Cellular Fatty Acid Methyl Esters Research Articles

Artificial neural network based identification of environmental bacteria by gas-chromatographic and electrophoretic data

Chemotaxonomic identification techniques are powerful tools for environmental micro-organisms, for which poor diagnostic schemes are available. Whole cellular fatty acid methyl esters (FAME) content is a stable bacterial profile, the analysis method is rapid, cheap, simple to perform and highly automated. Whole-cell protein is an even more powerful tool because it yields information at or below the species level. The description of new species and genera and subsequent continuous rearrangement provide large amounts of data, resulting in large databases. In order to set up suitable software tools to work on such large databases artificial neural network (ANN) based programs have been used to classify and identify marine bacteria at genus and species levels, starting from the fatty acid profiles and protein profiles respectively. We analysed 50 certified strains belonging to Halomonas, Marinomonas, Marinospirillum, Oceanospirillum and Pseudoalteromonas genera. Both supervised and unsupervised ANNs provide a correct classification of the marine strains analyzed. Moreover, a set of 73 marine fresh isolates were used as an example of identification using ANNs. We propose supervised and unsupervised ANNs as a reliable tool for classification of bacteria by means of their FAME and of whole-protein analyses and as a sound basis for a comprehensive artificial intelligence based system for polyphasic taxonomy.

Growth of a human intestinal Desulfovibrio desulfuricans in continuous cultures containing defined populations of saccharolytic and amino acid fermenting bacteria.

Ecological and physiological effects of the sulphate-reducing bacterium (SRB) Desulfovibrio desulfuricans on other intestinal organisms were investigated in anaerobic chemostats (dilution rate approximately 0.2 h-1). Reproducible defined bacterial communities were used in these experiments, comprising 14 different saccharolytic and amino acid fermenting species: Bifidobacterium longum, Bif. adolescentis, Bif. pseudolongum, Bif. infantis, Bacteroides thetaiotaomicron, Bact. vulgatus, Lactobacillus acidophilus, Enterococcus faecalis, Ent. faecium, Escherichia coli, Clostridium perfringens, Cl. butyricum, Cl. innocuum, Cl. bifermentans. Lactobacillus and Cl. bifermentans populations never rose above minimum detection limits (log10 2.0 and 4.0, respectively) under the experimental conditions employed in these studies. Inclusion of Des. desulfuricans in bacterial cultures (c. log10 8.4 viable cells ml-1) resulted in marked reductions (i.e. greater than 1 log) in planktonic cell population densities of several species, particularly Bif. longum, Cl. perfringens and Bif. pseudolongum. The two bacteroides species were unaffected by Des. desulfuricans, while numbers of Cl. butyricum increased. Extensive wall growth developed in the SRB culture, consisting mainly of Des. desulfuricans (log10 9.2 viable cells ml-1), Bact. thetaiotaomicron and Bact. vulgatus, with lesser numbers of facultative anaerobes, Cl. perfringens and Bif. longum. Wall growth was associated with a reduction in planktonic cell mass and increased acid production by the cultures. Chemotaxonomic study of chemostat microbiotas, on the basis of cellular fatty acid methyl ester (FAME) analyses, showed the existence of characteristic bacteroides (C15) and bifidobacterial (C18) markers, but desulfovibrio markers (i-C15:0, C16:0, i-C17:1) could be identified. The metabolic activities of saccharolytic organisms were altered in the SRB chemostat, including synthesis of a number of hydrolytic enzymes involved in carbohydrate breakdown, such as alpha-galactosidase, alpha-glucosidase and beta-galactosidase, together with several mucinolytic enzymes. High concentrations of sulphide (8.2 mmol 1-1) were detected in the SRB chemostat, suggesting that this metabolite may have been inhibitory to some species. Saccharolytic organisms growing in the SRB fermenter utilized more starch, but less galactose-containing polymers, which correlated with the observed glycosidase activities. Profound differences were also recorded with respect to fermentation product formation in the chemostats, where a major switch to acetate production occurred in the SRB culture, with concomitant reductions in propionate, butyrate and lactate, which is an important electron donor for desulfovibrios.

