Accuracy, Reproducibility, and Interpretation of Fatty Acid Methyl Ester Profiles of Model Bacterial Communities

We determined the accuracy and reproducibility of whole-community fatty acid methyl ester (FAME) analysis with two model bacterial communities differing in composition by using the Microbial ID, Inc. (MIDI), system. The biomass, taxonomic structure, and expected MIDI-FAME profiles under a variety of environmental conditions were known for these model communities a priori. Not all members of each community could be detected in the composite profile because of lack of fatty acid “signatures” in some isolates or because of variations (approximately fivefold) in fatty acid yield across taxa. MIDI-FAME profiles of replicate subsamples of a given community were similar in terms of fatty acid yield per unit of community dry weight and relative proportions of specific fatty acids. Principal-components analysis (PCA) of MIDI-FAME profiles resulted in a clear separation of the two different communities and a clustering of replicates of each community from two separate experiments on the first PCA axis. The first PCA axis accounted for 57.1% of the variance in the data and was correlated with fatty acids that varied significantly between communities and reflected the underlying community taxonomic structure. On the basis of our data, community fatty acid profiles can be used to assess the relative similarities and differences of microbial communities that differ in taxonomic composition. However, detailed interpretation of community fatty acid profiles in terms of biomass or community taxonomic composition must be viewed with caution until our knowledge of the quantitative and qualitative distribution of fatty acids over a wide variety of taxa and the effects of growth conditions on fatty acid profiles is more extensive.     

Use of Fatty Acid Methyl Ester Profiles for Discrimination of Bacillus cereus T-Strain Spores Grown on Different Media

