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Flavobacterium meningosepticum was isolated from the cerebrospinal fluid of an adult immunodeficient female. In spite of prompt therapy the patient succumbed to the infection. The opportunistic role of the organism is discussed.
During the period 1966-77, 24 strains of Flavobacterium odoratum were identified from among strains of Gram-negative, non-fermentative bacteria submitted to the National Collection of Type Cultures for computer-assisted identification. The F. odoratum strains showed resistance to therapeutic levels of gentamicin, tobramycin, amikacin, and carbenicillin as well as to several other antimicrobial agents generally useful in the treatment of infections caused by Gram-negative, non-fermentative bacteria. Two strains isolated from amputation stumps and another three strains isolated in significant numbers from urine specimens were possibly opportunist pathogens. The biochemical characteristics of the 24 strains, the proposed neotype strain of F. odoratum, and three strains representative of a group, referred to at the Center for Disease Control, Atlanta as group M-4f, were compared with those of biochemically similar species which may be isolated from clinical material.
After an outbreak of flavobacterium meningitis in the newborn nursery of the University Hospital, an investigation was carried out to determine the possible sources of this organisms in the nursery. Various serotypes of the organism were recovered from a variety of sources such as wash basins, sinks, suction apparatus, and disinfectants in the nursery as well as the neighbouring wards. Colonisation of the pharynx with this organism was demonstrated in several clinically healthy babies in the nursery. The possible role of fomites, hospital staff, newborn babies, and their mothers in the transmission of the organism is considered together with the measures taken to reduce environmental contamination.
Pseudomonas paucimobilis and Flavobacterium multivorum (formerly Group IIK biotype 1 and biotype 2, respectively) showed lectin-binding activity with Helix aspersa and no activity with eight other well-characterized lectins. Microagglutination titrations and immunodiffusion precipitation revealed specific antibody activity to immunizing antigens. However, no major antigens were found to be common from crude antigen preparations of P. paucimobilis and F. multivorum when tested against opposing antisera.
Several yellow-pigmented species within the family Flavobacteriaceae are commonly associated with diseases in fish and are difficult to speciate due to their fastidious, slow-growing nature and cross-reactive antigens. Here we report the development of specific, antibody-diagnostic tests for Flavobacterium psychrophilum, the aetiological agent of rainbow trout fry syndrome and bacterial cold water disease. A unique antigen from F. psychrophilum, the lipopolysaccharide (LPS) O-polysaccharide (O-PS), formed the basis for the antibody test. LPS O-PS was purified and conjugated to keyhole limpet haemocyanin and bovine serum albumin for the generation of rabbit immune sera and the development of antibody-based diagnostic tests. Rabbit polyclonal anti-O-PS serum was highly specific for F. psychrophilum, without the need for prior cross-absorption with related bacteria and was the basis of an effective ELISA diagnostic test. Antibodies were purified from rabbit anti-O-PS serum and adsorbed onto coloured latex beads for the development of a specific, bead agglutination assay for F. psychrophilum.
Bacterial endocarditis, particularly involving the left side, has been shown to occur in patients in regular hemodialysis. We report a case of right-sided endocarditis characterized by a very torpid evolution. Although the diagnosis was suspected early in the course, confirmation was obtained 2 months after the onset. Flavobacterium odoratum was identified in the fourth month of evolution and only after multiple blood cultures had been obtained. We believe the very low infectivity of F. odoratum and its very slow growth in culture media prevented an early diagnosis.
Neonates are predisposed to serious infections such as meningitis, probably due to their immature host reaction to the pathogens. We have studied the intrathecal immune response in 2 newborns with Flavobacterium meningosepticum meningitis. They showed a significant elevation of immunoglobulin indices ( >1.10), also after the CSF had become sterile with a normalized cell count. In addition, an intrathecal increase and subsequent decrease of both C3dg and TCC (terminal complement complex) were observed in 1 patient. We conclude that immunoglobulin production and complement activation may occur in neonatal CSF.
Flavobacterium indologenes has recently been implicated in nosocomial or opportunistic infection. It has been isolated from lids, conjunctiva, and lacrimal system, and is resistant to most antibiotics. No previous cases of F. indologenes corneal ulcer have been reported in the literature. The natural habitat of Flavobacteria is soil, water, plants, and foodstuffs. In the hospital environment, these bacteria exist in water systems and on wet surfaces. They are not part of the normal ocular flora.
Bacterial diseases of the gills of commercially reared salmonids in freshwater are common problems. They accounted for 18% of all diagnostic submissions to the Atlantic Veterinary College from commercial fish hatcheries. Definitive diagnosis is difficult because of the growth characteristics of the putative bacteria in culture. Research into the pathogenesis of these diseases has also been similarly limited. Monoclonal antibodies (MAbs) were developed to 2 globally significant gill bacterial pathogens, Flavobacterium branchiophilum, the causative agent of bacterial gill disease, and Cytophaga columnaris, the causative agent of columnaris disease of salmonids. These MAbs were then used as the basis for an indirect fluorescent antibody test to assess archived cases of gill disease in our study region. Of the cases tentatively diagnosed based on histopathology as bacterial gill disease, 76.2% tested positively with the MAbs to F. branchiophilum. Also present within 18.7% of these cases were bacteria which reacted positively to the MAbs for C. columnaris. We conclude that the MAbs produced are valuable diagnostic and research probes for common bacterial disease of the gills of salmon and trout in Atlantic Canada. This study also adds further proof that F. branchiophilum acting alone can be sufficient cause for bacterial gill disease.
