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Gram-negative, anaerobic bacilli are unusual organisms to be isolated in cases of acute septic arthritis. We report the isolation of Fusobacterium necrophorum from joint aspirate in a case of acute septic arthritis, which presented 3 weeks after the drainage of a dental abscess.
This is a report of a twelve-year-old black male who presents a history of left neck pain, left shoulder pain, weight loss and minimal fever. The admission diagnosis was a malignancy, supported by radiographic findings of consolidation of left lower and left upper lobes and periosteal new bone formation along the inferior aspects of the left second, third and fourth ribs. Open biopsy of the chest wall revealed a large abscess cavity. Subsequent cultures on anaerobic blood agar plates grew Actinomyces and Fusobacterium. The patient was treated with penicillin and improved clinically. This case emphasizes the need to culture tissue specimens for a wide array of organisms, including those not commonly seen today. A high index of suspicion is required for the diagnosis of anaerobic infections. Mixed anaerobic infections, including actinomyces, although uncommon in children, should be considered in the etiology of an intrathoracic mass.
The purpose of this study was to examine the effect of an extract of Fusobacterium nucleatum on humoral and cell-mediated immunity and phagocyte functions in mice. The extract was obtained from the heat-inactivated supernatant of sonicated F. nucleatum. Mouse splenocytes were cultured with sheep red blood cells (SRBC), and antibody formation was assayed by counting the plaque-forming cells (PFC). After C3H mouse skin was grafted onto the BALB/c mouse, cytotoxicity of BALB/c mouse splenocytes to L-cells was enumerated, T- and B-cell blastogenesis was determined by the uptake of 3H-thymidine. In vivo phagocyte functions were measured using carbon clearance test, bactericidal, and acid phosphatase activity assays. The number of SRBC-specific and non-specific PFC in splenocytes stimulated with the extract increased 2.5-8.7-fold over that of unstimulated splenocytes. Cytotoxicity of splenocytes from the extract-injected mice decreased in a dose-dependent fashion at 12 days after grafting. T-cell mitogenicity of the extract was observed. The carbon clearance rate was reduced, and bactericidal and acid phosphatase activities were elevated by the extract injection of the mice. In conclusion, F. nucleatum extract has an adjuvant effect on humoral immunity, and a suppressive effect on cell-mediated immunity and activated phagocyte functions.
Fusobacterium nucleatum is a Gram-negative anaerobic rod-shaped bacterium frequently isolated from human dental plaque. It is capable of the desulfuration of cysteine and methionine, resulting in the formation of sulfide and thiol volatiles, respectively. Intact cells, as well as cell-free extracts produced by French pressure cell lysis of F. nucleatum, hydrolyzed radiolabeled cysteine to produce sulfide, pyruvic acid, and ammonia. The hydrolysis products of radiolabeled methionine were a volatile thiol, ketobutyrate, and ammonia. Both activities were associated with the cytoplasmic component, not the membrane. The desulfuration mechanisms are heat-labile, inhibited by the presence of excess substrate, and rates are dependent upon substrate concentration. These dissimilar pathways by F. nucleatum can account in part for the presence of sulfur-containing volatile products that occur in the mouth.
Western blot analysis that used antisera to the E. coli core enzyme and sigma factors was used for examination of the RNA polymerase of Actinobacillus actinomycetemcomitans, Streptococcus mutans, Fusobacterium nucleatum, and Porphyromonas gingivalis. Both antisera reacted with proteins in A. actinomycetemcomitans and S. mutans whole-cell extracts. Reactions were seen with some F. nucleatum proteins when the anti-core RNA polymerase antisera were used, but the cross-reacting proteins were not of an expected molecular weight for beta or beta'. No reaction with F. nucleatum proteins was seen when extracts were reacted with antisera to E. coli sigma factor. There were no cross-reacting proteins detected in P. gingivalis extracts with either antisera. These results suggest that E. coli RNA polymerase may not be sufficiently similar to P. gingivalis and F. nucleatum RNA polymerase for E. coli RNA polymerase to recognize P. gingivalis or F. nucleatum promoters. Partially purified P. gingivalis and F. nucleatum RNA polymerase exhibited a specificity for a P. gingivalis DNA template, while having a decreased activity from an E. coli DNA template. The antibiotic sensitivity profile of P. gingivalis and F. nucleatum RNA polymerase activity was shown to differ from that of E. coli, with these activities not being affected by rifampicin, streptovaricin, or streptolydigin. We conclude that the efficient cloning and expression of P. gingivalis and F. nucleatum genes in E. coli will require the use of promoter-containing expression vectors.
