SEARCH · PubMed Health
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
An unusual case of Flavobacterium meningosepticum pneumonia in an immunocompromised patient is reported. The necessity to recognize this opportunistic pathogen and its implication is discussed.
We describe a rapid, low-toxicity and simple method for the detection of the bacterial fish pathogen Flavobacterium psychrophilum. The method, based on the polymerase chain reaction (PCR), combined the electrophoresis of PCR products in a vertical agarose gel and a modified methylene blue stain. DNA was amplified directly either from bacterial suspensions or from tissues experimentally infected with F. psychrophilum, using different non-toxic commercial DNA extraction kits. The protocol allowed to detect 15 to 150 cells of the pathogen in bacterial suspension, without prior DNA extraction, and 7500 to 75,000 cells in seeded spleen tissue and ovarian fluid using Dynabeads DNA DIRECT extraction system. This method, which has the advantage of not using hazardous products, is proposed as a fast tool for routine identification of F. psychrophilum.
Cellular polyamines of eighteen genera belonging to the Flavobacterium-Flexibacter-Cytophaga complex were analysed by ion exchange liquid chromatography. Homospermidine was the major polyamine in the genera Bergeyella, Riemerella, Ornithobacterium, Weeksella, Capnocytophaga, Polaribacter and Psychroflexus belonging to the family Flavobacteriaceae. In the family Spirosomaceae, Runella, Spirosoma and Flectobacillus species contained spermidine whereas Cyclobacterium species contained homospermidine. Within a divergent cluster, Haliscomenobacter and Lewinella species contained spermidine whereas Saprospira grandis contained agmatine alone. The major polyamine of Chitinophaga and Sporocytophaga species was homospermidine. Flexithrix dorotheae contained spermidine. Microscilla marina, the type species of the genus Microscilla, contained spermidine and cadaverine. However, 'Microscilla sericea' contained homospermidine, 'Microscilla furvescens' contained spermidine, and 'Microscilla arenaria' lacked all polyamines. Polyamine profiles serve as a phenotypic chemotaxonomic marker for the reclassification of the genera belonging to the complex.
The total degradation of heparin by the joint action of a purified heparinase and a heparitinase from Flavobacterium heparinum is reported. The heparinase acts directly upon heparin, yielding 52% of a trisulfated disaccharide (O-(alpha-L-ido-4-enepyranosyluronic acid 2-sulfate)-(1leads to 4)-2sulfoamino-2-deoxy-D-glucose 6-sulfate) and 40% of a tetrasaccharide besides small amounts of hexa- and disaccharides. The tetrasaccharide is in turn completely degraded by the heparitinase, forming trisulfated disaccharide and disulfated disaccharide (O-(alpha-D-glyco-4-enepyranosyluronic acid)-(1leads to 4)-2-sulfoamino-2-deoxy-D-glucose 6-sulfate) in equal amounts. These and other results indicate that the tri- and disulfated disaccharides are linked alternately, in a proportion of 3:1, respectively. The primary structure of heparin and the mode of action of the heparinase and the heparitinase are proposed based on the analysis of the different products formed by the action of the enzymes.
Plasmid borne organophosphorus pesticide degrading (opd) gene of Flavobacterium balustinum has been amplified using polymerase chain reaction (PCR) and the resulting PCR product (1.25 Kb) was cloned in pUC18. Further, a detailed restriction map was determined to PCR product and subcloned as overlapping restriction fragments. The nucleotide sequence was determined for all subclones to obtain complete sequence of PCR amplified fragment. The sequence showed 98% similarity to opd genes cloned from other soil bacteria isolated from diversified geographical regions. The protein sequence predicted from the nucleotide sequence was almost identical to parathion hydrolase, a triesterase involved in hydrolysis of triester bond found in variety of op-pesticides. The signal sequence of parathion hydrolase contained recently discovered twin arginine transport (tat) motif. It appears that tat motif plays a critical role in membrane targeting of parathion hydrolase.
Flavobacterium columnare is the cause of columnaris disease, a serious condition affecting numerous freshwater fish species all over the world. Hitherto, only very scarce information is available on the pathogenesis of this bacterial disease, making it difficult to adopt a preventive approach to combat this pathogen. This study ambiates to shed more light on the way F. columnare interacts with its host. Since a number of difficulties occur in trying to isolate F. columnare from diseased fish, first of all, a selective medium was developed which enabled to recover various isolates from diseased live-bearing aquarium fish. In experimental infection studies in which these isolates were adopted, differences in virulence were noted. In a further stage, a clear correlation was made between virulence and the ability to adhere to the gill tissue, documenting the importance of the gills in the pathogenesis of columnaris disease. In this respect, a gill perfusion model was developed, by which means the adhesion of F. columnare to the gill tissue was partly pinpointed and various environmental factors influencing the adherence process elucidated. It may hence be stated that an important milestone has been achieved in the realisation of one of the most frequently used ways to prevent disease, that is through vaccination. Columnaris disease may also be opposed by ensuring good management practices eliciting adequate environmental circumstances.
