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Fusobacterium nucleatum T18 aggregates human mononuclear cells and inhibits their PHA-stimulated proliferation.

In previous studies Fusobacterium nucleatum has been shown to induce either stimulatory or inhibitory effects on human mononuclear cells. We examined the interaction of human mononuclear cells with human and cynomolgus monkey strains of F. nucleatum. Peripheral blood mononuclear cells (PBMCs) isolated from normal donors were aggregated in the presence of cells of F. nucleatum but not control bacteria. The aggregation of PBMCs and F. nucleatum T18 was inhibited by either L-arginine, L-lysine, or pretreatment of the bacterial cells with heat, but was unaffected by the presence of sugars or normal human serum. Strain T18 aggregated purified T-cells and monocytes at approximately equal concentrations. When F. nucleatum T18 was incubated with PHA-stimulated PBMCs, DNA synthesis in the PBMCs was significantly inhibited and detection of IL-2R alpha on the PBMCs was reduced. These studies indicate that F. nucleatum aggregates PBMCs, and that this interaction is associated with both an inhibition of PBMC proliferation and a decrease in IL-2 receptor expression. The ability of F. nucleatum to inhibit mononuclear cell proliferation may be significant in the pathogenesis of periodontal diseases.

Animals↗

Arbitrarily primed-polymerase chain reaction for identification and epidemiologic subtyping of oral isolates of Fusobacterium nucleatum.

BACKGROUND: Fusobacterium nucleatum is the most frequently isolated bacterium in periodontal disease and plays an important role in serious infections in other parts of the body. Arbitrarily primed-polymerase chain reaction (AP-PCR) was used to construct primers for specific identification and subtyping of F. nucleatum. Subtypes may differ in virulence and, hence, are important as periodontal pathogens. Subtypes also may differ in antibiotic susceptibility; therefore, knowing the subtypes may influence choice of treatment. METHODS: We analyzed 70 DNA samples of F. nucleatum isolated from patients with periodontal disease (PD) (N = 32) or AIDS-related PD (N = 8) and from healthy carriers (N = 30). From 90 AP-PCR primers screened, five amplification products were selected, cloned in pCR II vector, and sequenced. These sequences were used to design new pairs of specific primers. Sequences were compared to GenBank entries with BLAST and showed no significant matches. RESULTS: Three primer pairs produced bands of approximately 1 Kb (primer 5059S) or 0.5 Kb (primers FN5047 or M1211) with all F. nucleatum DNAs tested. PCR amplification using primer pair M8171 produced a 1 Kb band with isolates from 7 (22%) PD and 5 (63%) PD-AIDS patients and 9 (30%) healthy controls. Using the same primer pair, 2 other bands of approximately 0.5 Kb and 0.4 Kb were observed with DNA from isolates from 2 (6%) PD and all PD-AIDS patients, but were not observed with DNA samples from healthy controls (P<0.0001). All the primer pairs produced no or different amplicon profiles with DNA samples from bacterial species other than F. nucleatum. CONCLUSIONS: Our results suggest that PCR primer pairs 5059S, FN5047 or M1211 can be used to specifically identify F. nucleatum isolates and distinguish them from other bacteria. The primer pair M8171 could also be used to differentiate F. nucleatum isolated from periodontal patients or healthy individuals. These specific primers can be used in PCR analysis for specific identification of F. nucleatum and to distinguish it from other bacteria associated with human periodontitis. These approaches appear promising in facilitating laboratory identification, molecular subtyping, and taxonomy of putative periodontopathogens.

AIDS-Related Opportunistic Infections↗

Effect of Fusobacterium necrophorum leukotoxoid vaccine on susceptibility to experimentally induced liver abscesses in cattle.

The efficacy and the optimum dose of Fusobacterium necrophorum crude leukotoxoid vaccine required to immunize and protect steers against experimentally induced liver abscesses were evaluated. The vaccine consisted of cell-free culture supernatant of a high leukotoxin-producing strain of F. necrophorum, inactivated with formalin and homogenized with an adjuvant. Twenty-five steers were assigned randomly to the following five treatment groups: control; three doses (1.0, 2.0, and 5.0 mL) of the culture supernatant; and 2.25 mL of the concentrated supernatant (equivalent to 5 mL of the original supernatant). Vaccine was injected subcutaneously on d 0 and 21. Blood samples were collected weekly to monitor antileukotoxin antibody titers. Three weeks after the second vaccination (d 42), all steers were injected intraportally with F. necrophorum culture to induce liver abscesses. Three weeks later (d 63), steers were euthanatized and necropsied; livers were examined and protection assessed. Antileukotoxin antibody titers in the control steers generally did not differ from the baseline (wk 0) titers. The titers in the vaccinated groups increased, more so after the second injection, and the increase was generally dose-dependent. Necropsy examination revealed that all steers in the control group had abscesses in the liver. In the vaccinated groups, two of five steers in the 1.0-mL group and one each in the 2.0-, 5.0-, and 2.25-mL (concentrated) groups had liver abscesses. Antileukotoxin antibody titers were higher (P < .05) in steers that did not develop abscesses than in steers that developed abscesses. The difference suggested a protective effect of antileukotoxin antibodies against experimentally induced liver abscesses.

