Metagenomic next-generation sequencing reveals co-infection with Legionella pneumophila and Fusobacterium necrophorum in a patient with severe pneumonia: a case report.

BACKGROUND: Legionella pneumonia is one of the most severe types of atypical pneumonia, impairing multiple organ systems, posing a threat to life. Diagnosing Legionella pneumonia is challenging due to difficulties in culturing the bacteria and limitations in immunoassay sensitivity and specificity. CASE PRESENTATION: This paper reports a rare case of sepsis caused by combined infection with Legionella pneumophila and Fusobacterium necrophorum, leading to respiratory failure, acute kidney injury, acute liver injury, myocardial damage, and electrolyte disorders. In addition, we systematically reviewed literature on patients with combined Legionella infections, analyzing their clinical features, laboratory results and diagnosis. CONCLUSIONS: For pathogens that require prolonged incubation periods and are less sensitive to conventional culturing methods, metagenomic next-generation sequencing (mNGS) can be a powerful supplement to pathogen screening and plays a significant role in the auxiliary diagnosis of complex infectious diseases.

Lemierre's syndrome complicating multiple organ failure caused by Fusobacterium necrophorum subsp. funduliforme F1260: Case report and review.

We described a case of a 24-year-old man with multiple organ failure caused by Fusobacterium necrophorum subsp. funduliforme F1260. This is the first described case of Lemierre's syndrome with multiple organ failure due to F. necrophorum subsp. funduliforme F1260 in an adult in China. Our study highlights that there may be a risk of misdiagnosis based solely on typical manifestations of internal jugular vein thrombophlebitis, metastatic lesions, and F. necrophorum isolated from blood cultures or normally sterile sites. Clinicians should be cognizant of the potential utility of metagenomic next-generation sequencing in facilitating early pathogen detection in severe infections, thus enabling timely and appropriate administration of antibiotics to reduce mortality rates and improve prognosis.

Case report: Metagenomics next-generation sequencing in the diagnosis of septic shock due to Fusobacterium necrophorum in a 6-year-old child.

Fusobacterium necrophorum (F. necrophorum) infection is rare in pediatrics. In addition, the detection time of F. necrophorum by blood culture is long, and the positive rate is low. Infection with F. necrophorum bacilli usually follows rapid disease progression, resulting in high mortality. In previous reports of F. necrophorum-related cases, the most dangerous moment of the disease occurred after the appearance of Lemierre's syndrome. We report an atypical case of a 6-year-old female patient who developed septic shock within 24 h of admission due to F. necrophorum infection in the absence of Lemierre's syndrome. F. necrophorum was identified in a blood sample by metagenomics next-generation sequencing (mNGS) but not by standard blood culture. The patient was finally cured and discharged after receiving timely and effective targeted anti-infection treatment. In the present case study, it was observed that the heightened virulence and invasiveness of F. necrophorum contribute significantly to its role as a primary pathogen in pediatric septic shock. This can precipitate hemodynamic instability and multiple organ failure, even in the absence of Lemierre's syndrome. The use of mNGS can deeply and rapidly identify infectious pathogens, guide the use of targeted antibiotics, and greatly improve the survival rate of patients.

Low usefulness of reporting tonsillar PCR Ct-values in pharyngeal infections with Fusobacterium necrophorum.

Tonsillar Fusobacterium necrophorum PCR Ct-values were higher in participants with asymptomatic tonsillar carriage than patients with pharyngeal infections. However, Ct-values were not associated with severity of disease or predictive of development of complications and hence lacked clinical usefulness. The reporting of F. necrophorum Ct-values in clinical samples is not recommended.

Unveiling the etiology of peritonsillar abscess using next generation sequencing.