The goal of this study was to determine if cellular fatty acid methyl ester (FAME) profiling could be used to distinguish among spore samples from a single species (Bacillus cereus T strain) that were prepared on 10 different medium formulations. To analyze profile differences and identify FAME biomarkers diagnostic for the chemical constituents in each sporulation medium, a variety of statistical techniques were used, including nonmetric multidimensional scaling (nMDS), analysis of similarities (ANOSIM), and discriminant function analysis (DFA). The results showed that one FAME biomarker, oleic acid (18:1 ω9c), was exclusively associated with spores grown on Columbia agar supplemented with sheep blood and was indicative of blood supplements that were present in the sporulation medium. For spores grown in other formulations, multivariate comparisons across several FAME biomarkers were required to discern profile differences. Clustering patterns in nMDS plots and R values from ANOSIM revealed that dissimilarities among FAME profiles were most pronounced when spores grown with disparate sources of complex additives or protein supplements were compared (R > 0.8), although other factors also contributed to FAME differences. DFA indicated that differentiation could be maximized with a targeted subset of FAME variables, and the relative contributions of branched FAME biomarkers to group dissimilarities changed when different media were compared. When taken together, these analyses indicate that B. cereus spore samples grown in different media can be resolved with FAME profiling and that this may be a useful technique for providing intelligence about the production methods of Bacillus organisms in a forensic investigation.In September 2001, letters containing spores of Bacillus anthracis, the causative agent for anthrax, were mailed to television and print media outlets, as well as two U.S. congressional offices, in an act of bioterrorism. Genetic tests identified a single strain of B. anthracis, Ames, in all evidence samples. Since then, considerable effort has gone into developing techniques that can be used to analyze microbiological evidence recovered from a crime scene. Because the Ames strain used in the 2001 attacks was difficult to distinguish genetically from several commonly used Ames strains (20), many recently developed techniques have concentrated on nongenetic signatures associated with the cell that are unique to the methods that were used to culture an organism. Examples include assays that detect the presence of residual agar on spores (59), C/N isotope ranges for different medium components (22, 30, 31), and detection of heme in spores grown on blood-containing media (56). Phenotypic signatures such as these that indicate specific metabolic substrates, characteristic compounds, or defining features of an organism''s production process could help in the attribution of a biocrime by providing leads or excluding suspects during an investigation (40, 59).One phenotypic system that has not been fully tested in a forensic context is fatty acid methyl ester (FAME) analysis. FAMEs have long been recognized as useful biochemical markers for bacterial classification and characterization (38, 53, 54). The types and relative abundances of fatty acids produced within a cell are largely determined by an organism''s genotype and can be used for identification of different species (50) and strains (34, 51) and for discriminating between free spores and vegetative cells (43, 47). Commercial systems that streamline fatty acid extraction and detection procedures (42) have facilitated the widespread use of fatty acid profiling to identify bacteria in clinical, agricultural, and biodefense settings (29, 53, 55).Besides aiding in the identification of bacterial species, FAME profiling can potentially provide information on the methods used to grow microorganisms of forensic interest. Within the Bacillus group, the amino acid content or the type of complex additives used in the cultivation media can significantly affect the fatty acid composition of bacterial cultures. The relative proportions of branched fatty acids (iso-odd, iso-even, and anteiso), which are prevalent in Bacillus spp. (33, 36a), are heavily dependent on the ratio of amino acid precursors (leucine, valine, and isoleucine) and the corresponding α-keto acids present in the growth media (12, 27, 28, 32). Accordingly, the complex additives and protein sources that supply these amino acid precursors in growth media also affect the fatty acid compositions of Bacillus cultures. For example, it has been reported that inclusion of components such as yeast extract, beef extract, or casein hydrolysate in growth medium formulations can change the relative ratios of iso and anteiso fatty acids in Bacillus cereus cultures (24, 25). Brain heart infusion (BHI) has been observed to have a similar effect on the relative proportions of branched fatty acids in Bacillus caldolyticus (52). Despite the clear relationship between the fatty acid compositions of vegetative cells and the formulations of growth media, no study has tested whether this could be exploited for investigative purposes by determining whether diagnostic FAME signatures for growth media exist within spores of a forensically relevant organism.To test whether FAME signatures can be used to infer the compositional characteristics of the sporulation medium, we examined fatty acid profiles among Bacillus cereus T-strain (BcT) spores grown on 10 different media, spanning nutrient formulations that varied primarily in the source of protein, either in the form of complex additives (yeast extract, beef extracts, brain-heart solids, etc.) or direct protein supplements (peptone, tryptone, or gelatin digest). Formulation pairs that differed in other medium attributes, such as the presence of supplemental sugars, the physical state (agar versus broth), or blood supplements, were included to compare the resulting FAME profile differences with the variation that derives from complex additive/protein components.The effects that protein components in the sporulation medium have on FAME profiles were specifically targeted in the experimental design because of the direct biosynthetic relationship between amino acids and the three structure classes of branched fatty acids in Bacillus (24, 27). In addition, there are a limited number of common or commercially available complex additive and protein sources used for microbiological media. Identifying forensic signatures based on a reduced number of defining components rather than the myriad of possible medium formulations makes comprehensive surveys feasible, an added advantage for any potential forensic marker.To further frame our study in the context of forensic or investigative applications, we chose B. cereus as a target organism because of its genetic, structural, and biochemical similarities to B. anthracis (6, 19, 26). Also, since evidence from the 2001 anthrax mailings was predominantly composed of Bacillus spores (3), we used spore preparations of B. cereus for all FAME analyses.Lastly, analysis of forensic signatures from fatty acid profiles is complicated by the large number of variables (typically >15) and complex interactions among different fatty acid structures during cellular biosynthesis (28). Therefore, FAME profiles were analyzed with orthogonal multivariate statistical techniques that first considered all variables simultaneously and analyzed the overall dissimilarities among spore FAME profiles and, second, maximized differentiation among groups using a subset of variables and extracted patterns in fatty acid differences that are diagnostic for specific medium formulations.     

我国霍乱弧菌的脂肪酸分型研究

目的 对脂肪酸分型方法在霍乱弧菌菌株鉴定、菌株相似性分析等方面的应用价值进行评价。方法 选取了分离自我国的两个主要致病血清群的194株霍乱弧菌菌株（1961年以来的El Tor型和1992年以来的O139群）,提取脂肪酸,应用MIDI公司的脂肪酸分型系统,进行数据分析。结果 检测的所有菌株都含有的脂肪酸成分有13种。霍乱弧菌的判断符合率为88.6%。二维聚类分析没有成明显可区分的群, O139群霍乱弧菌的脂肪酸组成与O1群的相似,产毒与非产毒霍乱弧菌的脂肪酸成分没有显著差异。结论 脂肪酸分型对弧菌种的快速鉴定有应用价值,对霍乱弧菌的现场分离鉴定有辅助意义,在小样本暴发资料的研究中能够反映菌株之间的亲缘关系,但其对霍乱弧菌种属内的各种特征性菌群不具有鉴别能力。     

