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Chronic inflammation caused by infections has been suggested to be one of the most important cause of cancers. It has recently been shown that there is correlation between intestinal bacteria and cancer development including metastasis. As over 700 bacterial species exist in an oral cavity, it has been concerning that bacterial infection may cause oral cancer. However, the role of bacteria regarding tumorigenesis of oral cancer remains unclear. Several papers have shown that Fusobacterium species deriving the oral cavities, especially, play a crucial role for the development of colorectal and esophageal cancer. F. nucleatum is a well-known oral bacterium involved in formation of typical dental plaque on human teeth and causing periodontal diseases. The greatest characteristic of F. nucleatum is its ability to adhere to various bacteria and host cells. Interestingly, F. nucleatum is frequently detected in oral cancer tissues. Moreover, detection of F. nucleatum is correlated with the clinical stage of oral cancer. Although the detailed mechanism is still unclear, Fusobacterium species have been suggested to be associated with cell adhesion, tumorigenesis, epithelial-to-mesenchymal transition, inflammasomes, cell cycle, etc. in oral cancer. In this review, we introduce the reports focused on the association of Fusobacterium species with cancer development and progression including oral, esophageal, and colon cancers.

Women with bacterial vaginosis (BV), an imbalance of the vaginal microbiome, are more likely to be colonized by potential pathogens such as Fusobacterium nucleatum, a bacterium linked with intrauterine infection and preterm birth. However, the conditions and mechanisms supporting pathogen colonization during vaginal dysbiosis remain obscure. We demonstrate that sialidase activity, a diagnostic feature of BV, promoted F. nucleatum foraging and growth on mammalian sialoglycans, a nutrient resource that was otherwise inaccessible because of the lack of endogenous F. nucleatum sialidase. In mice with sialidase-producing vaginal microbiotas, mutant F. nucleatum unable to consume sialic acids was impaired in vaginal colonization. These experiments in mice also led to the discovery that F. nucleatum may also "give back" to the community by reinforcing sialidase activity, a biochemical feature of human dysbiosis. Using human vaginal bacterial communities, we show that F. nucleatum supported robust outgrowth of Gardnerella vaginalis, a major sialidase producer and one of the most abundant organisms in BV. These results illustrate that mutually beneficial relationships between vaginal bacteria support pathogen colonization and may help maintain features of dysbiosis. These findings challenge the simplistic dogma that the mere absence of "healthy" lactobacilli is the sole mechanism that creates a permissive environment for pathogens during vaginal dysbiosis. Given the ubiquity of F. nucleatum in the human mouth, these studies also suggest a possible mechanism underlying links between vaginal dysbiosis and oral sex., Competing Interests: The authors declare that they have no relevant conflicts of interest.

Fusobacterium spp. are Gram-negative, anaerobic, opportunistic pathogens involved in multiple diseases, including a link between the oral pathogen Fusobacterium nucleatum and the progression and severity of colorectal cancer. The identification and characterization of virulence factors in the genus Fusobacterium has been greatly hindered by a lack of properly assembled and annotated genomes. Using newly completed genomes from nine strains and seven species of Fusobacterium , we report the identification and corrected annotation of verified and potential virulence factors from the type 5 secreted autotransporter, FadA, and MORN2 protein families, with a focus on the genetically tractable strain F. nucleatum subsp. nucleatum ATCC 23726 and type strain F. nucleatum subsp. nucleatum ATCC 25586. Within the autotransporters, we used sequence similarity networks to identify protein subsets and show a clear differentiation between the prediction of outer membrane adhesins, serine proteases, and proteins with unknown function. These data have identified unique subsets of type 5a autotransporters, which are key proteins associated with virulence in F. nucleatum However, we coupled our bioinformatic data with bacterial binding assays to show that a predicted weakly invasive strain of F. necrophorum that lacks a Fap2 autotransporter adhesin strongly binds human colonocytes. These analyses confirm a gap in our understanding of how autotransporters, MORN2 domain proteins, and FadA adhesins contribute to host interactions and invasion. In summary, we identify candidate virulence genes in Fusobacterium , and caution that experimental validation of host-microbe interactions should complement bioinformatic predictions to increase our understanding of virulence protein contributions in Fusobacterium infections and disease. IMPORTANCE Fusobacterium spp. are emerging pathogens that contribute to mammalian and human diseases, including colorectal cancer. Despite a validated connection with disease, few proteins have been characterized that define a direct molecular mechanism for Fusobacterium pathogenesis. We report a comprehensive examination of virulence-associated protein families in multiple Fusobacterium species and show that complete genomes facilitate the correction and identification of multiple, large type 5a secreted autotransporter genes in previously misannotated or fragmented genomes. In addition, we use protein sequence similarity networks and human cell interaction experiments to show that previously predicted noninvasive strains can indeed bind to and potentially invade human cells and that this could be due to the expansion of specific virulence proteins that drive Fusobacterium infections and disease., (Copyright © 2019 American Society for Microbiology.)

