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Considerable resources are being used to study associations between the human microbiota and malignancy. There is a particular interest in the connection between Fusobacterium animalis and colorectal cancer. In this paper, we correct recent taxonomic misconceptions of importance to this research and critically reassess proposed gene candidates for explaining F. animalis pathogenicity. We demonstrate the importance of strict adherence to taxonomic rules when discovering possibly novel phylogenetic groups and emphasize that genome references are still not available for all known bacteria. We reassess the phylogeny of the medically important Fusobacterium nucleatum group including F. animalis using contemporary approaches, provide a genome reference for Fusobacterium watanabei, and describe Fusobacterium paranimalis sp. nov. Our results dispute the concept of using a single closely related comparator phylogenetic group when searching for candidate genes potentially explaining species-specific pathogenicity and show that such comparative approaches can only be meaningful when all relevant related species are included.

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

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.

Background Fusobacterium species are anaerobic Gram-negative bacilli which are uncommon causes of bloodstream infection (BSI). This genus commonly colonises the gastrointestinal tract and can result in significant morbidity. Methods All blood cultures with growth of Fusobacterium species among residents of Queensland, Australia (population ≈ 5 million) were retrospectively identified over a 20-year period. Clinical, microbiological and outcome information was obtained from state-wide databases. Results 377 incident Fusobacterium species BSI among 375 individuals for an age and sex-standardised incidence of 4.4 per million residents per year. Median age was 47 years (IQR, 24.9–65.8) and 156 (42%) incident episodes were in females. There was a bimodal frequency distribution with respect to age with peaks occurring around 20 and 65 years, respectively. The most identified source of infection was the abdominal (17%), followed by head and neck (12%). 8% of patients had a septic thrombus present, and 4% had an abscess associated with their BSI. Most isolates were F. nucleatum (142, 38%) and F. necrophorum (140, 37%). 9% of isolates were resistant to penicillin. Older age (aHR 1.02, 95% CI 1.01–1.05), healthcare-associated hospital onset (aHR 3.16, 95% CI 1.35–7.40), and Charlson Comorbidity index (aHR 1.20, 95% CI 1.06–1.35) were all associated with 30-day all cause case-fatality. Oropharyngeal source appeared to be a protective factor ( P  = 0.02). Conclusions Fusobacterium species BSI results in significant morbidity and can cause death in vulnerable patient groups such as the elderly and those with malignancy. An identifiable oropharyngeal source identifies a favourable host.

Fusobacterium, a gram‐negative anaerobic bacillus in mouth, gastrointestinal tract and elsewhere, has long been considered as opportunistic pathogen. Increasing evidence indicate the association of Fusobacterium with human diseases, especially cancer. However, previous studies demonstrated contradictory prevalent features of Fusobacterium species in normal and patient population. To address this dissonance, we developed a high‐precision multiplex PCR assay that allows concurrent identification of five species of Fusobacterium (F. nucleatum, F. mortiferum, F. ulcerans, F. varium and F. necrophorum) and four subspecies of F. nucleatum (nucleatum, animalis, vincentii and polymorphum). By employing the PCR method, we investigated the prevalent features of Fusobacterium communities in Southern Chinese population and cancer patients. Surprisingly, we found F. nucleatum was dominant in both Southern Chinese population and cancer patients, and discovered the correlations of Fusobacterium species to host conditions. Moreover, F. mortiferum exhibited better diagnostic performance for cancers compared to other species, and the combination of F. mortiferum, F. nucleatum, body mass index and haemoglobin by a logistic regression model showed excellent diagnostic performances for cancers. Additionally, we determined the compositional features and loads of Fusobacterium communities in paired tumour, adjacent tissues and normal tissues of colorectal cancer and lung cancer. Hence, we developed a high‐precision multiplex PCR assay to profile Fusobacterium in human faeces and tumour, and demonstrate its prevalence in Southern Chinese population with correlations to host conditions and cancers. By large‐scale comparative genomics, we identified species and subspecies‐specific genetic markers in Fusobacterium, and developed a high‐precision multiplex PCR assay that allows concurrent identification of five species of Fusobacterium (F. nucleatum, F. mortiferum, F. ulcerans, F. varium and F. necrophorum) and four subspecies of F. nucleatum (nucleatum, animalis, vincentii and polymorphum).

Pyrosequencing of 16S rRNA genes allows for in-depth characterization of complex microbial communities. Although it is known that primer selection can influence the profile of a community generated by sequencing, the extent and severity of this bias on deep-sequencing methodologies is not well elucidated. We tested the hypothesis that the hypervariable region targeted for sequencing and primer degeneracy play important roles in influencing the composition of 16S pyrotag communities. Subgingival plaque from deep sites of current smokers with chronic periodontitis was analyzed using Sanger sequencing and pyrosequencing using 4 primer pairs. Greater numbers of species were detected by pyrosequencing than by Sanger sequencing. Rare taxa constituted nearly 6% of each pyrotag community and less than 1% of the Sanger sequencing community. However, the different target regions selected for pyrosequencing did not demonstrate a significant difference in the number of rare and abundant taxa detected. The genera Prevotella, Fusobacterium, Streptococcus, Granulicatella, Bacteroides, Porphyromonas and Treponema were abundant when the V1-V3 region was targeted, while Streptococcus, Treponema, Prevotella, Eubacterium, Porphyromonas, Campylobacter and Enterococcus predominated in the community generated by V4-V6 primers, and the most numerous genera in the V7-V9 community were Veillonella, Streptococcus, Eubacterium, Enterococcus, Treponema, Catonella and Selenomonas. Targeting the V4-V6 region failed to detect the genus Fusobacterium, while the taxa Selenomonas, TM7 and Mycoplasma were not detected by the V7-V9 primer pairs. The communities generated by degenerate and non-degenerate primers did not demonstrate significant differences. Averaging the community fingerprints generated by V1-V3 and V7-V9 primers provided results similar to Sanger sequencing, while allowing a significantly greater depth of coverage than is possible with Sanger sequencing. It is therefore important to use primers targeted to these two regions of the 16S rRNA gene in all deep-sequencing efforts to obtain representational characterization of complex microbial communities.