BACKGROUND: Peritonsillar abscess (PTA) is a severe deep neck space infection with an insufficiently characterized bacterial etiology. We aimed to reveal the bacteria associated with PTA applying next generation sequencing (NGS). Tonsil biopsies and pus samples of 91 PTA patients were analysed applying NGS method. RESULTS: Over 400 genera and 800 species belonging to 34 phyla were revealed. The most abundant species in both sample types were Streptococcus pyogenes, Fusobacterium necrophorum and Fusobacterium nucleatum. When present, S. pyogenes was normally a predominant species, although it was recovered as a minor population in some samples dominated by F. nucleatum and occasionally F. necrophorum. S. pyogenes and F. necrophorum were the predominant species (> 10% in a community) in 28 (31%) pus samples, while F. nucleatum in 21 (23%) and S. anginosus in 8 (9%) pus samples. We observed no substantial differences between the microbial findings in pus and tonsil biopsies. CONCLUSIONS: The most probable causative agents of PTA according to our NGS-study include Streptococcus pyogenes, Fusobacterium necrophorum and Fusobacterium nucleatum. Some other streptococci (S. anginosus) and anaerobes (Prevotella, Porphyromonas) may contribute to the infection as well. Pus of the peritonsillar abscess is more representative specimen for microbiological examination than the tonsillar tissue. Our results are important in the context of optimizing the handling of the PTA patients.

The outer membrane protein of Fusobacterium necrophorum, 43K OMP, stimulates inflammatory cytokine production through nuclear factor kappa B activation.

OBJECTIVE: Fusobacterium necrophorum causes bovine hepatic abscess, foot rot, mastitis, and endometritis. The 43 kDa outer membrane protein (43 K OMP) of F. necrophorum is a porin protein that plays an important role in infections by this bacterium, but the biological function and the pathogenesis of this protein are largely unknown. METHODS: In this study, we investigated the role of the 43 K OMP in bacterial infection of bovine mammary epithelial cells (MAC-T cells) by Tandem Mass Tag proteomic analysis. The RAW264.7 cells were incubated with recombinant 43 K OMP (12.5 µg/mL) for 2 h, 4 h, 6 h, and 12 h, and then the inflammatory related protein and inflammatory cytokine production were measured by Western blot analysis and ELISA, the mRNA expression levels of inflammatory cytokine were measured by Real-Time PCR. RESULTS: Proteomic analysis results demonstrated there were 224 differentially expressed proteins in the MAC-T cells stimulated with the 43 K OMP compared with control, and 118 proteins were upregulated and 106 proteins were downregulated. These differentially expressed proteins were mainly involved in NF-kappa B signaling, bacterial invasion of epithelial cells, cell adhesion, complement and coagulation cascades. The top six differentially expressed proteins were; MMP9, PLAU, STOM, PSMD13, PLAUR, and ITGAV, which were involved in a protein-protein interaction network. Furthermore, TLR/MyD88/NF-κB pathway related proteins and inflammatory cytokines (IL-6, TNF-α, and IL-1ß) were assessed by Western blot analysis and ELISA. Results showed the 43 K OMP to enhance the expression of TLR4 protein at 2 h (P < 0.01) and the MyD88 protein at 4 h (P < 0.05) post-stimulation, and to decrease IκBα expression at 4 h, 6 h and 12 h (P < 0.05) post-infection, as well as induce phosphorylation at Ser536 (P < 0.01). Levels of IL-6, IL-1ß, and TNF-α in the supernatants of mouse macrophages were increased (P < 0.05), as were mRNA expression levels of IL-6, IL-1ß, and TNF-α (P < 0.05), while IL-4 mRNA expression was decreased (P < 0.05). CONCLUSIONS: Taken together, these results suggested the important role for 43 K OMP in F. necrophorum infection, promoting the production of pro-inflammatory cytokines (IL-6 and TNF-α) by activation of the TLR/MyD88/NF-κB pathway. These findings provided a theoretical basis for a better understanding of the pathogenesis of F. necrophorum infection.

Comparative Genomic Analysis of Fusobacterium necrophorum Provides Insights into Conserved Virulence Genes.