The dysbiosis of human gut microbiota is strongly associated with the development of colorectal cancer (CRC). The dysbiotic features of the transition from advanced polyp to early-stage CRC are largely unknown. We performed a 16S rRNA gene sequencing and enterotype-based gut microbiota analysis study. In addition to Bacteroides- and Prevotella-dominated enterotypes, we identified an Escherichia-dominated enterotype. We found that the dysbiotic features of CRC were dissimilar in overall samples and especially Escherichia-dominated enterotype. Besides a higher abundance of Fusobacterium, Enterococcus, and Aeromonas in all CRC faecal microbiota, we found that the most notable characteristic of CRC faecal microbiota was a decreased abundance of potential beneficial butyrate-producing bacteria. Notably, Oscillospira was depleted in the transition from advanced adenoma to stage 0 CRC, whereas Haemophilus was depleted in the transition from stage 0 to early-stage CRC. We further identified 7 different CAGs by analysing bacterial clusters. The abundance of microbiota in cluster 3 significantly increased in the CRC group, whereas that of cluster 5 decreased. The abundance of both cluster 5 and cluster 7 decreased in the Escherichia-dominated enterotype of the CRC group. We present the first enterotype-based faecal microbiota analysis. The gut microbiota of colorectal neoplasms can be influenced by its enterotype.

Trueperella pyogenes is one of the most important microorganisms causing metritis in post-partum cattle. Co-infection with other bacterial species such as Escherichia coli or Fusobacterium necrofurom increases the severity of the disease and the persistence of bacteria in utero. The aim of this study was to investigate the frequency of T. pyogenes strains, and their virulence and antimicrobial resistant profiles in metritis cases. The study was carried out on 200 samples obtained from metritis discharges of postpartum cattle on 18 farms around Tehran, Iran. Sixty-five T. pyogenes isolates (32.5%) were identified, of which 16 isolates were detected as pure cultures and the other 49 isolates from cultures most commonly mixed with E. coli or F. necrofurom. In terms of diversity in biochemical characteristic of T. pyogenes strains, 8 different biotypes were identified among the isolates. Single or multi antimicrobial resistance was observed in 48 isolates (73.9%), which was mostly against trimethoprim sulfamethoxazole, azithromycin, erythromycin and streptomycin. The tetracycline resistance gene tetW and macrolide resistance genes ermB and ermX were detected in 30, 18 and 25 isolates, respectively. In the screening of genes encoding virulence factors, fimA and plo genes were identified in all tested isolates. Genes encoding nanP, nanH, fimC, fimG, fimE and cbpA were detected in 50, 54, 45, 40, 50 and 37 of isolates, respectively. Thirteen different genotypes were observed in these T. pyogenes isolates. A significant association between clonal types and virulence factor genes, biochemical profile, CAMP test result, severity of the disease and sampling time was detected.

Aim: To critically evaluate previous scientific evidence on Fusobacterium's role in colorectal neoplasia development., Methods: Two independent investigators systematically reviewed all original scientific articles published between January, 2000, and July, 2017, using PubMed, EMBASE, and MEDLINE. A total of 355 articles were screened at the abstract level. Of these, only original scientific human, animal, and in vitro studies investigating Fusobacterium and its relationship with colorectal cancer (CRC) were included in the analysis. Abstracts, review articles, studies investigating other colonic diseases, and studies written in other languages than English were excluded from our analysis. Ninety articles were included after removing duplicates, resolving disagreements between the two reviewers, and applying the above criteria., Results: Studies have consistently identified positive associations between Fusobacterium , especially Fusobacterium nucleatum ( F. nucleatum ), and CRC. Stronger associations were seen in CRCs proximal to the splenic flexure and CpG island methylator phenotype (CIMP)-high CRCs. There was evidence of temporality and a biological gradient, with increased F. nucleatum DNA detection and quantity along the traditional adenoma-carcinoma sequence and in CIMP-high CRC precursors. Diet may have a differential impact on colonic F. nucleatum enrichment; evidence suggests that high fiber diet may reduce the risk of a subset of CRCs that are F. nucleatum DNA-positive. Data also suggest shorter CRC and disease-specific survival with increased amount of F. nucleatum DNA in CRC tissue. The pathophysiology of enrichment of F. nucleatum and other Fusobacterium species in colonic tissue is unclear; however, the virulence factors and changes to the local colonic environment with disruption of the protective mucus layer may contribute. The presence of a host lectin (Gal-GalNAc) in the colonic epithelium may also mediate F. nucleatum attachment to CRC and precursors through interaction with an F. nucleatum protein, fibroblast activation protein 2 (FAP2). The clinical significance of detection or enrichment of Fusobacterium in colorectal neoplasia is ambiguous, but data suggest a procarcinogenic effect of F. nucleatum , likely due to activation of oncogenic and inflammatory pathways and modulation of the tumor immune environment. This is hypothesized to be mediated by certain F. nucleatum strains carrying invasive properties and virulence factors such as FadA and FAP., Conclusion: Evidence suggests a potential active role of Fusobacterium , specifically F. nucleatum , in CRC. Future prospective and experimental human studies would fill an important gap in this literature., Competing Interests: Conflict-of-interest statement: The authors do not have any relevant conflicts of interest (including relevant financial interests, activities, relationships, and/or affiliations).

Background: Anterior sacral meningoceles are rare, and usually occur with other malformations of the posterior lower spine. While these are more frequently reported in pediatric cohorts, we report a case in an elderly woman., Case Presentation: We report on a 71 year-old woman with a recently diagnosed colorectal adenocarcinoma who presented with a severe bacterial meningitis. The cerebrospinal fluid cell count revealed a pleocytosis of 80,000 cells/μl and a severe disturbance of the blood-brain-barrier. Fusobacterium nucleatum was cultured as the causing pathogen. A lumbar MRI showed, in addition to contrast-enhancing meninges as sign of inflammation, a presacral mass. In the next step, the mass was diagnosed as an anterior sacral meningocele connected to the gut. An adequate antibiotic was used to treat the leptomeningitis. The connection between gut and meningocele was closed surgically and the patient recovered well and underwent further treatment of her colorectal adenocarcinoma., Conclusion: We report on a case of meningitis with an anterior sacral meningocele that was connected to the gut in a patient with a infiltrative colorectal adenocarcinoma. Anatomic variants have to be considered as rare causes of meningitis with typical intestinal germs.

