-chain acids (i-C14:0 and i-C15:0), and small to moderate amounts of both branched- and straight-chain hydroxy acids (3-OH C15:0, i-3-OH C16:0, 3-OH C16:0, and i-3-OH C17:0). This fatty acid profile was unique as compared with the profiles of other bacteria we have previously tested but was most similar to the profiles of Capnocytophaga species.     

Assessing palatability of long-chain fatty acids from the licking behavior of BALB/c mice

Dietary oils such as corn oil, olive oil, and canola oil, which primarily contain triacylglycerol and small quantities of fatty acids, are highly palatable to animals. In a previous study, we examined the short-term (60 s) licking behavior of mice and observed that they exhibited a high licking response to a low concentration of fatty acid (linoleic acid), which is comparable to that observed for pure corn oil. This finding suggests that fatty acids contribute to the palatability of dietary oils. In order to supplement our knowledge of the fundamental features of fatty acid palatability in the oral cavity, we assessed the licking behavior of BALB/c mice to investigate the palatability of various types of long-chain fatty acids. The mice showed high licking responses to 1% unsaturated 16- and 18-carbon fatty acids (palmitoleic acid, 16:1; oleic acid, 18:1; linoleic acid, 18:2; and linolenic acid, 18:3), low licking responses to 16- and 20-carbon fatty acids (palmitic acid, 16:0 and arachidonic acid, 20:4), and no significant response to saturated fatty acids (stearic acid, 18:0 and arachidic acid, 20:0) or fatty acid derivatives (methyl linoleate and linole alcohol). Additionally, there were differences in the palatability of 18-carbon unsaturated fatty acids at very low concentrations. At fatty acid concentrations of 0.04% and 0.0625%, the mice showed significant preference for linoleic acid and linolenic acid, but not oleic acid, when compared with mineral oil. These results suggest that mice show high licking responses to 16- and 18-carbon unsaturated long-chain fatty acids at low concentrations. Further, we suggest that sensitivity to fatty acids is affected by the saturated state of the fatty acid, carbon chain length, and terminal carboxyl group.     

Unusual cellular fatty acids and distinctive ultrastructure in a new spiral bacterium (Campylobacter pyloridis) from the human gastric mucosa

Spiral bacteria, named Campylobacter pyloridis, were obtained from endoscopic biopsies of the gastric antrum of 14 patients with active chronic gastritis. Methyl esters of their cellular fatty acids were prepared by acid-catalysed transmethylation of whole cells. Their major fatty acids were tetradecanoic acid (14:0) and cis-9,10-methyleneoctadecanoic acid (19:0 delta), with a very small amount of hexadecanoic acid (16:0). This is markedly different from the fatty acids of other Campylobacter sp. whose major fatty acids are hexadecanoic, octadecenoic (18:1) and hexadecenoic acids (16:1). This is also different from other enterobacteria. Thin-section electronmicroscopy of gastric mucosal biopsies, and negative staining of cultured C. pyloridis, revealed features that differ from those of other campylobacters so far studied. C. pyloridis has a smooth not a rugose surface and multiple unipolar flagella of the sheathed type, each with a terminal bulb. Flagellar sheaths were in continuity with the unit membrane of the outer cell wall. The proposed species C. pyloridis does not belong among the spirochaetes and its DNA composition is incompatible with membership of the genera Spirillum or Vibrio but is compatible with Campylobacter. Thus C. pyloridis is either an atypical member of the genus Campylobacter, the limits of which may have to be redefined to accommodate the new species, or a representative of a new genus.     

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.