Once established, early-colonizing bacterial species tend to persist in the mouth. To obtain detailed information on the population dynamics of early-colonizing oral anaerobes, we examined the clonal diversity and persistence of clones among oral Fusobacterium nucleatum populations during the first 2 yrs of life. Consecutive salivary samples from 12 infants, collected at 2, 6, 12, 18, and 24 mos of age, yielded a total of 546 F. nucleatum isolates for clonal typing with arbitrarily primed PCR (AP-PCR). Up to 7 AP-PCR types were simultaneously detected in each sample. In 11 out of the 12 infants examined, AP-PCR types persisted for up to 1 yr. Strain turnover rate was high during the first year of life, but then the occurrence of persistent clones increased. This study indicates a wide genetic diversity within the species and provides evidence for the increasing persistence of F. nucleatum clones in the oral cavity with age.
The periodontal pathogen Fusobacterium nucleatum induces apoptosis in lymphocytes. We previously identified the autotransporter protein Fap2 in F. nucleatum strain PK1594 that induced apoptosis in lymphocytes when expressed in Escherichia coli. In this study, we identified protein homologs of Fap2 in the transformable F. nucleatum strain ATCC 23726, to determine their role in the induction of apoptosis in lymphocytes. We used a new gene-inactivation vector conferring thiamphenicol resistance (pHS31) to construct a mutant deficient in one of the homologs, aim1. Transcriptional analyses demonstrated disruption of aim1 expression, and phenotypic analyses revealed a 41% decrease in the ability of the mutant to induce apoptosis in Jurkat cells, as compared with the parental strain. These studies demonstrate, in the native host cell background, the contribution of aim1 to F. nucleatum induction of apoptosis and, to the best of our knowledge, represent the first report of a genetically defined and phenotypically characterized mutation in F. nucleatum.
Fusobacterium K-60, a ginsenoside Rb1-metabolizing bacterium, was isolated from human intestinal feces. From this Fusodobacterium K-60, a ginsenoside Rb1-metabolizing enzyme, beta-glucosidase, has been purified. The enzyme was purified to apparent homogeneity by a combination of butyl-Toyopearl, hydroxyapatite ultragel, Q-Sepharose, and Sephacryl S-300 HR column chromatographies with a final specific activity of 1.52 micromol/min/mg. It had optimal activity at pH 7.0 and 40 degrees C. The molecular mass of this purified enzyme was 320 kDa, with 4 identical subunits (80 kDa). The purified enzyme activity was inhibited by Ba++, Fe++, and some agents that modify cysteine residues. This enzyme strongly hydrolyzed sophorose, followed by p-nitrophenyl beta-D-glucopyranoside, esculin, and ginsenoside Rb1. However, this enzyme did not change 20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol (IH-901) to 20(S)-protopanaxadiol, while it weakly changed ginsenoside Rb1 to IH-901. These findings suggest that the Fusobacterial beta-glucosidase is a novel enzyme transforming ginsenoside Rb1.
The 16S-23S rRNA intergenic spacer regions (ISRs) of Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme were characterized. Products of two sizes, about 360 bp (small) and 530 bp (large), were generated by PCR amplification from the 16S-23S rRNA ISR of all the strains tested. The large and small 16S-23S rRNA ISRs of F. necrophorum exhibited a level of sequence similarity of 93.9% to 99.7% and 94.2% to 98.6% homologies within the species, respectively. Only the large spacer regions in these bacteria contained one or two tRNA genes. F. necrophorum subsp. necrophorum contains the isoleucine and alanine tRNA gene, whereas F. necrophorum subsp. funduliforme contains the isoleucine tRNA gene.
Three strains of Fusobacterium necrophorum biovar C were injected into mice intraperitoneally and intraportally. All the mice survived. In one mouse out of 15 mice injected intraperitoneally, a few focal abscesses were formed in the liver. The microorganisms were recovered from the liver abscess and the tissue of liver with abscess. No changes were observed in the organs of other 14 mice and no bacteria were recovered from them. In the 15 mice injected intraportally, no liver abscesses and no macroscopic changes in the organs were formed. However, the inoculated bacteria were recovered from the liver of four mice. The pathogenicity of F. necrophorum biovar C was weaker than that of other two biovars.
