Comparison of signature lipid methods to determine microbial community structure in compost

The microbial community structure changes substantially during the composting process and simple methods to follow these changes can potentially be used to estimate compost maturity. In this study, two such methods, the microbial identification (MIDI) method and the ester-linked (EL) procedure to determine the composition of long-chain fatty acids, were applied to compost samples of different age. The ability of the two methods to describe the microbial succession was evaluated by comparison with phospholipid fatty acid (PLFA) analysis on the same samples.Samples were taken from a 200-l laboratory compost reactor, treating source-separated organic household waste. During the initial stages of the process, the total concentration of fatty acids in compost samples treated with the EL and MIDI methods was many times higher than with the PLFA method. This was probably due to the presence of fatty acids from the organic material in the original waste. However, this substantial difference between PLFA and the other two methods was not found later in composting. Although the PLFA method gave the most detailed information about the growth and overall succession of the microbial community, the much simpler MIDI and EL methods also successfully described the shift from the initially dominating straight chain fatty acids to iso- and anteiso branched, 10 Me branched and cyclopropane fatty acids in the later stages of the process. Thus, the MIDI and EL extraction methods appear to be suitable for analysis of microbial FAME profiles in compost, particularly in the later stages of the process.     

Comparison of two total cellular fatty acid analysis protocols to differentiate Rhizoctonia oryzae and R. oryzae-sativae

Two fatty acid analysis protocols (the MIDI and a modified MIDI method) were investigated for their utility to characterize and differentiate Rhizoctonia oryzae and R. oryzae-sativae isolates from four countries. Only the modified MIDI method permitted a clear differentiation between the two species, regardless of the isolates' country of origin. The modified MIDI method gave the most consistent and reproducible fatty acid results. The failure of the MIDI method to differentiate between R. oryzae and R. oryzae-sativae isolates suggests that the 30 minutes saponification step is insufficient to completely break the cell wall of these two species. This study demonstrated that fatty acid profiles, obtained by the modified MIDI protocol, have the potential as a diagnostic tool for both R. oryzae and R. oryzae-sativae.     

Rapid sample processing and fast gas chromatography for identification of bacteria by fatty acid analysis

A commercially available system for microbial identification by fatty acid analysis (Microbial Identification System (MIS), MIDI, Newark, DE, USA) requires a four-step sample derivatization procedure in screw-cap test tubes. By using glass tubes in a 96-well format with multichannel pipetting, the time required for sample preparation can be greatly reduced. The standard gas chromatography column, 25 m long by 0.20 mm ID, is replaced with a 10 m long by 0.10 mm ID column, reducing the gas chromatography run time to one third of the standard time. Either or both of these procedures can be easily implemented in any laboratory using the MIDI system, resulting in faster identifications and higher sample throughput.     

Chemotaxonomic characterization of rice seedling blight complex using fatty acid methyl ester (FAME) profiles

Rice seedling blight is an important disease caused by a complex of fungi that include Fusarium, Rhizopus, Pythium, and Trichoderma species. A modified MIDI method was used for extraction of fatty acids from these causal pathogens, and fatty acid methyl ester (FAME) profiles were characterized. Factors that might affect fatty acid production, such as period of culture and saponification in extraction, were also evaluated. A total of 14 fatty acids were detected, and FAME profiles showed quantitative and qualitative variations by discriminant analysis and principal component analysis. Genus-specific FAME profiles consisting of the types of fatty acid produced and remarkable components of individual fatty acids were observed. The possibility of application as chemotaxonomic methods based on the FAME profiles for diagnosis of the rice seedling blight complex is also discussed.     

