Nucleatum Strains Research Articles

Antibiofilm efficacy of a calcium silicate-based intracanal medicament against Fusobacterium nucleatum strains

This study evaluated the antibiofilm efficacy of a calcium silicate-based intracanal medicament (Bio-C Temp) against Fusobacterium nucleatum (F. nucleatum). Dentin slices (n = 88) were inoculated with F. nucleatum biofilms and divided into experimental groups (n = 24 each) treated with Bio-C Temp, triple antibiotic paste (TAP), or Ca(OH)2 for 1 or 2 weeks. Untreated dentin slices exposed to saline served as a positive control, while sterile dentin slices incubated anaerobically served as a negative control. Bacterial viability was assessed using confocal laser scanning microscopy after staining with LIVE/DEAD®BacLight™ dye. Samples treated with TAP demonstrated the highest percentage of dead bacteria (95%). The antibiofilm effect of TAP and Bio-C Temp significantly increased from Week 1 to Week 2. At Week 2, samples treated with Bio-C Temp showed a significantly higher percentage of dead bacteria (75.25%) than those treated with Ca(OH)2 (58.65%). TAP showed superior efficacy against F. nucleatum biofilms. After an application time of 2 weeks, the antibiofilm efficacy of Bio-C Temp was superior to that of Ca(OH)2.

The Influence of Selected Titanium Alloy Micro-Texture Parameters on Bacterial Adhesion.

The colonization of microbes and the resulting formation of biofilms on dental implants are significant contributors to peri-implantitis and the failure of these implants. The aim of the research was to analyze the impact of density and depth of laser texturing of the Ti-6Al-7Nb alloy surface on the colonization of selected microorganisms and biofilm formation. Standard strains of Gram-negative and Gram-positive bacteria and yeasts from the American Type Culture Collection-ATCC-were used to demonstrate the ability to form single-species biofilms in vitro. The study evaluated three types of titanium samples with different texture density and depth. The colonization and biofilm formation abilities of the tested microorganisms were assessed. The obtained results were subjected to statistical analysis. Among the analyzed strains, L. rhamnosus showed the highest colonization of the tested surfaces. It was found that there is no relationship between the texture parameters and the number of colony-forming units (CFU/mL) for C. albicans, S. mutans, and L. rhamnosus. For the F. nucleatum strain, it was shown that the number of colony-forming bacteria is related to the texture density.

Progress of “Yiqi and Spleen Enhancement Method” in the Treatment of Colorectal Cancer based on Intestinal Flora

The intestinal microbiota is located close to the colorectal epithelium and consists of a large microbiota that interacts with host cells to regulate many physiological processes such as energy harvesting, metabolism and immune responses [1]. It corresponds to the functions of the “spleen” in Chinese medicine, which is responsible for the transportation of water and grains and the resistance to evil. The balance of intestinal flora directly reflects the functional status of the “spleen” in TCM. Sequencing studies have revealed the microbial composition and ecological changes in colorectal cancer (CRC) patients, and functional animal model studies have clarified the role of several bacteria in colorectal carcinogenesis, including Clostridium nucleatum, Escherichia coli, and Pseudomonas fragilis strains, which are closely related to CRC [2]. Based on this, this paper reviews the research progress on the mechanism of intestinal flora affecting CRC and the intervention of intestinal flora against CRC by Yiqi and Spleen Method, which provides ideas and references for the prevention of colorectal cancer by traditional Chinese medicine.

Oral inoculation of Fusobacterium nucleatum exacerbates ulcerative colitis via the secretion of virulence adhesin FadA

