Outer membrane proteins of Fusobacterium necrophorum subsp. necrophorum and subsp. funduliforme.

Fusobacterium necrophorum, classified into subsp. necrophorum (Fnn) and subsp. funduliforme (Fnf), is frequently associated with necrotic infections of animals and humans. The outer membrane proteins (OMP) of many Gram negative bacteria play an important role in bacterial adhesion and establishment of infection. The OMP profile of F. necrophorum has not been well characterized. We analyzed OMP of bovine strains of Fnn and Fnf and human strains of F. necrophorum. Electrophoretic separations of extracted OMP of Fnn and Fnf strains of cattle showed a total of 19 and 20 protein bands, respectively. The most prominent protein band was 40 kDa in Fnn and 37.5 kDa in Fnf. The four human clinical strains examined had more heterogeneous banding patterns and had different profiles than those of bovine Fnf strains. A total of 11 protein bands in Fnn and 13 protein bands in Fnf were recognized by sera from cattle with liver abscesses. The intensities of many of the bands in Fnn were higher than that of Fnf. We conclude that the two subspecies of F. necrophorum differ in their OMP profiles and the difference may account for differences in their virulence and involvement in the pathogenesis of necrotic infections.

Primary lumbar epidural abscess without spondylodiscitis caused by Fusobacterium necrophorum diagnosed by 16S rRNA PCR.

We report the case of a 71-year-old woman who presented a primary spinal epidural abscess caused by Fusobacterium necrophorum. This is the second report in the medical literature to associate this organism with a primary spinal epidural abscess without spondylodiscitis. After treatment with emergency laminectomy followed by 8 weeks of antibiotic treatment the patient was cured. Oral metronidazole (500 mg every 8 h) was the definitive choice of treatment. F. necrophorum spinal epidural abscess is rare, although samples for anaerobic culture should be collected in order to improve detection of anaerobic spinal infections. PCR amplification and sequencing of the 16S rRNA permits early diagnosis in anaerobic infections.

A molecular survey of a captive wallaby population for periodontopathogens and the co-incidence of Fusobacterium necrophorum subspecies necrophorum with periodontal diseases.

Periodontal diseases (PD) are diseases of polymicrobial aetiology and constitute major health problems in captive macropods. Increasing knowledge of the causal pathogens is therefore crucial for effective management and prevention of these diseases. PCR survey and sequence analyses of potential periodontopathogens in captive wallaby populations revealed a co-incidence of the diseases with the detection of Fusobacterium necrophorum subsp. necrophorum (Fnn) and its encoded leukotoxin (lktA) gene. Sequence analyses showed that the outer membrane protein of Fnn in the GenBank database shared significant homology (99%) with the Fnn encoded haemagglutinin-related-protein gene fragment identified in this study. In addition, this report suggests the existence of a variant of Fnn with no detectable lktA gene and thus warrants further studies. In contrast to reports associating Porphyromonas gingivalis and F. nucleatum with PD, this study revealed that PD in macropods are associated with Porphyromonas gulae and Fnn and raises the question: is there a possible host pathogen co-evolution in the pathogenesis of PD in animals and humans? These findings contribute to the understanding of the aetiology of periodontal disease in macropods as well as opening up a new direction of research into the microbial interactions involved in the pathogenesis of PD in macropods.

Fusobacterium necrophorum, and not Dichelobacter nodosus, is associated with equine hoof thrush.

The aim of this study was to determine which of the two species, Fusobacterium necrophorum or Dichelobacter nodosus, are associated with hoof thrush in horses. Fourteen hoof samples, collected from eight horses with thrush and 14 samples collected from eight horses with healthy hooves, were examined for the presence of F. necrophorum, Fusobacterium equinum and D. nodosus. Only isolates with phenotypic characteristics representing Fusobacterium could be cultured. Total DNA extracted from the 28 hoof samples was amplified by using DNA primers designed from gene lktA, present in F. necrophorum subsp. necrophorum, F. necrophorum subsp. funduliforme and F. equinum, and gene fimA, present in D. nodosus. The lktA gene was amplified from five of the 14 infected hoof samples and from one hoof sample without thrush. The DNA sequence of the amplified ltkA gene was identical to the lktA gene of the type strain of F. necrophorum (GenBank accession number AF312861). The isolates were phenotypically differentiated from F. equinum. No DNA was amplified using the fimA primer set, suggesting that F. necrophorum, and not D. nodosus, is associated with equine hoof thrush. Hoof thrush in horses is thus caused by F. necrophorum in the absence D. nodosus. This is different from footrot in sheep, goats, cattle and pigs, which is caused by the synergistic action of F. necrophorum and D. nodosus.

