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Proposal of three subspecies of Fusobacterium nucleatum Knorr 1922: Fusobacterium nucleatum subsp. nucleatum subsp. nov., comb. nov.; Fusobacterium nucleatum subsp. polymorphum subsp. nov., nom. rev., comb. nov.; and Fusobacterium nucleatum subsp. vincentii subsp. nov., nom. rev., comb. nov.

Heterogeneity among isolates of Fusobacterium nucleatum has been recognized for many years. The phenotypic properties of 340 strains considered to be F. nucleatum were examined. While these strains were phenotypically similar and fit the description of F. nucleatum, they could be differentiated into three groups on the basis of electrophoretic patterns of whole-cell proteins and DNA homology. Strains in groups I and II showed greater than 80% DNA homology within groups and less than 75% similarity between groups. Strains of group III demonstrated greater than 85% DNA homology to each other and less than 65% similarity to strains in groups I and II. We propose that Fusobacterium nucleatum be divided into the following three subspecies: Fusobacterium nucleatum subsp. nucleatum, with type strain ATCC 25586; Fusobacterium nucleatum subsp. polymorphum, with type strain ATCC 10953; and Fusobacterium nucleatum subsp. vincentii, with type strain ATCC 49256.

Bacterial Proteins↗

Proposal of two subspecies of Fusobacterium necrophorum (Flügge) Moore and Holdeman: Fusobacterium necrophorum subsp. necrophorum subsp. nov., nom. rev. (ex Flügge 1886), and Fusobacterium necrophorum subsp. funduliforme subsp. nov., nom. rev. (ex Hallé 1898).

The biological and biochemical properties, DNA base compositions, and levels of DNA-DNA homology of two biovars of Fusobacterium necrophorum were examined. Some differences were found between the two biovars in biological and biochemical properties. The G + C contents of DNAs from biovar A strains VPI 2891T (T = type strain), NCTC 10576, N167, Fn47, and Fn43, were 32, 30, 29, 28, and 31 mol%, respectively. The G + C contents of DNAs from biovar B strains Fn524T, 606, Fn49, Fn45, and 1260 were 30, 31, 27, 31, and 30 mol%, respectively. Labeled DNA from biovar A strain VPI 2891T exhibited 100 to 80% relatedness to DNAs from biovar A strains and 59 to 51% relatedness to DNAs from biovar B strains. Labeled DNA from biovar B strain Fn524T exhibited 100 to 81% relatedness to DNAs from biovar B strains and 71 to 60% relatedness to DNAs from biovar A strains. Therefore, the names Fusobacterium necrophorum subsp. necrophorum subsp. nov., nom. rev. (ex Flügge 1886), and Fusobacterium necrophorum subsp. funduliforme subsp. nov., nom. rev. (ex Hallé 1898), are proposed for Fusobacterium necrophorum biovars A and B, respectively. The type strain of F. necrophorum subsp. necrophorum is strain VPI 2891 (= JCM 3718 = ATCC 25286), and the type strain of F. necrophorum subsp. funduliforme is strain Fn524 (= JCM 3724).

Base Composition↗

Phylogenetic analysis of Fusobacterium necrophorum, Fusobacterium varium and Fusobacterium nucleatum based on gyrB gene sequences.

The nucleotide sequences of the DNA gyrase B subunit gene (gyrB) of Fusobacterium necrophorum subsp. necrophorum, F. necrophorum subsp. funduliforme and F. varium were determined and analyzed together with those of F. nucleatum subsp. nucleatum and F. nucleatum subsp. vincentii. On the phylogenetic tree constructed, the strains of each fusobacterial species formed distinct clusters with deep sublines. The degree of sequence similarity within each cluster was 93.2% or more, whereas similarities between clusters ranged from 70.1 to 72.7%. These clusters were recovered with 100% bootstrap probabilities and are in very good agreement with the species of Fusobacterium. These data suggest that gyrB is an accurate genealogical marker for the classification of the fusobacterial taxa considered in this study.

Base Sequence↗

Fusobacterium nucleatum subsp. fusiforme subsp. nov. and Fusobacterium nucleatum subsp. animalis subsp. nov. as additional subspecies within Fusobacterium nucleatum.

Using a variety of physiological, biochemical, and molecular systematic analyses, we have shown previously that there are four groups within the species Fusobacterium nucleatum. Two of these groups of strains correspond to the recently proposed taxa F. nucleatum subsp. nucleatum and F. nucleatum subsp. polymorphum. In this paper we show that the two remaining groups are distinct and formally propose that they should be recognized as F. nucleatum subsp. fusiforme (type strain, NCTC 11326) and F. nucleatum subsp. animalis (type strain, NCTC 12276). The tests which we used did not allow a full assessment of the status of F. nucleatum subsp. vincentii compared with F. nucleatum subsp. nucleatum.