Characterization and Differentiation of Filamentous Fungi Based on Fatty Acid Composition

Cellular fatty acid composition of 100 different filamentous fungi, including oomycetes, zygomycetes, ascomycetes, basidiomycetes, and sterile mycelia, was analyzed to determine if they can be differentiated from one another on this basis and how minor variations in culture temperature and age affect this characteristic. Many fungi were found to possess the same fatty acids but produced different relative concentrations of each. Some fungi differed in both the fatty acids produced and in the relative concentrations of others. Multivariate discriminant analysis demonstrated that all of the species included in this study had significantly different (P < 0.001) fatty acid profiles. Each of the three phyla from which representative species were analyzed and the sterile forms had distinctive fatty acid profiles. Significant differences in fatty acid composition were also found at the intraspecific level. Both culture temperature and age affected fatty acid composition in the fungi examined, but when these factors were held constant, variance in fatty acid composition was not a problem and fungal fatty acid profiles could be differentiated statistically.     

Hexadecenoic Fatty Acid Isomers in Human Blood Lipids and Their Relevance for the Interpretation of Lipidomic Profiles

Monounsaturated fatty acids (MUFA) are emerging health biomarkers, and in particular the ratio between palmitoleic acid (9cis-16:1) and palmitic acid (16:0) affords the delta-9 desaturase index that is increased in obesity. Recently, other positional and geometrical MUFA isomers belonging to the hexadecenoic family (C16 MUFA) were found in circulating lipids, such as sapienic acid (6cis-16:1), palmitelaidic acid (9trans-16:1) and 6trans-16:1. In this work we report: i) the identification of sapienic acid as component of human erythrocyte membrane phospholipids with significant increase in morbidly obese patients (n = 50) compared with age-matched lean controls (n = 50); and ii) the first comparison of erythrocyte membrane phospholipids (PL) and plasma cholesteryl esters (CE) in morbidly obese patients highlighting that some of their fatty acid levels have opposite trends: increases of both palmitic and sapienic acids with the decrease of linoleic acid (9cis,12cis-18:2, omega-6) in red blood cell (RBC) membrane PL were reversed in plasma CE, whereas the increase of palmitoleic acid was similar in both lipid species. Consequentially, desaturase enzymatic indexes gave different results, depending on the lipid class used for the fatty acid content. The fatty acid profile of morbidly obese subjects also showed significant increases of stearic acid (C18:0) and C20 omega-6, as well as decreases of oleic acid (9cis-18:1) and docosahexaenoic acid (C22:6 omega-3) as compared with lean healthy controls. Trans monounsaturated and polyunsaturated fatty acids were also measured and found significantly increased in both lipid classes of morbidly obese subjects. These results highlight the C16 MUFA isomers as emerging metabolic marker provided that the assignment of the double bond position and geometry is correctly performed, thus identifying the corresponding lipidomic pathway. Since RBC membrane PL and plasma CE have different fatty acid trends, caution must also be used in the choice of lipid species for the interpretation of lipidomic profiles.     

专一识别脱落酸甲酯的单克隆抗体的制备与应用

专一识别２－顺（Ｓ）ＡＢＡ甲酯的单克隆抗体来源于以ＡＢＡ分子中的１－ＣＯＯＨ为偶联位点合成的免疫原。它与游离态ＡＢＡ和结合态ＡＢＡ葡萄糖酯的交叉反应仅分别为１％与３．５％,而与ＡＢＡ类似物,如２－顺－黄质醛、紫黄质以及ＡＢＡ的２－反式异构体和（Ｒ）－对映体则无交叉反应。利用该抗体建立的高度灵敏和精确的ＡＢＡｍｅ酶联免疫测定法,其检测线性范围为０．０４８～１．５２ｐｍｏｌ。通过ＡＢＡｍｅＥＬＩＳＡ和ＧＡ１＋３ＥＬＩＳＡ分析可知羊蹄叶片衰老与内源ＧＡ１＋３／ＡＢＡ比值的下降有关。     