OBJECTIVE: To assess the role of anaerobic bacteria in acute exacerbation of chronic bronchitis (AECB). METHODS: The level of the Ig-A class to two organisms (Fusobacterium nucleatum and Prevotella intermedia) was determined in the sputum of 25 patients with AECB and 25 control patients. The presence and level of these antibodies were investigated by enzyme-linked immunosorbent assay. RESULTS: The median sputum antibody level for F nucleatum and P intermedia were significantly higher in patients with AECB as compared to control patients (p < 0.05). The IgA levels for F nucleatum was 3.5 times higher in patients with AECB and 3.8 times greater for P intermedia. CONCLUSIONS: This study demonstrates for the first time an elevated sputum antibody titers in patients with AECB to F nucleatum and P intermedia.
The antibacterial effect of visible light irradiation combined with photosensitizers has been reported. The objective of this was to test the effect of visible light irradiation without photosensitizers on the viability of oral microorganisms. Strains of Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mutans and Streptococcus faecalis in suspension or grown on agar were exposed to visible light at wavelengths of 400-500 nm. These wavelengths are used to photopolymerize composite resins widely used for dental restoration. Three photocuring light sources, quartz-tungsten-halogen lamp, light-emitting diode and plasma-arc, at power densities between 260 and 1300 mW/cm2 were used for up to 3 min. Bacterial samples were also exposed to a near-infrared diode laser (wavelength, 830 nm), using identical irradiation parameters for comparison. The results show that blue light sources exert a phototoxic effect on P. gingivalis and F. nucleatum. The minimal inhibitory dose for P. gingivalis and F. nucleatum was 16-62 J/cm2, a value significantly lower than that for S. mutans and S. faecalis (159-212 J/cm2). Near-infrared diode laser irradiation did not affect any of the bacteria tested. Our results suggest that visible light sources without exogenous photosensitizers have a phototoxic effect mainly on Gram-negative periodontal pathogens.
Phototoxicity of visible light laser on the porphyrin-producing bacteria, Porphyromonas gingivalis, in the absence of photosensitizers and under aerobic conditions was shown in previous studies. Recently, we found that the noncoherent visible light sources at wavelengths of 400-500 nm, commonly used in restorative dentistry, induced a phototoxic effect on P. gingivalis, as well as on Fusobacterium nucleatum, and to a lesser extent on the Streptococci sp. To elucidate the mechanism of this phototoxic effect, P. gingivalis and F. nucleatum were exposed to light (1) under aerobic and anaerobic environments and (2) in the presence of scavengers of reactive oxygen species (ROS). Phototoxic effect was not observed when the bacteria were exposed to light under anaerobic conditions. Dimethyl thiourea, a hydroxyl radical scavenger, was effective in reducing phototoxicity (P </= 0.05). Other scavengers, such as catalase, superoxide dismutase and ascorbic acid, were less effective when applied separately. These results support the assumption that the phototoxic effect of blue light on the periopathogenic bacteria is oxygen dependent and that hydroxyl radicals play an important role in this process.
Fusobacterium nucleatum is a strict anaerobe and is indigenous of the human oral cavity. This organism is commonly recovered from different monomicrobial and mixed infections in humans and animals. In this study, the plasmid profile, the plasmid stability and the penicillin-resistance association in oral F. nucleatum isolated from periodontal patients, healthy subjects and Cebus apella monkeys were evaluated. Forty-five F. nucleatum strains from patients, 38 from healthy subjects and seven from C. apella were identified and analyzed. Plasmid extraction was performed in all the isolated strains. These elements were found in 26.7% strains from patients and one strain from C. apella. Strains from healthy subjects did not show any plasmid. Most of strains showed two plasmid bands ranging from 4 to 16 Kb, but digestions with endonucleases showed that they belonged to a single plasmid. The plasmid profile was similar and stable in human and monkey strains. Also, plasmids were classified into three groups according to size. Two strains were positive to beta-lactamase production and no plasmid DNA-hybridization with a beta-lactamase gene probe was observed, suggesting a chromosomal resistance.
Fusobacterium nucleatum is a microorganism commonly cultured from periodontal disease sites. F. nucleatum isolates (120) were obtained from subgingival plaque samples taken from 27 clinically characterized sites using a selective culture medium. All isolates were verified by morphology, Gram-stain reactions, oxygen tolerance and biochemical reactions. A total of eight clinical isolates and two typed strains were used for further evaluation. In this study, there was no relationship found between GI and probing depth or between probing depth and frequency of isolation of Type I or Type II F. nucleatum colonies. There was a significant increase in isolation of Type II colonies with a GI of 2 (P less than 0.0001). All isolates tested shared lines of identity by double diffusion in agar and displayed similar ability to hemagglutinate sheep erythrocytes and a reduction in this hemagglutination activity by previous exposure to 50 mM D-galactose. All isolates tested showed similar protein patterns as determined by polyacrylamide gel electrophoresis. By the methods used, no differences were detected between Type I and Type II F. nucleatum; however, there was a statistically significant increase in Type II isolates with increasing levels of gingivitis.