Comparison of Whole-Cell Fatty Acid (MIDI) or Phospholipid Fatty Acid (PLFA) Extractants as Biomarkers to Profile Soil Microbial Communities

The whole-cell lipid extraction to profile microbial communities on soils using fatty acid (FA) biomarkers is commonly done with the two extractants associated with the phospholipid fatty acid (PLFA) or Microbial IDentification Inc. (MIDI) methods. These extractants have very different chemistry and lipid separation procedures, but often shown a similar ability to discriminate soils from various management and vegetation systems. However, the mechanism and the chemistry of the exact suite of FAs extracted by these two methods are poorly understood. Therefore, the objective was to qualitatively and quantitatively compare the MIDI and PLFA microbial profiling methods for detecting microbial community shifts due to soil type or management. Twenty-nine soil samples were collected from a wide range of soil types across Oregon and extracted FAs by each method were analyzed by gas chromatography (GC) and GC-mass spectrometry. Unlike PLFA profiles, which were highly related to microbial FAs, the overall MIDI-FA profiles were highly related to the plant-derived FAs. Plant-associated compounds were quantitatively related to particulate organic matter (POM) and qualitatively related to the standing vegetation at sampling. These FAs were negatively correlated to respiration rate normalized to POM (Resp_POM), which increased in systems under more intensive management. A strong negative correlation was found between MIDI-FA to PLFA ratios and total organic carbon (TOC). When the reagents used in MIDI procedure were tested for the limited recovery of MIDI-FAs from soil with high organic matter, the recovery of MIDI-FA microbial signatures sharply decreased with increasing ratios of soil to extractant. Hence, the MIDI method should be used with great caution for interpreting changes in FA profiles due to shifts in microbial communities.     

Changes in Bacteria Recoverable from Subsurface Volcanic Rock Samples during Storage at 4°C

The abundance of viable microorganisms recovered from deep subsurface volcanic rock samples increased after rock perturbation and storage for 1 week at 4°C, while the diversity and evenness of recoverable heterotrophic bacterial communities generally decreased. One sample of each morphologically distinct colony type, recovered both before and after storage of U12n rock samples, was purified and characterized by fatty acid methyl ester (MIDI) and API rapid NFT strips. As determined by MIDI cluster analysis, the composition of the recoverable microbial communities changed with storage of rock samples; some groups of organisms were recovered only before, only after, or at both sample times. In general, the isolates recovered only after storage of rock samples had a greater ability to utilize the carbohydrates included in API test strips and had faster generation times than isolates recovered only on initial plating. The nutritional versatility and faster growth rates of organisms recovered in higher proportions after sample storage provide evidence that some microbial community changes may be due to the proliferation of a few bacterial types. However, because some new genera are recovered only after storage, the possibility also exists that dormant bacterial types are resuscitated during sample perturbation and storage.     

Comparison of two fatty acid analysis protocols to characterize and differentiate Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici

The utility of fatty acid methyl ester (FAME) profiles for characterization and differentiation of isolates of Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici was investigated. Two fatty acid analysis protocols of the normal (MIDI) and a modified MIDI method were used for their utility. Only the modified MIDI method allowed a clear differentiation between F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicislycopersici. FAME profiles using the modified MIDI method gave the most consistent and reproducible analyzed fatty acid data. Evaluation of the FAME profiles based on cluster analysis and principal-component analysis revealed that FAME profiles from tested isolates were correlated with the same vegetative compatibility groups (VCGs) compared to the same races in F. oxysporum f. sp. lycopersici. Results indicated that FAME profiles could be an additional tool useful for characterizing isolates and forma species of F. oxysporum obtained from tomato.     

Changes in Bacteria Recoverable from Subsurface Volcanic Rock Samples during Storage at 4 degrees C

The abundance of viable microorganisms recovered from deep subsurface volcanic rock samples increased after rock perturbation and storage for 1 week at 4 degrees C, while the diversity and evenness of recoverable heterotrophic bacterial communities generally decreased. One sample of each morphologically distinct colony type, recovered both before and after storage of U12n rock samples, was purified and characterized by fatty acid methyl ester (MIDI) and API rapid NFT strips. As determined by MIDI cluster analysis, the composition of the recoverable microbial communities changed with storage of rock samples; some groups of organisms were recovered only before, only after, or at both sample times. In general, the isolates recovered only after storage of rock samples had a greater ability to utilize the carbohydrates included in API test strips and had faster generation times than isolates recovered only on initial plating. The nutritional versatility and faster growth rates of organisms recovered in higher proportions after sample storage provide evidence that some microbial community changes may be due to the proliferation of a few bacterial types. However, because some new genera are recovered only after storage, the possibility also exists that dormant bacterial types are resuscitated during sample perturbation and storage.     

