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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↗

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↗

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↗

Complicated otitis media caused by Fusobacterium necrophorum.

Fusobacterium necrophorum is implicated as an aetiological agent in a variety of necrotic diseases, such as Lemièrre's syndrome (LS) in humans. LS was initially described as septic thrombophlebitis of the internal jugular vein secondary to an acute oropharyngeal infection. Other primary sources of infection include parotitis, otitis media, sinusitis, odontogenic infection and mastoiditis. In the pre-antibiotic era LS carried a high mortality. This has been reduced as a result of the widespread use of antibiotics, but there is still a definite morbidity and mortality associated with infection with this virulent organism. We report three cases of complicated otitis media caused by Fusobacterium necrophorum. The patients were treated successively with intravenous metronidazole and surgery.

Anti-Infective Agents↗

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↗

Cloning, sequencing, and expression of the leukotoxin gene from Fusobacterium necrophorum.

Fusobacterium necrophorum is a gram-negative, rod-shaped, anaerobic bacterium that is a primary or secondary etiological agent in a variety of necrotic purulent infections in animals and humans. Included are diseases of cattle such as liver abscesses and foot rot, which have economically important consequences for the cattle industry. The major virulence factor of this bacterium is leukotoxin, a secreted protein of high molecular weight active against leukocytes from ruminants. The screening of a genomic DNA library with polyclonal antisera raised against native affinity-purified leukotoxin and further extension of the sequence using inverse PCR led to the cloning of the entire leukotoxin gene. The leukotoxin gene open reading frame (ORF; lktA) consists of 9,726 bp and encodes a protein of 3,241 amino acids with an overall molecular weight of 335,956. The leukotoxin does not have sequence similarity with any other bacterial leukotoxin. Five truncated overlapping polypeptides covering the whole lktA ORF were used to immunize rabbits. In Western blot assays, polyclonal antisera raised against all five truncated polypeptides recognized affinity-purified leukotoxin from F. necrophorum culture supernatant in a Western blot assay. Antisera directed against two of the five polypeptides had neutralizing activity against the toxin. The entire leukotoxin ORF was expressed in Escherichia coli. Flow-cytometric analysis showed that the recombinant leukotoxin was active against bovine polymorphonuclear leukocytes and was inhibited with antiserum raised against the F. necrophorum leukotoxin. Southern blot hybridization analysis revealed different patterns of lktA hybridizing bands between isolates of the two subspecies of F. necrophorum.

Animals↗

Bovine platelet aggregation by Fusobacterium necrophorum.

Fusobacterium necrophorum aggregated bovine platelets. The aggregation was paralleled by the haemagglutinating ability of the organism. Treatment of the bacterial cells with antiserum to the homologous purified haemagglutinin reduced the degree of platelet aggregation. Scanning electronmicroscopy revealed that little lysis of the affected platelets occurred during the 1-h incubation period. Purified haemagglutinin became bound to the surfaces of the platelet cells as shown by immunofluorescence microscopy. These observations suggest that platelet aggregation is mediated by the haemagglutinin and may be related to the pathogenicity of the bacterium.

Animals↗

Human infections with Fusobacterium necrophorum.

Fusobacterium necrophorum is a Gram-negative anaerobic bacillus that can be a primary pathogen causing either localised abscesses and throat infections or systemic life-threatening disease. Systemic infections due to F. necrophorum are referred to as either Lemierre's disease/syndrome, post-anginal sepsis or necrobacillosis, but in the context of this mini-review, all are included under the umbrella term of 'invasive F. necrophorum disease' (IFND). Although IFND has been well documented for over a century, it is quite a rare condition and modern-day clinicians of various medical disciplines are frequently unaware of this organism and the severity of symptoms that it can cause. IFND classically occurs in previously healthy young people although the factors that trigger the invasive process are not fully understood. There are countless descriptive case histories and small series of cases of IFND disease in the literature and although commonly referred to as a 'forgotten' disease, in truth, it is probably best described as a repeatedly 'discovered' disease, as it may not always be included in medical curricula, and neither is it mentioned in some major medical textbooks. There is some evidence that IFND may be on the increase, particularly in the UK. The potential reasons for this are considered in this review along with an historical overview, and updates on disease incidence, patient demography, pathogenesis and laboratory diagnosis.

Adolescent↗

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↗

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↗

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↗

Immunogenicity and protective effects of truncated recombinant leukotoxin proteins of Fusobacterium necrophorum in mice.