Fusobacterium nucleatum (Fn) is an important tumour-associated bacterium in colorectal cancer (CRC). The antioxidant protein alkyl hydroperoxide reductase subunit C (AhpC) can induce strong antibacterial immune response during various pathogen infections. Our study aimed to evaluate the efficacy of Fn-AhpC as a candidate vaccine. In this work, by western blot analysis, we showed that Fn-AhpC recombinant protein could be recognized specifically by antibodies present in the sera of CRC patients; using the mouse Fn-infection model, we observed that systemic prophylactic immunization with AhpC/alum conferred significant protection against infection in 77.3% of mice. In addition, we measured the anti-AhpC antibody level in the sera of CRC patients and found that there was no obvious increase of anti-AhpC antibodies in the early-stage CRC group. Furthermore, we treated Fn with the sera from both immunized mice and CRC patients and found that sera with high anti-AhpC antibodies titre could inhibit Fn growth. In conclusion, our findings support the use of AhpC as a potential vaccine candidate against inhabitation or infection of Fn in the intestinal tract, which could provide a practical strategy for the prevention of CRC associated with Fn infection.

Colorectal cancer is a leading cause of morbidity and mortality worldwide in both men and women. The gut microbiome is increasingly recognized as having an important role in human health and disease. Fusobacterium has been identified in former studies as a leading gut bacterium associated with colorectal cancer, but it is still not clear if it plays an oncogenic role. In the current study, fecal samples were collected prior to bowel preparation from participants of screening colonoscopy in the German BliTz study. Using 16S rRNA gene analysis, we examined the presence and relative abundance of Fusobacterium in fecal samples from 500 participants, including 46, 113, 110 and 231 individuals with colorectal cancer, advanced adenomas, non-advanced adenomas and without any neoplasms, respectively. We found that the abundance of Fusobacterium in feces was strongly associated with the presence of colorectal cancer (P-value < 0.0001). This was confirmed by PCR at the species level for Fusobacterium nucleatum. However, no association was seen with the presence of advanced adenomas (P-value = 0.80) or non-advanced adenomas (P-value = 0.80), nor were there any associations observed with dietary or lifestyle habits. Although a causal role cannot be ruled out, our observations, based on fecal microbiome, support the hypothesis that Fusobacterium is a passenger that multiplies in the more favorable conditions caused by the malignant tumor rather than a causal factor in colorectal cancer development., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The relevance of specific microbial colonisation to colorectal cancer (CRC) disease pathogenesis is increasingly recognised, but our understanding of possible underlying molecular mechanisms that may link colonisation to disease in vivo remains limited. Here, we investigate the relationships between the most commonly studied CRC-associated bacteria (Enterotoxigenic Bacteroides fragilis, pks+ Escherichia coli, Fusobacterium spp., afaC+ E. coli, Enterococcus faecalis & Enteropathogenic E. coli) and altered transcriptomic and methylation profiles of CRC patients, in order to gain insight into the potential contribution of these bacteria in the aetiopathogenesis of CRC. We show that colonisation by E. faecalis and high levels of Fusobacterium is associated with a specific transcriptomic subtype of CRC that is characterised by CpG island methylation, microsatellite instability and a significant increase in inflammatory and DNA damage pathways. Analysis of the significant, bacterially-associated changes in host gene expression, both at the level of individual genes as well as pathways, revealed a transcriptional remodeling that provides a plausible mechanistic link between specific bacterial colonisation and colorectal cancer disease development and progression in this subtype; these included upregulation of REG3A, REG1A and REG1P in the case of high-level colonization by Fusobacterium, and CXCL10 and BMI1 in the case of colonisation by E. faecalis. The enrichment of both E. faecalis and Fusobacterium in this CRC subtype suggests that polymicrobial colonisation of the colonic epithelium may well be an important aspect of colonic tumourigenesis., Competing Interests: The authors have declared that no competing interests exist.

