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A Flavobacterium meningosepticum outbreak among intensive care patients.

A Flavobacterium meningosepticum outbreak, involving 12 infected and 47 colonized intensive care patients during the months of February through July 1990, was investigated. F. meningosepticum was isolated from tap water and ice, but these environmental strains eventually proved to be distinct from those colonizing patients. A review of newly colonized patients' charts revealed that a common factor among the patients was daily changes of ventilator tubing pasteurized in the hospital's central sterile department. More than 90% of patients in the outbreak had been on ventilators that used the pasteurized tubing. An investigation of the pasteurization process found that two pasteurizer tanks had been operating at suboptimal temperatures (< 62 degrees C). Cultures of water from the tanks and droplets of water found in the pasteurized tubing grew species of Acinetobacter, Moraxella, and Pseudomonas but did not grow F. meningosepticum. After deficiencies in the pasteurization process were corrected, the outbreak terminated. Despite the failure to culture F. meningosepticum, an analysis of gram-negative bacillary isolates showed that the deficiency in the pasteurization process was a major contributor to colonization of ventilated patients by bacteria ubiquitous in tap water.

Central Supply, Hospital↗

Structure of chondroitin sulfates. Analyses of the products formed from chondroitin sulfates A and C by the action of the chondroitinases C and AC from Flavobacterium heparinum.

The structures of chondroitin sulfate A from whale cartilage and chondroitin sulfate C from shark cartilage have been examined with the aid of the chondroitinases AC and C from Flavobacterium heparinum. The analyses of the products formed from the chondroitin sulfates by the action of the chondroitinases have shown that three types of oligosaccharides compose the structure of chondroitin sulfate A, namely, a dodeca-, hexa- and a tetra-saccharide, containing five, two and one 4-sulfated disaccharides per 6-sulfated disaccharide residue, respectively. The polymer contains an average of 3 mol of each oligosaccharide per mol of chondroitin sulfate A. Each mol of chondroitin sulfate C contains an average of 5 mol of 4-sulfated disaccharide units. A tetra-saccharide containing one 4-sulfated disaccharide and one 6-sulfated disaccharide C indicating that the 4-sulfated disaccharides are not linked together in one specific region but spaced in the molecule.

Chondroitin↗

On the structure of heparitin sulfates. Analyses of the products formed from heparitin sulfates by two heparitinases and a heparinase from Flavobacterium heparinum.

The analyses of the products formed from heparitin sulfates by the action of two heparitinases and a heparinase from Flavorbacterium heparinum is reported. Heparitin sulfates A and B are degraded by heparitinase I yielding two disaccharides, one of them composed of N-acetylucosamine and an unsaturated uronic, joined by alpha(1 lead to 4) linkage, and the other, with the same composition but with an O-sulfate at the hexosamine moiety. A third disaccharide is also formed from heparitin sulfate B, by the action of the same enzyme, composed of glucosamine N-sulfate and an unsaturated uronic acid joined probably by alpha(1 lead to 4) linkage. Besides these three disaccharides, heparitin sulfate B yields, by the action of heparitinase I, an oligosaccharide (with an average molecular weight of 6000) which is completely degraded by the heparitinase II yielding a disaccharide composed of glucosamine 2,6-disulfate and unsaturated uronic acid. All the disaccharides are further degraded by alpha-glycuronidase from Flavobacterium heparinum yielding the respective monosaccharides. Based on these and other analyses the possible structures of the heparitin sulfates are proposed.

Acetylglucosamine↗

Kinetic control of phosphoglycerate mutase from Flavobacterium sp. grown on ethylene glycol.

A species of Flavobacterium able to oxidise ethylene glycol to pyruvate via glyoxylate, glycerate, 2-phosphoglycerate and phosphoenolpyruvate exploits phosphoglycerate mutase to initiate gluconeogenesis. Partially purified phosphoglycerate mutase from this bacterium is independent of adenylate charge control but is activated by phosphoenolpyruvate. The possible significance of this regulation is discussed.

Acetyl Coenzyme A↗

Isolation and characterization of an induced chondroitinase ABC from Flavobacterium heparinum.