Characterization of Buttiauxella and Kluyvera Species by Analysis of Whole Cell Fatty Acid Patterns

The cellular fatty acid methyl ester (FAME) composition of Buttiauxella spp. (66 strains, including all 7 recognized genomospecies), and Kluyvera spp. (17 strains, including all 4 recognized genomospecies) was determined by gas-liquid chromatographic analysis. All 83 strains contained 14:0, 16:0, 17:0 cyclo, summed feature 3 (16:1 iso I and/or 14:0 3OH), summed feature 3 (16:1 ω7c and/or 15:0 iso 2OH), and summed feature 7 (18:1 ω7c, 18:1 ω9t, and/or 18:1 ω12t). The homogeneity of both genera as revealed by biochemical reactions and by deoxyribonucleic acid-deoxyribonucleic acid hybridization was also reflected in the fatty acid composition. Only small interspecies differences were detected. Although the FAME profiles were very similar, quantitative variations in summed feature 7 and 15:0 could be helpful to differentiate the genera. In some cases, a species differentiation also was possible.

Genetic relatedness of Burkholderia (Pseudomonas) cepacia isolates from five cystic fibrosis centers in Michigan

Burkholderia cepacia isolates from patients with cystic fibrosis (CF) attending five CF centers were studied for relatedness by cellular fatty acid methyl esters (FAME) and by chromosomal DNA restriction analysis. Twenty-eight of 32 (87·5%) isolates tested were grouped in cluster group 1 based on their FAME profiles. DNA analysis revealed that 29 of 32 (90·6%) B. cepacia isolates from five CF centers had one closely related DNA pattern. To examine strain variation over a time period, FAME profiles and DNA patterns of isolates from serial cultures on seven patients from center D were studied. For four patients, all serial B. cepacia isolates belonged to a single FAME cluster group; for the remaining three patients, all serial isolates belonged to any two of the four cluster groups. On serial culture isolates, a single DNA pattern (pattern A) was found in 31 of 32 isolates demonstrating a close genetic relatedness. These data corroborate the observations that the majority of patients colonised with B. cepacia in a CF center harbor strains genetically closely related as determined by FAME profiles and DNA patterns.

Microbial Succession during a Field Evaluation of Phenol and Toluene as the Primary Substrates for Trichloroethene Cometabolism

Microbial community composition and succession were studied in an aquifer that was amended with phenol, toluene, and chlorinated aliphatic hydrocarbons to evaluate the effectiveness of these aromatic substrates for stimulating trichloroethene (TCE) bioremediation. Samples were taken after the previous year's field studies, which used phenol as the primary substrate, and after three successive monthly treatments of phenol plus 1,1-dichloroethene (1,1-DCE) plus TCE, phenol plus TCE, and toluene plus TCE. Dominant eubacteria in the community were assessed after each of the four treatments by characterizing isolates from the most dilute most-probable-number tubes and by extracting DNA from aquifer samples. The succession of dominant phenol- and toluene-degrading strains was evaluated by genomic fingerprinting, cellular fatty acid methyl ester (FAME) analysis, and amplified ribosomal DNA restriction analysis (ARDRA). 1,1-DCE was found to drastically reduce microbial growth and species richness, which corresponded to the reduction in bioremediation effectiveness noted previously for this treatment (G. D. Hopkins and P. L. McCarty, Environ. Sci. Technol. 29:1628-1637, 1995). Only a few gram-positive isolates could be obtained after treatment with 1,1-DCE, and these were not seen after any other treatments. Microbial densities returned to their original levels following the subsequent phenol-TCE treatment, but the original species richness was not restored until after the subsequent toluene-TCE treatment. Genomic fingerprinting and FAME analysis indicated that six of the seven originally dominant microbial groups were still dominant after the last treatment, indicating that the community is quite resilient to toxic disturbance by 1,1-DCE. FAME analysis indicated that six microbial taxa were dominant: three members of the (beta) subclass of the class Proteobacteria (Comamonas-Variovorax, Azoarcus, and Burkholderia) and three gram-positive groups (Bacillus, Nocardia, and an unidentified group). ARDRA revealed that the dominant community members were stable during the three nontoxic treatments and that virtually all of the bands could be accounted for by isolates from five of the dominant taxa, indicating that the isolation protocol used likely recovered most of the dominant members of this community.