In order to identify current trends in anaerobic bacteraemia, a 10-year retrospective study was performed in the University Hospital Brussel, Belgium. All clinically relevant bacteraemia detected from 2004 until 2013 were included. Medical records were reviewed in an attempt to define clinical parameters that might be associated with the occurrence of anaerobic bacteraemia. 437 of the isolated organisms causing anaerobic bacteraemia were thawed, subcultured and reanalyzed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). There were an average of 33 cases of anaerobic bacteraemia per year during 2004-2008 compared to an average of 27 cases per year during 2009-2013 (P = 0.017), corresponding to a decrease by 19% between the first and the latter period. Also, the total number of cases of anaerobic bacteraemia per 100,000 patient days decreased from 17.3 in the period from 2004 to 2008 to 13.7 in the period 2009 to 2013 (P = 0.023). Additionally, the mean incidence of anaerobic bacteraemia decreased during the study period (1.27/1000 patients in 2004 vs. 0.94/1000 patients in 2013; P = 0.008). In contrast, the proportion of isolated anaerobic bacteraemia compared to the number of all bacteraemia remained stable at 5%. Bacteroides spp. and Parabacteroides spp. accounted for 47.1% of the anaerobes, followed by 14.4% Clostridium spp., 12.6% non-spore-forming Gram-positive rods, 10.5% anaerobic cocci, 8.2% Prevotella spp. and other Gram-negative rods and 7.1% Fusobacterium spp. The lower gastrointestinal tract (47%) and wound infections (10%) were the two most frequent sources for bacteraemia, with the origin remaining unknown in 62 cases (21%). The overall mortality rate was 14%. Further studies focusing on the antimicrobial susceptibility and demographic background of patients are needed to further objectify the currently observed trends., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Recently, bacterial infection causing periodontal disease has attracted considerable attention as a risk factor for pancreatic cancer. Fusobacterium species is an oral bacterial group of the human microbiome. Some evidence suggests that Fusobacterium species promote colorectal cancer development; however, no previous studies have reported the association between Fusobacterium species and pancreatic cancer. Therefore, we examined whether Fusobacterium species exist in pancreatic cancer tissue. Using a database of 283 patients with pancreatic ductal adenocarcinoma (PDAC), we tested cancer tissue specimens for Fusobacterium species. We also tested the specimens for KRAS, NRAS, BRAF and PIK3CA mutations and measured microRNA-21 and microRNA-31. In addition, we assessed epigenetic alterations, including CpG island methylator phenotype (CIMP). Our data showed an 8.8% detection rate of Fusobacterium species in pancreatic cancers; however, tumor Fusobacterium status was not associated with any clinical and molecular features. In contrast, in multivariate Cox regression analysis, compared with the Fusobacterium species-negative group, we observed significantly higher cancer-specific mortality rates in the positive group (p = 0.023). In conclusion, Fusobacterium species were detected in pancreatic cancer tissue. Tumor Fusobacterium species status is independently associated with a worse prognosis of pancreatic cancer, suggesting that Fusobacterium species may be a prognostic biomarker of pancreatic cancer.

A total of 23 clinical isolates of Fusobacterium spp. were recovered at necropsy over a 2-year period from the respiratory tract of white-tailed deer (Odocoileus virginianus). Isolates were identified as Fusobacterium varium (18/23), Fusobacterium necrophorum subsp. funduliforme (3/23), and Fusobacterium necrophorum subsp. necrophorum (2/23). Using polymerase chain reaction-based detection of virulence genes, all F. necrophorum isolates were positive for the promoter region of the leukotoxin operon and the hemagglutinin-related protein gene, while all F. varium isolates were negative. The presence of the leukotoxin gene in F. necrophorum isolates and the absence of this gene in F. varium isolates were confirmed by Southern hybridization using 2 separate probes. Toxicity to bovine polymorphonuclear leukocytes was observed with all F. necrophorum isolates, but was not observed in any F. varium isolates. Susceptibility to antimicrobials was markedly different for F. varium as compared to F. necrophorum. In summary, no evidence of leukotoxin production was detected in any of the 23 F. varium isolates used in the current study. The data suggests that F. varium, the most common species isolated, may be a significant pathogen in deer with a different virulence mechanism than F. necrophorum.

The tumor microenvironment of colorectal carcinoma is a complex community of genomically altered cancer cells, nonneoplastic cells, and a diverse collection of microorganisms. Each of these components may contribute to carcinogenesis; however, the role of the microbiota is the least well understood. We have characterized the composition of the microbiota in colorectal carcinoma using whole genome sequences from nine tumor/normal pairs. Fusobacterium sequences were enriched in carcinomas, confirmed by quantitative PCR and 16S rDNA sequence analysis of 95 carcinoma/normal DNA pairs, while the Bacteroidetes and Firmicutes phyla were depleted in tumors. Fusobacteria were also visualized within colorectal tumors using FISH. These findings reveal alterations in the colorectal cancer microbiota; however, the precise role of Fusobacteria in colorectal carcinoma pathogenesis requires further investigation.

An association between the gram-positive anaerobe Filifactor alocis and periodontal disease has recently emerged; however, possible pathogenic mechanisms have not been investigated. In this study we examined the responses of primary cultures of gingival epithelial cells (GECs) to infection with F. alocis. Secretion of the pro-inflammatory cytokines interleukin-1β, interleukin-6 and tumor necrosis factor-α from GECs was stimulated by F. alocis infection. F. alocis also induced apoptosis in GECs through pathways that involved caspase-3 but not caspase-9. Apoptosis was coincident with inhibition of mitogen-activated protein kinase kinase (MEK) activation. These results show that F. alocis has characteristics in common with established periodontal pathogens and has the potential to contribute to periodontal tissue destruction., (© 2011 John Wiley & Sons A/S.)

