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PubMed1982

Comparative in vitro leukotoxin production of three bovine strains of Fusobacterium necrophorum.

The in vitro leukotoxic activity of 3 bovine isolates of Fusobacterium necrophorum which varied in pathogenicity were compared. Monolayers of mouse peritoneal macrophages were exposed to culture filtrates from each F necrophorum strain, and cell viability was determined, using the trypan blue dye exclusion test. Two methods were used for production of the leukotoxin: (1) medium M-1 continuous dialysis sac cultures and (2) brain-heart infusion agar plate cultures. Supernatant cultural fluids containing the leukotoxin were subjected to membrane-partition chromatography, using ultrafilters with approximate molecular weight (mol wt) exclusion limits of 100,000, 10,000, 2,000, and 500. All ultrafiltrates had a cytotoxic effect on the monolayers. Cytotoxic activity was not found in the ultrafilter residues or in the control media ultrafiltrates. Comparative study of leukotoxin production indicated that F necrophorum 2101, type A, produced the most leukotoxin; F necrophorum 2030, type AB, produced slightly less leukotoxin; and F necrophorum 2035, type B, produced small amounts of leukotoxin. Endotoxin activity, as demonstrated by the mouse lethality test, was found in the residues of the XM-100A ultrafilter (100,000 mol wt), but not in the filtrates. Culture supernatant fluids and the XM-100A ultrafiltrates were positive for endotoxin, using the limulus amebocyte lysate assay; however, the other ultrafiltrates with lower mol wt exclusion limits were negative.

Animals↗
PubMed1995

Local skin reaction in mice and guinea pigs induced by a single intradermal inoculation of Fusobacterium necrophorum lipopolysaccharide.

Dermal responses induced by Fusobacterium necrophorum strain VPI 2891 lipopolysaccharide (LPS) were studied using mice and guinea pigs. In ddY mice, the LPS elicited inflammatory, haemorrhagic lesions and an increase in local vascular permeability 24 h postinjection. Of the mouse strains, C3H/HeJ mice were less sensitive. The LPS induced erythema and haemorrhagic responses in guinea pig skin 24 h postinoculation. These responses were dose-dependent. The intensity of dermal inflammation-inducing activity of F. necrophorum LPS was similar to that of Escherichia coli strain 055:B5 LPS, but weaker than that of Salmonella typhimurium LPS. These findings suggest that the fusobacterial LPS may play an important role in contributing to produce the initial lesions in the bacterial infection.

Animals↗
PubMed1994

Biological and biochemical characterization of Fusobacterium necrophorum leukotoxin.

Biological and biochemical characteristics of the leukotoxin of Fusobacterium necrophorum were determined. Culture supernatant of F necrophorum was toxic to polymorphonuclear neutrophilic leukocytes from cattle and sheep, but not to those from pigs and rabbits. Culture supernatant and sonicated bacterial cell fractions had low hemolytic activity and did not cause dermonecrosis in a guinea pig. Supernatant-derived leukotoxin was inactivated at 56 C for 5 minutes and became unstable at pH > 7.8 or < 6.6. Chemical treatment with 0.1% sodium dodecyl sulfate, 0.25% sodium deoxycholate, 5.2% sodium sulfide, or 0.25 mM titanium (III) citrate markedly decreased leukotoxicity. Enzymatic treatment with protease, trypsin, and chymotrypsin inactivated the toxin completely, whereas amylase had no effect. Use of protease inhibitors failed to prevent loss of leukotoxin activity. Using membrane partition chromatography and gel filtration, the estimated molecular weight of the toxin was > 300,000. On reduction and denaturation, the toxin dissociated into several components by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Animals↗
PubMed1993

Partial characterization of HCl heat extracted antigen derived from Fusobacterium necrophorum.

The HCl heat extracted antigen derived from Fusobacterium necrophorum was partially purified by CM cellulose column chromatography and treatment by freeze-thawing. It demonstrated relatively high enzyme-linked immunosorbent assay values to anti-F. necrophorum sera. In sodium dodecyl sulphate polyacrylamide gel electrophoresis, the partially purified preparation exhibited one definite and two faint bands. The approximate molecular weight of the former was estimated as 16 kD.

Antigens, Bacterial↗
PubMed1977

Effects of Fusobacterium necrophorum leukotoxin on rabbit peritoneal macrophages in vitro.

