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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

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

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

Intrageneric relationships of members of the genus Fusobacterium as determined by reverse transcriptase sequencing of small-subunit rRNA.

The phylogenetic interrelationships of 14 members of the genus Fusobacterium were investigated by performing a comparative analysis of the 16S rRNA sequences of these organisms. The sequence data revealed considerable intrageneric heterogeneity. The four species Fusobacterium nucleatum (including F. nucleatum subsp. nucleatum, F. nucleatum subsp. polymorphum, "F. nucleatum subsp. fusiforme," and "F. nucleatum subsp. animalis"), Fusobacterium alocis, Fusobacterium periodonticum, and Fusobacterium simiae, which colonize oral cavities, exhibited high levels of sequence homology with each other and formed a distinct group within the genus. Fusobacterium mortiferum, Fusobacterium varium, and Fusobacterium ulcerans also formed a phylogenetically coherent group, as did the two species Fusobacterium gonidiaformans and Fusobacterium necrophorum. Fusobacterium russii and Fusobacterium necrogenes displayed no specific relationship with any of the other fusobacteria. The sequence data are discussed in the context of previous physiological and chemical findings.

Base Sequence

The relationship between Fusobacterium species and other flora in mixed infection.

Mixed infections with three Fusobacterium species and seven other bacterial species were studied in a subcutaneous abscess model in mice. Fifteen Fusobacterium isolates (eight F. nucleatum, four F. necrophorum, and three F. varium) and one isolate each of Bacteroides fragilis, B. asaccharolyticus, Staphylococcus aureus, Group A beta-haemolytic streptococcus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa were studied. Electronmicrographs showed the presence of a thin mucopolysaccharide wall before and after inoculation into mice in 12 isolates which included all of 11 Fusobacterium isolates that induced subcutaneous abscesses. After co-inoculation of Fusobacterium isolates with other species and selective therapy with antimicrobial agents, S. aureus and K. pneumoniae were found to be of equal or greater importance in abscess induction than were Fusobacterium isolates, while Fusobacterium isolates were found to be more important than Group A streptococci and E. coli. Mutual enhancement of the numbers of organisms in mixed infections was observed with Fusobacterium spp. and K. pneumoniae, P. aeruginosa or Bacteroides spp. Suppression of Fusobacterium spp. was noticed only when they were co-inoculated with Group A streptococci. The additive or synergistic capabilities of Fusobacterium species highlighted their potential pathogenicity in infection.

Abscess

Lack of constitutive beta-glucosidase (esculinase) in the genus Fusobacterium.

Esculin has been incorporated into both a medium and test with 20% bile for many years to differentiate Bacteroides from Fusobacterium organisms. After 24 to 48 h, all members of the Bacteroides fragilis group grow in 20% bile and hydrolyze esculin. Fusobacterium mortiferum can both grow in bile and hydrolyze esculin, thus limiting the use of the bile-esculin medium and test. The hypothesis that constitutive esculinase (beta-glucosidase) could differentiate Bacteroides from Fusobacterium organisms was investigated. Clinical isolates and American Type Culture Collection clones of the B. fragilis group and other species of Bacteroides and Fusobacterium were tested. All B. fragilis were positive within 30 min. In no case was a Fusobacterium organism positive for constitutive enzyme in a hydrolyzable substrate-based test. The percentage of positive results for other species of Bacteroides agreed with those published in the literature for the esculin test. The genus Fusobacterium can be separated from Bacteroides organisms based on a lack of constitutive beta-glucosidase in the former in a 30-min one-tube test.

Bacteroides

Fusobacterium: anaerobic occult amnionitis and premature labor.

Fusobacterium species are well established pathogens. Before the advent of effective anaerobic antimicrobial therapy, they were associated with prolonged, often fatal courses. Previously, fusobacterium had not been identified as a common perinatal pathogen. Three cases of occult amnionitis due to Fusobacterium are presented. Review of five series of occult amnionitis revealed 23 cases. In seven (30.4%), Fusobacterium was isolated. In 14 (60.8%), an anaerobic species was isolated. The average gestational age of patients from whom anaerobes were grown was 29.0 weeks. Of those that grew no anaerobes, the average gestational age was 32.3 weeks (P less than .05). The overall rate of maternal febrile morbidity was 35%. Fusobacterium accounted for 50% of the febrile cases while accounting for only 30.4% of the total cases.

