Role of the Filifactor alocis Hypothetical Protein FA519 in Oxidative Stress Resistance.

In the periodontal pocket, there is a direct correlation between environmental conditions, the dynamic oral microbial flora, and disease. The relative abundance of several newly recognized microbial species in the oral microenvironment has raised questions on their impact on disease development. One such organism, Filifactor alocis, is significant to the pathogenic biofilm structure. Moreover, its pathogenic characteristics are highlighted by its ability to survive in the oxidative-stress microenvironment of the periodontal pocket and alter the microbial community dynamics. There is a gap in our understanding of its mechanism(s) of oxidative stress resistance and impact on pathogenicity. Several proteins, including HMPRFF0389-00519 (FA519), were observed in high abundance in F. alocis during coinfection of epithelial cells with Porphyromonas gingivalis W83. Bioinformatics analysis shows that FA519 contains a "Cys-X-X-Cys zinc ribbon domain" which could be involved in DNA binding and oxidative stress resistance. We have characterized FA519 to elucidate its roles in the oxidative stress resistance and virulence of F. alocis. Compared to the wild-type strain, the F. alocis isogenic gene deletion mutant, FLL1013 (ΔFA519::ermF), showed significantly reduced sensitivity to hydrogen peroxide and nitric oxide-induced stress. The ability to form biofilm and adhere to and invade gingival epithelial cells was also reduced in the isogenic mutant. The recombinant FA519 protein was shown to protect DNA from Fenton-mediated damage with an intrinsic ability to reduce hydrogen peroxide and disulfide bonds. Collectively, these results suggest that FA519 is involved in oxidative stress resistance and can modulate important virulence attributes in F. alocis. IMPORTANCE Filifactor alocis is an emerging member of the periodontal community and is now proposed to be a diagnostic indicator of periodontal disease. However, due to the lack of genetic tools available to study this organism, not much is known about its virulence attributes. The mechanism(s) of oxidative stress resistance in F. alocis is unknown. Therefore, identifying the adaptive mechanisms utilized by F. alocis to survive in the oxidative stress environment of the periodontal pocket would lead to understanding its virulence regulation, which could help develop novel therapeutic treatments to combat the effects of periodontal disease. This study is focused on the characterization of FA519, a hypothetical protein in F. alocis, as a multifunctional protein that plays an important role in the reactive oxygen species-detoxification pathway. Collectively, our results suggest that FA519 is involved in oxidative stress resistance and can modulate important virulence attributes in F. alocis.

Structure of a laminarin-type ß-(1→3)-glucan from brown algae Sargassum henslowianum and its potential on regulating gut microbiota.

A homogeneous polysaccharide named SHNP with apparent molecular weight of 8.4 kDa was purified from brown algae Sargassum henslowianum using ethanol precipitation, ion-exchange chromatography, and gel-filtration column chromatography. Structural analyses reveal that SHNP is completely composed of glucose, and its backbone consists of ß-D-(1→3)-Glcp with side chains comprising t-ß-D-Glcp attached at the O-6 position. Thus, SHNP is a laminarin-type polysaccharide. In vitro fermentation test results showed that SHNP was digested by gut microbiota; the pH value in the fecal culture of SHNP was significantly decreased; and total short-chain fatty acids, acetic, propionic and n-butyric acids were significantly increased. Furthermore, SHNP regulated the intestinal microbiota composition by stimulating the growth of species belonging to Enterobacteriaceae while depleting Haemophilus parainfluenzae and Gemmiger formicilis. Taken together, these results indicate that SHNP has the potential for regulating gut microbiota, but its specific role in the regulation requires to be further investigated.

Paraclostridium dentum, a novel species with pathogenic features isolated from human dental plaque sample.