A method to demonstrate leukotoxic activity of Fusobacterium necrophorum in vitro is described. Continuous dialysis sac culture system, using reduced liquid medium was used to grow F necrophorum for 7-day periods. The continuous culture dialysis filtrate contained leukotoxic substance(s) which appeared to be less than 10,000 in molecular weight, heat resistant, and stable at 4 C for at least 10 days. Leukotoxic activity was demonstrated in vitro by determining the percentage of macrophages taking up trypan blue dye after these were exposed to a 1:2 solution of continuous culture dialysis filtrate and Eagle's minimal essential medium at 1, 2, 4, and 6 hours. Approximately 90% of these macrophages were destroyed during the 6-hour incubation period.

Animals↗
PubMed1995

Stable-isotope studies of glutamate catabolism in Fusobacterium nucleatum.

Catabolism of glutamate was investigated in Fusobacterium nucleatum, an anaerobic micro-organism that is strongly implicated in periodontal disease. The distribution of labels in acetate and butyrate derived from [13C]glutamates was determined by NMR spectroscopy and MS. The label from L-[5-13C]glutamate was not incorporated, whereas C-1 of acetate and butyrate were efficiently labelled by L-[1-13C]glutamate; these results indicated that the hydroxyglutarate pathway predominated. In butyrate, enrichment at C-3 was smaller than C-1; that this was not due to participation of the methylaspartate pathway was demonstrated by the incorporation of label from L-[4-13C]glutamate into only C-2 of acetate and C-4 (major)/C-2 (minor) of butyrate. The presence of label at a second site in butyrate was attributed to the synthesis of butyrate from acetate and verified by the incorporation of label from [1,2-13C2]- and [2H3]-acetate.

Acetates↗
PubMed1977

The effect of temperature on growth and survival of Fusobacterium necrophorum isolated from bovine liver abscesses.

The ability of Fusobacterium necrophorum to survive or grow in liquid nitrogen or at temperatures between -10 degrees and 59 degrees C was determined. The organism remained viable but did not grow in liquid nitrogen or between -10 degrees and 21 degrees C. It grew between 22 degrees and 43 degrees C. No isolate grew at temperatures above 43 degrees C and all three isolates survived for a minimum of 15 minutes and an average of 25 minutes at 59 degrees C. The optimum temperature for maximum growth was 37 degrees C. The organism survived in ampoules stored in liquid nitrogen for eight years. It survived in liver abscesses stored at -10 degrees C for five years and as cultures in screw capped tubes for three years.

Animals↗
PubMed1997

Some properties of gamma-glutamyl transpeptidase from Fusobacterium nucleatum.

A series of chromatographic techniques was used to purify gamma-glutamyl transpeptidase from Fusobacterium nucleatum ATCC 23726. The purified enzyme was homogenous with a molecular weight (MW) of approximately 101 kD consisting of two different subunits with MW of 67 kD and 31 kD. Its distribution by treatment of lysozyme-EDTA suggested that the enzyme was a periplasmic protein. The pl of the enzyme was 5.9 to 6.2, and the optimum pH of the transpeptidation and the hydrolytic reaction was 8.0. Glycylglycine, glycine and methionine were good acceptors, and the enzyme reaction, in the presence of glycylglycine, was especially efficient. Glutamic acid, glutamine and serine were poor acceptors, and the enzyme activity was inhibited by these amino acids. The apparent Km value for the gamma-glutamyl donor (gamma-glutamyl-p-nitroanilide) was 4.9 x 10(-4)M and that for the acceptor (glycylglycine) was 0.19 M. Affinity-labelling reagents, such as DON, azaserine and AT-125 strongly inhibited the enzyme activity, and its activity was inhibited by L-serine plus borate, as is mammalian gamma-glutamyl transpeptidase. The antibodies against gamma-glutamyl transpeptidase from bovine kidney reduced the activity of the bacterial enzyme by 65%. These results indicate that the catalytic sites in F. nucleatum gamma-glutamyl transpeptidase were similar to those in mammalian gamma-glutamyl transpeptidase.

Animals↗
PubMed1998

The behaviour of Fusobacterium nucleatum chemostat-grown in glucose- and amino acid-based chemically defined media.