Heparin lyase I has been purified from Flavobacterium heparinum and has been partially characterized (Yang, V. C., Linhardt, R. J., Berstein, H., Cooney, C. L., and Langer, R. (1985) J. Biol. Chem. 260, 1849-1857). There has been no report of the purification of the other polysaccharide lyases from this organism. Although all three of these heparin/heparan sulfate lyases are widely used, with the exception of heparin lyase I, there is no information on their purity or their physical and kinetic characteristics. The absence of pure heparin lyases and a lack of understanding of the optimal catalytic conditions and substrate specificity has stood in the way of the use of these enzymes as reagents for the specific depolymerization of heparin and heparan sulfate into oligosaccharides for structure and activity studies. This paper describes a single, reproducible scheme to simultaneously purify all three of the heparin lyases from F. heparinum to apparent homogeneity. Heparin lyase I (heparinase, EC 4.2.2.7), heparin lyase II (no EC number), and heparin lyase III (heparitinase, EC 4.2.2.8) have molecular weights (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric points (by isoelectric focusing) of M(r) 42,800, pI 9.1-9.2, M(r) 84,100, pI 8.9-9.1, M(r) 70,800, pI 9.9-10.1, respectively. Their amino acid analyses and peptide maps demonstrate that while these proteins are different gene products they are closely related. The kinetic properties of the heparin lyases have been determined as well as the conditions to optimize their activity and stability. These data should improve the application of these important enzymes in the study of heparin and heparan sulfate.
The gene coding for creatinase (creatine amidinohydrolase, EC 3.5.3.3) was isolated from Flavobacterium sp. U-188. The primary structure of creatinase deduced from the nucleotide sequence showed a protein (molecular weight, 42, 651) composed of 378 amino acids. The creatinase gene was over-expressed in Escherichia coli under the control of the lac promoter and the amount of this enzyme was over 20% of the soluble protein in the cell.
The beta-1,3-glucanase (1,3-beta-D-glucan glucanohydrolase, EC 3.2.1.6) gene from Flavobacterium dormitator var. glucanolyticae was cloned into Escherichia coli C600 with a vector plasmid, pBR322. The E. coli cells carrying a recombinant plasmid, pKU beta G1 (8.2 kb), showed a high beta-1,3-glucanase activity and a lytic activity on viable yeast cells. These activities were found in the periplasmic space of E. coli clone cells. Southern hybridization analysis showed that the cloned gene was derived from F. dormitator chromosomal DNA. The gene products were purified from the periplasmic fraction of E. coli by ammonium sulfate fractionation and ion-exchange chromatography. The purified enzymes were demonstrated to be identical with a lytic endo-beta-1,3-glucanase II and a nonlytic endo-beta-1,3-glucanase I from F. dormitator from their enzymological and immunological properties. In the E. coli cells, endo-beta-1,3-glucanase I was also formed by a proteolytic digestion of endo-beta-1,3-glucanase II during the cultivation as in F. dormitator. Thus, the only endo-beta-1,3-glucanase II was coded for in the cloned gene.
In most bacterial cell types studied, low intracellular free calcium is maintained by a variety of secondary exchangers which utilize transmembrane ion gradients. Prokaryotic calcium ATPases appear to be extremely uncommon, and none have been reported in Gram-negative organisms. We demonstrate ATP-dependent calcium uptake in everted membrane vesicles of Flavobacterium odoratum, a common Gram-negative soil and water bacterium. Calcium is transported with an apparent initial rate of 10 nmol/min mg of protein. It is inhibited by 20 microM orthovanadate, a specific P-type ATPase inhibitor, but significantly, it is unaffected by the addition of N-ethylmaleimide, N,N-dicyclohexylcarbodiimide, valinomycin, or nigericin. Because the Ca(2+)-ATPase makes up a high proportion of the total ATPase activity it is easily detected by a soluble ATP hydrolysis assay, with an initial rate for calcium-dependent ATPase activity in vesicles of 25-40 nmol/min.mg at pH 7.8 and 25 degrees C. The calcium-dependent activity is preferentially solubilized by the detergent C12E8 and can be precipitated at 55-80% ammonium sulfate in a fraction free of other contaminating ATPase activities. This partially purified fraction is enriched 15-fold and demonstrates an apparent Km for calcium of 2 microM, and for ATP of 130 microM. The IC50 for vanadate is 1.6 microM. These values are similar to those obtained for the eukaryotic sarcoplasmic reticulum calcium ATPase. The enzyme is rapidly phosphorylated by [gamma-32P]ATP in a calcium-dependent, vanadate-inhibitable manner. The phosphorylated species migrates with an apparent molecular mass of 60 kDa by NaDodSO4-polyacrylamide gel electrophoresis, and the phosphoryl group is sensitive to alkaline conditions, a characteristic of the acylphosphate linkage found in ATPases. These data demonstrate that the majority of calcium transport in F. odoratum is facilitated by a P-type ATPase.