A strictly anaerobic bacterial strain designated as SKVG24 was isolated from subgingival dental plaque samples of patients suffering from periodontitis. Cells were stained Gram-positive, rod shaped with endospore. The strain showed negative reaction to catalase and oxidase enzymes, but positive for gelatinase activity. Optimal growth was observed at 37 °C temperature and 7.0 pH. The 16S rRNA gene sequence BLAST analysis assigned strain SKVG24 to the genus Paraclostridium as it displayed 99.93% identity with P. benzoelyticum JC272T followed by P. bifermentans ATCC 638T (99.79%). However, average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) of the whole genome sequence showed <97% and <70% identity, respectively, with type strains of all closely related species. The G + C content of the DNA was 28.7 mol%. Total lipids profile showed presence of glycolipids as major lipids. Pathogenic features like hemolysis, gelatin hydrolysis and production of volatile sulfur compounds exhibited by strain SKVG24T were analogous to those observed in the established oral pathogenic strains. Further, whole genome sequence analysis confirmed the presence of genes encoding virulence factors and provided genomic insights on adaptation of the strain in oral environment. Based on the phenotypic and genetic differences with phylogenetic relatives, strain SKVG24T is proposed to represent a new species of the genus Paraclostridium with potential pathogenic ability, for which the name Paraclostridium dentum sp. nov., is suggested. The proposed type strain is SKVG24T (MTCC 12836T; = JCM 32760T).

TH1 cell-inducing Escherichia coli strain identified from the small intestinal mucosa of patients with Crohn's disease.

Dysbiotic microbiota contributes to the pathogenesis of Crohn's disease (CD) by regulating the immune system. Although pro-inflammatory microbes are probably enriched in the small intestinal (SI) mucosa, most studies have focused on fecal microbiota. This study aimed to examine jejunal and ileal mucosal specimens from patients with CD via double-balloon enteroscopy. Comparative microbiome analysis revealed that the microbiota composition of CD SI mucosa differs from that of non-CD controls, with an increased population of several families, including Enterobacteriaceae, Ruminococcaceae, and Bacteroidaceae. Upon anaerobic culturing of the CD SI mucosa, 80 bacterial strains were isolated, from which 9 strains representing 9 distinct species (Escherichia coli, Ruminococcus gnavus, Klebsiella pneumoniae, Erysipelatoclostridium ramosum, Bacteroides dorei, B. fragilis, B. uniformis, Parabacteroides distasonis, and Streptococcus pasteurianus) were selected on the basis of their significant association with CD. The colonization of germ-free (GF) mice with the 9 strains enhanced the accumulation of TH1 cells and, to a lesser extent, TH17 cells in the intestine, among which an E. coli strain displayed high potential to induce TH1 cells and intestinal inflammation in a strain-specific manner. The present results indicate that the CD SI mucosa harbors unique pro-inflammatory microbiota, including TH1 cell-inducing E. coli, which could be a potential therapeutic target.

Investigation of Filifactor alocis in primary and in secondary endodontic infections: A molecular study.

OBJECTIVE: Identification of specific bacteria in root canals (RCs) in distinct clinical conditions can support the comprehension of pathological processes. Thus, the objective of this clinical study was to investigate the presence of F. alocis in RCs of teeth with primary endodontic infection (PEI) and with persistent/secondary endodontic infection (SEI) by using molecular techniques. It was also aimed to associate its presence with the clinical features. In addition, the levels of F. alocis as well as the total bacterial cells in the samples were also quantitated. DESIGN: One hundred teeth (50 PEI and 50 SEI) were included. Microbial samples were performed using sterile paper points and assessed by using nested PCR and quantitative Real Time PCR (qPCR). The prevalence of F.alocis in RCs from PEI and SEI were compared by chi-square analysis. Fisher´s exact test or Pearson Chi-square, when appropriate, was used to test associations between clinical and radiographic features and the presence of F. alocis. Significance level was set at 5%. RESULTS: F. alocis was detected in 23 and 28 (PEI) and 12 and 11 (SEI) RCs using Nested PCR and qPCR, respectively. Statistically significant associations were found between the presence of F. alocis and PEI, pain, wet canals, swelling, abscess and purulent exudate (P < 0.05). Total bacterial count was similar in both conditions (P > 0.05). CONCLUSIONS: PEI harbour a significantly higher number of F. alocis than those with SEI. Filifactor alocis was significantly associated with clinical features in primary endodontic infections. Total bacterial count was similar in both clinical conditions.

A dysregulated bile acid-gut microbiota axis contributes to obesity susceptibility.