Fusobacterium nucleatum is a Gram-negative anaerobe, found in a number of different areas of human and animal bodies as part of the resident microbiota. However, it also appears to be involved in polymicrobial infections in such sites. It occurs in the oral cavity where it is a prominent member of various bacterial consortia associated with periodontal diseases. Like most fusobacteria, it derives energy via the fermentation of amino acids which it can obtain through the dissimilation of small peptides. However, the role of simple carbohydrates, such as glucose, in its growth and metabolism are not well understood. Accordingly, the aim of the present study was to study the behaviour ofF. nucleatum grown anaerobically in continuous culture in two different chemically-defined media (CDM); one containing only amino acids as the energy source, the other containing glucose as the predominant energy provider. At various dilution rates the culture were assayed for dry weight, intracellular polyglucose (IP) content, residual amino acids and glucose and acidic metabolic end-products. In the carbohydrate-free CDM the acidic end-products were a constant acetate : butyrate : formate of 1.5 : 1 : 0.4. The values of Y(max)amino acids, maximum yield of bacteria per mol of amino acids consumed, for two strains were estimated to be 15.2 and 18.6 g dry wt/mol, respectively. Them(amino acids), maintenance energy requirement for growth on amino acids, for the two strains was 0.81 and 0.94 mmol/g dry wt/h, respectively. Growth of one strain in the glucose-based CDM gave an estimated Y(max)glucose of 67.2 and an m(glucose) of 0.38; the acidic end-products were a fairly constant acetate : butyrate : formate : lactate of 0.7 : 1 : 0.3 : 2.5. Only at low growth rates, and then only in small amount, was IP produced in this medium. Overall, it was concluded that the occurrence of F. nucleatum in widely-differing oral niches may be explained, at least in part, by its metabolic versatility.

Journal Article↗
PubMed1998

Ribotyping of Fusobacterium necrophorum strains isolated from bovine and ovine hepatic abscesses.

A microbiological study was made of 100 strains of Fusobacterium necrophorum isolated from hepatic abscesses in bovine and ovine herds. Differences between the biological activity and ribotypes within the two F. necrophorum subspecies were studied. Conventional methods identified 89 isolates as F. necrophorum subsp. necrophorum and 11 as F. necrophorum subsp. funduliforme. For ribotyping, 50 strains (35 F.n. subsp. necrophorum, 11 F.n. subsp. funduliforme and 4 reference strains) were digested with restriction endonucleases (HindIII, EcoRI and BamHI) and examined after hybridization with digoxigenin-labelled cDNA probe transcribed from a 16 and 23S rRNAs from Escherichia coli. The most discriminating restriction endonuclease enzymes for ribotyping were EcoRI and BamHI. The presence or absence of two distinct band of 5 kb (EcoRI) and 10.5 kb (BamHI) differentiated the two subspecies. This technique also revealed genetic differences between isolates which could be used in the epidemiological study of clinical processes caused by F. necrophorum.

Journal Article↗
PubMed2003

Characterisation and subcellular localisation of the GroEL-like and DnaK-like proteins isolated from Fusobacterium nucleatum ATCC 10953.

Fusobacterium nucleatum is associated with periodontitis in humans, and is a central member of the dental biofilm. Heat shock proteins (HSPs) of many different bacteria have been considered to play important roles during inflammations and infections. We have identified and characterised the HSP60 and HSP70, the Escherichia coli GroEL and DnaK homologues, respectively, in F. nucleatum ATCC 10953. The N-terminal 22 amino acid residues of HSP60 exhibited up to 63.6% identity with members of the HSP60 heat shock protein family of some selected bacterial species, while the N-terminal of 25 residues of HSP70 revealed up to 80% identity with members of the HSP70 family. The subcellular localisation of HSP60 and HSP70 was analysed by immunoblotting of bacterial cell fractions and immunoelectron microscopy of whole cells. HSP60 and HSP70 were localised in the cytosol, associated with membranes and extracellular fractions. These results are consistent with localisation for HSPs found in other micro-organisms, which further lead to the suggestion of a potential role in the pathogenesis of infectious diseases.

Journal Article↗
PubMed2005

Fusobacterium necrophorum infections in animals: pathogenesis and pathogenic mechanisms.

Fusobacterium necrophorum, a Gram-negative, non-spore-forming anaerobe, is a normal inhabitant of the alimentary tract of animals and humans. Two subspecies of F. necrophorum, subsp. necrophorum (biotype A) and subsp. funduliforme (biotype B), have been recognized, that differ morphologically, biochemically, and biologically. The subsp. necrophorum is more virulent and is isolated more frequently from infections than the subsp. funduliforme. The organism is an opportunistic pathogen that causes numerous necrotic conditions (necrobacillosis), either specific or non-specific infections, in a variety of animals. Of these, bovine liver abscesses and foot rot are of significant concern to the cattle industry. Liver abscesses arise with the organisms that inhabit the rumen gaining entry into the portal circulation, and are often secondary to ruminal acidosis and rumenitis complex in grain-fed cattle. Foot rot is the major cause of lameness in dairy and beef cattle. The pathogenic mechanism of F. necrophorum is complex and not well defined. Several toxins or secreted products, such as leukotoxin, endotoxin, hemolysin, hemagglutinin, proteases, and adhesin, etc., have been implicated as virulence factors. The major virulence factor appears to be leukotoxin, a secreted protein of high molecular weight, active specifically against leukocytes from ruminants. The complete nucleotide sequence of the leukotoxin operon of F. necrophorum has been determined. The operon consists of three genes (lktBAC) of which the second gene (lktA) is the leukotoxin structural gene. The leukotoxin appears to be a novel protein and does not share sequence similarity with any other leukotoxin. F. necrophorum is also a human pathogen and the human strains appear to be different from the strains involved in animal infections.