During the investigation of alternative methods for the large scale preparation of chondroitinases AC, B and C from Flavobacterium heparinum, a new chondroitinase activity was observed. This new enzyme, like the other chondroitinases, acts as an eliminase, forming unsaturated sulfated disaccharides from dermatan and chondroitin sulfates. In contrast to the chondroitinases previously described, which are endoglycosidases, this chondroitinase ABC cleaves the glycosidic linkages in an exolytic fashion, beginning at the reducing end of the substrate molecules. The oligosaccharides formed as transient products by the action of either chondroitinases or testicular hyaluronidase upon dermatan and chondroitin sulfates are also rapidly degraded by the chondroitinase ABC, regardless of their size or the presence of delta-4,5 unsaturation in the terminal uronic acid residue. The maximum activity of the chondroitinase ABC occurs at 30 degrees C and at pH 6.0-7.5. Only 15% of the activity was observed at 37 degrees C, indicating that the enzyme is very sensitive to thermal denaturation. It is strongly inhibited by phosphate ions and is also inhibited by the unsaturated disaccharides formed.

Chondroitin Lyases↗

High level expression in Escherichia coli of isopenicillin N synthase genes from Flavobacterium and Streptomyces, and recovery of active enzyme from inclusion bodies.

A T7 promoter-based vector was used to express the isopenicillin N synthase (IPNS) genes of Flavobacterium sp. 12,154 and Streptomyces jumonjinensis in Escherichia coli. Most of the IPNS synthesized at 37 degrees C, and representing some 22% and 51% of the total cell protein respectively, occurred in an insoluble, enzymatically inactive form. Active IPNS was recovered in a rapid and simple two-step procedure in which the insoluble material was first denatured in 5 M urea and then refolded by passing the solubilized IPNS through a G-25 Sephadex sizing column. Further chromatography on DEAE-Sepharose resulted in highly active IPNS preparations. This procedure was found to be well suited for scaling up to produce large amounts of IPNS.

Cloning, Molecular↗

New restriction endonucleases from Flavobacterium okeanokoites (FokI) and Micrococcus luteus (MluI).

Two new restriction endonucleases have been isolated from Flavobacterium okeanokoites IFO12536 and Micrococcus luteus IFO12992 and named FokI and MluI, respectively. Based on analysis of the sequences around the restriction sites, the recognition sequences and cleavage sites of these endonucleases were deduced as below: FokI: (formula: see text). MluI introduces double-strand cleavages at unique sequences that are completely two-fold rotationally symmetric like most type II restriction endonucleases. FokI belongs to a class of restriction endonucleases that recognize specific but asymmetric nucleotide sequences and introduce staggered cleavages at appointed positions away from the recognition sequences.

Bacterial Proteins↗

Flavobacterium meningosepticum bacteremia in an adult with acute leukemia. Use of rifampin to clear persistent infection.

A case of Flavobacterium meningosepticum bacteremia complicating the course of a patient with leukemia is described. The patient was treated successfully when rifampin was added to the antibiotic therapy. Unusual organisms should be considered as agents of infection in immunocompromised hosts and susceptibility testing with drugs not commonly employed for gram-negative rods should be performed in complicated cases.

Acute Disease↗

[Composition of the complex lipids of 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.

Chemical Phenomena↗

Identification of Flavobacterium strains by gas liquid chromatographic analysis of volatile fatty acids produced in culture.

The classification previously established for 74 Flavobacterium strains by gas liquid chromatographic (GLC) analysis of volatile fatty acids (VFA) produced in culture allowed the recovery of 9 groups (J. gen. Microbiol., 1986, 132, 2723-2732). Since graphic representation of the strains based on the first 3 factors obtained by principal component analysis (PCA) clearly separated these groups, we tried to identify 80 new strains by comparing their positions with those of the 9 groups, on the basis of both hierarchical classification and PCA methods. Of the 153 strains studied, only 12 were not allocated to a group corresponding to their original biochemical identification. Thus, on the whole, this characterization method by GLC analysis seemed satisfactory, although it could not be established whether the method was adequate for routine identification, or would serve merely as a complement.

Chromatography, Gas↗

A novel meta-cleavage product hydrolase from Flavobacterium sp. ATCC27551.