BACKGROUND: The composition of the bile acid (BA) pool is closely associated with obesity and is modified by gut microbiota. Perturbations of gut microbiota shape the BA composition, which, in turn, may alter important BA signaling and affect host metabolism. METHODS: We investigated BA composition of high BMI subjects from a human cohort study and a high fat diet (HFD) obesity prone (HF-OP) / HFD obesity resistant (HF-OR) mice model. Gut microbiota was analysed by metagenomics sequencing. GLP-1 secretion and gene regulation studies involved ELISA, qPCR, Western blot, Immunohistochemistry, and Immunofluorescence staining. FINDINGS: We found that the proportion of non-12-OH BAs was significantly decreased in the unhealthy high BMI subjects. The HF-OR mice had an enhanced level of non-12-OH BAs. Non-12-OH BAs including ursodeoxycholate (UDCA), chenodeoxycholate (CDCA), and lithocholate (LCA) were decreased in the HF-OP mice and associated with altered gut microbiota. Clostridium scindens was decreased in HF-OP mice and had a positive correlation with UDCA and LCA. Gavage of Clostridium scindens in mice increased the levels of hepatic non-12-OH BAs, accompanied by elevated serum 7α-hydroxy-4-cholesten-3-one (C4) levels. In HF-OP mice, altered BA composition was associated with significantly downregulated expression of GLP-1 in ileum and PGC1α, UCP1 in brown adipose tissue. In addition, we identified that UDCA attenuated the high fat diet-induced obesity via enhancing levels of non-12-OH BAs. INTERPRETATION: Our study highlights that dysregulated BA signaling mediated by gut microbiota contributes to obesity susceptibility, suggesting modulation of BAs could be a promising strategy for obesity therapy.

Biological characteristics associated with virulence in Clostridioides difficile ribotype 002 in Hong Kong.

Clostridioides difficile infection (CDI) is a common cause of nosocomial diarrhea and can sometimes lead to pseudo-membranous colitis and toxic megacolon. We previously reported that the PCR ribotype 002 was a common C. difficile ribotype in Hong Kong that was associated with increased mortality. In this study, we assessed in vitro bacteriological characteristics and in vivo virulence of ribotype 002 compared to other common ribotypes, including ribotypes 012, 014 and 046. We observed significantly higher toxin A (p < 0.05) and toxin B (p < 0.05) production, sporulation (p < 0.001) and germination rates (p < 0.0001) in ribotype 002 than other common ribotypes. In a murine model of C. difficile infection, ribotype 002 caused significantly more weight loss (p < 0.001) and histological damage (p < 0.001) than other common ribotypes. These findings may have contributed to the higher prevalence and mortality observed, and provided mechanistic insights that can help public surveillance and develop novel therapeutics to combat against this infection.

Filifactor alocis and Dialister pneumosintes in a Mexican population affected by periodontitis and rheumatoid arthritis: An exploratory study.

Filifactor alocis and Dialister pneumosintes have been associated with the initiation and progression of periodontitis (PE). We determined and compared the frequency of both bacteria in patients with PE, rheumatoid arthritis (RA), and PE/RA simultaneously. Detection was performed by polymerase chain reaction in the subgingival biofilm. Bacteria were more frequent in patients with PE, and clinical periodontal parameters such as pocket depth (PD) and clinical attachment loss (CAL) were significantly higher in patients with PE/RA. F. alocis and D. pneumosintes could influence PD and CAL, hence participating in the initiation and progression of PE in patients with RA.

Ruminococcus gnavus, a member of the human gut microbiome associated with Crohn's disease, produces an inflammatory polysaccharide.

A substantial and increasing number of human diseases are associated with changes in the gut microbiota, and discovering the molecules and mechanisms underlying these associations represents a major research goal. Multiple studies associate Ruminococcus gnavus, a prevalent gut microbe, with Crohn's disease, a major type of inflammatory bowel disease. We have found that R. gnavus synthesizes and secretes a complex glucorhamnan polysaccharide with a rhamnose backbone and glucose sidechains. Chemical and spectroscopic studies indicated that the glucorhamnan was largely a repeating unit of five sugars with a linear backbone formed from three rhamnose units and a short sidechain composed of two glucose units. The rhamnose backbone is made from 1,2- and 1,3-linked rhamnose units, and the sidechain has a terminal glucose linked to a 1,6-glucose. This glucorhamnan potently induces inflammatory cytokine (TNFα) secretion by dendritic cells, and TNFα secretion is dependent on toll-like receptor 4 (TLR4). We also identify a putative biosynthetic gene cluster for this molecule, which has the four biosynthetic genes needed to convert glucose to rhamnose and the five glycosyl transferases needed to build the repeating pentasaccharide unit of the inflammatory glucorhamnan.

Genetic effects on the commensal microbiota in inflammatory bowel disease patients.