ABSTRACT Fusobacterium nucleatum (F. nucleatum), an anaerobic resident of the oral cavity, is increasingly recognized as a contributing factor to ulcerative colitis (UC). The adhesive properties of F. nucleatum are mediated by its key virulence protein, FadA adhesin. However, further investigations are needed to understand the pathogenic mechanisms of this oral pathogen in UC. The present study aimed to explore the role of the FadA adhesin in the colonization and invasion of oral F. nucleatum in dextran sulphate sodium (DSS)-induced colitis mice via molecular techniques. In this study, we found that oral inoculation of F. nucleatum strain carrying the FadA adhesin further exacerbated DSS-induced colitis, leading to elevated alveolar bone loss, disease severity, and mortality. Additionally, CDH1 gene knockout mice treated with DSS presented increases in body weight and alveolar bone density, as well as a reduction in disease severity. Furthermore, FadA adhesin adhered to its mucosal receptor E-cadherin, leading to the phosphorylation of β-catenin and the degradation of IκBα, the activation of the NF-κB signalling pathway and the upregulation of downstream cytokines. In conclusion, this research revealed that oral inoculation with F. nucleatum facilitates experimental colitis via the secretion of the virulence adhesin FadA. Targeting the oral pathogen F. nucleatum and its virulence factor FadA may represent a promising therapeutic approach for a portion of UC patients.

Draft genome sequence of a Fusobacterium nucleatum strain isolated from a patient in Kazakhstan with colorectal cancer.

Fusobacterium nucleatum is an invasive obligate anaerobe found in the oral microbiota and associated with colorectal cancer. Here, we announce the draft genome sequence of Fusobacterium nucleatum strain Fn11kaz from a patient with colorectal cancer in Kazakhstan.

Strain-level detection of Fusobacterium nucleatum in colorectal cancer specimens by targeting the CRISPR-Cas region.

Fusobacterium nucleatum is associated with colorectal cancer (CRC), and identical F. nucleatum strains seemed to be detected in 75% of patients who exhibited the presence of F. nucleatum in both CRC and saliva samples in our previous study; however, the validation of strain identity and the development of a method for strain-level discrimination are required for etiological studies. We confirmed that F. nucleatum isolates obtained from CRC and saliva samples derived from patients with CRC originated from their identical strains using whole-genome sequencing. We evaluated the hypervariable region of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CRISPR-Cas) system in F. nucleatum strains isolated from the CRC and saliva specimens of CRC patients to develop a method for genotyping this bacterium at the strain level. The developed method consisted of two simple PCR steps to amplify the different lengths and sequences of the CRISPR-Cas regions from F. nucleatum strains using specific but common primers. This method could successfully detect identical strains present in both cryopreserved CRC samples and saliva obtained from the same CRC patient. Dynamic monitoring of F. nucleatum strains in saliva obtained from patients with colorectal adenoma before and after oral care showed interindividual variability in F. nucleatum strains during oral care. This study provided a simple and rapid method for comprehensively identifying F. nucleatum at the strain level in clinical samples, leading to a paradigm shift in CRC research, such as the investigation of pathogenic F. nucleatum strains and monitoring of pathogenic strains in clinical trials for preventing CRC recurrence. (This study was registered in the UMIN Clinical Trials Registry under ID UMIN000016229.) IMPORTANCE Fusobacterium nucleatum is one of the predominant oral bacteria in humans. However, this bacterium is enriched in colorectal cancer (CRC) tissues and may be involved in CRC development. Our previous research suggested that F. nucleatum is present in CRC tissues originating from the oral cavity using a traditional strain-typing method [arbitrarily primed polymerase chain reaction (AP-PCR)]. First, using whole-genome sequencing, this study confirmed an exemplary similarity between the oral and tumoral strains derived from each patient with CRC. Second, we successfully developed a method to genotype this bacterium at the strain level, targeting the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated system, which is hypervariable (defined as F. nucleatum-strain genotyping PCR). This method can identify F. nucleatum strains in cryopreserved samples and is significantly superior to traditional AP-PCR, which can only be performed on isolates. The new methods have great potential for application in etiological studies of F. nucleatum in CRC.

Pangenomic Study of Fusobacterium nucleatum Reveals the Distribution of Pathogenic Genes and Functional Clusters at the Subspecies and Strain Levels.