Fusobacterium necrophorum is a Gram-negative, filamentous anaerobe prevalent in the mucosal flora of animals and humans. It causes necrotic infections in cattle, resulting in a substantial economic impact on the cattle industry. Although infection severity and management differ within F. necrophorum species, little is known about F. necrophorum speciation and the genetic virulence determinants between strains. To characterize the clinical isolates, we performed whole-genome sequencing of four bovine isolates (8L1, 212, B17, and SM1216) and one human isolate (MK12). To determine the phylogenetic relationship and evolution pattern and investigate the presence of antimicrobial resistance genes (ARGs) and potential virulence genes of F. necrophorum, we also performed comparative genomics with publicly available Fusobacterium genomes. Using up-to-date bacterial core gene (UBCG) set analysis, we uncovered distinct Fusobacterium species and F. necrophorum subspecies clades. Pangenome analyses revealed a high level of diversity among Fusobacterium strains down to species levels. The output also identified 14 and 26 genes specific to F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme, respectively, which could be essential for bacterial survival under different environmental conditions. ClonalFrameML-based recombination analysis suggested that extensive recombination among accessory genes led to species divergence. Furthermore, the only strain of F. necrophorum with ARGs was F. necrophorum subsp. funduliforme B35, with acquired macrolide and tetracycline resistance genes. Our custom search revealed common virulence genes, including toxins, adhesion proteins, outer membrane proteins, cell envelope, type IV secretion system, ABC (ATP-binding cassette) transporters, and transporter proteins. A focused study on these genes could help identify major virulence genes and inform effective vaccination strategies against fusobacterial infections. IMPORTANCE Fusobacterium necrophorum is an anaerobic bacterium that causes liver abscesses in cattle with an annual incidence rate of 10% to 20%, resulting in a substantial economic impact on the cattle industry. The lack of definite biochemical tests makes it difficult to distinguish F. necrophorum subspecies phenotypically, where genomic characterization plays a significant role. However, due to the lack of a good reference genome for comparison, F. necrophorum subspecies-level identification represents a significant challenge. To overcome this challenge, we used comparative genomics to validate clinical test strains for subspecies-level identification. The findings of our study help predict specific clades of previously uncharacterized strains of F. necrophorum. Our study identifies both general and subspecies-specific virulence genes through a custom search-based analysis. The virulence genes identified in this study can be the focus of future studies aimed at evaluating their potential as vaccine targets to prevent fusobacterial infections in cattle.

A genome-led study on the pathogenesis of Fusobacterium necrophorum infections.

Fusobacterium necrophorum causes a range of mild to life threatening infections and there is uncertainty in terms of diagnosis and treatment due to the lack of knowledge on their pathogenic mechanisms. This study characterised genomes of F. necrophorum to compare their virulence factors and investigate potential infection markers. 27 isolates of F. necrophorum from patients with pharyngotonsillitis were subjected to whole genome sequencing and compared with 42 genomes published in the NCBI database. Phylogenomics, pangemome, pan-GWAS and virulome were analysed to study strain variations with reference to virulence factors. Core genome based phylogenomic tree exhibited three clades of which Clade A belonged to F. necrophorum subsp necrophorum, clades B and C were F. necrophorum subsp funduliforme. Pan-GWAS and Pan-Virulome suggest some marker genes associated with clinical sources of isolation that needs further validation. Our study highlights some interesting features of the pathogenesis of F. necrophorum infections. Although the animal isolate genomes had some marker genes, the genomes of human isolates did not exhibit clear correlation to their clinical sources of isolation. This prompts to think of other mechanisms such as co-infections or host factors that can be involved in the pathogenesis.

Fusobacterium necrophorum Promotes Apoptosis and Inflammatory Cytokine Production Through the Activation of NF-κB and Death Receptor Signaling Pathways.

Fusobacterium necrophorum can cause liver abscess, foot rot in ruminants, and Lemire syndrome in humans, Also, its virulence factors can induce the apoptosis of macrophages and neutrophils. However, the detailed mechanism has not been fully clarified. This study investigated the mechanisms of apoptosis and inflammatory factor production in F. necrophorum-induced neutrophils and macrophages (RAW246.7). After infection of macrophages with F. necrophorum, 5-ethynyl-2'-deoxyuridine labeling assays indicated that F. necrophorum inhibited macrophage proliferation in a time- and dose-dependent manner. Hoechst staining and DNA ladder assays showed significant condensation of the nucleus and fragmentation of genomic DNA in F. necrophorum-infected macrophages, Annexin V (FITC) and propidium iodide (PI) assay confirmed the emergence of apoptosis in the macrophages and sheep neutrophils with F. necrophorum compared with the control. The group with significant apoptosis was subjected to RNA sequencing (RNA-Seq), and the sequencing results revealed 2581 up- and 2907 downregulated genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis of the differentially expressed genes showed that F. necrophorum drove apoptosis and production of inflammatory factors by activating genes related to the Nuclear Factor-κB (NF-κB) and death receptor pathways. Meanwhile, quantitative reverse transcription PCR and Western blot validation results were consistent with the results of transcriptome sequencing analysis. In conclusion, F. necrophorum induced apoptosis and production of pro-inflammatory factors through the NF-κB and death receptor signaling pathway, providing a theoretical basis for further mechanistic studies on the prevention and control of F. necrophorum disease treatment.