Adult

Bacteremia due to Fusobacterium species.

Twenty-six patients were identified as having bacteremia with Fusobacterium species over a five-year period at Boston City Hospital. They represented 0.9 percent of bacteremic patients and were equally divided as to sex. Bacteremia with Fusobacterium occurred primarily in young adults and in patients over 60 years of age and was not observed in children. In 16 patients (62 percent), Fusobacterium was the only blood culture isolate. The most common primary foci of infection were the female genital tract, the upper respiratory tract, the oral cavity, and the lower respiratory tract. Five patients had primary foci of infection that were initially occult. Three of these patients were found to have unappreciated oral and pharyngeal lesions, and one had a liver abscess; no primary infection was established in the remaining patient. Shock related to bacteremia developed in six patients (23 percent), four of whom had Fusobacterium species as the only blood culture isolate. Death occurred in three patients (12 percent), all of whom were over 60 years old. Metastatic infection occurred in only one patient in whom hematogenous osteomyelitis developed. Postpartum fusobacterial bacteremia was uniformly benign. Evaluation of bacteremia with Fusobacterium species in nonpostpartum patients, without an overt focus of infection, should be directed to a search for occult abscess, especially of the upper respiratory tract and oral cavity.

Adolescent

Studies on bacterial synergism in mice infected with Bacteroides intermedius and Fusobacterium necrophorum.

This study was undertaken to characterize the kinetics of possible bacterial synergy using a mouse model of mixed intraabdominal infection with Bacteroides intermedius and Fusobacterium necrophorum. Female CD-1 mice were injected intraperitoneally with B. intermedius, F. necrophorum or mixtures of both organisms. Generalized septic peritonitis developed within 24 hr, with abscess formation occurring after one to two wk in survivors with the mixed infection. Involvement of the reticuloendothelial system was evidenced by dose-dependent hepatosplenomegaly, which appeared during the first wk postinfection and progressed throughout the course of the experiment. Indirect immunofluorescence confirmed the presence of both species of bacteria in frozen sections of liver tissue. The median lethal dose (LD50) was 2.11 x 10(9) for the mixture, 3.03 X 10(9) for B. intermedius alone, and 1.07 X 10(9) for F. necrophorum alone. The median abscess-producing dose (AD50), the dose required to produce abscesses in fifty percent of the surviving mice at two wk, was approx. 1/100 of the LD50 dose. The AD50 for intrahepatic abscesses was 2.8 x 10(8) for the mixture, whereas the AD50 for intraabdominal abscesses occurring in any site was 5.14 X 10(7). Both Bacteroides and Fusobacterium persisted in tissue for at least 22 wk following mixed infection. The persistence of the Bacteroides in tissue represents a synergistic result of mixed infection with Fusobacterium and contributed to the chronicity of intraabdominal abscess formation. Bacteroides, injected alone, did not produce abscesses at any of the doses tested. However, when passaged (isolated from mixed infection hepatic abscesses) B. intermedius was used, the bacteria did induce abscesses.

Abscess

[Isolation and identification of oral spirochaetes and Fusobacterium in juvenile periodontitis].

Periodontal pocket specimens of 40 juveniles periodontitis (JP) and specimens from gingival sulcus in 40 normal juveniles (NP) were examined. 25 strains of anaerobic oral spirochaete (62.5%) (17 strains were Treponema microdentium, 3 strains macrodentium, 2 strains T orale, 3 strains Borrelia) and 21 strains of fusobacterium were isolated in 40 JP. In 40 NP, 2 strains of spirochaetes (5%) and 3 strains fusobacterium (7.5%) were found. This study shows that there is a relationship between the infection of spirochaeta, fusobacterium and JP.

Adolescent

[Pathogenicity of Fusobacterium. Apropos of 2 cases].