Fusobacterium nucleatum is a prevalent periodontal pathogen and is associated with many systemic diseases. Our knowledge of the genomic characteristics and pathogenic effectors of different F. nucleatum strains is limited. In this study, we completed the whole genome assembly of the 4 F. nucleatum strains and carried out a comprehensive pangenomic study of 30 strains with their complete genome sequences. Phylogenetic analysis revealed that the F. nucleatum strains are mainly divided into 4 subspecies, while 1 of the sequenced strains was classified into a new subspecies. Gene composition analysis revealed that a total of 517 "core/soft-core genes" with housekeeping functions widely distributed in almost all the strains. Each subspecies had a unique gene cluster shared by strains within the subspecies. Analysis of the virulence factors revealed that many virulence factors were widely distributed across all the strains, with some present in multiple copies. Some virulence genes showed no consistent occurrence rule at the subspecies level and were specifically distributed in certain strains. The genomic islands mainly revealed strain-specific characteristics instead of subspecies level consistency, while CRISPR types and secondary metabolite biosynthetic gene clusters were identically distributed in F. nucleatum strains from the same subspecies. The variation in amino acid sites in the adhesion protein FadA did not affect the monomer and dimer 3D structures, but it may affect the binding surface and the stability of binding to host receptors. This study provides a basis for the pathogenic study of F. nucleatum at the subspecies and strain levels. IMPORTANCE We used F. nucleatum as an example to analyze the genomic characteristics of oral pathogens at the species, subspecies, and strain levels and elucidate the similarities and differences in functional genes and virulence factors among different subspecies/strains of the same oral pathogen. We believe that the unique biological characteristics of each subspecies/strain can be attributed to the differences in functional gene clusters or the presence/absence of certain virulence genes. This study showed that F. nucleatum strains from the same subspecies had similar functional gene compositions, CRISPR types, and secondary metabolite biosynthetic gene clusters, while pathogenic genes, such as virulence genes, antibiotic resistance genes, and GIs, had more strain level specificity. The findings of this study suggest that, for microbial pathogenicity studies, we should carefully consider the subspecies/strains being used, as different strains may vary greatly.

Intestinal cancer development in response to oral infection with high-fat diet-induced Type 2 diabetes (T2D) in collaborative cross mice under different host genetic background effects.

Type 2 diabetes (T2D) is a metabolic disease with an imbalance in blood glucose concentration. There are significant studies currently showing association between T2D and intestinal cancer developments. High-fat diet (HFD) plays part in the disease development of T2D, intestinal cancer and infectious diseases through many biological mechanisms, including but not limited to inflammation. Understanding the system genetics of the multimorbidity of these diseases will provide an important knowledge and platform for dissecting the complexity of these diseases. Furthermore, in this study we used some machine learning (ML) models to explore more aspects of diabetes mellitus. The ultimate aim of this project is to study the genetic factors, which underline T2D development, associated with intestinal cancer in response to a HFD consumption and oral coinfection, jointly or separately, on the same host genetic background. A cohort of 307 mice of eight different CC mouse lines in the four experimental groups was assessed. The mice were maintained on either HFD or chow diet (CHD) for 12-week period, while half of each dietary group was either coinfected with oral bacteria or uninfected. Host response to a glucose load and clearance was assessed using intraperitoneal glucose tolerance test (IPGTT) at two time points (weeks 6 and 12) during the experiment period and, subsequently, was translated to area under curve (AUC) values. At week 5 of the experiment, mice of group two and four were coinfected with Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn) strains, three times a week, while keeping the other uninfected mice as a control group. At week 12, mice were killed, small intestines and colon were extracted, and subsequently, the polyp counts were assessed; as well, the intestine lengths and size were measured. Our results have shown that there is a significant variation in polyp's number in different CC lines, with a spectrum between 2.5 and 12.8 total polyps on average. There was a significant correlation between area under curve (AUC) and intestine measurements, including polyp counts, length and size. In addition, our results have shown a significant sex effect on polyp development and glucose tolerance ability with males more susceptible to HFD than females by showing higher AUC in the glucose tolerance test. The ML results showed that classification with random forest could reach the highest accuracy when all the attributes were used. These results provide an excellent platform for proceeding toward understanding the nature of the genes involved in resistance and rate of development of intestinal cancer and T2D induced by HFD and oral coinfection. Once obtained, such data can be used to predict individual risk for developing these diseases and to establish the genetically based strategy for their prevention and treatment.

Therapeutic Target Identification and Inhibitor Screening against Riboflavin Synthase of Colorectal Cancer Associated Fusobacterium nucleatum.