In order to investigate differences among infections due to Gram-negative anaerobic bacteria (Bacteroides, Prevotella and Fusobacterium spp.), clinical, epidemiological, and microbiological data were collected and evaluated from 206 anaerobic infections. The most frequently isolated species was Bacteroides fragilis. The majority of the cases were intra-abdominal infections (49%) followed by skin and soft tissue infections (24.7%). Logistic regression analysis showed that Bacteroides spp. strains were more often isolated from intra-abdominal infections (p = 0.002), whereas Prevotella spp. were isolated more frequently from cases with shorter duration of hospitalization (p = 0.026), and less frequently from bloodstream infections (p = 0.049). In addition, Bacteroides spp. were associated with coinfection due to Enterobacteriaceae species (p = 0.007), whereas Prevotella spp. were associated with coinfection due to Staphylococcus spp. (p = 0.002). Patients with an infection due to B. fragilis, were more frequently admitted in a general surgical ward (p = 0.017), or have been treated with a 2nd generation cephalosporin before anaerobic infection onset (p = 0.05). Total mortality was 10.9% and was associated with bacteremia (p = 0.026), and hematological (p = 0.028), or solid organ malignancy (p = 0.007). Metronidazole resistance was detected only among Prevotella spp. (16.2%) and B. fragilis group (0.8%) isolates. In conclusion, this study indicated differences between infections due to the most frequently isolated Gram-negative anaerobic species, differences that may affect the design and implementation of empirical antimicrobial chemotherapy guidelines., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

To analyze immunodominant regions of leukotoxin protein of Fusobacterium necrophorum strain H05, a series of truncated forms of leukotoxin gene were expressed in Escherichia coli using the vector pGEX-6p-1 or pPROEX HTa. The results of SDS-PAGE showed the truncated forms PL1, PL2, PL4, and PL5 were expressed in Escherichia coli using the vector pGEX-6p-1, and the truncated forms PL3 was expressed in Escherichia coli using the vector pPROEX HTa. These recombinant proteins were able to react with antisera against Fusobacterium necrophorum strain A25. In five recombinant proteins, the recombinant proteins PL1, PL3 and PL4 as vaccine were able to elicit formation of the better protective effects on mice against infection of Fusobacterium necrophorum strain A25.

We examined survival and replication of fusobacteria inside epithelial cells. Subconfluent cultures of HaCaT keratinocytes were infected with five bacterial strains representing three Fusobacterium species: F. nucleatum, F. necrophorum, and F. mortiferum. Adhesion and invasion of the bacteria were assayed before and after antibiotic treatment that killed the adhered and extracellular bacteria. The number of live fusobacteria was examined by bacterial culturing after sonication of the epithelial cells. The role of host cell cytoskeleton functions was examined by treating the epithelial cells with cell function inhibitors. Number of viable epithelial cells was measured with the CellTiter96 kit. The tested Fusobacterium species adhered to and invaded the epithelial cells, and multiplied intracellularly for several hours. Thereafter, the intracellular number of bacteria rapidly declined. Concomitantly, viable fusobacteria were detected in the culture medium. Treatment of the infected epithelial cells with an actin formation inhibitor markedly reduced the number of living intracellular fusobacteria. Newly formed actin filaments were seen by confocal microscopy in the epithelial cells associated with the invaded bacteria. Fusobacteria infection did not reduce the number of viable epithelial cells in culture. Thus, fusobacteria are able to adhere to and invade epithelial cells, and survive under aerobic conditions. This property may enable them to survive in mucosa and participate in various disease processes of oral and pharyngeal tissues.

Background and Objective: The purpose of this study was to determine the time of infection by anaerobic gram-negative rods associated with periodontal disease, and to clarify their transmission from mother to child., Material and Methods: Seventy-eight Japanese children (including 10 siblings), aged from 3 to 9 years, and 68 mothers, were enrolled in this study. Colonization by 11 periodontal bacterial species was determined using polymerase chain reaction amplification of samples of subgingival plaque obtained from the children and their mothers., Results: The detection rates of Porphyromonas gingivalis, Tannerella forsythensis and Treponema denticola increased in children after the age of 6 years. We found a high consistency in colonization by P. gingivalis, T. denticola, Prevotella intermedia and Prevotella nigrescens in 9 of the 10 siblings. The average number of bacterial species in plaque samples harboring Fusobacterium nucleatum and/or Fusobacterium periodonticum was significantly greater than in those without, in both children and mothers. Kappa statistical analysis revealed that the detection of Capnocytophaga gingivalis, Capnocytophaga ochracea, Campylobacter rectus and T. denticola in children was consistent with that in the mother., Conclusion: Periodontal bacterial colonization in Japanese children increased with age and was associated with F. nucleatum and/or periodonticum, and the bacterial flora in children was similar to that in their mothers.

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

Bacterial biofilms have been found to develop on root surfaces outside the apical foramen and be associated with refractory periapical periodontitis. However, it is unknown which bacterial species form extraradicular biofilms. The present study aimed to investigate the identity and localization of bacteria in human extraradicular biofilms. Twenty extraradicular biofilms, used to identify bacteria using a PCR-based 16S rRNA gene assay, and seven root-tips, used to observe immunohistochemical localization of three selected bacterial species, were taken from 27 patients with refractory periapical periodontitis. Bacterial DNA was detected from 14 of the 20 samples, and 113 bacterial species were isolated. Fusobacterium nucleatum (14 of 14), Porphyromonas gingivalis (12 of 14), and Tannellera forsythensis (8 of 14) were frequently detected. Unidentified and uncultured bacterial DNA was also detected in 11 of the 14 samples in which DNA was detected. In the biofilms, P. gingivalis was immunohistochemically detected in all parts of the extraradicular biofilms. Positive reactions to anti-F. nucleatum and anti-T. forsythensis sera were found at specific portions of the biofilm. These findings suggested that P. gingivalis, T. forsythensis, and F. nucleatum were associated with extraradicular biofilm formation and refractory periapical periodontitis.