A thermostable glycerol kinase (FGK) was purified 34-fold to homogeneity from Flavobacterium meningosepticum. The molecular masses of the enzyme were 200 kDa by gel filtration and 50 kDa by SDS-PAGE. The Km for glycerol and ATP were 0.088 and 0.030 mM, respectively. The enzyme was stable at 65 degrees C for 10 min and at 37 degrees C for two weeks. The enzyme gene was cloned into Escherichia coli and its complete DNA was sequenced. The FGK gene consists of an open reading frame of 1494-bp encoding a protein of 498 amino acids. The deduced amino acid sequence of the gene had 40-60% similarity to those of glycerol kinases from other origins and the amino acid sequence of the putative active site residue reported for E. coli GK is identical to the corresponding sequence of FGK except for one amino acid residue.
We found that a psychrophilic bacterium, Flavobacterium sp., characterized in this study, has a beta-mannanase (EC 3.2.1.78) activity in the culture medium. The mannanase activity was the highest in the culture medium, containing 1.0% (w/v) guar gum (as a carbon source), 0.3% (NH4)2SO4 (as a nitrogen source), and 0.06% (w/v) yeast extract, of five-days cultivation at 4 degrees C. No mannanase activity was found in the medium containing a monosaccharide or a disaccharide as a carbon source, although the psychrophile could use them. The enzyme activity was found only when the bacterium was cultured in the medium containing a polysaccharide. The enzyme preparation showed a single activity band on a washed gel of SDS-PAGE. The optimal temperature for the enzyme activity was 35 degrees C. When the reaction was done at 10 degrees C, the enzyme showed 25% of the optimal activity. The beta-mannanase preparation efficiently hydrolyzed guar gum, locust bean gum, and glucomannan as well as beta-mannan.
Flavobacterium johnsonae was isolated as a microorganism that produced a beta-glucosidase with hydrolytic activity of beta-glucosyl ester linkages in steviol glycosides. The enzyme was purified to homogeneity from a cell-free extract by streptomycin treatment, ammonium sulfate fractionation, and column chromatographies on S-Sepharose and phenyl-Toyopearl. The molecular mass of the purified enzyme was about 72 kDa by SDS-PAGE. An isoelectric point of pI 8.8 was estimated by isoelectric focusing. The enzyme was most active at pH 7.0, and was stable between pH 3.0 and 9.0. The optimum temperature was 45 degrees C, and the enzyme was stable below 35 degrees C. The enzyme hydrolyzed glucosyl ester linkages at site 19 of rebaudioside A, stevioside, and rubusoside, although it could not degrad beta-glucosidic linkages at site 13 of rebaudioside B or steviol bioside. The enzyme acted on aryl beta-glucosides such as p-nitrophenyl beta-glucoside, phenyl betaglucoside, and salicin, and glucobioses such as sophorose and laminaribiose. The enzyme activity on Rub was inactivated completely by Hg2+, and reduced by Fe3+, Cu2+, p-chloromercuric benzoate, and phenylmethylsulfonyl fluoride (residual activity; 67.9-84.8%). The pNPG hydrolysis was also inactivated to almost the same degrees. Kinetic behaviors in the mixed substrate reactions of rebaudioside A and steviol monoside, and of steviol monoglucosyl ester and phenyl beta-glucoside suggested the glucosidic and glucosyl ester linkages were hydrolyzed at a single active site of the enzyme.
The gene encoding the endo-beta-N-acetylglucosaminidase from Flavobacterium sp. (Endo-Fsp) was sequenced. The Endo-Fsp gene was overexpressed in Escherichia coli cells, and was purified from inclusion bodies after denaturation by 8 M urea. The renatured Endo-Fsp had the same optimum pH and substrate specificity as the native enzyme. Endo-Fsp had 60% sequence identity with the endo-beta-N-acetylglucosaminidase from Streptomyces plicatus (Endo-H), and the putative catalytic residues were conserved. Site-directed mutagenesis was done at conserved residues based on the three-dimensional structure and mutagenesis of Endo-H. The mutant of Glu-128, corresponding to Glu-132 in Endo-H and identified as an active site residue, was inactivated. Mutagenesis around the predicted active site of Endo-Fsp reduced the enzymatic activity. Moreover, the hydrolytic activity toward hybrid-type oligosaccharides was decreased compared to that toward high-mannose type oligosaccharides by mutagenesis of Asp-126 and Asp-127. Therefore, site-directed mutagenesis of some of these conserved residues indicates that the predicted active sites are essential to the enzymatic activity of Endo-Fsp, and may have similar roles in catalysis as their counterparts in Endo-H.