Electrophoresis↗

Phylogenetic analysis of Fusobacterium alocis and Fusobacterium sulci based on 16S rRNA gene sequences: proposal of Filifactor alocis (Cato, Moore and Moore) comb. nov. and Eubacterium sulci (Cato, Moore and Moore) comb. nov.

Genes encoding the 16S rRNA of Fusobacterium alocis ATCC 35896T and Fusobacterium sulci ATCC 35585T were sequenced. These sequences did not have any affinity with the 16S rRNA gene sequences of members of the genus Fusobacterium. Fusobacterium alocis ATCC 35896T and Fusobacterium sulci ATCC 35585T belonged to Clostridium cluster XI; the species most closely related to these strains were Filifactor villosus NCTC 11220T and Eubacterium infirmum W1471, respectively. Two new combinations are proposed: Filifactor alocis (Cato, Moore and Moore) comb. nov. (type strain ATCC 35896T) and Eubacterium sulci (Cato, Moore and Moore) comb. nov. (type strain ATCC 35585T).

Animals↗

New medium selective for Fusobacterium species and differential for Fusobacterium necrophorum.

Fusobacterium egg yolk agar is a new medium selective for Fusobacterium species and differential for Fusobacterium necrophorum. The medium is a Brucella Agar base (Difco Laboratories, Detroit, Mich.) containing vancomycin, neomycin, josamycin, and egg yolk. All species of fusobacteria grew with only minimal inhibition. The mean log difference in counts between Fusobacterium egg yolk agar and control media for 30 strains of seven species of fusobacteria was 0.1922. F. necrophorum typically showed a strong lipase reaction. Most other organisms were significantly inhibited by the medium.

Agar↗

Characterization of shared antigens of Fusobacterium nucleatum and Fusobacterium necrophorum.

Fusobacterium nucleatum and Fusobacterium necrophorum are gram-negative, non-spore-forming anaerobic rods, frequently isolated from the normal flora and diseased lesions of the human oral cavity, gastrointestinal and genitourinary tracts. F. necrophorum is also known to be an animal pathogen. Studies were undertaken with rabbit anti-F. nucleatum sera and with human adult periodontitis (AP) sera that demonstrated the sharing of antigens between the two species. Immunodiffusion and immunoelectrophoresis studies of Fusobacterium species with rabbit anti-F. nucleatum sera demonstrated the presence of shared antigen(s) between F. nucleatum and F. necrophorum. Adsorption studies of AP sera in an enzyme-linked immunosorbent assay demonstrated the presence of antibodies reacting with the shared antigens of the two species. Immunoblot (IB) analysis of a soluble protein preparation of the two species of Fusobacterium, when allowed to react with rabbit anti-F. nucleatum 10197 serum, demonstrated 53 kDa and 30 kDa bands present in members of the two species. Further, IB analysis of protein preparations of the two species with AP sera indicated the presence of antibodies reacting with the shared 53 kDa band and in some cases the 30 kDa band. During serological testing with antisera or host immune studies with human sera to these species, the presence of shared antigens must be considered.

Adult↗

Recognition of biovar C of Fusobacterium necrophorum (Flügge) Moore and Holdeman as Fusobacterium pseudonecrophorum sp. nov., nom. rev. (ex Prévot 1940).

The cellular morphology, colonial morphology, biochemical properties, DNA base compositions, and DNA-DNA homolgies of three biovars of Fusobacterium necrophorum were examined. Some differences were found among the three biovars in cellular morphology, colonial morphology, and biochemical properties. The guanine-plus-cytosine contents of DNAs from biovar C strains Fn521T (T = type strain), Fn522, and Fn520 were 30.4, 29.3, and 28.0 mol%, respectively, and the guanine-plus-cytosine contents of DNAs from strains VPI 2891 (biovar A) and VPI 6161 (biovar B) were 31.3 and 32.0 mol%, respectively. Labeled DNA from biovar C strain Fn521T exhibited 96 and 82% relatedness to DNAs from biovar C strains Fn522 and Fn520, respectively; however, it exhibited only about 10% relatedness to DNAs from strains of biovars A and B. Labeled DNAs from strains VPI 2891 and VPI 6161 exhibited more than 70% relatedness to each other, but about 6 to 20% relatedness to DNAs from biovar C strains. Therefore, Fusobacterium pseudonecrophorum sp. nov., nom. rev. (ex Prévot 1940) is proposed for Fusobacterium necrophorum biovar C. The type strain is strain Fn521 (= JCM 3722).

Animals↗

Molecular characterization of a 40-kDa outer membrane protein, FomA, of Fusobacterium periodonticum and comparison with Fusobacterium nucleatum.

