Involvement of Fusobacterium Species in Oral Cancer Progression: A Literature Review Including Other Types of Cancer.

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.

Utilizing Whole Fusobacterium Genomes To Identify, Correct, and Characterize Potential Virulence Protein Families.

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.IMPORTANCEFusobacterium 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.

Enterotype-based Analysis of Gut Microbiota along the Conventional Adenoma-Carcinoma Colorectal Cancer Pathway.

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.

Anterior sacral meningocele infected with Fusobacterium in a patient with recently diagnosed colorectal carcinoma - a case report.

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.

Immunization with alkyl hydroperoxide reductase subunit C reduces Fusobacterium nucleatum load in the intestinal tract.

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.

Fusobacterium and colorectal cancer: causal factor or passenger? Results from a large colorectal cancer screening study.

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.

Fusobacterium's link to colorectal neoplasia sequenced: A systematic review and future insights.

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.

Bacterially-Associated Transcriptional Remodelling in a Distinct Genomic Subtype of Colorectal Cancer Provides a Plausible Molecular Basis for Disease Development.

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.

Association of Fusobacterium species in pancreatic cancer tissues with molecular features and prognosis.

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.

Genomic analysis identifies association of Fusobacterium with colorectal carcinoma.

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.

Filifactor alocis interactions with gingival epithelial cells.

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-1ß, 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.

Epidemiological characteristics of infections caused by Bacteroides, Prevotella and Fusobacterium species: a prospective observational study.

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.

Intracellular replication of fusobacteria requires new actin filament formation of epithelial cells.

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.

The changing pattern of Fusobacterium infections in humans: recent experience with Fusobacterium bacteraemia.

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.

Bacteriocin-like activity of oral Fusobacterium nucleatum isolated from human and non-human primates

"Fusobacterium nucleatum" is indigenous of the human oral cavity and has been involved in different infectious processes. The production of bacteriocin-like substances may be important in regulation of bacterial microbiota in oral cavity. The ability to produce bacteriocin-like substances by 80 oral "F. nucleatum" isolates obtained from periodontal patients, healthy individuals and "Cebus apella" monkeys, was examinated. 17.5(per cent) of all tested isolates showed auto-antagonism and 78.8(per cent) iso- or hetero-antagonism. No isolate from monkey was capable to produce auto-inhibition. In this study, the antagonistic substances production was variable in all tested isolates. Most of the "F. nucleatum" showed antagonistic activity against tested reference strains. These data suggest a possible participation of these substances on the oral microbial ecology in humans and animals. However, the role of bacteriocins in regulating dental plaque microbiota "in vivo" is discussed

Volatile fatty acid, metabolic by-product of periodontopathic bacteria, induces apoptosis in WEHI 231 and RAJI B lymphoma cells and splenic B cells.

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.

Microbiologic and pathologic aspects of pulpal and periapical disease.

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.

Meningitis por fusobacterium mortiferum / Meningitis by fusobacterium mortiferum

Se presenta el caso de una mujer de 33 años, que ingresó al Hospital de Enfermedades Infecciosas de Santiago de Chile, con una meningitis purulenta de origen ótico, cuyo agente etiológico, aislado del LCR y sangre, fue Fusobacterium mortiferum. Se comentan las complicaciones del SNC derivadas de estas patologías oto-sinuales crónicas, tan frecuentes en nuestro medio, recomendándose buscar microorganismos anaerobios en estos enfermos