Dialister pneumosintes and Filifactor alocis have been recently considered as candidate endodontic pathogens. In this study, we devised a 16S rDNA-directed multiplex PCR protocol for simultaneous detection of these two bacterial species in endodontic infections. Samples were taken from infected root canals associated with asymptomatic periradicular lesions as well as from cases of acute periradicular abscesses. DNA extracted from the samples was used as template for simultaneous detection of D. pneumosintes and F. alocis through a multiplex PCR assay. Two fragments of the expected sizes, one specific for D. pneumosintes and the other for F. alocis, were simultaneously amplified from a mixture of reference genomic DNA containing DNA from both species. Clinical samples that were positive for the target species showed a single band of the predicted size for each species. D. pneumosintes was detected by multiplex PCR in 11 samples (7 asymptomatic and 4 abscesses) and F. alocis was identified in 9 cases (6 asymptomatic and 3 abscesses). Six samples (3 asymptomatic and 3 abscesses) shared the two species. Data from the present study confirmed that D. pneumosintes and F. alocis are common members of the microbiota present in primary endodontic infections and thereby may participate in the pathogenesis of periradicular lesions. The proposed multiplex PCR assay is a simple, rapid, and accurate method for the simultaneous detection of these two candidate endodontic pathogens.

A TaqMan-based real-time PCR assay was established to quantify the periodontopathic bacteria Tannerella forsythensis and Fusobacterium spp. With this assay, the prevalence and proportion of these bacteria in clinical specimens were evaluated. Our preliminary results suggest a positive colocalization of T. forsythensis and Fusobacterium spp. in periodontal pockets.

Bacterial cultures from 32 living and dead farmed white-tailed deer (Odocoileus virginianus) with necrobacillosis yielded Fusobacterium necrophorum from nine individuals, F. varium from six individuals, and Arcanobacterium pyogenes from 16 individuals. The isolates were characterized biochemically using automated identification systems. Gram-stained smears suggested the presence of Fusobacterium spp. in eight cases from which organisms were not cultured. Minimum inhibitory concentration determinations in 23 strains of gram-negative anaerobic bacteria detected resistance to enrofloxacin and clindamycin. Enrofloxacin resistance was detected in A. pyogenes isolates, and although biochemical profiling indicated that the deer strains of A. pyogenes could be grouped, it is uncertain whether these biochemical characteristics correlate with antigenic or virulence factors. Deer-specific or autogenous vaccines may provide a useful alternative to generic vaccines.

Recent molecular studies have expanded the list of suspected endodontic pathogens. The aim of this study was to evaluate the occurrence of Filifactor alocis in primary endodontic infections associated with different forms of periradicular diseases. Identification by nested polymerase chain reaction was performed in root canal samples from teeth associated with either asymptomatic periradicular lesions or acute apical periodontitis. Samples were also taken by aspiration of purulent exudate associated with acute apical abscesses. DNA extracted from the samples was initially amplified using universal 16S rDNA primers followed by a second round of amplification using the first PCR products to detect a specific fragment of F. alocis 16S rDNA. F. alocis was detected in 12/21 (57.1%) root canal samples from teeth showing asymptomatic periradicular lesions and in 3/10 (30%) samples taken from root canals associated with acute apical periodontitis. This species occurred in 8/19 (42.1%) pus aspirates obtained from abscessed teeth. In general, F. alocis was detected in 23/50 (46%) samples taken from endodontic infections. These findings suggest that F. alocis is involved in the etiology of different forms of periradicular diseases and has the potential to be an endodontic pathogen.

Background: Bacteria are implicated in certain forms of model chronic colitis but the identity and role of bacteria in human ulcerative colitis (UC) are uncertain., Aims: To isolate pathogenic bacteria from inflamed mucosa of patients with UC, to examine whether the bacteria have a toxin to Vero cells, and to determine whether the toxin induces UC-like lesions in animals., Methods: Bacteria were isolated from UC patients and supernatants from cultures were filtered and tested for cytotoxicity to Vero cells. Bacterial cells producing the cytotoxic supernatants were examined by polymerase chain reaction for verotoxin genes. Culture supernatants of cytotoxic strains were examined by high performance liquid chromatography for organic acid concentrations. Mice were given enemas containing organic acid at the mean concentration in the supernatants of cytotoxic strains to ascertain whether colonic lesions appear in UC., Results: Only supernatants from cultures of Fusobacterium varium killed Vero cells. Bacterial cells lacked verotoxin genes. Bacterial culture supernatants contained high concentrations of n-butyric acid and the mean concentration (32 mmol/l) was cytotoxic to Vero cells. Twenty four hours after mice were given enemas containing either butyric acid or F varium culture supernatants, colonic ulcers with crypt abscesses, inflammatory cell infiltration, and apoptotic changes were observed., Conclusions: Butyric acid in culture supernatants from cultures of F varium caused UC-like lesions in mice. This study indicates that F varium may be one of the elusive pathogenic factors in UC.

The involvement of fusobacteria in a wide range of human and animal infection has long been recognised. Slow-growing anaerobes, often in polymicrobial culture, they are not always identified but are present mainly in the oropharynx, from where they are bloodborne to other sites or aspirated into the lung. Fusobacterium nucleatum is commonly found in periodontal disease and produces tissue irritants such as butyric acid, proteases and cytokines. It has strong adhesive properties due to the presence of lectins, and these outer-membrane proteins mediate adhesion to epithelia and tooth surfaces, and coagglutination with other suspected pathogens. F. necrophorum may cause necrotising tonsillitis and septicaemia, leading to the spread of infection and the development of abscesses in the lung and brain--a form of Lemierre's syndrome. Calf diphtheria, foot rot and other infections in animals are well defined, with the pathogenic mechanisms involving leucotoxins, endotoxins and adhesins. A foul smell produced by butyric acid and other metabolic products is common to all fusobacterial infections. Identification using simple tests is within the scope of most laboratories.