Distinction of Prevotella intermedia and Prevotella nigrescens from endodontic bacteremia through their fatty acid contents

Prevotella nigrescens has recently been recognized as a new species distinct from Prevotella intermedia. The distinction is based largely on DNA-DNA hybridization, electrophoretic migration of malate and glutamate dehydrogenase, and peptidase and lipase activities of type strains. Gas chromatography of cellular fatty acids can be a useful adjunct for characterization and identification of bacterial species. In the present study, cellular fatty acid profiles were determined for seven strains of P. intermedia and six strains of P. nigrescens. Six of these 13 strains were isolated from the root canal and blood of three patients during endodontic therapy of teeth with Asymptomatic apical periodontitis. The bacteria were cultivated anaerobically in 10 mL prereduced anaerobically sterilized peptone-yeast extract-glucose broth for 24 h. Dried cells of each isolate were methanolysed and their fatty acid contents determined by the Microbial Identification System software package by MIDI. The data were treated by principal component analysis, which distinguished P. nigrescensfromP. intermedia. Cellular fatty acid profiles of these strains of the species in blood matched the profiles of their respective root canal isolates, as demonstrated by Euclidean Distance Square assessment. This suggested that the organisms in the root canal had spread to the bloodstream during endodontic treatment.     

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.     

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。     

Mining fatty acid databases for detection of novel compounds in aerobic bacteria

This study examines how the discriminatory power of an automated bacterial whole-cell fatty acid identification system can be significantly enhanced by exploring the vast amounts of information accumulated during 15 years of routine gas chromatographic analysis of the fatty acid content of aerobic bacteria. Construction of a global peak occurrence histogram based upon a large fatty acid database is shown to serve as a highly informative tool for assessing the delineation of the naming windows used during the automatic recognition of fatty acid compounds. Along the lines of this data mining application, it is suggested that several naming windows of the Sherlock MIS TSBA50 peak naming method may need to be re-evaluated in order to fit more closely with the bulk of observed fatty acid profiles. At the same time, the global peak occurrence histogram has put forward the delineation of 32 new peak naming windows, accounting for a 26% increase in the total number of fatty acid features taken into account for bacterial identification. By scrutinizing the relationships between the newly delineated naming windows and the many taxonomic units covered within a proprietary fatty acid database, all new naming windows were proven to correspond with stable features of some specific groups of microorganisms. This latter analysis clearly underscores the impact of incorporating the new fatty acid compounds for improving the resolution of the bacterial identification system and endorses the applicability of knowledge discovery in databases within the field of microbiology.     

A Soil and Rhizosphere Microorganism Isolation and Enumeration Medium That Inhibits Bacillus mycoides

A new solid medium has been developed for the enumeration and isolation of soil and rhizosphere microorganisms. This medium, named rhizosphere isolation medium, contains glucose and 15 of the 20 common amino acids. The absence of five other amino acids, namely, aspartic acid, asparagine, cysteine, proline, and threonine, inhibits the growth of Bacillus mycoides, a commonly encountered bacterium that rapidly spreads on agar media and complicates the isolation and enumeration of other microorganisms. Compared with a similar medium containing Casamino Acids, rhizosphere isolation medium had half as many colonies of B. mycoides, with each colony approximately half the diameter. The two media had similar total numbers of bacterial colonies. Isolates were divided into taxononomic groups, roughly corresponding to species and genus, by fatty acid methyl ester analysis and numerical methods. There were 24 genera and 41 species found in the isolates from rhizosphere isolation medium, while 19 genera and 35 species were found in the isolates from the medium prepared with Casamino Acids. No major group of bacteria was found to occur only on one medium or on the other, indicating that the five missing amino acids had no great effect on organisms other than B. mycoides. This medium may prove useful in soil and rhizosphere studies in which the growth of B. mycoides is undesirable.     