Significant pathogens in peritonsillar abscesses.

Peritonsillar abscesses (PTA) are polymicrobial infections, with a diverse aerobic and anaerobic flora. The aim of the present study is to compare bacteriologic culture results from patients with PTA to those from patients undergoing elective tonsillectomy (clinically non-infected tonsils), to better elucidate the pathogenic significance of various isolates. A prospective study was conducted on 36 PTA patients undergoing acute tonsillectomy and on 80 electively tonsillectomised patients. Fusobacterium necrophorum (FN) and Streptococcus group A (GAS) were isolated significantly more frequently from the tonsillar cores of PTA patients, from both the abscessed (p = 0.001 and p = 0.046, respectively) and non-abscessed sides (p < 0.001 and p = 0.046, respectively), than from the tonsillar cores of electively tonsillectomised patients. Our findings indicate that FN and GAS are the prominent pathogens in PTA. In patients with PTA, the incidence of FN and GAS isolated from the abscessed tonsil was the same as from the non-abscessed contralateral side, and the growth was comparable by a semi-quantitative approach. Our findings suggest that FN is also of pathogenic importance in acute tonsillitis, and that FN growth is not a subsequent phenomenon once an abscess has formed. Our findings further suggest that other factors influence the development of PTA.

[Can anaerobic culture of throat swabs prevent Lemierre's syndrome?]. / Kan anaerob dyrkning af svaelgpodning forhindre Lemierres syndrom?

Six cases of Lemierre's syndrome were reported from 2004 to 2007 at Viborg Hospital, corresponding to 33 cases per year in Denmark. All six patients were healthy younger persons presenting with a suspected bacterial tonsillitis which had been found strep A antigen negative. Fusobacterium necrophorum was found in throat swabs by anaerobic culture on selective media and/or by real-time PCR. We recommend that all patients 10 to 40 years of age with strep A antigentest negative bacterial tonsillitis have throat swabs anaerobically cultured on selective media. We believe early identification and therapy may prevent progression to Lemierre's syndrome.

Dichelobacter nodosus, Fusobacterium necrophorum and the epidemiology of footrot.

Footrot is a debilitating disease of sheep resulting in lameness, production losses and suffering. To study the basic bacteriology of the disease, a survey was initiated across commercial farms and non-commercial research flocks to compare the bacteriology of symptomatic footrot infected sheep with healthy asymptomatic sheep. Of the 80 farmers initially contacted, 14 collected hoof swabs and returned the swabs by post. Following DNA extraction, species-specific PCR was used to identify if Dichelobacter nodosus (D. nodosus) or Fusobacterium necrophorum (F. necrophorum) species were present on each swab. Of the 42 swabs taken from symptomatic footrot infected sheep, 17 were positive for both F. necrophorum and D. nodosus, two were positive for F. necrophorum only, two for D. nodosus only and 23 swabs were negative for both F. necrophorum and D. nod osus. Of the 50 swabs received from healthy asymptomatic sheep, one was positive for F. necrophorum only and 49 were negative for both D. nodosus and F. necrophorum. This suggests that both F. necrophorum and D. nodosus are linked to footrot in the field in a pastoral farming system. If these bacteria are linked together and collectively cause footrot, this may need to be considered when managing a footrot outbreak, or maintaining a quarantine.

Fusobacterium necrophorum: a ruminal bacterium that invades liver to cause abscesses in cattle.

Fusobacterium necrophorum, a Gram-negative, rod-shaped, and an aerotolerant anaerobe, is a normal inhabitant of the rumen of cattle. The organism is in ruminal contents and adherent to the ruminal wall. Its role in ruminal fermentation is to metabolize lactic acid and degrade feed and epithelial proteins. The ruminal concentration is higher in grain-fed than forage-fed cattle. From the rumen, the organism gains entry into the portal circulation and is trapped in the liver to cause abscesses. The organism is an opportunistic pathogen and a primary causative agent of liver abscesses, an economically important disease of grain-fed cattle. Liver abscesses are often secondary to ruminal acidosis and rumenitis in grain-fed cattle. Two subspecies of F. necrophorum, subsp. necrophorum (biotype A) and subsp. funduliforme (biotype B), are recognized that can be differentiated based on morphological, biochemical, biological and molecular characteristics. The subsp. necrophorum is more virulent and is isolated more frequently from infections than the subsp. funduliforme. Several toxins or secreted products have been implicated as virulence factors. The major factors contributing to ruminal colonization and invasion into the liver are hemagglutinin, endotoxin and leukotoxin, of which leukotoxin is the protective antigen. In some conditions, the organism synergistically interacts with Arcanobacterium pyogenes, a facultative anaerobic organism and a secondary etiologic agent, to cause liver abscesses.