The present study was undertaken to determine if mucosal presentation of the periodontopathic bacterium Fusobacterium nucleatum could induce systemic tolerance. Two separate protocols of mucosal priming were carried out. In the first, mice were gastrically intubated on 2 consecutive days; this was repeated 5 days later. In the second protocol, mice were similarly primed but received another priming dose after a further 7 days. Positive control mice were similarly primed with sheep red blood cells (SRBC) while negative control animals were sham-primed with saline. Following mucosal priming, mice were systemically sensitized with the respective antigen and then subsequently challenged in the left hind footpad. The right footpad was challenged with saline and served as a negative control. Serum antibody levels were measured using enzyme-linked immunoabsorbent assay (ELISA) and haemagglutination assays. Mucosal priming with F. nucleatum was found to suppress the local delayed type hypersensitivity reaction as determined by footpad measurements. Sham-priming did not suppress the local response. On the other hand, the levels of serum antibodies were not influenced by mucosal priming. These results suggest that under the experimental conditions used, mucosal presentation of F. nucleatum can induce a degree of split tolerance in which T cell responses are suppressed while B cell responses remain intact. The implication of this finding to human periodontal disease is yet to be determined.
To learn more about colonization of the oral epithelium by Fusobacterium nucleatum and the role of fibronectin in mediating adhesion of this microorganism, we studied attachment of this bacterium to cultured gingival epithelial cells that were coated with exogenous, purified plasma fibronectin. The three strains of F. nucleatum studied adhered in large numbers to epithelial cells that had been coated with fibronectin, compared with buffer-coated control cells. Bacterial adherence was also enhanced when epithelial cells were coated with whole human saliva. However, cells coated with saliva depleted of fibronectin did not facilitate adhesion of bacteria. Bacterial adhesion was restored when purified fibronectin was added back. We also tested adherence of bacteria to coverslips coated with fibronectin, saliva, and saliva depleted of fibronectin. The bacteria adhered to coverslips coated with fibronectin or whole human saliva, but did not adhere to coverslips coated with fibronectin-depleted saliva. Bacterial adhesion to coverslips was restored upon addition of purified fibronectin to the fibronectin-depleted saliva. Bacterial attachment to fibronectin-coated coverslips was found to be temperature-dependent, with maximal adhesion observed at 37 degrees C. Pre-treatment of F. nucleatum with soluble fibronectin inhibited attachment of the bacteria by 92%, whereas pre-treatment with bovine serum albumin had no effect. Pre-treatment of bacteria with laminin or type IV collagen caused moderate inhibition of attachment by 60% and 50%, respectively. Treatment of fibronectin-coated coverslips with Fab fragments of anti-fibronectin IgG blocked the attachment of F. nucleatum by 93%. Fab fragments of the other antisera tested had no inhibitory effect.(ABSTRACT TRUNCATED AT 250 WORDS)
Prevotella intermedia and fusobacterium nucleatum are associated with various forms of periodontal disease. The purpose of the present study was to infect the clinical isolates of these periodontopathic bacteria and to induce a significant loss of alveolar bone in specific pathogen-free (SPF) rats in the absence of ligatures. P. intermedia YKD8 and F. nucleatum YKZ5 were isolated from a prepubertal periodontitis patient, while P. gingivalis MWB13 was from a patient with juvenile periodontitis. At first, SPF Sprague-Dawley rats (70 days of age, male) were infected with A. viscosus Ny1R and subsequently superinfected with P. gingivalis MWB13, P. intermedia YKD8, or F. nucleatum YKZ5, respectively. The control group was infected with A. viscosus Ny1R alone. All rats were killed and periodontal bone levels were assessed morphometrically 135 days after the first infection with A. viscosus. P. intermedia YKD8 was recovered frequently from rats, with serum antibody levels remaining highly elevated throughout the experiment. Significant loss of alveolar bone was found in rats infected with P. intermedia YKD8, the virulence of which was equivalent to that of P. gingivalis MWB13. F. nucleatum YKZ5 also induced alveolar bone loss, but not significantly when compared with rats infected with A. viscosus Ny1R alone.