Protection of mice from lethal endotoxemia by use of an ornithine-containing lipid or a serine-containing lipid.

The effects of an ornithine-containing lipid [alpha-N-(3-acyloxyacyl)-ornithine (Orn-L)] or a serine-containing lipid [alpha-N-(3-acyloxyacyl)-serine (Ser-L)] from Flavobacterium meningosepticum on lethal endotoxemia in mice were examined. When 500 micrograms of Orn-L was intravenously administered 1 h before intravenous administration of a lethal dose of endotoxin, none of the mice died. The protective effect of Ser-L was weaker than that of Orn-L. Light and electron microscopic studies demonstrated that necrosis of hepatocytes caused by endotoxin was prevented by pretreatment with Orn-L. Furthermore, Kupffer cells were activated morphologically 1 h after the administration of Orn-L or Ser-L, and the liposomes of the lipoamino acids were incorporated into phagolysosomes in activated Kupffer cells. The activity of tumor necrosis factor in sera of endotoxin-treated mice was decreased markedly by pretreatment of mice with Orn-L. In vitro, the lipoamino acids suppressed endotoxin-induced tumor necrosis factor generation but did not suppress tumor necrosis factor generation induced by zymosan and whole cells of Staphylococcus aureus. These results suggested that Orn-L and Ser-L can be used as specific blocking agents against endotoxin. The blocking mechanism may be antagonistic, because of the structural similarities between the lipoamino acids and endotoxin lipid A.

Pneumonia and meningitis caused by a new nonfermentative unknown gram-negative bacterium.

Seven isolates of an unclassified bacterium resembling Flavobacterium spp. were characterized by growth requirements, microscopic examination, biochemical characteristics, antimicrobial susceptibility tests, protein profile analysis, and serologic data. The unclassified isolates were differentiated from Flavobacterium meningosepticum, Flavobacterium odoratum, Flavobacterium balustinum, Flavobacterium strain IIb, Chromobacterium violaceum, Aquaspirillum serpens, and Pseudomonas spp. The bacterium was a gram-negative rod with a polar flagellum. Protein profile analysis demonstrated two major protein bands present in the unclassified isolates that were absent from the Flavobacterium and Pseudomonas controls but present in the Aquaspirillum and Chromobacterium controls. However, no serologic cross-reactions were observed. Our results showed that the unclassified bacterium was distinct from any previously known genus of bacterium.

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.

[Composition of the complex lipids of Flavobacterium meningosepticum]. / Composition des lipides complexes de Flavobacterium meningosepticum.

The free lipids of Flavobacterium meningosepticum were separated by thin layer chromatography, and the main lipid fractions were analysed by FAB (fast atom bombardment) mass spectrometry. The major products were di-iso-C15- and iso-C15-iso-C17-phosphatidylethanolamine, and two ninhydrin + and phosphorus- fractions. The structures of the latter two fractions were established as ornithine lipids by using MIKE (mass ions kinetic energy) mass spectrometry, GC/MS (gas chromatography coupled with mass spectrometry) and conventional methods. The presence of small amounts of sphingolipids with C17- and C16-sphinganines was demonstrated. F. meningosepticum can be distinguished from F. multivorum and F. spiritivorum by easy characterization of the ornithine lipids by thin layer chromatography.

Various kinds of lipoamino acids including a novel serine-containing lipid in an opportunistic pathogen Flavobacterium. Their structures and biological activities on erythrocytes.