The abscess forming abilities of "Streptococcus milleri" strains (Streptococcus constellatus, Streptococcus anginiosus, and Streptococcus intermedius) isolated from dentoalveolar abscesses and the synergistic effect of Fusobacterium nucleatum co-inoculated with the isolates were examined on a mouse subcutaneous abscess model. Five days after inoculation, all S. milleri strains formed abscesses, which showed less pathological spread to surrounding connective tissues than those formed by Staphylococcus aureus 209P strain and were similar to those by F. nucleatum ATCC25586. When each S. milleri strain and F. nucleatum were co-inoculated, abscess sizes and each bacterial number recovered from abscesses increased in comparison to those treated by bacterial mono-inoculation of each S. milleri strain or F. nucleatum alone. The strongest synergistic effect was observed in the combination of S. constellatus and F. nucleatum. In a time course experiment with this combination, the recovery of S. constellatus subsequently decreased after the decrement of F. nucleatum, and it appeared that the association with F. nucleatum maintained the bacterial number of S. constellatus in the abscess. The cell-free supernatant of F. nucleatum had a tendency to increase the abscess size caused by S. constellatus in this model. When S. constellatus was cultured with F. nucleatum culture supernatant in vitro, growth enhancement in the early phase was observed. Furthermore, the phagocytic killing of S. constellatus by human polymorphonuclear leukocytes (PMNs) was significantly suppressed and the PMN membranes appeared to be injured by addition of the F. nucleatum culture supernatant. These results suggest that the pathogenicity of S. milleri strains in odontogenic infections may be enhanced by the co-existence of F. nucleatum.

The ability of butyric acid, an extracellular metabolite from periodontopathic bacteria, to induce apoptosis in murine WEHI 231 cells, splenic B cells, and human RAJI cells was examined. The culture filtrate of Porphyromonas gingivalis, Prevotella loescheii, and Fusobacterium nucleatum, which contains high a percentage of butyric acid, induced DNA fragmentation in WEHI 231 cells. Volatile fatty acid, especially butyric acid, significantly suppressed B-cell viability in a concentration-dependent fashion. The DNA fragmentation assay indicated that butyric acid rapidly induced apoptosis in WEHI 231 cells (with 1.25 mM butyric acid and 6 h after treatment), splenic B cells (with 1.25 mM butyric acid), and RAJI cells (with 2.5 mM butyric acid). Incubation of WEHI 231 cells with butyric acid for 16 h resulted in the typical ladder pattern of DNA fragmentation and the apoptoic change such as chromatin condensation and hypodiploid nuclei. Cell cycle analysis implied that butyric acid arrested the cells at the G1 phase. The inhibitory assay suggested that butyric acid-induced apoptosis of WEHI 231 and splenic B cells was inhibited by W-7, a calmodulin inhibitor. These results suggest that calmodulin-dependent regulation is involved in the signal transduction pathway of butyric acid.

The greatest cause of endodontic and periapical pathosis is microbial infection of the pulp. Most odontogenic infections are of a polymicrobial nature. With advances in anaerobic isolation and culturing techniques, much has been learned about the presence of pathogenic organisms such as Porphyromonas and Prevotella species (formerly classified as black-pigmented Bacteroides species) in infected root canals. This review provides a summary of recent developments in endodontic microbiology, virulence factors, and host defense systems as they relate to the pathogenesis of pulpal and periapical inflammatory lesions.

Five virulent strains of Fusobacterium necrophorum resembled a single strain examined earlier by possessing little or no immunogenicity: severe subcutaneous infections cured with metronidazole failed to increase the resistance of mice to subcutaneous challenge 22 days after the cessation of treatment.

It had already been shown with a single virulent strain (A42) of Fusobacterium necrophorum that suspension of the fusobacteria in sub-lethal doses of broth cultures of other bacteria reduced the minimum infective dose (greater than 10(6) organisms) for mice by subcutaneous inoculation, sometimes to less than 10 organisms. The present study extended the known range of bacteria with strong infectivity-enhancing properties to include Bacillus cereus, Klebsiella oxytoca and Staphylococcus aureus; and those with weaker effect to include Bacillus subtilis, 'Bacteroides melaninogenicus', Clostridium sporogenes, Pasteurella haemolytica, and Proteus mirabilis. The study also showed that five further virulent strains of F. necrophorum closely resembled A42 in respect of striking susceptibility to infectivity enhancement by Escherichia coli. Actinomyces (Corynebacterium) pyogenes and S. aureus. One further strain (A6) of F. necrophorum resembled A42 in respect of strong infectivity enhancement by A. pyogenes, S. aureus, B. cereus and K. oxytoca but differed from it and the other five strains in being only slightly affected by E. coli. This work was a necessary prelude to the development of a method, based on infectivity enhancement, for the detection and isolation of F. necrophorum present in small numbers in heavily contaminated material such as faeces.