Interaction of 43K OMP of Fusobacterium necrophorum with fibronectin mediates adhesion to bovine epithelial cells.

Fusobacterium necrophorum, a Gram-negative anaerobe, is an important bovine pathogen that causes hepatic abscesses, foot rot, mastitis and endometritis. We have previously shown that the 43 kDa outer membrane protein (43 K OMP) of F. necrophorum is a porin protein that plays an important role in bacterial infections; however, the molecular mechanisms by which this protein mediates adhesion remain unclear. In this study, we investigated the role of 43 K OMP in F. necrophorum adhesion to bovine epithelial cells using 43 K OMP-deficient mutants, and identified the protein that interacts with 43 K OMP by immunoprecipitation-mass spectrometry. Our results indicated that the native 43 K OMP and recombinant 43 K OMP could bind to the cell membrane of MAC-T or bovine endometrial epithelial cells (BEECs). When F. necrophorum was preincubated with antibodies against the recombinant 43 K OMP or bovine epithelial cells were preincubated with 43 K OMP, the adhesion of F. necrophorum to MAC-T or BEECs decreased significantly (P<0.01). We successfully constructed a 43 K OMP-deficient strain (A25Δ43 K OMP) and bacterial attachment to MAC-T or BEECs was significantly higher with the F. necrophorum A25 strain than with mutant strain A25Δ43 K OMP (P<0.01). The deficiency of 43 K OMP reduced the binding of F. necrophorum to bovine epithelial cells by 90.5 %-94.9 %. Among the 39 potential differential proteins, fibronectin, collagen and myosin were selected as the target proteins, and direct interaction between 43 K OMP of F. necrophorum and fibronectin was demonstrated. Taken together, these results suggest that 43 K OMP plays a key role in adhesion of F. necrophorum to bovine epithelial cells through its interaction with fibronectin. These findings provide a theoretical basis for the pathogenic mechanism of F. necrophorum.

Ovine footrot in Southern Portugal: Detection of Dichelobacter nodosus and Fusobacterium necrophorum in sheep with different lesion scores.

The Mediterranean climate region of Alentejo in the Southern of Portugal is an important sheep production centre but little is known about the presence and characteristics of Dichelobacter nodosus in association with Fusobacterium necrophorum in the different footrot lesion scores. DNA from 261 interdigital biopsy samples, taken from 14 footrot affected flocks and from three non-affected flocks, were analysed for the presence of D. nodosus and F. necrophorum by real-time PCR. Both virulence and serogroup were determined for 132 and 53 D. nodosus positive biopsy samples, respectively. The co-infection with both bacteria was the commonest epidemiological finding associated with a greater disease severity. There was a statistically significant association (p = 0.002) between footrot-affected flocks and the presence of D. nodosus. Most D. nodosus positive samples were virulent (96.2 %) and belonged to serogroup B (90 %).

Should the risk of Fusobacterium necrophorum pharyngotonsillitis influence prescribing empiric antibiotics for sore throats in adolescents and young adults?

Fusobacterium necrophorum, a gram-negative anaerobe, causes pharyngotonsillitis primarily in adolescents and young adults (approximately 15-30 years old). The same age group has the highest incidence of peritonsillar abscess and the Lemierre syndrome. The same organism, F. necrophorum, is the most common cause of peritonsillar abscess in this age group and causes at least 80% of Lemierre syndrome cases. We outline the case for empiric antibiotic treatment of some patient in this age group who have a significant probability that F. necrophorum is the cause of their pharyngotonsillitis.

Leukotoxin production by Fusobacterium necrophorum strains in relation to severity of liver abscesses in cattle.