Colorectal cancer (CRC) ranks third among all cancers in terms of prevalence. There is growing evidence that gut microbiota has a role in the development of colorectal cancer. Fusobacterium nucleatum is overrepresented in the gastrointestinal tract and tumor microenvironment of patients with CRC. This suggests the role of F. nucleatum as a potential risk factor in the development of CRC. Hence, we aimed to explore whole genomes of F. nucleatum strains related to CRC to predict potential therapeutic markers through a pan-genome integrated subtractive genomics approach. In the current study, we identified 538 proteins as essential for F. nucleatum survival, 209 non-homologous to a human host, and 12 as drug targets. Eventually, riboflavin synthase (RiS) was selected as a therapeutic target for further processing. Three different inhibitor libraries of lead-like natural products, i.e., cyanobactins (n = 237), streptomycins (n = 607), and marine bacterial secondary metabolites (n = 1226) were screened against it. After the structure-based study, three compounds, i.e., CMNPD3609 (−7.63) > Malyngamide V (−7.03) > ZINC06804365 (−7.01) were prioritized as potential inhibitors of F. nucleatum. Additionally, the stability and flexibility of these compounds bound to RiS were determined via a molecular dynamics simulation of 50 ns. Results revealed the stability of these compounds within the binding pocket, after 5 ns. ADMET profiling showed compounds as drug-like, non-permeable to the blood brain barrier, non-toxic, and HIA permeable. Pan-genomics mediated drug target identification and the virtual screening of inhibitors is the preliminary step towards inhibition of this pathogenic oncobacterium and we suggest mouse model experiments to validate our findings.

Structure of the lipopolysaccharide O-antigens from Fusobacterium nucleatum strains HM-994, HM-995, HM-997

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. It was also found in colorectal cancer tissues and is linked with pregnancy complications, including pre-term and stillbirths. Cell surface structures of the bacterium could be implicated in pathogenesis. Here we report the structure of the lipopolysaccharide O-chain (OPS) of three strains of F. nucleatum HM-994, HM-995, and HM-997, isolated from cancerous tissues:-3-β-D-ManNAc4Lac-4-β-D-Glc6OAc-3-β-D-FucNAc4N- HM-994.-4-α-L-GalNHBuA-3-α-D-QuiNAc4NHBu-3-α-L-Rha-6-α-D-GalN- HM-995.-3-[α-L-GulNAcA-4-]-β-D-Glc-4-β-D-ManNAcAN-3-β-D-FucNAc4N-3- HM-997.where HBu is 3-hydroxybutyryl, ManNAc4Lac is 4-O-(1-carboxyethyl)-2-acetamido-2-deoxy-mannose. All monosaccharides are in the pyranose form. The structures were determined using standard NMR (2D homo- and hetero-nuclear techniques), MS and chemical methods following gtypical LPS isolation and purification methods. In some cases polymeric material was further degraded in order to produce compounds that gave improved NMR spectra that were easier to be fully interpreted. Structure of the OPS from strain HM-994 was identical to the OPS from F. nucleatum strain MJR 7757 B. Structures of the OPS from HM-995 and HM-997 are novel and to our knowledge have not been previously reported and include the often observed 6-deoxy- sugars found in several F. nucleatum strains and butyrate rather than acetate modifications in the HM-995 strain. This structural knowledge adds to the ever increasing variation found in LPS O-antigen structures from F. nucleatum strain from both oral and cancerous origin and suggests that there may be a multitude of different LPS O-antigen structures elaborated by this organism that may present challenges to any serotyping efforts.

The structure of the LPS O-chain from five Fusobacterium nucleatum strains CTX47T, CC2_6JVN3, CC2_3FMU1, CC2_1JVN3, HM-996, containing alditol and phosphate in the main chain and development of mouse monoclonal antibodies specific to the O-antigens

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. It was also found in colorectal cancer tissues and is linked with pregnancy complications, including pre-term and stillbirths. Cell surface structures of the bacterium could be implicated in pathogenesis. Here we report four new structures of the lipopolysaccharide O-chain (OPS) from five strains of F. nucleatum CTX47T, CC2_6JVN3, CC2_3FMU1, CC2_1JVN3, HM-996, isolated from cancerous tissues. Three of the four structures have a common sequence of hexose-diaminofucose-hexitol-phosphate in the main chain:

Abstract 3052: Fusobacterial-dominant colorectal cancer biofilms are associated with high levels of the polyamine N1,N12-diacetylspermine

Abstract Background: Mucus-invasive colonic bacterial biofilms are dense, polymicrobial communities that are in direct contact with the epithelium. Prior data from our group has demonstrated that these invasive biofilms are prevalent in colorectal cancer, are directly tumorigenic in germ-free and specific pathogen-free ApcMin/+ mouse models, and are associated with enrichment of the polyamine N1,N12-diactylspermine (DAS) in patient samples. However, whether the composition of these biofilms impacts their pro-tumorigenic behavior and/or metabolism is presently unknown. Methods: Carnoy’s-fixed CRC resections from 115 patients from the University of Malaya in Malaysia were stained with the all-bacterial EUB338 probe and screened for mucus-invasive biofilms (BF). All BF+ samples were subsequently stained with oligonucleotide probes targeting Bacteroidetes, Fusobacteria, Lachnospiraceae, and γ/β-Proteobacteria. Imaging was performed on a Zeiss780 confocal microscope with linear unmixing. Untargeted metabolomics was performed on 5-10 samples of each biofilm type using a Waters UPLC Xevo GS-XS quadrupole time-of-flight (QTOF) mass spectrometer. Five Fusobacterium nucleatum (Fn) strains derived from CRC cohorts were singly gavaged into germ-free ApcMin/+ mice and colons were harvested for tumors 11 wk later. Results: Seventy-nine of 115 Malaysian tumors (69%) were BF+ using EUB338 staining. As with our prior US CRC cohort, BFs were more prevalent on the tumors proximal to the hepatic flexure (35/39, 90%) vs. those that were distal (44/76, 58%) (Chi-square p < 0.001). Of the BF+ tumors (BF+T), 55% were polymicrobial (predominantly Bacteroidetes/Lachnospiraceae), 40% were polymicrobial with fusobacterial blooms, and 5% were Proteobacteria dominant. Untargeted metabolomics of BF- healthy biopsies, BF-T, and BF+T with various BF composition subtypes revealed a gradient in levels of the polyamine DAS, with BF+T with fusobacterial blooms having the highest levels (p = 0.055 vs. Proteo-dominant BF+T, p = 0.043 vs. polymicrobial BF+T, p = 0.015 vs. BF-T, p < 0.001 vs. BF- healthy biopsies), suggesting that fusobacteria may drive proliferation and polyamine production in CRC tissues. However, inoculation of 5 different CRC-derived Fn strains including a strain isolated from a Malaysia CRC patient did not promote tumorigenesis in germ-free ApcMin/+ mice. Conclusions: Fusobacterial-dominant biofilms are prevalent in a Malaysian CRC cohort and are associated with high levels of the polyamine N1,N12-diacetylspermine. However, F. nucleatum strains were not tumorigenic in germ-free ApcMin/+ mice, suggesting that the association between Fn, DAS, and tumorigenesis may require other bacteria in order to confer a procarcinogenic state. Citation Format: Julia L. Drewes, Jessica Queen, Jada C. Domingue, Caroline R. Wensel, Yuping Cai, Jane Wanyiri, April C. Roslani, Jamuna Vadivelu, Caroline H. Johnson, Cynthia L. Sears. Fusobacterial-dominant colorectal cancer biofilms are associated with high levels of the polyamine N1,N12-diacetylspermine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3052.

Structure of the lipopolysaccharide O-antigens from Fusobacterium nucleatum strains SB-106CP and HM-992 and immunological comparison to the O-antigen of strain 12230