Fusobacterium necrophorum, a gram-negative, anaerobic and rod-shaped bacterium, is generally an opportunistic pathogen and causes a wide variety of necrotic infections in animals and humans. Leukotoxin, a secreted protein, is a major virulence factor. The gene encoding the leukotoxin (lktA) in F. necrophorum has been cloned, sequenced and expressed in Escherichia coli. Because of low expression levels, problems associated with purifying full-length recombinant protein, and of the physical instability of the protein, five overlapping leukotoxin gene truncations were constructed. The recombinant polypeptides (BSBSE, SX, GAS, SH, and FINAL) were expressed in E. coli and purified by nickel-affinity chromatography. The objectives were to investigate the effectiveness of the purified truncated polypeptides to induce protective immunity in mice challenged with F. necrophorum. The polypeptides, individually or in combination, and inactivated native leukotoxin or culture supernatant of F. necrophorum were homogenized with an adjuvant and injected into mice on days 0 and 21. Blood samples were collected to measure serum anti-leukotoxin antibody titers on days 0, 21 and 42 and on day 42, mice were experimentally challenged with F. necrophorum. All polypeptides were immunogenic, with GAS polypeptide eliciting the least antibody response. Two polypeptides (BSBSE and SH) induced significant protection in mice against F. necrophorum infection. Protection was better than the full-length native leukotoxin or inactivated supernatant.The study demonstrated that the leukotoxin of F. necrophorum carries epitopes that induce protective immunity against experimental fusobacterial infection, thus providing further evidence to the importance of leukotoxin as a major virulence factor.

Animals↗

Partial characterization of leukocidin from Fusobacterium necrophorum.

Leukocidin from Fusobacterium necrophorum was produced in the diffusate of a dialysis culture. It was free from deoxyribonuclease, fibrinolysin, gelatinase, haemolysin, lipase, caseinase and endotoxin. The leukocidin had a molecular weight between 10,000 and 5,000 as estimated by membrane partition chromatography. It formed precipitin lines with anti-leukocidin-serum in double immunodiffusion tests. Mouse peritoneal cells were characteristically damaged by the leukocidin, as revealed by scanning electron microscopy. The damaged cells lost microvilli and suffered partial destruction of their cell membranes.

Animals↗

Meningitis due to Fusobacterium necrophorum in an adult.

BACKGROUND: Fusobacterium necrophorum may cause a number of clinical syndromes, collectively known as necrobacillosis. Meningitis is a significant cause of mortality, rarely reported in the adult population. CASE PRESENTATION: We report a fatal case of meningitis, caused by Fusobacterium necrophorum, secondary to otitis media in an alcoholic male. Diagnosis was delayed due to the typical slow growth of the organism. The clinical course was complicated by encephalitis and by hydrocephalus. The patient failed to respond to metronidazole and penicillin. The patient died on day 12 from increased intracranial pressure and brain stem infarction. CONCLUSIONS: This case emphasizes the need for a high index of clinical suspicion to make the diagnosis of Fusobacterium necrophorum meningitis. We recommend the use of appropriate anaerobic culture techniques and antimicrobial coverage for anaerobic organisms when the gram stain shows gram negative bacilli.

Brain↗

Correlations between leukocidin production and virulence of two isolates of Fusobacterium necrophorum.

Leukocidin production by Fusobacterium necrophorum was suggested to be an important element in the development of intraabdominal and liver abscesses in mice. Leukocidin production by cultures of F necrophorum was demonstrated by an in vitro assay. One of two isolates of F necrophorum was demonstrated to produce leukocidin. The leukocidin-producing strain was observed to be more infective than the nonleukocidin-producing strain (as demonstrated by abscess formation following intraperitoneal injection of immune-suppressed and normal mice). The infectivity of the leukocidin-producing strain was increased by successive passage in immune-suppressed mice. A simultaneous increase in leukocidin production was also demonstrated. The nonleukocidin-producing strain could not be passed effectively and was relatively noninfective for mice.

Animals↗

Multiple brain abscesses and bacteremia in a child due to Fusobacterium necrophorum.

A case of Fusobacterium necrophorum bacteremia and multiple brain abscesses in a 6-year-old child following a Mycoplasma pneumoniae infection is described. Brain abscess due to this microorganism has only been described in three other cases since 1977. The clinical picture was consistent with postanginal septicemia, with septic metastatic complications normally only seen in teenagers and young adults. The patient was successfully treated with a 2-month course of parenteral penicillin G and metronidazole.

Bacteremia↗

Stability and stabilization of Fusobacterium necrophorum hemolysin.

The stability and stabilization of the hemolytic activity of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme were monitored over a period of four weeks using culture supernatants. The hemolytic activity was completely lost after one week at room temperature and 37 degrees C. After a two-week storage at 4 degrees C and -80 degrees C only trace activity was detected with -80 degrees C being the better of the two conditions. The addition of cysteine monohydrochloride, bovine serum albumin or Tween 80 as stabilizers, however, led to the detection of a considerable amount of the hemolytic activity in the sample stored at 4 degrees C and - 80 degrees C throughout the period investigated. The hemolytic activity appeared to be more stable in the presence of Tween 80 at -80 degrees C. Cysteine monohydrochloride was found to crystallize at - 80 degrees C and was therefore ineffective as a stabilizer at this temperature. Hemoglobin was also ineffective as a stabilizer.

Albumins↗

Partial characterization of Fusobacterium necrophorum protease.

Partial characterization of Fusobacterium necrophorum protease was investigated. The protease was partially purified by gel filtration with Toyopearl HW 55. The final preparation was inactivated completely by heating at 60 degrees C for 30 min and inhibited by ascorbic acid, sodium thioglycollate and p-hydromercuribenzoate. Antibody response to the protease was demonstrated in mice receiving 10(4) CFU of F. necrophorum.

Antibodies, Bacterial↗