Ilia Brondz

Development of fatty acid analysis by high-performance liquid chromatography, gas chromatography, and related techniques

Abstract This review surveys current knowledge of fatty acids (FAs) analysis, with an emphasis on the analysis of fatty acids in microorganisms in relation to microbial taxonomy, chemotaxonomy, and clinical practice. Aspects of fatty acid analysis in foods, drugs, and other products are also discussed, and a general perspective on the history of chromatography is presented.

Rapid Two-Step Procedure for Large-Scale Purification of Pediocin-Like Bacteriocins and Other Cationic Antimicrobial Peptides from Complex Culture Medium

ABSTRACT A rapid and simple two-step procedure suitable for both small- and large-scale purification of pediocin-like bacteriocins and other cationic peptides has been developed. In the first step, the bacterial culture was applied directly on a cation-exchange column (1-ml cation exchanger per 100-ml cell culture). Bacteria and anionic compounds passed through the column, and cationic bacteriocins were subsequently eluted with 1 M NaCl. In the second step, the bacteriocin fraction was applied on a low-pressure, reverse-phase column and the bacteriocins were detected as major optical density peaks upon elution with propanol. More than 80% of the activity that was initially in the culture supernatant was recovered in both purification steps, and the final bacteriocin preparation was more than 90% pure as judged by analytical reverse-phase chromatography and capillary electrophoresis.

Microbial chemotaxonomy : Chromatography, electrophoresis and relevant profiling techniques

This review deals with the chemistry of marker substances used in microbial classification and identification, their isolation and purification and their biomedical application. A critical evaluation of current methods is also included. The information presented is partly based on personal experience, partly derived from more than 300 publications in this rapidly expanding field of science. Much has been done to improve the recognition of microorganisms by GC, but there are also a series of other techniques available that can assist in bacterial classification and identification. Some of these techniques have been made available to the clinical microbiologist through commercial systems, e.g., assessment of bacterial fatty acids. A fingerprint library has been developed by Hewlett-Packard for the analysis of fatty acids from approximately 6000 different bacteria. Other chemotaxonomic methods require great personal expertise and advanced equipment. Efforts should therefore be made to adapt and simplify such methods for application in the routine clinical laboratory. Chemical markers will probably have a great impact on future microbial taxonomy, particularly in cases where conventional methods fail to give satisfactory classifications. In order to make taxonomy more objective, there seems to be a need for screening of chemical markers in bacterial species and for compiling chemotaxonomic fingerprints in clinical manuals.

The relative stereochemistry of hyperforin - an antibiotic from L.

Abstract The relative stereochemistry of hyperforin, an antibiotic from Hyperium perforatum has been determined by X-ray diffraction measurements.

Analysis of fatty acids. A new method for characterization of moulds

Aspergillus flavus, A. oryzae, A. fumigatus, Penicillium roqueforti, P. verrucosum, P. viridicatum, Mucor hiemalis, M. plumbeus and M. piriformis were cultivated on standardized media. The spores were harvested and the fatty acids of the spores transesterified. The fatty acid methyl esters were analysed by gas chromatography, and the fatty acids data were subjected to multivariate statistical analyses by SIMCA (Soft Independent Modelling of Class Analogies) pattern recognition. The results showed that this method enables various species of the moulds to be identified in a reproducible way.

Differentiation between Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus based on carbohydrates in lipopolysaccharide

In the present study, the closely related facultative, Gram-negative rods, Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus, were distinguished taxonomically by means of their carbohydrate composition in phenol-extracted lipopolysaccharide. Both A. actinomycetemcomitans and H. aphrophilus lipopolysaccharide contained rhamnose, fucose, galactose, glucose, L-glycero-D-mannoheptose, galactosamine, and glucosamine. The content of galactose was approximately twice as high in lipopolysaccharide from H. aphrophilus as in lipopolysaccharide from A. actinomycetemcomitans. D-Glycero-D-mannoheptose was detected exclusively in lipopolysaccharide from A. actinomycetemcomitans where it constituted 11.8-16.7% of the sugar content. This aldoheptose may therefore serve as a marker for chemotaxonomic differentiation between A. actinomycetemcomitans and H. aphrophilus. The present study also describes fragmentation of methylheptoside derivatives of trifluoroacetic acid (D-glycero- and L-glycero-D-mannoheptose) from A. actinomycetemcomitans as suggested by mass spectrometry.