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. It was also found in colorectal cancer tissues and is linked with pregnancy complications, including pre-term and stillbirths. Cell surface structures of the bacterium could be implicated in pathogenesis. Here we report the structures of the lipopolysaccharide O-chain (OPS) of two strains of F. nucleatum, SB-106CP and HM-992, both isolated from cancerous tissues. These strains elaborate the same sugar chain, differing only by their N-acylation pattern:-6-α-D-GlcNAc-4-β-D-GlcNHBu3NABuA-3-β-D-QuiNAc4NABuAc- SB-106CP-6-α-D-GlcNAc-4-β-D-GlcNHBu3NABuA-3-β-D-QuiNAc4NAc- HM-992ABu = (R)-3-amino-butyrylAbuAc = (R)-3-N-acetyl-3-aminobutyrylHBu = (R)-3-hydroxy-butyrylAll monosaccharides are in the pyranose form.Previously we published the structure of the OPS from F. nucleatum 12230, a transtracheal isolate, which had similar sugar chain, differing by replacement of GlcNAc with Glc and a different acylation pattern:-6-α-d-Glc-4-β-d-GlcNHBu3NHBuA-3-β-d-QuiNAc4NABu-A mouse monoclonal antibody specific for the 12230 O-antigen did not cross react with the LPS of strains SB-106CP and HM-992 confirming the structural differentiation.

Transcriptome analysis of Fusobacterium nucleatum reveals differential gene expression patterns in the biofilm versus planktonic cells

As a chronic infectious disease, periodontitis can cause gum recession, loss of alveolar bone, loosening of teeth, and even loss of teeth. Dental plaque biofilm is the initiating factor for the occurrence and development of periodontitis. Fusobacterium nucleatum (F. nucleatum) plays a vital role in the structure and ecology of dental plaque biofilms. It is a bridge between early and late colonization bacteria in dental plaque. Understanding the molecular mechanism of F. nucleatum during biofilm development is essential to control periodontitis. This study aimed to determine gene expression profiles of the F. nucleatum strain, ATCC 25586, in the planktonic and biofilm phase through RNA-sequencing approach. The results were confirmed by quantitative reverse transcriptase PCR (RT-qPCR). The results clearly illustrate the difference in gene expression of F. nucleatum under planktonic and biofilms. A total of 110 genes were differentially expressed by F. nucleatum in the biofilm state compared with the planktonic state. The 25 upregulated genes in the biofilm state were mainly related to carbohydrate and amino acid metabolism, while the 85 downregulated genes were primarily associated with cell growth, division, and oxidative stress; most of the upregulated genes of F. nucleatum involved in virulence and oral malodor. Furthermore, the transcriptome analysis and antibacterial activity test also identified Lysine might exhibit the antibacterial and antibiofilm activity of F. nucleatum for the first time. These new findings could provide caveats for future studies on the regulation and maintenance of plaque biofilm and the development of biomarkers for periodontitis.

Expression, Purification, and Characterization of the Recombinant, Two-Component, Response Regulator ArlR from Fusobacterium nucleatum.

Fusobacterium nucleatum is associated with the incidence and development of multiple diseases, such as periodontitis and colorectal cancer (CRC). Until now, studies have proved only a few proteins to be associated with such pathogenic diseases. The two-component system is one of the most prevalent forms of bacterial signal transduction related to intestinal diseases. Here, we report a novel, recombinant, two-component, response regulator protein ArlR from the genome of F. nucleatum strain ATCC 25,586. We optimized the expression and purification conditions of ArlR; in addition, we characterized the interaction of this response regulator protein with the corresponding histidine kinase and DNA sequence. The full-length ArlR was successfully expressed in six E. coli host strains. However, optimum expression conditions of ArlR were present only in E. coli strain BL21 CodonPlus (DE3) RIL that was later induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) for 8h at 25°C. The SDS-PAGE analysis revealed the molecular weight of the recombinant protein as 27.3kDa with approximately 90% purity after gel filtration chromatography. Because ArlR was biologically active after its purification, it accepted the corresponding phosphorylated histidine kinase phosphate group and bound to the analogous DNA sequence. The binding constant between ArlR and the corresponding histidine kinase was about 2.1μM, whereas the binding constant between ArlR and its operon was 6.4μM. Altogether, these results illustrate an effective expression and purification method for the novel two-component system protein ArlR.

Siglec-7 Mediates Immunomodulation by Colorectal Cancer-Associated Fusobacterium nucleatum ssp. animalis.