Non -nucleatum Fusobacterium may play a nonnegligible role in colorectal cancer (CRC) and certain Fusobacterium lineages (namely, L1 and L5) have shown specific associations with CRC. We aim to clarify the complex connections between Fusobacterium and CRC. We found that the widely adopted quantitative PCR (qPCR) method could overestimate F. nucleatum abundance and, in fact, reflect L1 levels in clinical samples. A lineage-specific qPCR assay targeting L1/L5 was developed and validated using mock and clinical samples. Its application in independent cohorts confirmed that L1 was overabundant in CRC, whereas L5 correlated with lymphovascular invasion. Importantly, faecal L1 abundance was more predictive of CRC than F. nucleatum , supported also by cross-population metagenomic data. CRC-associated virulence and colonisation genes were found in various L1 species other than F. nucleatum . Our results highlight the clinical importance of L1/L5 in CRC with high-diversity Fusobacterium contexts and suggest that non- nucleatum Fusobacterium may also contribute to CRC.

Periodontitis is a progressive disease of the periodontium with a complex, polymicrobial etiology. Recent Next-Generation Sequencing (NGS) studies of the microbial diversity associated with periodontitis have revealed strong, community-level differences in bacterial assemblages associated with healthy or diseased periodontal sites. In this study, we used NGS approaches to characterize changes in periodontal pocket bacterial diversity after standard periodontal treatment. Despite consistent changes in the abundance of certain taxa in individuals whose condition improved with treatment, post-treatment samples retained the highest similarity to pre-treatment samples from the same individual. Deeper phylogenetic analysis of periodontal pathogen-containing genera Prevotella and Fusobacterium found both unexpected diversity and differential treatment response among species. Our results highlight how understanding interpersonal variability among microbiomes is necessary for determining how polymicrobial diseases respond to treatment and disturbance.

Studies on the possible association between bacteria and oral squamous cell carcinoma (OSCC) remain inconclusive, largely due to methodological variations/limitations. The objective of this study was to characterize the species composition as well as functional potential of the bacteriome associated with OSCC. DNA obtained from 20 fresh OSCC biopsies (cases) and 20 deep-epithelium swabs (matched control subjects) was sequenced for the V1-V3 region using Illumina’s 2 × 300 bp chemistry. High quality, non-chimeric merged reads were classified to species level using a prioritized BLASTN-algorithm. Downstream analyses were performed using QIIME, PICRUSt, and LEfSe. Fusobacterium nucleatum subsp . polymorphum was the most significantly overrepresented species in the tumors followed by Pseudomonas aeruginosa and Campylobacter sp . Oral taxon 44, while Streptococcus mitis , Rothia mucilaginosa and Haemophilus parainfluenzae were the most significantly abundant in the controls. Functional prediction showed that genes involved in bacterial mobility, flagellar assembly, bacterial chemotaxis and LPS synthesis were enriched in the tumors while those responsible for DNA repair and combination, purine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, ribosome biogenesis and glycolysis/gluconeogenesis were significantly associated with the controls. This is the first epidemiological evidence for association of F . nucleatum and P . aeruginosa with OSCC. Functionally, an “inflammatory bacteriome” is enriched in OSSC.

Fusobacteria have been associated to different diseases, including colorectal cancer (CRC), but knowledge of which taxonomic groups contribute to specific conditions is incomplete. We analyzed the genetic diversity and relationships within the Fusobacterium genus. We report recent and ancestral recombination in core genes, indicating that fusobacteria have mosaic genomes and emphasizing that taxonomic demarcation should not rely on single genes/gene regions. Across databases, we found ample evidence of species miss-classification and of undescribed species, which are both expected to complicate disease association. By focusing on a lineage that includes F. periodonticum/pseudoperiodonticum and F. nucleatum , we show that genomes belong to four modern populations, but most known species/subspecies emerged from individual ancestral populations. Of these, the F. periodonticum/pseudoperiodonticum population experienced the lowest drift and displays the highest genetic diversity, in line with the less specialized distribution of these bacteria in oral sites. A highly drifted ancestral population instead contributed genetic ancestry to a new species, which includes genomes classified within the F. nucleatum animalis diversity in a recent CRC study. Thus, evidence herein calls for further evolutionary and phylogenomic analyses based on more Flavobacterium nucleatum genome sequences. More generally, our data inform future molecular profiling approaches to investigate the epidemiology of Fusobacterium -associated diseases. Parsing the genetic diversity within the Fusobacterium genus provides ample evidence of species miss-classification and of undescribed species, and calls for a re-analysis of F. nucleatum features associated to colorectal cancer.