Fusobacterium necrophorum, a Gram-negative anaerobe, is the primary etiologic agent of liver abscesses of beef cattle. The bacterium, a member of the microbial community of the rumen, travels to the liver via portal circulation to cause abscesses. The severity of liver abscesses vary from mild with one or two small abscesses to severe with medium to large multiple abscesses. Leukotoxin, a secreted protein, is the critical virulence factor involved in the infection. Our objective was to compare leukotoxin production between strains of F. necrophorum isolated from mild and severe liver abscesses collected from slaughtered cattle. The quantification of leukotoxin was based on assays to measure cytotoxicity and protein antigen concentration. One-hundred strains, 50 from mild and 50 from severe abscesses, were utilized in the study. Cell-free supernatants were prepared from cultures grown in anaerobic broth at 9 and 24 h incubations. The leukotoxic activity was quantified by measuring cytotoxicity based on the release of lactic dehydrogenase from bovine lymphocyte cells, BL3, treated with the culture supernatant. Leukotoxin protein concentration was quantified by a sandwich ELISA assay with a leukotoxin-specific monoclonal antibody as the capture antibody. The leukotoxin activity and concentration were highly variable among the strains within each severity of liver abscesses. Although the leukotoxic activity was unaffected by incubation time, leukotoxin protein concentration was consistently higher at 24 h compared to 9 h incubation. Strains from severe liver abscesses had significantly higher leukotoxic activity and higher protein concentration compared to strains from mild liver abscesses (P < 0.0001) at both 9 and 24 h culture supernatants. Across all strains, the correlation coefficients between leukotoxic activity and leukotoxin concentration at 9 and 24 h were 0.14 (P = 0.17) and 0.47 (P < 0.0001), respectively. In conclusion, strains isolated from severe liver abscesses had significantly higher leukotoxic activities and leukotoxin protein concentrations compared to strains isolated from mild liver abscesses.

Oral and pulmonary necrobacillosis in a juvenile reticulated giraffe.

A 1-mo-old reticulated giraffe had progressive anorexia and died at the Ordos Zoo. Autopsy revealed necrotic stomatitis with severe bilateral necroulcerative lesions at the base of the tongue and of the cheeks near the commissures of the mouth. There was also severe bilateral confluent bronchopneumonia with a pronounced bronchial pattern and multifocal fibrinous pleuritis. Histologically, there was serofibrinous-suppurative bronchopneumonia with necrosuppurative bronchiolitis and necrotic arteritis. Filamentous bacteria with morphology consistent with Fusobacterium necrophorum were observed at the advancing edge of the necrotic tissue in the tongue and cheeks, as well as in the affected alveolar spaces and bronchioles. Aggregates of slender, gram-negative, rod-like or filamentous bacteria were identified in the lung impression smear. PCR results of 16S rDNA of the tongue and lung lesions had 100% homology to the F. necrophorum subsp. funduliforme B35 sequence (EF447425.1). The gross, histologic, Gram stain, and PCR product sequencing features in our case were consistent with oral and pulmonary necrobacillosis in ruminants, a rare disease of giraffes.

Novel ulcerative leg lesions in yearling lambs: Clinical features, microbiology and histopathology.

Here we report an outbreak of an atypical, ulcerative dermatitis in North Country mule lambs, located in South Gloucestershire, UK. The lesions, which appeared to be contagious, occured between the coronary band and the carpal joint as a focal, well demarcated, circular, ulcerative dermatitis. Histopathological examination of the lesion biopsies revealed areas of ulceration, epidermal hyperplasia, suppurative dermatitis and granulation tissue. Clumped keratohyalin granules and intracellular keratinocyte oedema (ballooning degeneration) were evident within lesion biopsies, consistent with an underlying viral aetiology. A PCR-based microbiological investigation failed to detect bovine digital dermatitis-associated treponeme phylogroups, Dichelobacter nodosus, Staphylococcus aureus, Dermatophilus congolensis or Chordopoxvirinae virus DNA. However, 3 of the 10 (30 %) and 6 of 10 (60 %) lesion samples were positive for Fusobacterium necrophorum and Streptococcus dysgalactiae DNA, respectively. Contralateral limb swabs were negative by all standard PCR assays. To better define the involvement of F. necrophorum in the aetiology of these lesions, a qPCR targeting the rpoB gene was employed and confirmed the presence of F. necrophorum DNA in both the control and lesions swab samples, although the mean F. necrophorum genome copy number detected in the lesion swab samples was ∼19-fold higher than detected in the contralateral control swab samples (245 versus 4752 genome copies/µl, respectively; P < 0.001). Although we have not been able to conclusively define an aetiological agent, the presence of both F. necrophorum and S. dysgalactiae in the majority of lesions assayed supports their role in the aetiopathogenesis of these lesions.