Animals↗

Partial characterization of a peptidoglycan-protein complex from Fusobacterium nucleatum Fev1.

A protein in the cell wall of Fusobacterium nucleatum Fev1 remained associated with the peptidoglycan during extraction with various detergents and organic solvents. On digestion of this peptidoglycan-protein complex (PPC) with murein hydrolases, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed polypeptide bands with apparent molecular weights (MWs) in the range of 3000 to 40,000. After reaction with maleic anhydride the electrophoretic mobilities of these polypeptide bands increased to those of MWs 3000 to 12,000. The PPC protein showed a limited susceptibility toward trypsin, giving polypeptides that migrated in SDS-PAGE as a diffuse band with MW in the range of 3000 to 6000. The amino acid composition of all polypeptide bands eluted from SDS-PAGE was very similar, whichever enzyme was used for the solubilization of the PPC, and was nearly identical to that found for the protein moiety of the PPC. On the basis of a MW of 3000 for a protein unit, about one molecule of protein was found per five peptidoglycan subunits. Lanthionine was not found associated with released polypeptide, and muramic acid and glucosamine were either absent or present in amounts less than one molecule per protein unit. The PPC was immunogenic in rabbits, and purified anti-PPC IgG reacted with murein hydrolase-released protein separated on SDS-PAGE but preferentially with bands of MWs greater than 18,000.

Bacterial Proteins↗

Cysteine conjugate beta-lyase in the gastrointestinal bacterium Fusobacterium necrophorum.

A cysteine conjugate beta-lyase from the anaerobic gastrointestinal bacterium Fusobacterium necrophorum was purified 51-fold by heat treatment, ammonium sulphate fractionation, gel-filtration chromatography, and anion-exchange chromatography. This enzyme catalyses the cleavage of the thioether linkage in cysteine conjugates of the following S-alkyl- or S-aryl-linked compounds: cysteine conjugate of propachlor (2-S-cysteinyl-N-isopropylacetanilide); 1,2-dihydro-1-hydroxy-2-S-cysteinylnaphthalene and S-(2-benzothiazolyl)cysteine. 2-Mercapto-N-isopropylacetanilide, pyruvic acid and ammonia were produced from the beta-lyase cleavage of the cysteine conjugate of propachlor in equimolar ratios. The apparent Km values for the cysteine conjugate of propachlor and S-(benzothiazolyl)cysteine were 1.1 and 1.0 mM, respectively. Pyridoxal phosphate was required for enzymic activity. Ammonium ion activated enzymic activity, while hydroxylamine completely inhibited the enzyme. Dithiothreitol and bovine serum albumin had no effect on enzymic activity.

Carbon-Sulfur Lyases↗

Septicemia and meningitis caused by Fusobacterium aquatile.

A case of septicemia and meningitis caused by Fusobacterium aquatile is described. The insidious onset of the megningitis and occurrence of multiple cranial nerve affections were outstanding features. In spite of adequate antibiotic treatment the course was protracted, and the outcome was permanent damage to 3 of the affected cranial nerves.

Adolescent↗

Haemagglutination and haemolysis by oral Fusobacterium nucleatum.

Haemagglutination and haemolytic activity of 80 Fusobacterium nucleatum isolates from human and animal origin, on different human blood types was evaluated. All the isolates were able to agglutinate erythrocytes and the most were either alpha-haemolytic or beta-haemolytic. No specificity between haemolysin or haemagglutinin and blood type was observed. Haemagglutination activity was inhibited when D-galactose, D-lactose or D-raffinose were used. Haemagglutination and haemolysis may be important factors in the pathogenesis of human and animal periodontal diseases.

Animals↗

Location of haemagglutinin in bacterial cells of Fusobacterium necrophorum subsp. necrophorum.