The 40 kDa-outer membrane protein FomA of Fusobacterium periodonticum ATCC 33693 was found to exhibit heat modifiable properties, typical for a porin, and N-terminal sequencing indicated a close relationship to the porin FomA of Fusobacterium nucleatum. A polymerase chain reaction approach was therefore applied for sequencing the fomA gene of F. periodonticum, and nucleotide and deduced amino acid sequences were aligned and compared with the corresponding sequences of different strains of F. nucleatum. In all strains we found a common protein upstream of the fomA gene. The noncoding area upstream of the putative -35 region of the F. periodonticum fomA gene exhibited little sequence similarity with the F. nucleatum gene. The transcriptional unit of FomA, on the other hand, was very similar, with the similarities concentrated in domains that were interspersed with hypervariable regions. A topology model was made and compared with those made for F. nucleatum. This indicated that the great similarities reside in the membrane-spanning segments of the protein, while most cell surface exposed loops were hypervariable. The results strongly support the proposed model for FomA and also indicate that these taxa are related but on a lower level than the subspecies level. The codon usage of F. periodonticum is comparable to that of F. nucleatum, and the triplet AGA is the only codon used for arginine.

Amino Acid Sequence↗

The changing pattern of Fusobacterium infections in humans: recent experience with Fusobacterium bacteraemia.

A retrospective study was conducted of 26 adult cases of fusobacterium bacteraemia that occurred between 1998 and 2003 at Center Hospitalier Universitaire de Grenoble, France. Most patients presented with pre-existing adverse medical conditions, including evolving malignant diseases (eight patients), recent surgery (four patients), and chronic organ failure (six patients). Only one patient presented with a classic Lemierre's syndrome. These results suggest an opportunistic pattern of modern fusobacterium infections.

Adolescent↗

Bone resorption stimulated by lipopolysaccharides from Bacteroides, Fusobacterium and Veillonella, and by the lipid A and the polysaccharide part of Fusobacterium lipopolysaccharide.

Lipopolysaccharides (LPS) isolated from oral strains of Veillonella, Fusobacterium and Bacteroides stimulated the release of 45Ca from prelabeled fetal rat bones in culture. There was a typical dose-response relationship between the quantities of released 45Ca and LPS used for stimulation. Bacteroides-LPS proved to be the less active inducer of 45Ca release. LPS had no stimulating effect on the release of 45Ca from devitalized bone. The stimulated 45Ca release was paralleled by an increase in the culture medium of hydroxyproline and lactate. This, together with the findings of numerous osteoclasts in stained histological specimens of the experimental bones, indicates that LPS stimulated the osteoclasts to bone resorption. Heparin, which did not directly induce 45Ca release, potentiated the bone resorption stimulating capability of LPS. The lipid A and the polysaccharide portion of Fusobacterium LPS also stimulated bone resorption and, remarkably, the polysaccharide portion showed the greatest activity. This may explain the mode of action of LPS lacking a typical lipid A. It is suggested that stimulation of osteoclasts by LPS may result from activation of complement components by lipid A or its polysaccharide portion.

Animals↗

Similarities between Fusobacterium nucleatum and bacteroides fragilis studied by two DNA probes derived from Fusobacterium nucleatum.

A polymerase chain reaction (PCR)-amplified oligonucleotide DNA probe corresponding to a Fusobacterium nucleatum Fevl DNA region coding for a 40-kDa major outer-membrane protein (OMP) and a randomly cloned 2.1 kb DNA probe were found to recognize DNA from the Gram-negative bacteria Fusobacterium nucleatum and Bacteroides fragilis on Southern blots and slot blots. The results indicate sequence similarity within the DNA fragments studied. Immunoblots tested with polyclonal antibodies against whole cells of F. nucleatum revealed only weak antigen similarity between these species.

Aggregatibacter actinomycetemcomitans↗

Comparison of extracellular enzymes of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme.

A total of 10 strains each of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme were tested for the production of 13 extracellular enzymes. DNase, alkaline phosphatase, and lipase were predominantly associated with all the strains of F. necrophorum subsp. necrophorum, with DNase not detected in any of the strains of F. necrophorum subsp. funduliforme. In addition, the strains of F. necrophorum subsp. necrophorum were generally more hemolytic than those of F. necrophorum subsp. funduliforme. Lecithinase, beta-lactamase, elastase, hyaluronidase, chondroitin sulfatase, and coagulase were not detected in any of the strains. DNase may be used to differentiate between the two subspecies.

Alkaline Phosphatase↗

The leukotoxin operon of Fusobacterium necrophorum is not present in other species of Fusobacterium.

