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Immunization with bacterial antigens: Flavobacterium and Flexibacter infections.

Seven bacterial species belonging to the Flavobacterium-Cytophaga group are currently considered to be pathogenic for fish. Because they were only recently described and/or because the disease they provoke has little economic significance, no study has been performed concerning immunization against Chryseobacterium scophthalmum, Flavobacterium johnsoniae and Flexibacter ovolyticus. Immunization with Flexibacter maritimus has not been investigated, and fish surviving a natural infection appear to remain equally susceptible to the disease during subsequent outbreaks. On the other hand, interesting data are available concerning immunization against Flavobacterium branchiophilum, Flavobacterium columnare and Flavobacterium psychrophilum because these bacterial species have been known for many years and are responsible for heavy losses in many countries. Surviving fish are usually protected against further infection by the same pathogens. Extensive serological studies have been performed and virulence mechanisms have also been investigated. The review of immunization trials against these three bacterial species shows important variations depending on the species of fish and the route of administration, but in several cases vaccinated fish were successfully protected by high titre of specific antibodies. However, no vaccine is commercially available.

Animals↗

Reclassification of [Flavobacterium] ferrugineum as Terrimonas ferruginea gen. nov., comb. nov., and description of Terrimonas lutea sp. nov., isolated from soil.

Strain DY(T), which was isolated from garden soil in Japan, was subjected to a polyphasic taxonomic study. Sequence analysis of the 16S rRNA gene and the GyrB protein revealed that the closest relative of strain DYT was [Flavobacterium] ferrugineum Sickles and Shaw 1934, with 94.8 and 90.1 % similarity, respectively. The two strains had similar chemotaxonomic characteristics, with menaquinone 7 as the major quinone system, 47.2-48.9 mol% DNA G+C content and 15 : 0 iso, 15 : 1 iso, 17 : 0 iso 3-OH and summed feature 3 as the major fatty acids. Based on genotypic and phenotypic characteristics, [Flavobacterium] ferrugineum IAM 15098T could be clearly differentiated from other members of the genus Flavobacterium. Strain DYT and [Flavobacterium] ferrugineum IAM 15098T could be easily distinguished from neighbouring taxa by morphological features (non-motile, non-gliding and non-filamentous single cells). Therefore, it is proposed that [Flavobacterium] ferrugineum IAM 15098T and strain DYT represent two separate species of a new genus, Terrimonas gen. nov., with the names Terrimonas ferruginea comb. nov. (type species; type strain IAM 15098T=ATCC 13524T) and Terrimonas lutea sp. nov. (type strain DYT=IAM 15284T=CCTCC AB205006T), respectively.

Bacterial Typing Techniques↗

Study of the mechanism of Flavobacterium sp. for hydrolyzing organophosphate pesticides.

The biotransformation by Flavobacterium sp. of the following organophosphate pesticides was experimentally and theoretically studied: phorate, tetrachlorvinphos, methyl-parathion, terbufos, trichloronate, ethoprophos, phosphamidon, fenitrothion, dimethoate and DEF. The Flavobacterium sp. ATCC 27551 strain bearing the organophosphate-degradation gene was used. Bacteria were incubated in the presence of each pesticide for a duration of 7 days. Parent pesticides were identified and quantified by means of a gas-chromatography mass spectrum system. Activity was considered as the amount (micromol) of each pesticide degraded by Flavobacterium sp. Also, structural parameters obtained by means of the CAChe program package for biomolecules, the reactivity index of phosphorus, of oxygen at the P = O function and of sulfur at the P = S function, and lipophilicity (log Poct) (ALOGPS v. 2.0) were obtained for each pesticide. Pesticides were hydrolyzed at the bond between phosphorous and the heteroatom, producing phosphoric acid and three metabolites. Enzymatic activity was significantly explained by the following multiple linear relationship: Enzymatic activity = 162.2 - 9.5(dihedral angle energy) - 25.0(Total energy) - 0.51(Molecular weight). Finally, a mechanism of Flavobacterium sp. to hydrolyze pesticides was proposed.

Bacteriological Techniques↗

Substrate specificity of endo-beta-galactosidases from Flavobacterium keratolyticus and Escherichia freundii is different from that of Pseudomonas sp.