There is increasing evidence that access to patent dentinal tubules by bacteria and their products rather than trauma from restorative materials is responsible for subsequent pulpitides. The purpose of this study was to compare the relative cytotoxicity of centrifugal fractions of two bacteria, Fusobacterium nucleatum and Treponema denticola, on L929 cells in monolayer cultures and in the "in vitro pulp chamber." Neutrophilic chemotaxis assays and Limmulus assays were performed to verify biological activity of the various fractions of these bacteria. It was found that T. denticola inhibits new protein synthesis in cultured cells to a much greater extent than F. nucleatum, but that only F. nucleatum fractions are chemoattractive for human neutrophils in the absence of serum. While the chemical nature and molecular weights of the "toxic" materials were not determined, it appeared that eukaryotic protein synthesis inhibition caused by the T. denticola pellet fraction in the in vitro pulp chamber was at least 1000 times less than that caused by the same concentrations in monolayer cultures.

Most of the bacteria involved in the endodontic infection can be considered as "highly specific", for they appear to be almost irrelevant in the large field of the other human disease from infection. Namely the most important anaerobia responsible for endodontic infection are: Bacteroides, Fusobacterium, Peptostreptococcus, and Veillonella. The group of Bacteroides is composed by several species with different metabolic and genetic characteristics. They are Gram-, non-motile, non-spore-forming rods. Fusobacterium are spindle-shaped, Gram-, non motile, non-spore-forming bacilli. Peptostreptococci are round-shaped, Gram+, non-motile cocci. Veillonella are small, Gram-, non-motile cocci.

The pathogenicity of 20 strains belonging to nine bacterial species isolated from acute dentoalveolar abscesses was assessed individually and in two species combinations by subcutaneous inoculation of mice. Infections were produced by all the bacteria although variations were seen both in the type of lesion produced and the subsequent recovery of viable bacteria. Anaerobic gram-negative bacilli were recovered more often (p less than 0.05) at high concentrations (10(6)-10(9) cfu/ml) and produced a localised abscess with peripheral necrosis more frequently (p less than 0.001) than either Streptococcus milleri or anaerobic gram-positive cocci. Lesions induced by a combination of bacteria comprising anaerobic gram-negative bacillus and any other species yielded both strains at high concentration more often (p less than 0.001) than a combination comprising anaerobic gram-positive cocci and S. milleri. It is concluded that anaerobic gram-negative bacilli are major pathogens in acute dentoalveolar abscesses.

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.

The literature dealing with the pathogenicity of anaerobic gram-negative rods in humans is reviewed. Knowledge concerning definite pathogenic mechanisms is, at best, cursory. There is evidence that encapsulation plays a role in the pathogenicity of Bacteroides fragilis and some of the black-pigmented Bacteroides. A range of enzymes, among them collagenase and IgA protease, are produced by several Bacteroides species. Supernatants of Fusobacterium necrophorum cultures may be leukotoxic. Synergism between anaerobic gram-negative rods and other bacterial species has been demonstrated in experimental animals.

Bacteroides sp. (Bacteroides melaninogenicus, Bacteroides oralis and Bacteroides fragilis), peptostreptococci and Fusobacterium sp. are important pathogens in upper respiratory tract infections. A recent increase in numbers of beta-lactamase-producing strains of anaerobic Gram-negative bacteria in upper respiratory tract infections has been associated with increased failure rates of penicillins in eradication of these infections. These infections include chronic otitis media, chronic sinusitis and mastoiditis, chronic recurrent tonsillitis and lung abscesses. The indirect pathogenicity of these organisms is apparent through their ability not only to survive penicillin therapy but also to protect penicillin-susceptible pathogens from the drug. These direct and indirect virulence characteristics of anaerobic bacteria require the administration of appropriate antimicrobial therapy directed against all pathogens in mixed infections.

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.

Fusobacterium nucleatum expresses lectinlike adherence factors which mediate binding to a variety of human tissue cells. Adherence is selectively inhibited by galactose, lactose, and N-acetyl-D-galactosamine. In this study, adherence of F. nucleatum to human peripheral blood polymorphonuclear neutrophils (PMNs) was investigated. The results indicated that the fusobacteria adhered to live and metabolically inactivated or fixed PMNs. Adherence of F. nucleatum resulted in activation of PMNs as determined by PMN aggregation, membrane depolarization, increased intracellular free Ca2+, superoxide anion production, and lysozyme release. Transmission electron micrographs showed that F. nucleatum was phagocytized by the PMNs. Microbicidal assays indicated that greater than 98% of F. nucleatum organisms were killed by PMNs within 60 min. Adherence to and activation of PMNs by F. nucleatum were inhibited by N-acetyl-D-galactosamine or lactose greater than galactose, whereas equal concentrations of glucose, N-acetyl-D-glucosamine, mannose, and fucose had little or no effect on F. nucleatum-PMN interactions. Pretreatment of the fusobacteria with heat (80 degrees C, 20 min) or proteases inhibited adherence to and activation of PMNs, but superoxide production was also stimulated by heated bacteria. The results indicate that interaction of F. nucleatum with PMNs is lectinlike and is probably mediated by fusobacterial proteins which bind to other human tissue cells. Adherence of F. nucleatum to PMNs in the absence of serum opsonins, such as antibodies and complement, may play an important role in PMN recognition and killing of F. nucleatum in the gingival sulcus and in the subsequent release of PMN factors associated with tissue destruction.

A range of anaerobic bacteria may be pathogenic to man. Those of particular clinical significance are reviewed and prospects for their control by antibiotic therapy discussed.