The organophosphate degrading (opd) gene cluster of plasmid pPDL2 of Flavobacterium sp. ATCC27551 contains a novel open-reading frame, orf243. This was predicted to encode an alpha/beta hydrolase distantly related to the meta-fission product (MFP) hydrolases such as XylF, PhnD, and CumD. By homology modeling Orf243 has most of the structural features of MFP hydrolases including the characteristic active site catalytic triad. The purified protein (designated MfhA) is a homotetramer and shows similar affinity for 2-hydroxy-6-oxohepta-2,4-dienoate (HOHD), 2-hydroxymuconic semialdehyde (HMSA), and 2-hydroxy-5-methylmuconic semialdehyde (HMMSA), the meta-fission products of 3-methyl catechol, catechol, and 4-methyl catechol. The unique catalytic properties of MfhA and the presence near its structural gene of cis-elements required for transposition suggest that mfhA has evolved towards encoding a common hydrolase that can act on meta-fission products containing either aldehyde or ketone groups.

Amino Acid Sequence↗

Purification and characterization of ascorbic acid 2-kinase from Flavobacterium devorans ATCC 10829.

Maximum activity for phosphorylating C(2)-OH of the ascorbic acid was observed at the time of 16 h incubation from the culture of Flavobacterium devorans ATCC 10829. The enzyme was purified 1.178-fold, via ammonium sulfate fractionation, Fast Q anion exchange, and phenyl agarose chromatography. Gel chromatography and SDS-polyacrylamide electrophoresis experiments showed that the enzyme is a tetramer with subunit MW of 29 kDa. Among available second substrates, pyrophosphate showed the highest activity. Optimum temperature and pH were 45 degrees C and 5.5, respectively. The enzyme was chemically modified only by diethylpyrocarbonate and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), indicating that histidine and carboxylate are in the active site. pH studies showed that two histidines are involved in the binding of the substrates and a carboxylate in catalysis. Therefore, the chemical mechanism of the enzyme is likely that two histidines bind to pyrophosphate and carboxylate, respectively, and a carboxylate acts as a general base.

Ascorbic Acid↗

Rapid direct determination using combined separation by prepared immunomagnetic and flow cytometry of Flavobacterium psychrophilum.

Flavobacterium psychrophilum, the causative agent of bacterial cold-water disease (BCWD), was originally isolated from coho salmon Oncorhychus kisutch in the USA. Bacterial cold-water disease has since been spreading throughout Japan and has caused serious damage to populations of ayu Plecoglossus altivel in many farms and rivers. The rapid method of detecting for F. psuchrophilum is requested, however, traditional methods are laborious because of complicated assay procedures. In this study, a rapid method of detecting F. psychrophilum was developed using a modified method of flow cytometry (FCM) analysis and immunomagnetic separation (IMS). Magnetic iron, in small particles, was prepared by the reaction of a mixture of ferric and ferrous ions under alkaline conditions. The particles were coated with antiserum against F. psychrophilum by dextran. Polyclonal antibodies (anti-F. psychrophilum) conjugated with fluorescein isothiocyanate (FITC) were reacted with F. psychrophilum, and then prepared immunomagnetic were applied using IMS, followed by FCM determination. A good correlation was observed between the cell numbers determined by the FCM method and the traditional method in the range of 10(2)-10(8) cells ml(-1). The FCM analysis could count cells within 1min, and the total analysis time, including sample preparation, was less than 2 h.

Cell Separation↗

The structure of the glycopeptides from the fish pathogen Flavobacterium columnare.

Proteolytic digestion of the phenol-water extraction product of the fish pathogen Flavobacterium columnare afforded a mixture of glycopeptides in which the oligosaccharide moiety was an unusual hexasaccharide composed of 4-O-methyl-2-acetamido-2-deoxy-D-glucuronic acid (GlcNAcA), D-glucuronic acid (D-GlcA), 2,3-di-O-acetyl-D-xylose (D-Xyl), 2-O-methyl-D-glucuronic acid (D-GlcA), D-mannose (D-Man), and 2-O-methyl-L-rhamnose (L-Rha). By the application of high-resolution 1D and 2D NMR, mass spectrometry, and chemical analysis, the hexasaccharide structure was determined to be: [carbohydrate structure--see text] where all monosaccharides have the D-configuration except for 2-O-methyl-L-rhamnose; and were in the pyranose form. Only one carbohydrate structure was found. The peptide part was represented by tri- to hepta-peptides with a minimal common tripeptide fragment Asp-Ser-Ala, extended with Ala and Val.