Lemierre's syndrome and other disseminated Fusobacterium necrophorum infections in Denmark: a prospective epidemiological and clinical survey.

In a 3-year prospective study, all cases of disseminated Fusobacterium necrophorum infections found in Denmark from 1998 to 2001 were analysed, with the aim of describing the epidemiology and clinical features of the variants of Lemierre's syndrome and disseminated non-head-and-neck-associated F. necrophorum infections. Fifty-eight cases of Lemierre's syndrome were reported in previously healthy persons, with an incidence of 14.4 cases per million per year in youngsters aged 15-24 years old. There was no increase during the study period. Lemierre's syndrome originating from an oropharyngeal infection was seen in 37 youngsters. An otogenic variant of Lemierre's syndrome was seen in 5 children with dissemination to nearby regions, and other variants of Lemierre's syndrome, e.g. from the sinuses and teeth, were seen in 16 adults. Patients often had metastatic infections already on admission and needed prolonged hospitalisation. The overall mortality of Lemierre's syndrome was 9%. Forty-two elderly patients had disseminated F. necrophorum infections originating from foci in lower parts of the body. They frequently had predisposing diseases, e.g. abdominal or urogenital cancers, which contributed to the high mortality of 26%. This study shows that the incidence of Lemierre's syndrome is higher than that previously found and has a characteristic age distribution. Early suspicion of the diagnosis, several weeks of antibiotic therapy, often combined with surgical drainage, is mandatory to lower the mortality. In disseminated non-head-and-neck-associated F. necrophorum infections, underlying cancers must be considered.

Detection of Fusobacterium necrophorum subsp. funduliforme in tonsillitis in young adults by real-time PCR.

Throat swabs from 61 patients, aged 18-32 years, with non-streptococcal tonsillitis (NST) and 92 healthy controls were examined for the presence of Fusobacterium necrophorum DNA using a novel TaqMan-based real-time quantitative PCR assay for F. necrophorum subspecies. The assay was based on the gyrB subunit gene, and detected F. necrophorum DNA in 48% of patients with NST and in 21% of controls (p <0.001). F. necrophorum subsp. funduliforme was the only subspecies found in both patients and controls. The load of F. necrophorum DNA on swabs from patients with NST was significantly higher than that on swabs from controls (p <0.001). Furthermore, patients with recurrent NST had a significantly higher load of F. necrophorum DNA compared to patients with acute NST (p 0.04). In addition, 26 patients with tonsillitis and group C streptococci (GCS) had a significantly higher load of F. necrophorum DNA compared to the NST group (p <0.001). It was concluded that F. necrophorum subsp. funduliforme is present in small numbers as part of the normal human throat flora, and that F. necrophorum in large quantities may cause tonsillitis, especially recurrent tonsillitis. In addition, the study suggests that the concomitant presence of GCS may aggravate F. necrophorum tonsillitis.

The two major subspecies of Fusobacterium necrophorum have distinct leukotoxin operon promoter regions.

Fusobacterium necrophorum, a gram-negative, non-spore-forming anaerobe, is a normal inhabitant of the alimentary tract of animals and humans. Two types of F. necrophorum, subspecies necrophorum (biotype A) and funduliforme (biotype B), have been recognized, which differ morphologically, biochemically and biologically. The organism is an opportunistic pathogen that causes numerous necrotic conditions (necrobacillosis) such as bovine hepatic abscesses and ruminant foot abscesses. Subspecies necrophorum strains are considered to be more virulent for cattle and have been shown to produce greater amounts of leukotoxin than subspecies funduliforme strains. The leukotoxin operon of F. necrophorum consists of three genes (lktBAC) of which the leukotoxin structural gene (lktA) is the second gene in the operon. In this study, the promoter regions of the leukotoxin operons from the two subspecies were identified and their nucleotide sequence compared. The promoter regions were found to differ in sequence, in length of the sequence between the upstream determinant (oppF) and the first gene of the leukotoxin operon (lktB), and in promoter strength as assayed in Escherichia coli host cells.