Surveying bovine digital dermatitis and non-healing bovine foot lesions for the presence of Fusobacterium necrophorum, Porphyromonas endodontalis and Treponema pallidum.

BACKGROUND: Non-healing bovine foot lesions, including non-healing white line disease, non-healing sole ulcer and toe necrosis, are an increasingly important cause of chronic lameness that are poorly responsive to treatment. Recent studies have demonstrated a high-level association between these non-healing lesions and the Treponema phylogroups implicated in bovine digital dermatitis (BDD). However, a polymicrobial aetiology involving other gram-stain-negative anaerobes is suspected. METHODS: A PCR-based bacteriological survey of uncomplicated BDD lesions (n=10) and non-healing bovine foot lesions (n=10) targeting Fusobacterium necrophorum, Porphyromonas endodontalis, Dichelobacter nodosus and Treponema pallidum/T. paraluiscuniculi was performed. RESULTS: P. endodontalis DNA was detected in 80.0% of the non-healing lesion biopsies (p=<0.001) but was entirely absent from uncomplicated BDD lesion biopsies. When compared to the BDD lesions, F. necrophorum was detected at a higher frequency in the non-healing lesions (33.3% vs 70.0%, respectively), whereas D. nodosus was detected at a lower frequency (55.5% vs 20.0%, respectively). Conversely, T. pallidum/T. paraluiscuniculi DNA was not detected in either lesion type. CONCLUSION: The data from this pilot study suggest that P. endodontalis and F. necrophorum should be further investigated as potential aetiological agents of non-healing bovine foot lesions. A failure to detect syphilis treponemes in either lesion type is reassuring given the potential public health implications such an infection would present.

Invasive infections with Fusobacterium necrophorum including Lemierre's syndrome: an 8-year Swedish nationwide retrospective study.

OBJECTIVES: We aimed to evaluate the nationwide incidence and a potential increase in invasive infections with Fusobacterium necrophorum. Secondly, we aimed to describe epidemiology, clinical characteristics and outcomes for the different presentations: Lemierre's syndrome (LS), invasive head and neck-infection without LS and invasive non-head and neck-infection. METHODS: A retrospective multicentric population-based study of all invasive infections with F. necrophorum diagnosed in Sweden from 2010 to 2017 with 6 months of follow-up was performed through reviews of medical records. Invasive infections were defined and identified by a positive blood culture or sequencing of 16S rDNA, targeted PCR or culture from normally sterile sites. Incidence calculations were performed, including comparisons between 2010-13 and 2014-17, age groups and clinical presentations. Patient and infection characteristics, treatment and clinical outcomes were analysed. RESULTS: Invasive infections with F. necrophorum were diagnosed in 300 cases in Sweden 2010-17. The incidence increased from 2.9 to 5.0 cases/million/year from 2010-13 to 2014-17 (p 0.001). A total of 104/300 (35%) patients developed LS, 102/300 (34%) invasive head and neck infection without LS and 94/300 (31%) invasive non-head and neck infection. The median age was 20, 25 and 64 years, respectively. Among patients with LS 72/96 (75%) had thrombocytopenia on admission, 86/104 (83%) had sepsis, 19/104 (18%) developed septic shock and 45/104 (43%) needed intensive care. 30-day mortality in LS was 2/104 (2%). CONCLUSION: We describe an increased incidence of invasive infections with F. necrophorum in Sweden and highlight its full spectrum of invasive clinical presentations. LS, in particular, causes considerable morbidity in young and previously healthy patients.

Differences in mortality in Fusobacterium necrophorum and Fusobacterium nucleatum infections detected by culture and 16S rRNA gene sequencing.