Significance of cellular fatty acids and sugars in defining the genus Porphyromonas

It has recently been proposed that three asaccharolytic species forming pigmented colonies on blood agar should be transferred from the genus Bacteroides to the new genus Porphyromonas. In the taxonomy of the genus Bacteroides cellular fatty acid profiles obtained by gas chromatography and mass spectrometry after methylation and derivatization of whole cells have proved to be useful. In this study cellular fatty acids and sugars were analyzed in strains of Porphyromonas and Bacteroides species, and the resulting multivariate data were investigated by using principal component analysis. This analysis clearly separated the Porphyromonas spp. strains from the strains ofBacteroides fragilis, Bacteroides melaninogenicus, Bacteroides intermedius, and Bacteroides levii. In the description of the genus Porphyromonas the presence of various hydroxylated fatty acids was not discussed. In this study we show that these cellular components are useful taxonomic markers.

Multivariate analysis of quantitative chemical and enzymic characterization data in classification of Actinobacillus, Haemophilus and Pasteurella spp.

Chemotaxonomic data for strains of Actinobacillus, Haemophilus and Pasteurella spp. were analysed using three multivariate statistical strategies: principal components, partial least squares discriminant, and soft independent modelling of class analogy. The species comprised Actinobacillus actinomycetemcomitans. Haemophilus aphrophilus, H. paraphrophilus, H. influenzae, Pasteurella multocida, P. haemolytica and P. ureae. Strains were characterized by cell sugar and fatty acid composition, lysis kinetics during EDTA and EDTA plus lysozyme treatment, and methylene blue reduction. In total 23 quantitative variables were compiled from chemotaxonomic analyses of 25 strains. A. actinomycetemcomitans and H. aphrophilus formed distinct classes which differed from those of H. paraphrophilus, H. influenzae and Pasteurella spp. All characterization variables, except those describing fatty acid content, contributed significantly to inter-species discrimination.

Chemotaxonomy of selected species of the Actinobacillus—Haemophilus—Pasteurella group by means of gas chromatography, gas chromatography—mass spectrometry and bioenzymatic methods

Instrumental analytical and bioenzymatic methods were used to differentiate between species of the Actinobacillus-Haemophilus-Pasteurella group. Long-chain fatty acids were analysed directly with gas chromatography (GC) without derivatization. GC of trifluoroacetylated whole-cell methanolysates was a rapid method for differentiation. Cellular sugars were more suitable for differentiation than fatty acids. D-Glycero-D-mannoheptose, the major localization of which was lipopolysaccharide, distinguished H. aphrophilus from A. actinomycetemcomitans, H. paraphrophilus, H. influenzae type b, P. haemolytica, P. multocida, and P. ureae. GC of single colonies, which is a new chemotaxonomic method, was preferable to GC of liquid-grown cells. Lysozyme-and EDTA-induced bacteriolysis and reduction of methylene blue by cellular hydrogenase served as additional criteria for differentiation.

Multivariate analyses of fatty acid data from whole-cell methanolysates of Prevotella, Bacteroides and Porphyromonas spp.

The genus Bacteroides contains a number of biochemically and physiologically heterogeneous groups of organisms and needs taxonomic revision. In this study cellular fatty acids from a number of Bacteroides spp. were identified and quantified using gas chromatography and gas chromatography-mass spectrometry. The chemical data were then subjected to principal components analysis. In B. fragilis, which is the type species of the genus Bacteroides, C3-OH-iso17 was the predominant fatty acid (38.0%) and Cante15 was present in higher amounts (32.7%) than Ciso15 (14.6%). B. fragilis thus differed from all the other species examined: Prevotella (Bacteroides) buccae, P. (B.) oralis, P. (B.) oris, P. (B.) disiens, P. (B.) veroralis, P. (B.) heparinolytica and Porphyromonas (Bacteroides) endodontalis. Principal components analysis also enabled the closely related P. buccae, P. oralis and P. oris to be differentiated.