The substrate specificity of endo-beta-galactosidase of Pseudomonas sp. was found to differ from that of Flavobacterium keratolyticus or Escherichia freundii, based on the following experimental results. The endo-beta-galactosidases from these three bacteria released 6-O-sulfo-GlcNAc beta 1-3Gal as one of the major products from keratan sulfates from different sources. In addition to the sulfated disaccharide, Flavobacterium and Escherichia enzymes produced GlcNAc beta 1-3Gal, which is also an integral repeating unit of keratan sulfate, whereas the Pseudomonas enzyme did not release any non-sulfated disaccharide. Tetrasaccharides were prepared from the teleost skin keratan sulfate by digestion with Pseudomonas enzyme followed by gel filtration on Sephadex G-50 chromatography. A part of the tetrasaccharide fraction was hydrolyzed by Flavobacterium enzyme to produce 6-O-sulfo-GlcNAc beta 1-3Gal and GlcNAc beta 1-3Gal, whereas the fraction was completely resistant to retreatment with the Pseudomonas enzyme. Endo-beta-galactosidases from F. keratolyticus and E. freundii hydrolyzed the internal beta-1,4-galactosyl linkage of various neolacto-type glycosphingolipids to produce glucosylceramides. However, these glycosphingolipids were completely resistant to the Pseudomonas enzyme. These findings clearly show that the sulfation on the N-acetylglucosamine adjacent to galactose in the lactosaminoglycans is essential for expression of the Pseudomonas enzyme, but not for that of the Flavobacterium or Escherichia enzyme.

Chromatography, Thin Layer↗

Flavobacterium hibernum sp. nov., a lactose-utilizing bacterium from a freshwater Antarctic lake.

Four freshwater Antarctic lakes were examined for the presence of beta-galactosidase-producing bacteria using mineral medium enrichments and lactose. Enrichments from only one of the lakes produced growth and two strains were isolated that were very similar in phenotype and fatty acid profile, and shared considerable homology in their DNA (DNA-DNA hybridization = 93 +/- 7%). The strains were psychrotrophic with theoretical Tmax, Tmin and Topt of 30-31, -7 degrees and 26 degrees C, respectively. The beta-galactosidase in cell extracts had an optimal activity at 39 degrees C. The strains were Gram-negative rods, showed gliding motility, contained branched and hydroxy fatty acids, and menaquinone 6 as the major respiratory quinone. The strains did not form microcysts and utilized lactose while using ammonium ions as a source of nitrogen, and a range of other sugars. The G + C content of the DNA was 34 mol%. Phylogenetic analysis of one of the strains, by comparison of 16S rDNA sequences, showed that it was most similar, but not identical to, Flavobacterium columnare and '[Sporocytophaga] cauliformis'. Both species could be differentiated phenotypically from the Antarctic isolates. DNA-DNA hybridization of the Antarctic isolate with six different members of the Flavobacterium 16S rDNA cluster showed no strain with greater than 18% relatedness. The nearest type species to the Antarctic isolate in the phylogenetic analysis was Flavobacterium aquatile. The name Flavobacterium hibernum is proposed for the Antarctic strains, and the type strain is ATCC 51468T (= ACAM 376T).

Antarctic Regions↗

Flavobacterium croceum sp. nov., isolated from activated sludge.

A Gram-negative, non-motile, rod-shaped bacterium, designated strain EMB47(T), was isolated from activated sludge performing enhanced biological phosphorus removal in a sequencing batch reactor. Growth was observed between 10 and 40 degrees C (optimum, 25-35 degrees C) and between pH 5.0 and 8.5 (optimum, pH 7.5-8.0). The predominant fatty acids of strain EMB47(T) were iso-C(16 : 0) 3-OH, iso-C(15 : 1) G, C(15 : 0), iso-C(15 : 0), iso-C(14 : 0) and iso-C(16 : 0) and it contained phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylcholine as polar lipids. The G+C content of the genomic DNA was 40.8 mol% and the major quinone was MK-6. Comparative 16S rRNA gene sequence analyses showed that strain EMB47(T) formed a distinct phyletic line within the genus Flavobacterium. The levels of 16S rRNA gene sequence similarity with respect to Flavobacterium species were below 94.7 %. On the basis of the phenotypic, chemotaxonomic and molecular data, strain EMB47(T) represents a novel species within the genus Flavobacterium, for which the name Flavobacterium croceum sp. nov. is proposed. The type strain is EMB47(T) (=KCTC 12611(T)=DSM 17960(T)).

Bacterial Typing Techniques↗

Studies on a collection of strains of the genus Flavobacterium. 1. Biochemical studies.