Comparison of Fatty Acid Methyl Ester Analysis with the Use of API 20E and NFT Strips for Identification of Aquatic Bacteria

Aquatic bacteria grown on MacConkey agar and modified nutrient agar were identified by using API 20E and NFT strips and fatty acid methyl ester (FAME) analysis. Identifications agreed at the species level 35.7% of the time when API 20E strips and FAME analysis were used and in 4.3% of the cases when API NFT strips and FAME analysis were used. These techniques require further development before extended use in ecological studies.     

Characterization of Benthic Microbial Community Structure by High-Resolution Gas Chromatography of Fatty Acid Methyl Esters

Fatty acids are a widely studied group of lipids of sufficient taxonomic diversity to be useful in defining microbial community structure. The extraordinary resolution of glass capillary gas-liquid chromatography can be utilized to separate and tentatively identify large numbers of fatty acid methyl esters derived from the lipids of estuarine detritus and marine benthic microbiota without the bias of selective methods requiring culture or recovery of the microbes. The gas-liquid chromatographic analyses are both reproducible and highly sensitive, and the recovery of fatty acids is quantitative. The analyses can be automated, and the diagnostic technique of mass spectral fragmentation analysis can be readily applied. Splitless injection on glass capillary gas chromatographic columns detected by mass spectral selective ion monitoring provides an ultrasensitive and definitive monitoring system. Reciprocal mixtures of bacteria and fungi, when extracted and analyzed, showed progressive changes of distinctive fatty acid methyl esters derived from the lipids. By manipulating the environment of an estuarine detrital microbial community with antibiotics and culture conditions, it was possible to produce a community greatly enriched in eucaryotic fungi, as evidenced by scanning electron microscopic morphology. The fatty acid methyl esters from the lipids in the fungus-enriched detritus showed enrichment of the C₁₈ dienoic and the C₁₈ and C₂₀ polyenoic esters. Manipulation of the detrital microbiota that increased the procaryotic population resulted in an absence of large structures typical of fungal mycelia or diatoms, as evidenced by scanning electron microscopy, and a significantly larger proportion of anteiso- and isobranched C₁₅ fatty acid esters, C₁₇ cyclopropane fatty acid esters, and the cis-vaccenic isomer of the C₁₈ monoenoic fatty acid esters. As determined by these techniques, a marine settling community showed greater differences in bacterial as contrasted to microeucaryotic populations when compared with the microbial communities of benthic cores.     

Fatty Acid Methyl Ester (FAME) Succession in Different Substrates as Affected by the Co-Application of Three Pesticides

Introduction

In intensive agriculture areas the use of pesticides can alter soil properties and microbial community structure with the risk of reducing soil quality.

Materials and Methods

In this study the fatty acid methyl esters (FAMEs) evolution has been studied in a factorial lab experiment combining five substrates (a soil, two aged composts and their mixtures) treated with a co-application of three pesticides (azoxystrobin, chlorotoluron and epoxiconazole), with two extraction methods, and two incubation times (0 and 58 days). FAMEs extraction followed the microbial identification system (MIDI) and ester-linked method (EL).

Results and Discussion

The pesticides showed high persistence, as revealed by half-life (t_1/2) values ranging from 168 to 298 days, which confirms their recalcitrance to degradation. However, t_1/2 values were affected by substrate and compost age down to 8 days for chlorotoluron in S and up to 453 days for epoxiconazole in 12M. Fifty-six FAMEs were detected. Analysis of variance (ANOVA) showed that the EL method detected a higher number of FAMEs and unique FAMEs than the MIDI one, whereas principal component analysis (PCA) highlighted that the monosaturated 18:1ω9c and cyclopropane 19:0ω10c/19ω6 were the most significant FAMEs grouping by extraction method. The cyclopropyl to monoenoic acids ratio evidenced higher stress conditions when pesticides were applied to compost and compost+soil than solely soil, as well as with final time.