A prolyl endopeptidase was purified from Flavobacterium meningosepticum. It was digested with trypsin. Two oligonucleotides, based on tryptic peptide sequences and used in PCR experiments, amplified a 300-base pair (bp) fragment. A 2.4-kilobase EcoRI fragment that hybridized to the 300-bp probe was cloned in lambda ZAP and sequenced from both strands. It contains a reading frame of 2115 bp, encoding the complete protein sequence of 705 amino acids. Ion-spray mass spectrometry experiments demonstrated the presence of an NH2-terminal signal peptide: the periplasmic mature protease is 685 residues in length for a molecular mass of 76784 Da. The prolyl endopeptidase showed no general sequence homology with known protein sequences except with that of porcine brain prolyl endopeptidase. In order to identify the active-site serine, the prolyl endopeptidase was labeled with [3H]diisopropyl fluorophosphate. One labeled peptide was purified and sequenced. The active-site serine was located in position 536 within the sequence GRSNGG. This sequence is different from the active-site sequence of the trypsin (GDSGGP) and subtilisin (GTSMAS) families.
A full-length insert for the endo-beta-N-acetylglucosaminidase (Endo) F1 gene was located on a 2,200-base pair EcoRI fragment of genomic DNA and cloned into the plasmid vector Bluescript. Transformed Escherichia coli cells expressed Endo F1 activity very well, but the enzyme apparently was not processed and secreted into the medium as it normally is in Flavobacterium meningosepticum. DNA sequencing revealed an open reading frame of 1,017 nucleotides encoding a putative 50-amino acid signal sequence, and a mature protein (31,667 Da) of 289 amino acids. The deduced amino acid sequence was verified by direct Edman microsequencing of 88% of the purified protein as tryptic and V8 protease peptides. Alignment of Endo F1 (289 amino acids) with the established amino acid sequence of Streptomyces plicatus Endo H (271 amino acids) revealed a 32% structural identity over the entire sequence and a high degree of conservative replacements. Potential catalytic domains identified in other proteins that hydrolyze the beta 1,4 glycosidic linkage between N-acetylglucosamine residues are also conserved for amino acid identity and relative spacing in Endo F1.
Flavobacterium meningosepticum endo-beta-acetyl-glucosaminidase F preparations have been resolved by hydrophobic interaction chromatography on TSK-butyl resin into at least three activities designated endo F1, endo F2 and endo F3 each with a unique substrate specificity. The 32-kDa endo F1 protein is the principle component representing in excess of 95% of most earlier and currently available commercial endoglycosidase preparations, the remainder being a mixture of five proteins from 32 to 43 kDa. Substrate specificity studies reveal endo F1 and endo H from Streptomyces plicatus to have nearly identical capacities to hydrolyze high-mannose oligosaccharides with a minimum Man1 alpha 3Man1 alpha 6Man1 beta 4GlcNAc1 beta 4GlcNAc structure. Although endo H will hydrolyze fucose-containing hybrid oligosaccharides at rates approaching comparable high-mannose forms, core-linked fucose reduces the hydrolysis rate of endo F1 by over 50-fold relative to high-mannose structures. Neither homogeneous endo F1 nor endo H hydrolyze complex multi-antennary glycans. The biantennary cleaving activity previously reported for endo F preparations (Tarentino, A. L., Gómez, C. M., and Plummer, T. H., Jr. (1985) Biochemistry 24, 4665-4671) is a characteristic of the contaminating endo F2 activity.
The purification of two heparitinases and a heparinase, in high yields from Flavobacterium heparinum was achieved by a combination of molecular sieving and cation-exchange chromatography. Heparinase acts upon N-sulfated glucosaminido-L-iduronic acid linkages of heparin. Substitution of N-sulfate by N-acetyl groups renders the heparin molecule resistant to degradation by the enzyme. Heparitinase I acts on N-acetylated or N-sulfated glucosaminido-glucuronic acid linkages of the heparan sulfate. Sulfate groups at the 6-position of the glucosamine moiety of the heparan sulfate chains seem to be impeditive for heparitinase I action. Heparitinase II acts upon heparan sulfate producing disulfated, N-sulfated and N-acetylated-6-sulfated disaccharides, and small amounts of N-acetylated disaccharide. These and other results suggest that heparitinase II acts preferentially upon N,6-sulfated glucosaminido-glucuronic acid linkages. The total degradation of heparan sulfate is only achieved by the combined action of both heparitinases. The 13C NMR spectra of the disaccharides formed from heparan sulfate and a heparin oligosaccharide formed by the action of the heparitinases are in accordance to the proposed mode of action of the enzymes. Comparative studies of the enzymes with the commercially available heparinase and heparitinase are described.