Animals↗

Immunization of eyed channel catfish, Ictalurus punctatus, eggs with monovalent Flavobacterium columnare vaccine and bivalent F. columnare and Edwardsiella ictaluri vaccine.

The efficacy of a modified live monovalent Flavobacterium columnare vaccine and bivalent F. columnare and Edwardsiella ictaluri vaccines were evaluated following immersion vaccination of eyed channel catfish (Ictalurus punctatus) eggs. The modified live F. columnare vaccine was grown in modified Shieh broth and administered at 1.35 x 10(7) CFU/ml for 15 min exposure (1l water). Booster immunization was conducted at day 34 with 2.17 x 10(7) CFU/ml for 15 min. Bivalent vaccines consisted of a 1:1 ratio of the modified live F. columnare and AQUAVAC-ESC vaccine for the 15 min exposure (1l immersion bath). Non-vaccinated controls were held in 1l water without vaccine for 15 min. Fish were challenged with F. columnare (ALG-00-530) by immersion at days 109, 116, and 137 post-primary immunization or E. ictaluri (AL-93-75) by immersion at day 116 (bivalent vaccine group). Efficacy of monovalent modified live F. columnare vaccine administered singly or with a booster vaccination was shown to be protective with relative percent survival (RPS) values ranging from 50.0 to 76.8. Some variation was seen in RPS values following bivalent immunization, ranging from 33.0 to 59.7 in the fish challenged with F. columnare and 44.5 to 66.7 in fish challenged with E. ictaluri. However, the RPS values were not statistically different. The results suggest that administration of live bivalent vaccine at the eyed-egg stage is safe and elicits protection upon single pathogen challenge.

Animals↗

Concomitant exposure of rainbow trout fry to Gyrodactylus derjavini and Flavobacterium psychrophilum: effects on infection and mortality of host.

Although rainbow trout fry mortality syndrome caused by the bacterium Flavobacterium psychrophilum is widespread in fish farms it is difficult to reproduce infection of rainbow trout in the laboratory using immersion exposure with bacterial suspensions. It has therefore been speculated that ectoparasites could act as enhancers of bacterial infections under natural conditions. In the present study rainbow trout fry were exposed to infections with F. psychrophilum (immersion for 30 min or 10 h) alone, exposed to the ectoparasitic monogenean Gyrodactylus derjavini alone or exposed to both pathogens in combination. Infection levels and host mortality were subsequently monitored to elucidate if the ectoparasitic monogeneans could enhance infection of fish with the bacterium. Immersion of fish in bacterial suspensions alone did not result in infection. Only one fish became infected with the bacterium and this fish belonged to the combination exposure group. The parasite populations increased differently in the various groups and it was found that host mortality was correlated to gyrodactylid infection levels (r=0.94) but not to bacterial exposure. The results emphasise the pathogenicity of the parasite G. derjavini, the relative resistance of intact fish to direct exposure to F. psychrophilum but provide only a weak indication of a possible enhancement of bacterial invasion due to ectoparasitic infections. It cannot be excluded that higher parasite burdens and/or prolonged immersion (more than 10 h) in bacterial suspensions may result in bacterial invasion.

Animals↗

Dextran alpha-(1 yields 2)-debranching enzyme from Flavobacterium Sp. M-73. Properties and mode of action.

The general properties and specificity of a dextran alpha-(1 yields 2)-debranching enzyme from Flavobacterium have been examined in order to apply this enzyme to the structural analysis of highly branched dextrans. The optimum pH range and temperature were pH 5.5-6.5, and 45 degrees, respectively. The enzyme was stable up to 40 degrees on heating for 10 min, and over a pH range of 6.5-9.0 on incubation at 4 degrees for 24 h. The effects of various metal ions and chemical reagents have also been examined. The debranching enzyme has a strict specificity for the (1 yields 2)-alpha-D-glucosidic linkage at branch points of dextrans and related branched oligosaccharides, and produces D-glucose as the only reducing sugar. The degree of hydrolysis of the dextrans by this enzyme and the Km value (mg/mL) were as follows: B-1298 soluble, 25.2%, 0.21; B-1299 soluble, 31.5%, 0.27; and B-1397, 11.8%, 0.91. the debranching enzyme thus has a novel type of specificity as a dextranhydrolase. We have termed this enzyme as dextran alpha-(1 yields 2)-debranching enzyme, and its systematic name is also discussed.

Cations↗