Fusobacterium necrophorum as the cause of recurrent sore throat: comparison of isolates from persistent sore throat syndrome and Lemierre's disease.

OBJECTIVE: Fusobacterium necrophorum is a well established cause of Lemierre's disease (LD); a syndrome characterised by severe sore throat, septicaemia, multiple abscesses and jugular vein thrombosis. There is no published data concerning the role of F. necrophorum in recurrent sore throats. As the result of an index case of persistent sore throat attributable to this organism being diagnosed in our laboratory, a subsequent case controlled study (not yet published) isolated F. necrophorum from 21% (P=0.0001) of cases of persistent, recurrent and chronic sore throats. The object of this study was to compare isolates of F. necrophorum from cases of systemic disease with isolates from cases of persistent sore throat syndrome (PSTS) to ascertain whether strains of similar type were responsible for both throat and systemic disease or whether different strains were involved in these presentations. METHODS: Throat swabs were cultured on GN anaerobe medium (Oxoid) and incubated at 37 degrees C for 5 days. Seventeen PSTS isolates were identified phenotypically. These were compared to 17 strains isolated from blood cultures which were referred to the Anaerobe Reference Unit, (ARU) cardiff, using enterogenic repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). The control strains Fusobacterium necrophorum ssp. necrophorum (JCM 3718(T)) and Fusobacterium necrophorum ssp. funduliforme (JCM 3724(T)) from the Japanese Collection of Microrganisms (JCM) were tested in parallel with the clinical isolates. RESULTS: At least 12 separate types were identified. Four of 17 PSTS isolates and seven of 17 blood culture isolates grouped together with the F. necrophorum ssp. funduliforme control strain. There were also similarities between other proposed strains and clinical types but no comparison with the F. necrophorum ssp. necrophorum control. CONCLUSION: These results show that clinical disease caused by F. necrophorum has a wider spectrum than first anticipated. Similar strains are able to cause either chronic local or acute systemic disease suggesting that genetic factors such as those relating to major histocompatibility complex (MHC) class may be influencing the outcome of the disease in each patient. Further work is required to produce a more accurate typing scheme and to ascertain the mechanisms of disease caused by this organism. An age correlation between the high risk groups for onset of infectious mononucleosis (IM), peritonsillar abscess (PTA), LD and PSTS has been noted in adolescents and young adults. Further work is required to investigate whether IM is associated with the onset of PTA caused by F. necrophorum which may lead to either PSTS or LD.

Detection of pathogens from periodontal lesions.

OBJECTIVE: To comparatively detect A. actinomycetemcomitans and F. nucleatum from periodontal and healthy sites. METHODS: Subgingival clinical samples from 50 periodontitis adult patients and 50 healthy subjects were analyzed. Both organisms were isolated using a trypticase soy agar-bacitracin-vancomycin (TSBV) medium and detected by PCR. Conventional biochemical tests were used for bacteria identification. RESULTS: A. actinomycetemcomitans and F. nucleatum were isolated in 18% and 20% of the patients, respectively, and in 2% and 24% of healthy subjects. Among A. actinomycetemcomitans isolates, biotype II was the most prevalent. Primer pair AA was 100% sensitive in the detection of A. actinomycetemcomitans from both subject groups. Primers ASH and FU were also 100% sensitive to detect this organism in healthy subject samples. Primer pair FN5047 was more sensitive to detect F. nucleatum in patients or in healthy samples than primer 5059S. Primers ASH and 5059S were more specific in the detection of A. actinomycetemcomitans and F. nucleatum, respectively, in patients and in healthy subject samples. CONCLUSIONS: PCR is an effective tool for detecting periodontal pathogens in subgingival samples, providing a faster and safer diagnostic tool of periodontal diseases. The method's sensitivity and specificity is conditioned by the choice of the set of primers used.

Specific detection and differentiation of two subspecies of Fusobacterium necrophorum by PCR.

The phylogenic relationships of two subspecies of Fusobacterium necrophorum were investigated by randomly amplified polymorphism DNA-polymerase chain reaction (RAPD-PCR). With each of the 12 random primers, the DNA fingerprints generated were subjected to cluster analysis for dendrograms. The analysis indicated that twelve strains were organized into two major clusters, and that all strains of each subspecies were confined to one cluster. Furthermore, two of the random primers examined each generated a unique band in F. n. necrophorum strains. We cloned these specific bands and determined the nucleotide sequences. A search for amino acid sequence homologies revealed that the two specific fragments had significant homology to the rpoB gene of Lactococcus lactis subsp. lactis and the hemagglutinin-related protein gene of Ralstonia solanacearum, respectively. New specific primers designed for the rpoB gene were able to amplify 900bp fragments from both subspecies. However, the specific primers designed for the hemagglutinin-related protein gene amplified only a 250bp fragment of the genome of the F. n. necrophorum strains, suggesting that this gene is unique to F. n. necrophorum. These results were further confirmed by dot blot hybridization. Finally, a one-step duplex PCR technique in a single tube for the rapid detection and differentiation of the F. necrophorum subspecies was developed.