Lipoamino acids were found to represent 60% of the total extractable cellular lipids in Flavobacterium meningosepticum and F. indologenes which are known to be opportunistic pathogens. The structures of the lipoamino acids were resolved by chemical and physicochemical methods. Three kinds of lipoamino acids were included in the lipid of Flavobacterium: 3-hydroxyisoheptadecanoic acid amide-linked to serine and esterified to isopentadecanoic acid, and 3-hydroxyisoheptadecanoic acid amide-linked to ornithine and esterified to isopentadecanoic acid or 2-hydroxyisopentadecanoic acid. This type of serine-containing lipid is a novel, rare substance and exhibited high hemagglutinating activity (minimum hemagglutinating concentration was 0.25-0.5 micrograms/ml) with very good, stable dispersion of its liposomes. We called the serine-containing lipid 'flavolipin', based on the genus name of the bacteria. Of the two types of the ornithine-containing lipids, the one that had a nonpolar terminal fatty acid showed higher hemagglutinating activity than the one that had a polar terminal fatty acid. The reconstituted liposomes whose lipid composition was similar to that of the original bacterial cell membrane exhibited definite hemagglutinating activity. As soon as a terminal fatty acid of the lipoamino acids was lost, their biological activity on erythrocytes changed from hemagglutinating to hemolytic, being accompanied by the disappearance of animal species specificity. The mechanism of both the hemagglutination and the hemolysis was discussed.

Analysis of the microbiological particulates in municipal drinking-water by scanning electron microscopy/X-ray energy spectroscopy.

Scanning electron microscopy and X-ray energy spectroscopy (SEM/XES) were used to survey the biological and nonbiological particles in two different municipal drinking-water systems. Microbiological particles could be differentiated from non-biological by their qualitative elemental compositions and this information was used as the basis for an automated detection scheme. Automated SEM/XES analyses were used to demonstrate microbiological differences between well-water and surface-water in distribution systems.

Chemical characterization of Flavobacterium odoratum, Flavobacterium breve, and Flavobacterium-like groups IIe, IIh, and IIf.

The cellular fatty acid, sphingolipid, and isoprenoid quinone compositions of Flavobacterium odoratum, Flavobacterium breve, and Flavobacterium-like groups IIe, IIh, and IIf were determined, using thin-layer, gas-liquid, and reverse-phase high-performance liquid chromatography. The fatty acid data showed that groups IIe, IIh, and IIf were similar to recognized Flavobacterium species by the presence of relatively large amounts of iso-branched hydroxy and nonhydroxy acids. Groups IIe and IIh were essentially identical in fatty acid composition but were distinguished from group IIf, F. breve, and F. odoratum on the basis of minor qualitative and quantitative differences. All strains tested contained menaquinone 6 as the major isoprenoid quinone, and all lacked sphingolipids. Overall, the chemical data suggest that groups IIe, IIh, and IIf are additional Flavobacterium species and are different from sphingobacteria, which contain sphingolipid and menaquinone 7 as the major quinone.

Endotoxicity of newly recognized gram negative organism: a collective review.

A comprehensive picture of the in part personal investigations concerning an Endotoxin-like activity associated with some recently recognized Gram negative significant pathogens has been drawn. Suspensions of heat-killed organisms (or endotoxic lipopolysaccharide when available) have been tested using as markers of endotoxicity the Limulus assay and the skin Shwartzman reaction in rabbit. Some general considerations are finally set forth on the possible role of the surface structures in mediating (with other virulence factors) the damage in the host.

Separation of bacterial ubiquinones by reverse-phase high-pressure liquid chromatography.

A procedure was developed for the separation of ubiquinones by high-pressure liquid chromatography on a reverse-phase C18 column. Ubiquinones Q6 through Q14 were resolved in 20 min and were distinguished from menaquinones by comparing UV spectra at 248 and 275 nm.

The similarities between Pseudomonas paucimobilis and allied bacteria derived from analysis of deoxyribonucleic acids and electrophoretic protein patterns.