The location of haemagglutinin (HA) of Fusobacterium necrophorum subsp. necrophorum VPI 2891 strain was investigated by immunofluorescence, confocal laser scan microscopy and immunoelectron microscopy. The immunofluorescence study demonstrated the fluorescence specific for the HA on the bacterial cells and confocal laser scan microscopy indicated similar fluorescence around the cross section of the bacterial cell. The immunoelectron microscopic study also revealed that the protein A-gold conjugates were located around the bacterial surfaces. These findings suggest that HA is one of the components of the cell surfaces of F. necrophorum subsp, necrophorum.

Fluorescent Antibody Technique, Indirect↗

[Septic thrombophlebitis of the internal jugular vein due to Fusobacterium necrophorum (Lemierre's syndrome): case report and review of literature].

Lemierre's syndrome is an uncommon clinical entity. It is characterized by oropharyngeal infection followed by septic thrombophlebitis of the jugular vein with embolization to the lungs and other organs. It is usually due to Fusobacterium necrophorum. Although Lemierre's syndrome is rare, it is potentially fatal. It is important for clinicians to recognize and treat it appropriately. With prompt recognition, abscess drainage with possible ligation of the internal jugular vein and appropriate antibiotic coverage complete recovery can be achieved in most patients.

Bacteremia↗

Adherence of Fusobacterium necrophorum subspecies necrophorum to different animal cells.

The adherence of Fusobacterium necrophorum subsp. necrophorum to the surfaces of animal cells was studied in order to elucidate the differences between the bacterial appearance in clinical specimens from various animals. The bacterial cells had a strong affinity for murine and rabbit cheek cell surfaces. The bacterium showed a moderate affinity for goat cells, whereas it adhered not so well to canine, feline, human or porcine cells. Treatment of the bacterial cells with haemagglutinin antiserum prior to the binding assay reduced the degree of attachment to murine and rabbit cells. Scanning electron microscopy revealed that the adherent fusobacteria often penetrated into murine and rabbit cell membranes. These observations indicate that the bacterial attachment contributes to the establishment of the infection in mice and rabbits. It is suggested that the weak binding ability resulted in a low incidence of the bacterium in canine, feline and porcine lesions.

Adult↗

Actin degradation concomitant with Fusobacterium necrophorum subsp. necrophorum adhesion to bovine portal cells.

The effects of Fusobacterium necrophorum subsp. necrophorum on cellular actin were investigated using tissue-cultured bovine portal cells. Fluorescence studies revealed the appearance of intense fluorescent spots on the cellular actin and the spots increased in a time dependent manner. Sodium dodecyl sulphate polyacrylamide gel electrophoresis profiles manifested partial or complete degradation of actin preparation after treatment with the bacterial cells. These findings suggest that the bacterial cell wall may contribute to the degradation of the cellular actin during the initial stage of the infection caused by F. necrophorum subsp. necrophorum.

Actins↗

Chemical composition of endotoxins produced by Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme.

The endotoxins from two recently-classified subspecies of Fusobacterium, namely F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme, were compared. Chemical analysis of the isolated endotoxins revealed that they were clearly different. Distinct levels of polysaccharides were demonstrated. The endotoxins isolated were devoid of heptose and 3-deoxy-D-manno-octulosonate (KDO). The endotoxins of F. n. necrophorum and F. n. funduliforme contained lipid A in a ratio of 4:1 which may account for the variations in their virulence.

Endotoxins↗

Effects of Fusobacterium necrophorum subspecies necrophorum on extracellular matrix of tissue-cultured bovine kidney cells.

The effects of Fusobacterium necrophorum subsp. necrophorum on the extracellular matrix were investigated. The toxic preparation from the culture induced reduction in the number of tissue-cultured bovine kidney cells. The exposed cells often manifested partial loss of cytoplasm and were morphologically irregular. Scanning electron microscopy demonstrated partial loss of the microvilli on the exposed cells and roughness of the cell surfaces. Finally, sodium dodecyl sulphate polyacrylamide gel electrophoresis profiles revealed complete degradation of bovine collagen type 1 after treatment with the toxic preparation. This degradation was inhibited by the addition of homologous antiserum. These findings indicate that the degradation may contribute to the establishment of the infection caused by F.n. subsp. necrophorum.

Animals↗

Influence of antimicrobial subinhibitory concentrations on hemolytic activity and bacteriocin-like substances in oral Fusobacterium nucleatum.