Genotypic and Chemotaxonomic Description of Two Subgroups in the Species Aeromonas eucrenophila and Their Affiliation to A. encheleia and Aeromonas DNA Hybridization Group 11

We describe the results of a polyphasic taxonomic study that aimed to elucidate the genotypic heterogeneity observed previously among the eight original reference strains of the species Aeromonas eucrenophila . In addition, this investigation also included representatives of the neighbouring taxa Aeromonas encheleia and Aeromonas DNA hybridization group (HG) 11, as well as a collection of 14 mainly aquatic Aeromonas sp. isolates which were previously found to be closely related to A. eucrenophila . All 28 strains were subjected to ribotyping, whole-genome analysis by AFLP fingerprinting, electrophoretic profiling of soluble cellular proteins, and gas-liquid chromatographic analysis of cellular fatty acid methyl esters. Cluster analysis of the data uniformly confirmed our previous findings and delineated two wellseparated subgroups in A. eucrenophila . Subgroup I was represented by the type strain of this species (LMG 3774) and four other original reference strains, of which one strain (LMG 13687) displayed a somewhat atypical fatty acid profile. This subgroup could be extended by the inclusion of four Aeromonas isolates that were unambiguously classified in A. eucrenophila subgroup I. The three remaining reference strains originally described as A. eucrenophila were allocated to subgroup II, but could not be differentiated from members of A. encheleia and HG11. Consequently, a clear-cut classification of poorly identified or unknown Aeromonas isolates into one of the latter three taxa was not possible on genotypic or chemotaxonomic basis. In conclusion, the current report has provided substantial polyphasic evidence to support an emended description of the species A. eucrenophila . Moreover, the high genotypic and chemotaxonomic resemblance of A. eucrenophila subgroup II, HG11, and A. encheleia revealed in this study suggests that these three groups may actually belong to the same genomic species.

Differentiation of Salmonella gallinarum and Salmonella pullorum by their whole-cell fatty acid methyl ester profiles.

The whole-cell fatty acid methyl ester (FAME) profiles of Salmonella (S.) gallinarum and Salmonella (S.) pullorum strains were compared in the computer-linked gas-liquid chromatography. The profiles of whole cellular FAMEs allow the separation and identification S. gallinarum and S. pullorum by the Microbial Identification System and so can be used for their differentiation.

Influence of temperature and process technology on the occurrence of Aeromonas species and hygienic indicator organisms in drinking water production plants

The occurrence of Aeromonas spp. and hygienic indicator organisms in raw and treated waters of five drinking water production plants in Flanders (Belgium) was surveyed over a period of 17 months. Aeromonads were isolated on ampicillin-dextrin agar (ADA) and further identified by gas-liquid chromatographic analysis of their cellular fatty acid methyl ester (FAME) content. ADA medium was found to be highly specific for the enumeration of Aeromonas spp. In general, Aeromonas counts were very low in untreated groundwater but numbered 10(4)-10(6) colony-forming units per liter in open storage reservoirs for surface water. Aeromonas spp. were seasonally distributed with maximal densities occurring during the summer. The ecology of Aeromonas in the different waters was studied in relation to the physical, chemical, and microbiological water characteristics. Strongly positive correlations were observed between Aeromonas densities and heterotrophic plate counts, whereas a clearly negative relationship was found with dissolved oxygen. On average, 99.7% of the aeromonads were removed by flocculation-decantation followed by breakpoint chlorination, whereas 98.9% were removed by slow sand filtration. Flocculation-decantation without breakpoint chlorination did not reduce the microbial numbers. At three of four drinking water production plants tested, rapid sand filtration decreased the number of aeromonads and hygienic indicator organisms. At one plant, however, the numbers of Aeromonas and hygienic indicator organisms were high in the sand filter effluents. Increased numbers of aeromonads were also counted in the effluent of the activated carbon filters. Hence, inactivation of Aeromonas spp. by the current process technology appears not sufficient to exclude postchlorination. The survival of aeromonads in certain filter systems may be due to the growth of these bacteria on biodegradable organic material, provided by the decomposition from bacteria, algae, or other sources.