The complete nucleotide sequence of the leukotoxin operon of Fusobacterium necrophorum has been determined. The operon consists of three genes (lktBAC) of which the leukotoxin structural gene is the middle determinant. Southern and western blot analyses and flow cytometry analysis for biological activity of the culture supernatants were carried out to determine if the leukotoxin is present in other species of the genus Fusobacterium. Only the two subspecies of F. necrophorum were found to possess the leukotoxin locus and produce the toxin. The human periodontal pathogen, F. nucleatum does not produce detectable leukotoxin. The F. necrophorum leukotoxin was found to be active against human neutrophils.

Journal Article↗

Utilization of D-amino acids by Fusobacterium nucleatum and Fusobacterium varium.

The utilization of D- and L-amino acids with acidic, basic or polar side chains was demonstrated by HPLC. Two species of the anaerobe Fusobacterium utilized D-lysine and the L isomers of glutamate, glutamine, histidine, lysine and serine. Only F. varium used L-arginine, D-glutamate and D-serine as substrates, whereas F. nucleatum specifically utilized D-histidine and D-glutamine. D-Glutamate accumulated in F. nucleatum cultures supplemented with D-glutamine, and ornithine was detected when either DL- or L-arginine was included in F. varium cultures. Based on literature precedents, D-glutamate and D-histidine are isomerized to their L isomers prior to degradation, but separate catabolic pathways are possible for each enantiomer of lysine and serine.

Amino Acids↗

Intergeneric coaggregation of oral Treponema spp. with Fusobacterium spp. and intrageneric coaggregation among Fusobacterium spp.

A total of 22 strains of Treponema spp. including members of all four named human oral species were tested for coaggregation with 7 strains of oral fusobacteria, 2 strains of nonoral fusobacteria, and 45 strains of other oral bacteria, which included actinobacilli, actinomyces, capnocytophagae, eubacteria, porphyromonads, prevotellae, selenomonads, streptococci, and veillonellae. None of the treponemes coaggregated with any of the latter 45 oral strains or with the two nonoral fusobacteria. All treponemes, eight Treponema denticola strains, eight T. socranskii strains, four oral pectinolytic treponemes, one T. pectinovorum strain, and one T. vincentii strain coaggregated with at least one strain of the fusobacteria tested as partners. The partners consisted of one strain of Fusobacterium periodonticum, five F. nucleatum strains including all four subspecies of F. nucleatum, and a strain of F. simiae obtained from the dental plaque of a monkey. In the more than 100 coaggregations observed, the fusobacterial partner was heat inactivated (85 degrees C for 30 min), while the treponemes were unaffected by the heat treatment. Furthermore, the fusobacteria were usually inactivated by proteinase K treatment, and the treponemes were not affected. Only the T. denticola coaggregations were inhibited by lactose and D-galactosamine. None were inhibited by any of 23 other different sugars or L-arginine. Intragenic coaggregations were seen among the subspecies of F. nucleatum and with F. periodonticum, and none were inhibited by any of the sugars tested or by L-arginine. No intrageneric coaggregations were observed among the treponemes. These data indicate that the human oral treponemes show a specificity for oral fusobacteria as coaggregation partners. Such cell-to cell contact may facilitate efficient metabolic communication and enhance the proliferation of each cell in the progressively more severe stages of periodontal disease.

Endopeptidase K↗

Comparison of the 16S-23S rRNA intergenic spacer regions between Fusobacterium varium and "Fusobacterium pseudonecrophorum" strains.

The 16S-23S rRNA intergenic spacer regions (ISRs) of five strains of "Fusobacterium pseudonecrophorum" which had been proposed as a new species, were compared with those of F. varium ATCC 8501T. All the strains of "F. pseudonecrophorum" exhibited of sequence similarities of 97.7% to 100% to the strain of F. varium in their 16S-23S rRNA ISR sequences. This indicates that the strains of "F. pseudonecrophorum" and the type strain of F. varium are identical at the species level.

Base Sequence↗

Comparison of haemolytic activity between Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme in vitro and in vivo.

Haemolytic activity of two subspecies of Fusobacterium necrophorum was compared in vitro and in vivo. F. necrophorum subsp. necrophorum (Fnn) showed a stronger activity than F. necrophorum subsp. funduliforme (Fnf) in vitro. Haemolytic activity of Fnn and Fnf was 57.97%+/-1.90 and 17.33%+/-1.44, respectively, compared to complete haemolysis by distilled water. In the mice injected with Fnn, haemolysin was detected in the liver at a titre of from 1 : 16 to 1 : 128, and Fnn was recovered from all mice at a viable bacterial count of 10(5) to 10(6) cells per gram liver tissue. In the mice injected with Fnf, haemolysin titre was <1 : 2 to 1 : 32. No liver abscess was formed. The viable count of recovered bacteria was 10(3) to 10(5) cells per gram, except for two mice in which no Fnf was detected. The results suggest that haemolysin might be a virulence factor in this species.

Animals↗