The genus Flavobacterium has undergone a number of changes during the last years resulting in a revised description of the genus in which restriction of Flavobacterium to certain low G + C content species is proposed. The purpose of the present study has been to see how a collection of 184 clinical and environmental strains of Flavobacterium-like organisms isolated in Denmark would fit into this latest revision. It was found that most of the different biochemical patterns represented gradually merged into each other, with certain patterns, however, occurring more frequently than others and corresponding to the already described taxa: F. meningosepticum, Flavobacterium group IIb, F. breve, F. odoratum, F. multivorum and F. spiritivorum. The lately proposed subdivision of the genus will need either revision or extension or both to cope satisfactorily with the situation revealed in this study.

Animals↗

Airway colonization by Flavobacterium in an intensive care unit.

A total of 195 patients admitted to a respiratory-surgical intensive care unit became colonized with species of Flavobacterium during a 70-month prospective study. By biochemical, cultural, and morphological criteria and a comparison of antibiotic susceptibilities, all patient isolates of Flavobacterium were apparently related. The origin of these organisms was sought. Flavobacterium were recovered from different water-associated areas of the hospital and from the hands of respiratory-surgical intensive care care unit staff. The organisms were also found in university dormitory sinks. The isolation of these organisms from tap water led to their recovery from reservoirs supplying drinking water to the city of Boston and surrounding communities. These organisms are resistant to chlorine concentrations found in municipal water. There was no proven case of pneumonia caused by Flavobacterium in 2,329 consecutive patients studied in our respiratory-surgical intensive care unit.

Air Microbiology↗

Classification and identification of Flavobacterium species by carbon source utilization.

Carbon substrates used as the sole source of carbon and energy were tested for the classification and identification of species of Flavobacterium: Flavobacterium meningosepticum, F. breve, F. odoratum, F. multivorum, F. thalpophilum, and Flavobacterium sp. group IIb. Hierarchical classification and stepwise discriminant analysis revealed three F. meningosepticum, two F. breve, two F. odoratum, and two Flavobacterium sp. group IIb subgroups. Glucose, histidine, asparagine, tryptophan, maltose, citric acid, and glycine were selected as the most useful substrates to differentiate between the groups and subgroups.

Alcohols↗

[Isolation, identification and clinical significance of species of the Flavobacterium genus].

Strictly aerobic Gram negative bacteria are found more and more often in human pathological specimens and in the environment. Amongst these bacteria, Flavobacterium poses problems of bacteriological diagnosis as their precise taxonomy is still recent. In this study, conducted on 321 strains of Flavobacterium from various sources, but essentially from patients in the intensive care unit, the authors define the methods and features of identification and the clinical significance of the species of the Flavobacterium genus. One species is particularly important in medical bacteriology, Flavobacterium meningosepticum which can cause septicaemia and neonatal meningitis which are difficult to treat.

Anti-Bacterial Agents↗

Molecular cloning and sequence analysis of Flavobacterium meningosepticum glycosylasparaginase: a single gene encodes the alpha and beta subunits.

A full-length insert for the Flavobacterium meningosepticum N4-(N-acetyl-beta-glucosaminyl)-L-asparagine amidase gene was located on a 2500-bp HindIII fragment and cloned into the plasmid vector pBluescript. DNA sequencing revealed an open reading frame of 1020 nucleotides encoding a putative 45-amino-acid leader sequence and a deduced precursor polypeptide of 295 amino acids. In F. meningosepticum this precursor polypeptide undergoes proteolytic processing by an as yet unknown mechanism to generate an alpha-subunit and a beta-subunit, which constitute the active form of the heterodimeric mature glycosylasparaginase. The Flavobacterium glycosylasparaginase gene was expressed in Escherichia coli and found to be enzymatically active. The recombinant enzyme was purified from crude lysates and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to consist of the typical alpha- and beta-subunits. The recombinant beta-subunit cross-reacted to antibody specific for the rat liver beta-subunit, and Edman analysis demonstrated that its amino-terminus corresponded exactly to that of the mature native glycosylasparagine beta-subunit. A comparison of the Flavobacterium glycosylasparaginase with a mammalian glycosylasparaginase revealed 30% structural identity and 60% overall similarity between the prokaryotic and eukaryotic forms of the enzyme. Even more striking was the conservation of the amino acid sequence in both proteins where the post-translational cleavage to generate the active enzyme occurs. Our data demonstrate that deglycosylation of asparagine-linked glycans via hydrolysis of the AspNHGlcNAc linkage is an important reaction which has been preserved during evolution.