The chromosomal DNA was isolated and purified from 17 strains of Pseudomonas paucimobilis, and from the type or reference strains of Flavobacterium capsulatum, F. devorans, F. multivorum, 'Chromobacterium lividum', Xanthomonas campestris and seven species of Pseudomonas. The DNA base compositions (mol% G + C) of P. paucimobilis strains were between 62.2 and 68.6%, and typical strains had a mean value of 65.3 +/- 0.4 mol%, determined from thermal denaturation temperature. DNA-DNA molecular hybridization with 3H-labelled probe DNA from NCTC 11030 P. paucimobilis (the type strain) indicated that the species comprised a core of 13 closely related strains (74 to 96%), which included F. devorans NCIB 8195 (= ATCC 10829). Four P. paucimobilis strains displayed lower levels of hybridization (less than or equal to 38%). The hybridization results showed that P. paucimobilis was not closely related to allied yellow-pigmented bacteria or to other reference pseudomonads. The electrophoretic protein patterns of representative strains were analysed by computer-assisted techniques and similarity coefficients were calculated. A high degree of congruence was obtained with the DNA hybridization data, and the protein analyses indicated that the four atypical P. paucimobilis strains were heterogeneous and not a single group within the species. The generic affinities of P. paucimobilis, F. capsulatum, 'P. azotocolligans' and P. echinoides are uncertain, but available chemotaxonomic data indicate these species could provide the basis for a new genus.

Gas-liquid chromatography as an analytical tool in microbiology.

The use of gas-liquid chromatography (GLC) in clinical and diagnostic bacteriology laboratories has increased significantly in recent years. GLC analysis of metabolic products from bacterial growth and chemical components of bacterial cells has provided useful information for rapid detection and identification of several bacterial groups or species. The use of short-chain acid products and cellular fatty acid composition for identifying and classifying Pseudomonas species and other medically important gram-negative non-fermentative bacteria is illustrated. Application of the flexible, fused, silica-glass, capillary column for increased resolution of bacterial fatty acids is also discussed.

Deoxyribonucleic acid relatedness of some menaquinone-producing Flavobacterium and Cytophaga strains.

Nine menaquinone-forming strains of the Flavobacterium--Cytophaga complex with DNA base compositions between 35 and 45 moles percent guanine-plus-cytosine were investigated for genome sizes and DNA relatedness by DNA:DNA hybridization in vitro, using the optically recorded initial reassociation kinetics. Two strains representing C. hutchinsonii and C. marinoflava proved to be related on the 50 percent binding level, i.e. on a level of DNA relatedness commonly found within well-classified conventional genera of bacteria. Strains of C. johnsonae, F. heparinum, F. meningosepticum, F. odoratum, F. pectinovorum, and an unnamed Flavobacterium--Cytophaga strain were found to be interrelated, and linked to the genus Cytophaga, on the 30, or 20 percent binding levels, respectively. These findings indicate that the organisms in question are related to Cytophaga. They therefore should be transferred into the family Cytophagaceae.

Cellular fatty acids of Flavobacterium meningosepticum and Flavobacterium species group IIb.

The cellular fatty acid profiles of Flavobacterium meningosepticum and Flavobacterium species group IIb were markedly different from those of related bacteria. The profiles were characterized by the presence of 13-methyl-tetradecanoate and three uncommon acids: 2-hydroxy-13-methyl-tetradecanoate, 15-methyl-hexadecanoate, and 3-hydroxy-15-methyl-hexadecanoate.

Isolation of a novel sphingoglycolipid containing glucuronic acid and 2-hydroxy fatty acid from Flavobacterium devorans ATCC 10829.

A new acidic sphingoglycolipid has been isolated from a Gram-negative, glucose-non-fermentative (obligatory aerobic) bacterium, Flavobacterium devorans ATCC 10829, by thin-layer chromatography on silica gel after mild alkaline hydrolysis of the cellular lipids. Chemical degradation studies, thin-layer chromatographic behavior, IR and mass-spectrometric analysis of the original and reduced glycolipid with LiA1H4 revealed that the lipid contained glucuronic acid, long-chain bases, and fatty acids in a molar ratio of approximately 1:1:1. The major long-chain bases were identified by gas chromatography-mass spectrometry as dihydrosphingosine (d-18 :0) and longer homologues, while the N-acyl group was exclusively 2-hydroxy myristic acid. The most probable structure of this glycolipid appeared to be a ceramide glucuronic acid (N-acyl dihydrosphingosine 1-glucuronic acid).