Diversity of Aeromonas sp. in Flemish drinking water production plants as determined by gas-liquid chromatographic analysis of cellular fatty acid methyl esters (FAMEs).

Gas-liquid chromatography of cellular fatty acid methyl esters (FAMEs) was used to determine the phenotypic and genotypic diversity among 489 presumptive Aeromonas strains isolated from five Flemish drinking water production plants. FAME profiles were compared with the predetermined library profiles of a representative database, AER48C, which contains the mean FAME data of all 14 currently established hybridization groups (HGs) or genospecies within Aeromonas. Using AER48C, more than 93% (457 strains) of all presumptive aeromonads isolated on ampicillin-dextrin agar were unequivocally identified as belonging to this genus. Moreover, 85.5% and 73.5% of these strains could be assigned to a particular phenospecies or HG, respectively. Raw and treated surface water samples were dominated by members of the Aer. hydrophila complex (38.8%, comprising HGs 1-3), followed by the Aer. caviae complex (22.7%, comprising HGs 4-6) and the Aer. sobria complex (16.7%, comprising HGs 7-9). HGs 3, 5A/B and 8 were the most prominent genospecies in this type of water. On the other hand, it was found that raw and treated phreatic groundwater samples displayed a much more limited species diversity since these were almost entirely dominated (95.8%) by strains belonging to HGs 2 and 3 of the Aer. hydrophila complex. In general, flocculation-decantation and sand filtration were not shown to influence the overall species distribution in any of the plants examined.

Cellular lipid and fatty acid compositions of Burkholderia pseudomallei strains isolated from human and environment in Viet Nam.

Viet nam is known as an endemic area of melioidosis but its etiologic agent originated in Viet nam was not extensively studied. For the first time, we analyzed the cellular lipid and fatty acid compositions of 15 Vietnamese isolates of Burkholderia pseudomallei, 10 from humans and 5 from the environment. Cellular lipid compositions were analyzed by two-dimensional thin-layer chromatography on silica gel G plates. Cellular fatty acid methyl esters were analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The major lipids in all the isolates were phosphatidylglycerol (PG), two forms of phosphatidylethanolamine (PE-1 and PE-2), and two forms of ornithine-containing lipid (OL-1 and OL-2). PE-1 contained non-hydroxy fatty acids at both sn-1 and -2 positions, while PE-2 possessed 2-hydroxy fatty acids and non-hydroxy fatty acids in a ratio of 1:1. Since snake venom phospholipase A2 digestion of PE-2 liberated 2-hydroxy fatty acids, it was confirmed that these acids are at the sn-2 position of glycerol moiety. In both OL-1 and OL-2, amide-linked fatty acid was 3-hydroxy palmitic acid (3-OH-C16:0), while ester-linked fatty acids were non-hydroxy acids in OL-1 and 2-hydroxy acids in OL-2. The total cellular fatty acid compositions of the test strains were characterized by the presence of 2-hydroxy palmitic (2-OH-C16:0), 2-hydroxy hexadecenoic (2-OH-C16:1), 2-hydroxy octadecenoic (2-OH-C18:1), 2-hydroxy methylene octadecanoic (2-OH-C19CPA), 3-hydroxy myristic (3-OH-C14:0) and 3-hydroxy palmitic (3-OH-C16:0) acids. There were significant differences in the concentration of hexadecenoic (C16:1), methylene hexadecanoic (C17CPA), octadecenoic (C18:1) and methylene octadecanoic (C19CPA) acids among the Vietnamese isolates of B. pseudomallei. However, no significant difference was observed in cellular lipid and fatty acid components between strains of human and environmental origins.