Conclusion

Overall, FAMEs profiles showed the importance of the extraction method for both substrate and incubation time, the t_1/2 values highlighted the effectiveness of solely soil and the less mature compost in reducing the persistence of pesticides.     

Cellular Fatty Acid and Ester Formation by Brewers′ Yeast

The activity of alcohol acetyltransferase, bound to the cell membrane and responsible for the formation of acetate esters, was affected by the fatty acid composition of the cell membrane. When saturated fatty acids, which only slightly inhibit alcohol acetyltransferase activity, were in-corporated into the cell membrane, the enzyme activity and ester formation were only slightly affected. On. the other hand, when unsaturated fatty acids, which strongly inhibit the enzyme activity, accumulated in the cell membrane, ester formation was suppressed with inhibition of the enzyme activity. The mechanism of formation of acetate esters by brewers′ yeast was explained by the alcohol acetyltransferase activity under the influence of the fatty acid composition of the cell membrane.     

Synthesis and Immunomodulating Activity of New Amino Acid Derivatives of Glycyrrhizic Acid and Its Methyl Ester

New amino acid derivatives of glycyrrhizic acid and its methyl ester were selectively synthesized using active N-succinimide esters. The compounds with residues of glycine ethyl ester and alanine methyl and butyl esters increased the level of agglutinins and hemolysins in blood serum of mice two- to threefold in comparison with the control upon parenteral administration at a dose of 2 mg/kg for 14 days.     

Fatty Acid Toxicity and Methyl Ketone Production in Aspergillus niger

Vegetative hyphae of Aspergillus niger rapidly converted caproic acid into 2-pentanone. More caproic acid was required for maximal ketone production at alkaline as compared to acidic pH values. Further increases in caproate concentrations at each pH value tested (4.5, 5.5, 6.5, 7.5, and 8.5) resulted in inhibition of ketone production and O₂ uptake. At alkaline pH values (8.5 and 7.5), oxygen uptake above the endogenous level and the production of 2-pentanone were parallel. This relationship did not hold at acidic pH values. At these pH values, ketone production continued (pH 6.5) or attained a maximum (pH 5.5 and 4.5) at caproate concentrations at which oxygen uptake was inhibited below endogenous levels. These data indicate that endogenous oxygen uptake was not inhibited by caproate at alkaline pH values at concentrations which did inhibit caproate oxidation and 2-pentanone production. Conversely, at acidic pH values, endogenous oxygen uptake was vigorously inhibited by caproate at concentrations at which exogenous fatty acid oxidation and 2-pentanone production were less affected. Simon-Beevers plots of these data showed that the undissociated acid was the permeant form of caproic acid. The fatty anion appeared to be the active or inhibitory form of caproate within the cell. Vegetative hyphae of A. niger were poorly buffered. Once the hyphae were washed and resuspended in phosphate buffer, they were well buffered towards inhibitory concentrations of caproic acid. These findings suggest that the primary mechanism(s) by which caproate inhibits oxygen uptake and ketone formation does not involve a change in the intracellular pH.     

Characterization of 19 Genes Encoding Membrane-Bound Fatty Acid Desaturases and their Expression Profiles in Gossypium raimondii Under Low Temperature

To produce unsaturated fatty acids, membrane-bound fatty acid desaturases (FADs) can be exploited to introduce double bonds into the acyl chains of fatty acids. In this study, 19 membrane-bound FAD genes were identified in Gossypium raimondii through database searches and were classified into four different subfamilies based on phylogenetic analysis. All 19 membrane-bound FAD proteins shared three highly conserved histidine boxes, except for GrFAD2.1, which lost the third histidine box in the C-terminal region. In the G. raimondii genome, tandem duplication might have led to the increasing size of the FAD2 cluster in the Omega Desaturase subfamily, whereas segmental duplication appeared to be the dominant mechanism for the expansion of the Sphingolipid and Front-end Desaturase subfamilies. Gene expression analysis showed that seven membrane-bound FAD genes were significantly up-regulated and that five genes were greatly suppressed in G. raimondii leaves exposed to low temperature conditions.     