Amino Acid Sequence↗

Verification of the role of PCP 4-monooxygenase in chlorine elimination from pentachlorophenol by Flavobacterium sp. strain ATCC 39723.

The bacterial enzyme PCP 4-monooxygenase from Flavobacterium sp. strain ATCC 39723 catalyzes the oxygenolytic removal of the first chlorine from pentachlorophenol. PCP 4-monooxygenase is an FAD binding, NADPH requiring oxygenase, with similar functional domains as other bacterial flavoprotein monooxygenases specific for phenolic substrates. However, the definitive proof for the singular role of an oxygenolytic elimination of the primary chlorine from pentachlorophenol by Flavobacterium sp. has awaited the development of a genetic system whereby targeted mutagenesis via allelic exchange could be carried out with the corresponding gene from PCP 4-monooxygenase, pcpB. We report the development of a genetic system for Flavobacterium sp. strain ATCC 39723, and its application in targeted mutagenesis of the pcpB allele for elimination of PCP 4-monooxygenase activity.

Blotting, Southern↗

Heparinase III from Flavobacterium heparinum: cloning and recombinant expression in Escherichia coli.

Heparinase III (E.C. 4.2.2.8), formerly heparinase I, produced by Flavobacterium heparinum is an enzyme that specifically cleaves heparan sulfate-rich regions of acidic polysaccharides. In this study, we report the cloning of the heparinase III gene using polymerase chain reaction (PCR). Two degenerate oligonucleotides, based on amino acid sequences derived from tryptic peptides of purified heparinase III were used to generate a approximately 1100-bp probe by PCR amplification using Flavobacterium genomic DNA as the template. The PCR-derived probe was used to screen a Flavobacterium genomic DNA library in lambda ZAP II. The open reading frame of the heparinase III gene is 1980 bp in length, encoding a precursor protein of 75,950 Da; 10 of the tryptic peptides mapped onto the open reading frame which corresponded to approximately 18% of the protein. Recombinant heparinase III was expressed in Escherichia coli using the T7 polymerase pET expression system. This is the first report of the cloning and recombinant expression of an enzyme primarily degrading heparan sulfate.

Amino Acid Sequence↗

Formation of methylantimony species by an aerobic prokaryote: Flavobacterium sp.

Pure cultures of an aerobically grown Flavobacterium sp. were shown by hydride generation-cold trap-atomic absorption spectrometry to biomethylate inorganic antimony (III) supplied as potassium antimony tartrate. Growth inhibition of the Flavobacterium sp. by antimony (III) over the range 0-30 mg Sb l(-1) was assessed by optimising parameters within an extended logistic growth model. Antimony (III) concentrations over this range influenced both the extent of antimony biomethylation (up to 4.0 microg l(-1)) and the relative proportions of the involatile mono-, di, and trimethylantimony species formed. Provision of inorganic arsenic (III) alongside antimony (III) enhanced formation of the involatile methylantimony species up to eight-fold. The data are consistent with accumulation of involatile intermediates from an antimony or arsenic biomethylation pathway in culture supernatants. Low yields of methylantimony species (<0.03%) suggest that antimony biomethylation by the Flavobacterium sp. was a fortuitous rather than a primary resistance mechanism for this element. These findings demonstrate that anaerobiosis is not an obligate requirement for methylantimony formation in prokaryotes, thus broadening the range of habitats for potential formation of methylantimony species in nature.

Aerobiosis↗

Secreted beta-galactosidase from a Flavobacterium sp. isolated from a low-temperature environment.

The bacterial strain Flavobacterium sp. 4214 isolated from Greenland was found to express beta-galactosidase (EC 3.2.1.23) at temperatures below 25 degrees C. A chromosomal library of Flavobacterium sp. 4214 was constructed in Escherichia coli, and the gene gal4214-1 encoding a beta-galactosidase of 1,046 amino acids (114.3 kDa) belonging to glycosyl hydrolase family 2 was isolated. This was the only gene encoding beta-galactosidase activity that was identified in the chromosomal library. Expression levels in both Flavobacterium sp. 4214 and in initial recombinant E. coli strains were insufficient for biochemical characterization. However, a combination of T7 promoter expression and introduction of an E. coli host that complemented rare transfer RNA genes yielded 15 mg of beta-galactosidase per liter of culture. Gal4214-1-His protein was found to be active in monomeric conformation. The protein was secreted from the cytoplasm, probably through an N-terminal signaling sequence. The Gal4214-1-His protein was found to have optimum activity at a temperature of 42 degrees C, but with short-term stability at temperatures above 25 degrees C.