Fusobacterium nucleatum is considered for its role in colonization of initial and late microorganisms in dental plaque and for its coaggregation with other bacterial species. It is known that action of different antimicrobial substances may interfere with either virulence factors or with host-bacteria interaction. The goal of this study was to examine the influence of subinhibitory concentrations of chlorhexidine, triclosan, penicillin G and metronidazole on hemolytic activity and bacteriocin-like substance production of oral F. nucleatum. A high resistance to penicillin G was observed and 63% of the isolates were beta-lactamase positive. All the tested isolates were susceptible to metronidazole. F. nucleatum isolates grown with or without antimicrobials were alpha-hemolytics. Bacteriocin-like substance production was increased in isolates grown with penicillin G. Impaired production of hemolytic or antagonic substances can suggest a role in the regulation of oral microbiota.

Adult↗

Endotoxin from Fusobacterium necrophorum of bovine hepatic abscess origin.

The endotoxic activity of Fusobacterium necrophorum bov 5 was investigated. The supernatant (S) fluid and cell wall (CW) preparation, obtained after differential centrifugation of the ruptured cell mass, were lethal for mice. The toxicity of the S fluid was stable during prolonged storage, treatment with formalin, and heating for 15 minutes at 80, 100, and 121 C, but was destroyed by alkaline hydrolysis with 0.25 N NaOH. The toxic factor was found in a high molecular weight (MW) fraction after gel filtration. The properties exhibited by the toxic S fluid resembled those of endotoxic lipopolysaccharide (LPS). Extracted and partially purified LPS (endotoxin) from F necrophorum bov 5 demonstrated a mouse median lethal dose (mouse LD50) of 16.8 mg/kg of body weight. The toxic LPS material, a high molecular weight moiety as estimated by gel filtration, was resistant to ribonuclease (RNase), deoxyribonuclease (DNase), and pronase treatment. A positive Shwartzman reaction (median skin lesion dose (SLD50) equal to 3.32 mug/kg of body weight) and biphasic fever response (minimal dose required to produce a fever index of 40 sq cm which falls on the linear portion of dose-response curve (FL40) equal to 0.41 mug/kg of body weight) further indicated the toxin was endotoxin in nature. The LPS from F necrophorum bov 5 was less toxic than Salmonella typhimurium LPS; but had considerable toxicity for experimental animals. The toxic activity of the partially purified F necrophorum bov 5 endotoxin was separated into 2 fraction regions by diethylaminoethyl (DEAE)-cellulose chromatography. The data provide evidence for the production of a potent endotoxin, possibly composed of more than one toxic component, which may be released upon cell disruption.

Animals↗

Collagenolytic activity of a cell wall preparation from Fusobacterium necrophorum subsp. necrophorum.

A collagenolytic preparation of Fusobacterium necrophorum subsp. necrophorum was derived from the bacterial cell. It was further treated for gel permeation with Toyopearl HW 50, followed by Sepharose 4B column chromatography. In sodium dodecyl sulphate polyacrylamide gel electrophoresis, the final preparation exhibited one definite band and at least one faint band. It was inactivated completely by adjusting the pH to 4.0 or by heating at 80 degrees C for 30 min.

Animals↗

Fusobacterium necrophorum and Bacteroides melaninogenicus as etiologic agents of foot rot in cattle.

Fusobacterium necrophorum (Sphaerophorus necrophorus) and Bacteroides melaninogenicus were the predominant bacteria isolated from biopsy specimens of lesions in cattle affected with foot rot. Mixed inoculums of the 2 bacteria, applied to the scarified interdigital skin or inoculated intradermally into the interdigital skin of test cattle, induced typical lesions of foot rot. Both bacteria were reisolated in large numbers from the induced lesions.

Administration, Topical↗

[Ribotyping Fusobacterium nucleatum isolates from healthy and diseased periodontium].

Fusobacterium nucleatum is one of the most frequently cultured bacteria from subgingival plaque. It can be isolated from active periodontal lesions as well as from healthy sites. Currently, F. nucleatum has been divided into five subspecies. An early hypothesis suggested an association between certain subspecies and periodontal disease or health. On the other hand, a broad heterogeneity between F. nucleatum isolates has been suggested, so that the subspeciation scheme and therefore the proposed association may not be valid. The purpose of the present study was to analyze if clonal differences exist between human F. nucleatum isolates from either periodontally healthy or diseased individuals. 23 F. nucleatum isolates from periodontally healthy and 17 isolates from periodontally diseased individuals were analysed by ribotyping. A broad genetic heterogeneity was present, although some of the isolates from periodontally healthy individuals were genotypically identical. However, no clonal differences between isolates from periodontally healthy or diseased individuals was detected.

Adolescent↗