The thermostability of Flavobacterium meningosepticum glycerol kinase was increased by the change from Ser329 to Asp [Protein Eng., 14, 663-667 (2001)]. Based on a three-dimensional structure model of the mutant, we have postulated that a new charged-neutral hydrogen bond was formed between Asp329 and Ser414, and the formation of the hydrogen bond contributed to the stabilization of the tertiary structure and increased thermostability of the mutant enzyme. If the postulation is the case, FGK thermostabilization would be possible similarly by the single amino acid substitution from Ser414 to another amino acid which could form the hydrogen bond with Ser329. We did a single amino acid substitution of the wild-type enzyme from Ser414 to Asn. As we expected, S414N showed comparable thermostability to that of S329D. On the other hand, a difference in kinetic properties for ATP between S414N and S329D was observed.
We purified the psychrophilic and thermolabile malate dehydrogenase to homogeneity from a novel psychrotolerant, Flavobacterium frigidimaris KUC-1, isolated from Antarctic seawater. The enzyme was a homotetramer with a molecular weight of about 123 k and that of the subunit was about 32 k. The enzyme required NAD(P)(+) as a coenzyme and catalyzed the oxidation of L-malate and the reduction of oxalacetate specifically. The reaction proceeded through an ordered bi-bi mechanism. The enzyme was highly susceptible to heat treatment, and the half-life time at 40 degrees C was estimated to be 3.0 min. The k(cat)/K(m) (microM(-1).s(-1)) values for L-malate and NAD(+) at 30 degrees C were 289 and 2,790, respectively. The enzyme showed pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of the coenzyme. The enzyme contained 311 amino acid residues and much lower numbers of proline and arginine residues than other malate dehydrogenases.
The gene encoding the FokI methyltransferase from Flavobacterium okeanokoites was cloned into an Escherichia coli vector. The transcriptional start sites were mapped as well as putative -10 and -35 regions of the fokIM promoter. Enzyme overproduction was ensured by cloning the fokIM gene under the phi 10 promoter of phase T7. M.FokI was purified using a two-step chromatography procedure. M.FokI is a monomeric protein with a M(r) = 76,000 +/- 1,500 under denaturing conditions. It contains 21 Arg residues, and at least one of which is required for activity as shown by inhibition using 2,3-butanedione. Deletion mutants in the N- and C-terminus of M.FokI were isolated and characterized. The N-terminal derivative (M.FokIN) methylates the adenine residue within the sequence 5'-GGATG-3', whereas the C-terminal derivative (M.FokIC) modifies the adenine residue within the sequence 5'-CATCC-3'. Substrate-protection studies, utilizing chemical modification combined with data on the effect of divalent cations and pH on methylation activity, proved the existence of two catalytic centers within the FokI methyltransferase molecule. M.FokI and its truncated derivatives require S-adenosyl-L-methionine as the methyl-group donor, and they are strongly inhibited by divalent cations (Mg2+, Ca2+, Ba2+, Mn2+, and Zn2+) and S-adenosyl-L-homocysteine. The Km values for the methyl donor, S-adenosyl-L-methionine are 0.6 microM (M.FokI), 0.4 microM (M.FokIN), and 0.9 microM (M.FokIC) while the Km values for substrate lambda DNA are 1.2 nM (M.FokI), 1.4 nM (M.FokIN), and 1.3 nM (M.FokIC).
Flavobacterium heparinum is a soil bacterium that produces several mucopolysaccharidases such as heparinase, heparitinases I and II, and chondroitinases AC, B, C and ABC. The purpose of the present study was to optimize the preparation of F. heparinum chondroitinases, which are very useful tools for the identification and structural characterization of chondroitin and dermatan sulfates. We observed that during the routine procedure for cell disruption (ultrasound, 100 kHz, 5 min) some of the chondroitinase B activity was lost. Using milder conditions (2 min), most of the chondroitinase B and AC protein was solubilized and the enzyme activities were preserved. Tryptic soy broth without glucose was the best culture medium both for bacterial growth and enzyme induction. Chondroitinases AC and B were separated from each other and also from glucuronidases and sulfatases by hydrophobic interaction chromatography on HP Phenyl-Sepharose. A rapid method for screening of the column fractions was also developed based on the metachromatic shift of the color of dimethylmethylene blue.
As one of the attempts to find clues on precursors or intermediates of the vitamin B6 biosynthetic pathway, the resting cell system of Flavobacterium sp. 238-7, a bacterium producing a high amount of the vitamin, was employed. Among various compounds tested as an additive to the reaction mixture, L-glutamate and nucleoside derivatives increased the formation of vitamin B6, respectively. The possible mechanism of the participation of these compounds in vitamin B6 biosynthesis is discussed. The identification of vitamin B6 formed in the reaction mixture was done using column chromatography.