Amino Acid Sequence↗

An isoamylase with neutral pH optimum from a Flavobacterium species: cloning, characterization and expression of the iam gene.

The gene encoding an isoamylase with neutral pH optimum (iam) from a Flavobacterium species was cloned using a PCR probe generated from highly conserved regions of amylolytic enzymes. Active isoamylase was expressed from a 4.9-kb Pst I fragment in Escherichia coli, and was detected in the extracellular medium by a plate assay. The iam nucleotide sequence has an open reading frame of 2334 nucleotides (778 amino acids) with a GC content of 69%. Sequence analysis suggests that transcriptional control of the Flavobacterium sp. iam gene is mediated through the product of a malT regulatory gene. The deduced amino acid sequence of iam contained an N-terminal signal peptide of 32 amino acids, and was 61% homologous with Pseudomonas amyloderamosa isoamylase. The mature enzyme, which was engineered for overexpression in E. coli and purified to homogeneity, has a relative molecular mass of 83 kDa, a pH optimum of 6-7, and a highest rate of hydrolysis for glycogen (but did not cleave pullulan). Polyclonal antiserum generated from purified donor isoamylase cross-reacted with crude and purified recombinant isoamylase from E. coli. This is the first report of the cloning, characterization, and sequence of an novel isoamylase that has a neutral pH optimum. A comparison of the sequence of Flavobacterium sp. iam with acidic isoamylase from Pseudomonas sp. identified putative residues which may be associated with the pH for optimal activity of isoamylases.

Amino Acid Sequence↗

Members of the Cytophaga-Flavobacterium-Bacteroides phylum as intracellular bacteria of acanthamoebae: proposal of 'Candidatus Amoebophilus asiaticus'.

Three Gram-negative, rod-shaped bacteria that were found intracellularly in two environmental and one clinical Acanthamoeba sp. isolates were analysed. Two endocytobionts showing a parasitic behaviour were propagated successfully outside their amoebal host cells and were identified subsequently by comparative 16S rRNA sequence analysis as being most closely affiliated with Flavobacterium succinicans (99% 16S rRNA sequence similarity) or Flavobacterium johnsoniae (98% 16S rRNA sequence similarity). One endocytobiont could neither be cultivated outside its original Acanthamoeba host (Acanthamoeba sp. TUMSJ-321) nor transferred into other amoebae. Electron microscopy revealed that the amoebal trophozoites and cysts were almost completely filled with cells of this endosymbiont which are surrounded by a host-derived membrane. According to 16S rRNA sequence analysis, this endosymbiont could also be assigned to the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum, but was not closely affiliated to any recognized species within this phylogenetic group (less than 82% 16S rRNA sequence similarity). Identity and intracellular localization of this endosymbiont were confirmed by application of a specific fluorescently labelled 16S rRNA-targeted probe. Based on these findings, we propose classification of this obligate Acanthamoeba endosymbiont as 'Candidatus Amoebophilus asiaticus'. Comparative 18S rRNA sequence analysis of the host of 'Candidatus Amoebophilus asiaticus' revealed its membership with Acanthamoeba 18S rDNA sequence type T4 that comprises the majority of all Acanthamoeba isolates.

Acanthamoeba↗

Indwelling arterial catheters as a source of nosocomial bacteremia. An outbreak caused by Flavobacterium Species.

Between mid-May and mid-October, 1973, 49 blood cultures from 14 patients in an intensive care unit were positive for flavobacterium species, Group II-b. We conducted an investigation to determine how patients were being infected with this unusual organism. Comparison of the 14 infected patients with 37 controls associated indwelling arterial catheters with subsequent flavobacterium bacteremia (p = 0.005). Risk of infection was greatest during the period in which blood gas determinations were done most frequently (the first three days of catheterization) and in which infected patients had more blood gas determinations than control patients with arterial catheters (p less than 0.05). Flavobacterium species was cultured from in-use arterial catheters, from stopcocks, and from ice in the intensive-care unit's ice machine; the catheters were probably contaminated by syringes that were cooled in ice before being used to obtain arterial specimens for blood gas determination. This outbreak calls attention to arterial monitoring systems as a potential source of nosocomial infection.

Arteries↗