Elastolytic enzyme was purified and crystallized from culture fluid of Flavobacterium immotum No. 9-35. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight was determined by Sephadex G-100 gel filtration to be 13,000. The isoelectric point was between pH 8.3 and 8.9. The optimum pH of the enzyme was 7.2 for elastolytic activity. The purified enzyme showed not only elastolytic activity, but also non-specific proteolytic activity against various other proteins. Milk-clotting activity was also observed. The enzyme did not act on keratin, collagen, or fourteen amino acid esters, including N-benzoyl-L-alanine methyl ester, N-benzoyl-L-arginine ethyl ester, and N-acetyl-L-tyrosine ethyl ester, which were typical substrates of pancreatic elastase [EC 3.4.21.11], trypsin [EC 3.4.21.4], and chymotrypsin [EC 3.4.21.1], respectively. However, the enzyme selectively hydrolyzed elastin when both elastin and albumin were present in the reaction mixture. The enzyme was inhibited by o-phenanthroline and various heavy metals such as cadmium, lead, zinc, and mercury. Various inhibitors, such as diisopropyl phosphofluoridate, tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylalanine chloromethyl ketone, trypsin inhibitor, iodoacetamide, etc., had no effect on the elastolytic activity.
A chondroitinase that acts upon chondroitin sulfate C and hyaluronic acid was isolated from Flavobacterium heparinum. This enzyme was seperated from constitutional chondroitinase AC and an induced chondroitinase B also present in extracts of F. heparinum previously grown in the presence of chondroitin sulfates A, B or C. The enzyme acts upon chondroitin sulfate C producing tetrasaccharide plus an unsaturated 6-sulfated disaccharide (delta Di-6S), and upon hyaluronic acid producing unsaturated nonsulfated disaccharide (delta Di-OS). Chondroitin sulfate A is also degraded producing oligosaccharides and delta Di-6S but not delta Di-4S. The chondroitinase C is also distinguished from the chondroitinases B and AC by several properties, such as effect of ions, temperature for optimal activity, and susceptibility to increasing salt concentrations. The substrate specificity of the chondroitinase C is different from that of any other chondroitinase or hyaluronidase described so far.
18 neonates with bacteriologically confirmed Flavobacterium meningitis and ventriculitis were treated with various antibiotic regimens, including the use of intraventricular antibiotics. During the course of treatment, four patients died. 8/14 patients developed progressive hydrocephalus which required insertion of ventriculo-peritoneal shunts. The remainder 6/14 patients had normal ventricles or only mild ventriculomegaly. 5/8 patients with progressive hydrocephalus and 5/6 patients with normal or mildly dilated ventricles were followed up for at least 24 months. 4/5 of the patients with progressive hydrocephalus had severe bilateral hearing loss and delayed milestones. All the 5 patients with normal or mildly dilated ventricles had normal hearing although 2 of them had gross motor delay due to spastic paraplegia. Patients with progressive hydrocephalus received effective antibiotic treatment more than 8 days after the onset of infection while those with normal or mildly dilated ventricles within 8 days of infection. Onset of ventricular dilatation was associated with ventriculitis. Daily ultrasound scanning of the ventricles in the early stage helped to determine the need for early instillation of intraventricular antibiotics. Combined use of intravenous rifampicin, moxalactam and piperacillin showed promise as an effective antibiotic regimen in treating patients with normal or mildly dilated ventricles. Once significant ventriculomegaly has occurred, concomitant intravenous and intraventricular administration of antibiotics, to which the organisms were sensitive, was necessary to eradicate the infection.
A 3-day-old male infant, weighing 3,413 g and a gestational age of 38 weeks developed neonatal meningitis due to Flavobacterium meningosepticum. Treatment with cefmetazole and cefotaxime led him to a complete recovery without neurologic deficit. Of 82 previously published cases under 1 year old, 41 cases died and 16 of survivors developed hydrocephalus because the organism was resistant to many antibiotics. Therapy of meningitis due to the organism has not been standardized but the early laboratory identification and the choice of effective and safe antibiotics determined by antimicrobial sensitivity test improve the outcome.