Identification and serotyping of atypical Legionella pneumophila strains isolated from human and environmental sources

AIMS: To validate identification methods for Legionella pneumophila strains that cannot be serotyped into the known serogroups and to characterize their antigenic diversity. METHODS AND RESULTS: Fifty L. pneumophila strains that could not be serogrouped, but which had been confirmed as L. pneumophila by mip gene sequencing, were further identified phenotypically. We used (i) MONOFLUO anti-Legionella Staining Reagent (Bio-Rad) (50/50), (ii) an in-house prepared immunoblot assay for the detection of L. pneumophila- specific Mip protein epitope (50/50), (iii) fatty acid analysis using the Microbial Identifications System (MIDI) (47/50) and (iv) Oxoid agglutination tests (44/50). The serological diversity was further characterized by testing with five serogroup-cross-reactive monoclonal antibodies, resulting in nine phenons. CONCLUSIONS: The division of L. pneumophila into 15 serogroups does not reflect the serogroup heterogeneity. Results of these tests indicate that there are more serogroups. SIGNIFICANCE AND IMPACT OF THE STUDY: MONOFLUO anti-Legionella Staining Reagent is the only commercially available tool for identifying atypical strains of L. pneumophila. If necessary for epidemiological purposes, the antigenic heterogeneity of these strains can be analysed by monoclonal antibodies.     

Dereplication of<Emphasis Type="Italic"> Streptomyces</Emphasis> soil isolates and detection of specific biosynthetic genes using an automated ribotyping instrument

The discrimination of distinct cultures among morphologically similar Streptomyces soil isolates (dereplication) and the detection of specific biosynthetic pathways in these strains are important steps in the selection of microorganisms to include in a natural products library. We have developed methods for analysis of actinomycetes using the RiboPrinter microbial characterization system, an automated instrument that performs ribotyping on bacterial samples. To evaluate our dereplication method, 26 Streptomyces isolates, obtained from soil samples collected in Maui, Hawaii, were ribotyped and compared with each other, using the RiboPrinter. The strains were also compared by 16S rDNA sequence analysis, MIDI fatty acid analysis, and LC-MS profiling of fermentation extracts. The RiboPrinter was able to identify closely related isolates and to discriminate between morphologically similar isolates with unique genetic, fatty acid and fermentation profiles. For the detection of biosynthetic genes, a 1,006-bp probe containing a portion of an adenylation domain of a non-ribosomal peptide synthetase (NRPS) was employed. Using this alternate probe in place of the standard ribosomal probe, the RiboPrinter was able to detect NRPS genes in several strains of Streptomyces. These results demonstrate that the RiboPrinter has multiple applications in a natural products research program.     

Selectivity and Thermal Properties of Various Starches Incorporating Free Fatty Acids

The effects of free fatty acids on the selectivity and thermal properties of starch samples incorporating free fatty acids were examined by DSC. An analysis of the free fatty acid values incorporated into cassava starch and potato starch shows that myristic acid was the highest and linoleic acid was the lowest, while the free fatty acid values of corn starch were significantly higher than those of the other starches. DSC measurements on corn starch show an initial peak and another peak in a higher-temperature region, this second peak differing according to the incorporated free fatty acid. It is thus considered that the state of the complex of each free fatty acid with amylose might be better understood by observing the respective DSC characteristics.     