Update on the taxonomy and clinical aspects of the genus fusobacterium.

The genus Fusobacterium currently includes 13 species. Fusobacterium nucleatum, the most frequently encountered species in humans, is heterogeneous and currently includes 5 subspecies. A potentially new subspecies of F. nucleatum that is intrinsically quinolone-resistant and phylogenetically separate from the other 5 subspecies has been identified from dog and cat oral flora. Two subspecies have been described for Fusobacterium necrophorum, and a new species, Fusobacterium equinum, which is related to F. necrophorum, has been described from horse oral flora. Additional molecular studies have characterized Fusobacterium ulcerans as separate from the phenotypically similar Fusobacterium mortiferum and Fusobacterium varium. Fusobacterium sulci and Fusobacterium alocis have been reclassified as Eubacterium sulci and Filifactor alocis, respectively. Fusobacterium prausnitzii is phylogenetically related to the Eubacterium-like organisms and will likely be reclassified in the future. The status of the remaining species is unchanged.

Differentiation of Fusobacterium necrophorum subspecies from bovine pathological lesions by RAPD-PCR.

Nineteen strains from bovine abscesses identified as Fusobacterium necrophorum by the VPI method were examined by other methods. The API 20A test kit characterized all 19 strains as F. necrophorum. Seven of the strains had haemagglutinating activity and were classified as F. necrophorum subspecies necrophorum, and the remaining, 12 nonhaemagglutinating strains, were classified as F. necrophorum subspecies funduliforme. We used RAPD-PCR with a 10-mer oligonucleotide primer, W1L-2, to confirm this differentiation of the two subspecies. These results suggest that random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) with a suitable primer can be used as a new tool for the differentiation of F. necrophorum subspecies isolated from bovine pathological lesions.

Electrophoretic mobility anomalies associated with PCR amplification of the intergenic spacer region between 16S and 23S ribosomal RNA genes of Fusobacterium necrophorum.

PCR amplification of the intergenic spacer region (ISR) between 16S and 23S rRNA genes among subspecies of the anaerobic bacterium Fusobacterium necrophorum gave identical patterns, with two forms of ISR identified. However, extra bands resulting from anomalous electrophoretic mobility of amplified DNA fragments with certain primer combinations were encountered. Therefore, PCR assays relying solely on banding patterns may be unreliable, and supporting sequence analysis is essential for correct culture identification.

Fusobacterium equinum sp. nov., from the oral cavity of horses.

Two strains of gram-negative, anaerobic, non-sporulating rod that were isolated from the normal oral cavity and oral-associated disease from horses and which phenotypically resembled Fusobacterium necrophorum were characterized by sequencing of the 16S rRNA gene, phylogenetic analysis, DNA-DNA hybridization and phenotypic characterization. The results placed the novel strains as distinct members of the genus Fusobacterium. The novel species Fusobacterium equinum sp. nov. is proposed, with strain VPB 4027T (= NCTC 13176T = JCM 11174T) as the type strain.

Human necrobacillosis, with emphasis on Lemierre's syndrome.

Lemierre's syndrome is the classical presentation of human necrobacillosis. It is characterized by a primary infection in the head in a young, previously healthy person who subsequently develops persistent high fever and disseminated metastatic abscesses, frequently including a septic thrombophlebitis of the internal jugular vein. The main pathogen is Fusobacterium necrophorum, an obligate anaerobic, pleomorphic, gram-negative rod. Clinical microbiologists have a key role in alerting clinicians and advising proper antibiotic treatment when the characteristic microscopic morphology of the pleomorphic F. necrophorum is seen in Gram stains from positive anaerobic cultures of blood and pus. Early diagnosis and prolonged appropriate antibiotic treatment with good anaerobic coverage are crucial to reduce morbidity and mortality. F. necrophorum also causes human necrobacillosis with foci caudal to the head, mainly in elderly patients with high mortality related to age and predisposing diseases, such as cancers of the primary focus.