Journal Article↗
PubMed2003

Susceptibility of Fusobacterium nucleatum to killing by peroxidase-iodide-hydrogen peroxide combination in buffer solution and in human whole saliva.

Some Gram-negative anaerobic bacteria have been associated with the infection of tooth supporting tissues, i.e. periodontitis. Of these bacteria, Fusobacterium nucleatum is sensitive to lactoperoxidase/myeloperoxidase-iodide-hydrogen peroxide system in vitro, but salivary concentrations of thiocyanate abolishes the bactericidality. These bacteria are located in periodontal pockets, on oral mucosa and in saliva. Although F. nucleatum most probably does not belong to the group of main periodontal pathogens, it sustains its proportion in the periodontal flora when gingivitis progresses to periodontitis. In this study, the sensitivity of F. nucleatum to different horseradish peroxidase-iodide-hydrogen peroxide combinations was tested both in buffer and in sterilized human whole saliva. Horseradish peroxidase was chosen because it does not bind thiocyanate at pH > or = 6. After 1h incubation at 37 degrees C, the cell viability was estimated by plate count and with flow cytometer using LIVE/DEAD BacLight kit (Molecular Probes, USA). In saliva, the horseradish peroxidase (50 microg/mL)-iodide (2.5 mM)-hydrogen peroxide (2.5 mM) combination decreased the amount of viable bacteria to 37% compared to 85% in the control without any of the components when measured with flow cytometer. Replacement of buffer by saliva decreased the bactericidality of the peroxidase system. However, in buffer less iodide and hydrogen peroxide was needed to produce significant decrease in the number of viable bacteria when measured by plate count than with flow cytometer. Our study shows that horseradish peroxidase-iodide-hydrogen peroxide combination is able to kill F. nucleatum cells in saliva. Horseradish peroxidase-iodide-hydrogen peroxide combination may be useful to diminish the degree of re-colonization of periodontitis-associated bacteria after periodontal therapy and to inhibit the transmission of these bacteria via saliva.

Journal Article↗
PubMed1981

Lysozyme digestion and chemical characterization of the peptidoglycan of Fusobacterium nucleatum Fev 1.

The amino acids in the peptidoglycan of Fusobacterium nucleatum Fev 1 are D-glutamic acid, meso-lanthionine, and D- (42%) and L-alanine (58%). About 70% of the lanthionine residues were not susceptible to dinitrophenylation, evidently because they are involved in cross-linkages. Consequently, lysozyme digestion of the peptidoglycan yielded 20 to 25% uncross-linked disaccharide tri- and tetrapeptides. A chemical analysis of isolated glycopeptides indicated that the structure of the building block of this peptidoglycan is N-acetylgucosamine-N-acetylmuramic acid-L-alanine-D-glutamic acid-meso-lanthionine(-D-alanine). I present evidence which supports the classification of the F. nucleatum Fev 1 peptidoglycan as a new A1 delta, directly cross-linked, meso-lanthionine-containing peptidoglycan.

Alanine↗
PubMed2006

Effects of sonicated Prevotella intermedia, Fusobacterium nucleatum and Lactobacillus casei extracts on interleukin-8 production by human dental pulp cells.

The objective of this study was to determine the effects of Prevotella intermedia, Fusobacterium nucleatum and Lactobacillus casei on the production of IL-8 by human dental pulp cells. Human dental pulp cells from teeth of young patients (aged 18-25 years) were cultured and tested with sonicated P. intermedia ATCC 25611, F. nucleatum ATCC 25586 and L. casei ATCC 4646 extracts. IL-8 secreted into the culture supernatants were measured at 6, 12 and 24 hours using a quantitative sandwich enzyme immunoassay technique. Cell viability was evaluated using trypan blue exclusion technique. IL-8 production by human dental pulp cells increased significantly at 12 and 24 hours after exposure to P. intermedia and F. nucleatum, whereas L. casei extract exhibited low IL-8 production. The sonicated bacterial extracts did not significantly affect viability or total number of dental pulp cells.

Adolescent↗