Two cases of severe septico-pyohaemia due to Fusobacterium necrophorum are reported. Clinical data from human cases and experimental results from infections of laboratory animal with Fusobacterium necrophorum suggest: the important role of individual factors which enhance anaerobic growth (i.e. decrease of tissular redox potential); the involvement of thrombophlebitis with septic thrombi increasing the frequency of metastases; the early and long-lasting bacterial localisation in tissues, particularly in the liver where the proliferation of Fusobacterium necrophorum in Küpffer cells impairs macrophage function.

Adult

Outer membrane proteins as major antigens of Fusobacterium nucleatum.

The immunochemical reactions of rabbit polyclonal antibodies directed to different preparations of Fusobacterium nucleatum i.e, whole cells, peptidoglycan associated proteins, a peptidoglycan-protein complex and a purified 40 kiloDalton (kDa) protein, were investigated on outer membrane preparations of Fusobacterium species and a restricted number of Leptotrichia buccalis after their separation on sodium dodecyl sulphate polyacrylamide gels and electrotransfer to nitrocellulose. All F. nucleatum strains had identical reaction patterns with the immune sera tested. Surface exposed parts of a restricted number of proteins with apparent molecular weights at 70 kDa (a doublet band), 60 kDa, 55 kDa and 40 kDa seemed to be major immunogens. Antigenic related proteins either of identical or slightly deviating electrophoretic mobilities to the 40-kDa protein were observed with the other members of Bacteroidaceae tested. The characteristic 70-kDa protein doublet seemed to be restricted to F. nucleatum although single protein bands of near identical molecular weights belonging to the other species tested also reacted. The data also indicate that the 60-kDa and 55-kDa polypeptides might be present in other species of Fusobacterium.

Animals

Use of synthetic oligonucleotide DNA probes for the identification of different strains of Fusobacterium nucleatum.

The ability of three different oligonucleotide probes to identify different oral strains of Fusobacterium nucleatum was tested. Probes 119 and 214 were designed on the basis of known amino acid sequences from the N-terminal end of an outer membrane protein of F. nucleatum strain Fev1. Probe H 2.1, was a randomly cloned chromosomal DNA fragment of 2.1 kbp. The specificities of the probes were examined by testing each of them against a panel of five other species of Fusobacterium (six strains), oral and non-oral, and also against seven different oral species of six other genera. The chromosomal DNA of the bacteria to be examined was digested to completion using the restriction enzymes HincII, HindIII, EcoRI, EcoRV and Sau3AI. Digests were run on agarose gels and subjected to southern blotting before being hybridized with the probe in question. The results were confirmed by running a slot blot of genomic DNA from all of the 19 strains and hybridizing with a probe H 2.1. Probe H 2.1 turned out to be the most specific and useful of the three probes investigated. By use of this probe, interstrain identification of the six different strains of F. nucleatum could be performed and the strains could be distinguished from other oral species found in the oral cavity, such as Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Capnocytophaga sputigena, Porphyromonas (Bacteroides) gingivalis, Eikenella corrodens and Leptotrichia buccalis. The probe reacted weakly with other non-oral species of the genus Fusobacterium, and there was no problem in distinguishing between the different strains.

Amino Acid Sequence

Cell-wall-defective variants of Fusobacterium.

The activity of antimicrobial agents against Fusobacterium species has been reported as variable in the literature. For some strains, the inconsistency arises from difficulty in determining the endpoint of growth in agar dilution susceptibility tests. Certain strains persist as a subtle haze beyond the levels of antibiotic that permit conventional colonial growth. We have determined by light and electron microscopy that this haze represents the colonial growth of cell-wall-defective (CWD) variants of the parent Fusobacterium. The CWD forms could be propagated indefinitely in hypertonic medium containing the antibiotic inducing agent. However, when the antibiotic was eliminated, the organisms would revert to their native morphology. Formation of CWD variants was observed in the presence of cell-wall-active drugs (e.g., beta-lactam agents) but not with drugs that work by a different mechanism (e.g., clindamycin or chloramphenicol). Fourteen of 22 F. varium strains, 8 of 11 F. mortiferum strains, 2 of 10 F. gonidiaformans strains, and 1 of 4 of F. necrophorum strains could be induced to a CWD form in vitro in the usual agar dilution susceptibility test. Although the clinical significance of CWD variants of Fusobacterium is unknown, they may be a source of confusion in interpreting agar dilution susceptibility tests.

Anti-Bacterial Agents