Fusobacterium nucleatum is involved in the development of colorectal cancer (CRC) through innate immune cell modulation. However, the receptors of the interaction between F. nucleatum ssp. and immune cells remain largely undetermined. Here, we showed that F. nucleatum ssp. animalis interacts with Siglecs (sialic acid–binding immunoglobulin-like lectins) expressed on innate immune cells with highest binding to Siglec-7. Binding to Siglec-7 was also observed using F. nucleatum-derived outer membrane vesicles (OMVs) and lipopolysaccharide (LPS). F. nucleatum and its derived OMVs or LPS induced a pro-inflammatory profile in human monocyte-derived dendritic cells (moDCs) and a tumour associated profile in human monocyte-derived macrophages (moMϕs). Siglec-7 silencing in moDCs or CRISPR-cas9 Siglec-7-depletion of U-937 macrophage cells altered F. nucleatum induced cytokine but not marker expression. The molecular interaction between Siglec-7 and the LPS O-antigen purified from F. nucleatum ssp. animalis was further characterised by saturation transfer difference (STD) NMR spectroscopy, revealing novel ligands for Siglec-7. Together, these data support a new role for Siglec-7 in mediating immune modulation by F. nucleatum strains and their OMVs through recognition of LPS on the bacterial cell surface. This opens a new dimension in our understanding of how F. nucleatum promotes CRC progression through the generation of a pro-inflammatory environment and provides a molecular lead for the development of novel cancer therapeutic approaches targeting F. nucleatum-Siglec-7 interaction.

Epidemiology and antibiotic susceptibility in anaerobic bacteraemia: a 15-year retrospective study in South-Eastern Hungary

Introduction Bloodstream infections are considered as important factors of morbidity and represent a remarkable number of death, especially in developed countries. Methods In a 15-year retrospective study in the South-Eastern region of Hungary, the microbiological results of anaerobic blood culture (BC) bottles were analysed. The incubation of BCs was performed in the automated incubation system, and the isolates were cultured in an anaerobic chamber. The identification of the isolates was based on their biochemical profile (2004–2012) and mass spectrometry analyses (2013–2018). Antibiotic susceptibility testing was performed with minimal inhibitory concentration gradient diffusion method. Results The majority of the samples were submitted by the Department of Internal Medicine (37.7%), Intensive Care Unit (31.6%) and the Emergency Department (16.4%), and the number of strict anaerobic isolates per year showed an increasing tendency throughout the study. More than half of the isolates were Cutibacterium acnes (54.4%), but this bacterium was considered as a contaminant. The most frequent species upon Cutibacterium acnes were Bacteroides fragilis (9.2%), Clostridium perfringens (6.4%), other Clostridium species (4.8%), other Cutibacterium species (3.5%) and other Bacteroides species (2.5%). Penicillin, amoxicillin/clavulanic acid, imipenem, clindamycin, metronidazole and tigecyclin were very effective against Bacteroides fragilis, Clostridium perfringens, Fusobacterium nucleatum, Gemella morbillorum, Parvimonas micra and Eggerthella lenta strains. Conclusion We performed a 15-year period, retrospective investigation in South-Eastern Hungary with the participation of more than 1000 patients. The results of antibiotic susceptibility tests are in line with previous reports in the literature, and the results from the different geographical regions of the world suggest similar resistance mechanisms.

Interaction of periodontitis and orthodontic tooth movement\u2014an in vitro and in vivo study

ObjectivesThe aim of this in vitro and in vivo study was to investigate the interaction of periodontitis and orthodontic tooth movement on interleukin (IL)-6 and C-X-C motif chemokine 2 (CXCL2).Materials and methodsThe effect of periodontitis and/or orthodontic tooth movement (OTM) on alveolar bone and gingival IL-6 and CXCL2 expressions was studied in rats by histology and RT-PCR, respectively. The animals were assigned to four groups (control, periodontitis, OTM, and combination of periodontitis and OTM). The IL-6 and CXCL2 levels were also studied in human gingival biopsies from periodontally healthy and periodontitis subjects by RT-PCR and immunohistochemistry. Additionally, the synthesis of IL-6 and CXCL2 in response to the periodontopathogen Fusobacterium nucleatum and/or mechanical strain was studied in periodontal fibroblasts by RT-PCR and ELISA.ResultsPeriodontitis caused an increase in gingival levels of IL-6 and CXCL2 in the animal model. Moreover, orthodontic tooth movement further enhanced the bacteria-induced periodontal destruction and gingival IL-6 gene expression. Elevated IL-6 and CXCL2 gingival levels were also found in human periodontitis. Furthermore, mechanical strain increased the stimulatory effect of F. nucleatum on IL-6 protein in vitro.ConclusionsOur study suggests that orthodontic tooth movement can enhance bacteria-induced periodontal inflammation and thus destruction and that IL-6 may play a pivotal role in this process.Clinical relevanceOrthodontic tooth movement should only be performed after periodontal therapy. In case of periodontitis relapse, orthodontic therapy should be suspended until the periodontal inflammation has been successfully treated and thus the periodontal disease is controlled again.