诺卡氏菌型放线菌细胞中脂肪酸的气相色谱分析

采用Sherolock全自动微生物鉴定系统,用气相色谱法,分析测定了4株诺卡氏菌型放线菌分离菌株的脂肪酸成分和含量,结果表明,该法具有分辨率高,稳定、重现性好,简便易行等特点,在一定程度上与16S rRNA基因序列比较结果相一致,能在种及菌株水平上反映出放线菌的基因型,系统发育和分类关系,是一种较好的脂肪酸定量测定方法,尤其适用于分析大量的菌株或分离株,可应用于放线菌种水平的分类和快速鉴定.     

Single step thin-layer chromatographic method for quantitation of enzymatic formation of fatty acid anilides

The activity of the enzyme involved in catalyzing the formation of fatty acid anilides can be measured by quantitating the fatty acid anilides formed. We have shown earlier that oleic acid is the most preferred substrate among other fatty acids studied for the conjugation with aniline. The reaction product (oleyl anilide) could be separated by thin-layer chromatography (TLC) and then quantified by reversed-phase high-performance liquid chromatography (HPLC). Using [1-¹⁴C]oleic acid as substrate, the fatty acid anilide forming activity can be determined in a single step by TLC analysis. The conventional TLC methods used for the separation of the fatty acid esters, however, could not resolve oleyl anilide from the residual [1-¹⁴C]oleic acid. Therefore, a simple and reliable TLC method was developed for the separation of oleyl anilide from oleic acid using a freshly prepared solvent consisting of petroleum ether–ethyl acetate–ammonium hydroxide (80:20:1, v/v). Using this solvent system the relative flow (R_f) values were found to be 0.54 for oleyl anilide and 0.34 for aniline, whereas oleic acid remained at the origin. The TLC procedure developed in the present study could be used to determine the fatty acid anilide forming activity using [1-¹⁴C]oleic or other fatty acids as substrate and was also found suitable for the analysis of fatty acid anilides from the biological samples.     

Evaluation of several economical computational methods for geometry optimization of phosphorus acid derivatives

Several economical methods for geometry optimisation, applicable to larger molecules, have been evaluated for phosphorus acid derivatives. MP2/cc-pVDZ and B3LYP/6-31+G(d) geometry optimizations are used as reference points, results from geometry optimizations for other methods and their subsequent single point energy calculations are compared to these references. The geometries from HF/MIDI! optimizations were close to those of the references and subsequent single point energies with B3LYP/6-31+G(d,p) or EDF1/6-31+G(d) gave a mean average deviation (MAD) of less than 0.5 kcal mol-1 from those obtained with the reference geometries.     

Characterization of the microbiology within a 21 m3section of rock from the deep subsurface

The distribution of aerobic chemoheterotrophic microorganisms within a 21 m3 section of deep subsurface rock was determined. Nineteen samples for microbiological analysis were aseptically taken by hand from the walls of a 400 m deep subsurface tunnel after an alpine miner created fresh rock faces 0.76, 1.52, 2.28, and 3.04 m into the tunnel wall. The direct counts were several orders of magnitude greater than viable counts in all samples. One of each morphologically distinct bacterial type from each sample was purified and analyzed for fatty acid methyl esters (FAME) using the Microbial Identification System (MIDI). Numbers of bacterial types, diversity, and equitability of recoverable microbial communities were the same or similar using either morphotype or FAME analyses as the basis for distinguishing between bacterial types. Twenty-nine genera (Euclidean distance of 25) were found within the rock section, while 28 of the 210 bacterial types isolated were nonculturable under the growth regime required for cluster analysis. Most isolates clustered at the genus level with Arthrobacter, Gordona, and Acinetobacter. Two genera, containing 16 isolates, were unmatched to known organisms within the MIDI data base and clustered with other isolates at a Euclidean distance greater than 50. While some species (Euclidean distance 10) were recovered from multiple sites within the rock section, most were found at 1–3 sites and usually without a definitive pattern of distribution. Offprint requests to: P. S. Amy.