脂肪酸甲酯法检测空心莲子草入侵影响土壤微生物群落结构的初步研究

空心莲子草是我国2003年公布的第一批外来入侵物种。为了进一步了解该植物的入侵机制,采集湖北咸宁、仙桃和武汉三地的土样,采用土壤脂肪酸甲酯谱图分析的方法探讨该植物入侵对土壤微生物的影响。结果显示：空心莲子草入侵后土壤可培养细菌、真菌的数量显著增加,而放线菌的数量显著下降。脂肪酸分析表明土壤微生物群落结构发生一定程度的改变,但其变化因土壤的不同而有差异。     

Effect of trans Fatty Acid Intake on LC-MS and NMR Plasma Profiles

Background

The consumption of high levels of industrial trans fatty acids (TFA) has been related to cardiovascular disease, diabetes and sudden cardiac death but the causal mechanisms are not well known. In this study, NMR and LC-MS untargeted metabolomics has been used as an approach to explore the impact of TFA intake on plasma metabolites.

Methodology/Principal Findings

In a double-blinded randomized controlled parallel-group study, 52 overweight postmenopausal women received either partially hydrogenated soybean oil, providing 15.7 g/day of TFA (trans18:1) or control oil with mainly oleic acid for 16 weeks. Subsequent to the intervention period, the subjects participated in a 12-week dietary weight loss program. Before and after the TFA intervention and after the weight loss programme, volunteers participated in an oral glucose tolerance test. PLSDA revealed elevated lipid profiles with TFA intake. NMR indicated up-regulated LDL cholesterol levels and unsaturation. LC-MS profiles demonstrated elevated levels of specific polyunsaturated (PUFA) long-chain phosphatidylcholines (PCs) and a sphingomyelin (SM) which were confirmed with a lipidomics based method. Plasma levels of these markers of TFA intake declined to their low baseline levels after the weight loss program for the TFA group and did not fluctuate for the control group. The marker levels were unaffected by OGTT.

Conclusions/Significance

This study demonstrates that intake of TFA affects phospholipid metabolism. The preferential integration of trans18:1 into the sn-1 position of PCs, all containing PUFA in the sn-2 position, could be explained by a general up-regulation in the formation of long-chain PUFAs after TFA intake and/or by specific mobilisation of these fats into PCs. NMR supported these findings by revealing increased unsaturation of plasma lipids in the TFA group. These specific changes in membrane lipid species may be related to the mechanisms of TFA-induced disease but need further validation as risk markers.

Trial registration

Registered at clinicaltrials.gov as {"type":"clinical-trial","attrs":{"text":"NCT00655902","term_id":"NCT00655902"}}NCT00655902     

Synthesis and Biological Activity of Nitrilic Derivatives of the Methyl Ester of Maleopimaric Acid

An effective method of a modification of the anhydride ring of the maleopimaric acid methyl ester by means of the cyanoethylation reaction was developed. A primary screening of a cytotoxic activity in vitro demonstrated an ability of the cyanoethyl derivative of the maleopimaric acid methyl ester to induce the death of the PC-3 cells of prostate cancer.     

Identification of Rhizobium leguminosarum and Rhizobium sp. (Cicer) strains using a custom Fatty Acid Methyl Ester (FAME) profile library

The potential of using fatty acid methyl ester (FAME) profiles of Rhizobium leguminosarum bv. viceae , phaseoli and trifolii , and Rhizobium sp. ( Cicer ) strains, for the identification of unknown isolates was assessed. This was achieved by developing a Rhizobium FAME library using 16 different Rhizobium strains of Rh. leguminosarum bv. viceae ( n  ₌ 5), Rh. leguminosarum bv. phaseoli ( n  ₌ 5), Rh. leguminosarum bv. trifolii ( n  ₌ 1) and Rhizobium sp. ( Cicer ) ( n  ₌ 5). Although there were considerable differences between Rh. leguminosarum biovars and strains and Rhizobium sp. ( Cicer ) strains, the variation within a particular biovar of Rh. leguminosarum was not high. Nevertheless, the feature FAME profiles of the various groups in the library allowed 75 putative rhizobia obtained from surface-sterilized nodules of field-grown lentil and pea plants to be identified.