Cellular Fatty Acid Composition as a Chemotaxonomic Marker for the Differentiation of Phenospecies and Hybridization Groups in the Genus Aeromonas

Ninety genotypically characterized Aeromonas strains, including members of all 14 currently established genospecies, were studied by performing gas-liquid chromatographic analysis of their cellular fatty acid methyl esters (FAMEs). A total of 44 fatty acids and two alcohols were found in members of the genus Aeromonas. All 90 strains contained 12:0, 13:0 iso, 14:0, 15:0 iso 30H, 16:0, 16:1 ω7c, 17:0 iso, iso 17:1 ω9c, summed feature 3 (16:1 iso I and/or 14:0 30H), and summed feature 7 (18:1 ω7c, 18:1 ω9t, and/or 18:1 ω12t), whereas all but one strain (99%) also contained 15:0 iso. Although the FAME profiles were very similar, minor quantitative variations could be used to differentiate phenospecies and/or hybridization groups. A cluster analysis of the mean data revealed five FAME clusters, which were compared with phenotypic and genotypic groups identified in the genus Aeromonas. Hybridization groups that constituted the Aeromonas hydrophila complex, the Aeromonas caviae complex, and the Aeromonas sobria complex were basically grouped into distinct FAME clusters. The taxonomic positions of hybridization groups 7 and 11 in these clusters, however, remained unclear. All of our results were highly reproducible. A new database of Aeromonas FAME fingerprints was generated, and this database can be used for rapid identification of unknown aeromonads. Using a large set of well-characterized aeromonads, we demonstrated for the first time that gas-liquid chromatographic FAME analysis can be used to differentiate the majority of the phenospecies and/or hybridization groups in the genus Aeromonas.

Cellular fatty acid methyl ester profiles as a tool in the differentiation of members of the genus Mycobacterium

The cellular fatty acid profiles of 84 strains belonging to 53 different species of the genus Mycobacterium were determined by gas liquid chromatography-mass spectrometry (GCMS). Two main types and four subtypes of fatty acid profiles were recognizable. The first main type is the G type, nominally referring to M. gordonae, members of which lack tuberculostearic acid or other 10-methyl branched-chain fatty acids, but contain normal saturated and unsaturated fatty acids. This type is further subdivided into the G alpha subtype that is characterized by 2-methyl tetradecanoic acid (2-Me-C14:0) as the only 2-methyl branched fatty acid. Strains belonging to the second main type, the T type, nominally referring to tuberculosis, contain tuberculostearic acid and other 10-methyl branched acids in addition to the normal saturated and unsaturated ones. This type has been further subdivided into three subtypes: the T alpha subtype that does not contain any 2-methyl branched fatty acids; the T beta subtype that contains both 2-methyl tetradecanoic (2-Me-C14:0) and 2,4-dimethyl tetradecanoic (2,4-DMe-C14:0) acids as 2-methyl branched fatty acids; the T gamma subtype which contains 2-methyl dodecanoic (2-Me-C12:0), 2,4-dimethyl dodecanoic (2,4-DMe-C12:0) and 2,4-dimethyl tetradecanoic (2,4-DMe-C14:0) acids as 2-methyl branched-chain acids. Fatty acid analysis showed a great homogeneity within the genus and the profiles produced were not very helpful in distinguishing between members of the genus Mycobacterium except for the identification of M. gordonae, M. kansasii, and M. gastri.