Fusobacterium nucleatum drives a pro-inflammatory intestinal microenvironment through metabolite receptor-dependent modulation of IL-17 expression

ABSTRACT The colorectal cancer (CRC)-associated microbiota creates a pro-tumorigenic intestinal milieu and shapes immune responses within the tumor microenvironment. However, how oncomicrobes – like Fusobacterium nucleatum, found in the oral cavity and associated with CRC tissues– affect these distinct aspects of tumorigenesis is difficult to parse. Herein, we found that neonatal inoculation of ApcMin/+ mice with F. nucleatum strain Fn7-1 circumvents technical barriers preventing its intestinal colonization, drives colonic Il17a expression prior to tumor formation, and potentiates intestinal tumorigenesis. Using gnotobiotic mice colonized with a minimal complexity microbiota (the altered Schaedler’s flora), we observed that intestinal Fn7-1 colonization increases colonic Th17 cell frequency and their IL-17A and IL-17F expression, along with a concurrent increase in colonic lamina propria Il23p19 expression. As Fn7-1 stably colonizes the intestinal tract in our models, we posited that microbial metabolites, specifically short-chain fatty acids (SCFA) that F. nucleatum abundantly produces in culture and, as we demonstrate, in the intestinal tract, might mediate part of its immunomodulatory effects in vivo. Supporting this hypothesis, we found that Fn7-1 did not alter RORγt+ CD4+T cell frequency in the absence of the SCFA receptor FFAR2. Taken together, our work suggests that F. nucleatum influences intestinal immunity by shaping Th17 responses in an FFAR2-dependent manner, although further studies are necessary to clarify the precise and multifaceted roles of FFAR2. The potential to increase intestinal Th17 responses is shared by another oncomicrobe, enterotoxigenic Bacteroides fragilis, highlighting a conserved pathway that could potentially be targeted to slow oncomicrobe-mediated CRC.

High-salt and glucose diet attenuates alveolar bone recovery in a ligature-induced rat model of experimental periodontitis

Excessive intake of sodium caused by high salt diet promotes the expression of inflammatory cytokines and differentiation of helper T cells resulting in inflammatory responses. High-glucose diet also contributes to the pathogenesis of periodontitis by inducing changes in the oral microbiome and reducing salivation. However, the effect of a high-salt and glucose diet (HSGD) on the prognosis of periodontitis remains unclear. In this study, a rat model of experimental periodontitis was established by periodic insertion of absorbable sutures containing Porphyromonas gingivalis and Fusobacterium nucleatum strains into the right gingival sulcus to analyze the effect of HSGD on the incidence and progression of periodontitis. The alveolar bone heights (ABH) was measured with microcomputed tomography imaging of the HSGD- and general diet (GD)-treated groups. The right ABH was significantly decreased compared to the left in both groups at 4 weeks after induction of inflammation; however, no significant difference was noted between the groups. Notably, the ABH in the HSGD-treated group was significantly decreased at 8 weeks after induction of inflammation, whereas in the GD-treated group, an increase in the ABH was observed; a significant difference of the ABH was noted between the two groups (p < 0.05). At 12 weeks, recovery of the alveolar bone was observed in both groups, with no significant differences in ABH between the two groups. These findings indicate that the intake of excessive sodium attenuates the recovery rate of the alveolar bone even after the local infectant is removed. In addition, this study demonstrates the use of HSGD in establishing a new animal model of periodontitis.