Fusobacterium species are components of the normal microbiota of the oral cavity, gastrointestinal tract, and female genital tract. They are increasingly recognized as causative agents of oral, laryngeal, and tonsillar infections. Several fusobacterial species are involved in infections, with F. necrophorum and F. nucleatum being the most commonly cultured subtypes. In this study, we aimed to investigate clinical and prognostic differences in terms of mortality and association with malignancy between F. necrophorum and F. nucleatum detected by culture and 16S rRNA gene sequencing. This is a systematic, comparative, retrospective, non-interventional study. Data were extracted from the Department of Clinical Microbiology, Region Zealand, Denmark: all patients with F. necrophorum or F. nucleatum detected by culture or 16S rRNA gene sequencing from 1st of January 2010 to 30th of June 2015 were included. In total, F. necrophorum was detected in samples from 75 patients, and F. nucleatum in samples from 68 patients (total: n = 143). Thirteen patients had a current cancer diagnosis at the time of fusobacterial sampling. Multivariate analyses revealed a significant association of "current cancer" with 30-day mortality. Fusobacterial subtype was not associated with mortality neither in overall nor in subgroups with or without current cancer. Despite differences in clinical disease pattern between F. necrophorum and F. nucleatum, mortality was unaffected by fusobacterial subtype. Mortality was significantly related to comorbidity, especially a current diagnosis of cancer. Our data highlights the current debate whether fusobacterial involvement in cancer may have disease-altering properties, rather than being opportunistic pathogens secondary to cancer disease.

Real-time PCR investigation of the prevalence of Fusobacterium necrophorum in patients with pharyngitis in Japan.

PURPOSE: Recent data suggest an association between Fusobacterium necrophorum infection and pharyngotonsillitis among adolescents and adults. However, existing reports are only from North America and Europe. We aimed to identify and compare the prevalence of F. necrophorum among patients with pharyngitis and asymptomatic controls in Japan and clarify the epidemiological characteristics of pharyngitis. METHODS: Patients aged ≥16 years with pharyngitis and asymptomatic controls were prospectively included. F. necrophorum was detected by using both conventional culture methods and real-time F. necrophorum-specific PCR targeting the rpoB gene. The prevalence of ß-hemolytic streptococci was also identified and compared between groups. RESULTS: Forty-four pharyngitis patients and 31 asymptomatic controls were included. F. necrophorum was identified using PCR in 6 (13.6%) pharyngitis cases and 2 (6.5%) controls, with no significant difference (p = 0.457). The median bacterial load of F. necrophorum identified with real-time PCR was significantly higher in pharyngitis cases than in controls (p = 0.046). Patients with a high Centor Score tended to have a higher bacterial load than those with a low Centor Score and controls. In cases of pharyngitis, the prevalence of F. necrophorum was similar to that of Streptococcus pyogenes (F. necrophorum-positive: 6 [13.6%] vs. S. pyogenes-positive: 5 [11.4%], p = 0.99). CONCLUSION: F. necrophorum was similarly prevalent among pharyngitis cases as S. pyogenes in Japan. The association of higher F. necrophorum bacterial load with symptomatic pharyngitis in accordance with the previous findings from a different geographical region suggests that F. necrophorum is an important causative agent of bacterial pharyngitis.

Fusobacterium necrophorum Subsp. funduliforme in Tonsils from Various Patient Populations in Japan.

Fusobacterium necrophorum has recently been suggested to be associated with tonsillopharyngitis, peritonsillar abscess, and recurrent tonsillitis. Between the 2 subspecies of F. necrophorum, subsp. funduliforme is known to be a major human pathogen. To better understand the epidemiology of F. necrophorum subsp. funduliforme (FNSF), we studied the prevalence of FNSF in the tonsils of patients undergoing elective tonsillectomy (TE) for different indications. Adult patients who underwent elective TE from October 2014 to November 2015 were included. The tonsils were sent for aerobic and anaerobic tissue culture within 30 min of excision; the presence of FNSF was detected using PCR with gyrB primers and 16S rRNA. A total of 32 patients were enrolled. The prevalence of FNSF identified by either culture or gyrB PCR did not significantly differ between infectious and noninfectious TE indications. The constant presence of FNSF might not be associated with recurrent pharyngotonsillitis.