Several bacteria in the gut microbiota have been shown to be associated with inflammatory bowel disease (IBD), and dozens of IBD genetic variants have been identified in genome-wide association studies. However, the role of the microbiota in the etiology of IBD in terms of host genetic susceptibility remains unclear. Here, we studied the association between four major genetic variants associated with an increased risk of IBD and bacterial taxa in up to 633 IBD cases. We performed systematic screening for associations, identifying and replicating associations between NOD2 variants and two taxa: the Roseburia genus and the Faecalibacterium prausnitzii species. By exploring the overall association patterns between genes and bacteria, we found that IBD risk alleles were significantly enriched for associations concordant with bacteria-IBD associations. To understand the significance of this pattern in terms of the study design and known effects from the literature, we used counterfactual principles to assess the fitness of a few parsimonious gene-bacteria-IBD causal models. Our analyses showed evidence that the disease risk of these genetic variants were likely to be partially mediated by the microbiome. We confirmed these results in extensive simulation studies and sensitivity analyses using the association between NOD2 and F. prausnitzii as a case study.

Clostridioides difficile-related toxic megacolon after Cryptococcus neoformans cellulitis: A complex of two rare infections in an immunocompromised host.

A 76-year-old Japanese woman was admitted due to uncontrolled cellulitis of the right lower leg. She had deep vein thrombosis on the right limb. Moreover, she had a long history of rheumatoid arthritis treated with corticosteroids. Skin biopsy and lumbar puncture were performed to diagnose disseminated cryptococcosis. She was administered antifungal agents (liposomal amphotericin B and 5-fluorocytosine). On treatment day 14, debridement was performed, and cryptococcosis was controlled. However, she developed toxic megacolon due to Clostridioides difficile infection (CDI). On day 32, she was transferred to the intensive care unit due to severe acidosis and acute kidney injury secondary to CDI-related toxic megacolon. Vancomycin, metronidazole, and tigecycline were administered for treatment of CDI. After several weeks of intensive care, toxic megacolon was improved, but renal replacement therapy was discontinued according to the patient's will. On day 73, she died of renal failure. We experienced a complex of rare diseases, Cryptococcus neoformans cellulitis and Clostridioides difficile-related toxic megacolon. Both diseases were presumed to be the result of corticosteroid and methotrexate use. Hence, careful monitoring is required when treating immunocompromised hosts to reduce the risk of developing complications.

Paraclostridium bifermentans exacerbates pathosis in a mouse model of ulcerative colitis.

Although it has been recognized that intestinal bacteria play an important role in the pathology of human ulcerative colitis (UC), specific pathogenic bacteria for UC have not been identified. We investigated the influence of Paraclostridium bifermentans PAGU1678 strain on the pathology of a UC mouse model and found it increased UC pathosis scores such as loose and bloody stools, reduced diversity of fecal flora, disappearance of the crypt structure of distal colon tissue, destruction of intestinal epithelial cells, and atrophy of the colon. Furthermore, we observed an increase in COX-2, TNF-α, IL-6, IL-1, and IL-17 expression and a decrease in Foxp3 and SOCS3 expression, as inflammation-related factors and inflammatory cytokines, a decrease in the concentration of short chain fatty acids (acetic acid, propionic acid, and butyric acid) in feces, and an increase of intestinal mucosal myeloperoxidase activity. These results suggest that P. bifermentans PAGU1678 is a pathology-exacerbating factor in a mouse model of UC. This study is the first to demonstrate exacerbation of the pathological condition in a mouse model of UC by a single bacterial strain.

Accelerated dysbiosis of gut microbiota during aggravation of DSS-induced colitis by a butyrate-producing bacterium.

Butyrate-producing bacteria (BPB) are potential probiotic candidates for inflammatory bowel diseases as they are often depleted in the diseased gut microbiota. However, here we found that augmentation of a human-derived butyrate-producing strain, Anaerostipes hadrus BPB5, significantly aggravated colitis in dextran sulphate sodium (DSS)-treated mice while exerted no detrimental effect in healthy mice. We explored how the interaction between BPB5 and gut microbiota may contribute to this differential impact on the hosts. Butyrate production and severity of colitis were assessed in both healthy and DSS-treated mice, and gut microbiota structural changes were analysed using high-throughput sequencing. BPB5-inoculated healthy mice showed no signs of colitis, but increased butyrate content in the gut. In DSS-treated mice, BPB5 augmentation did not increase butyrate content, but induced significantly more severe disease activity index and much higher mortality. BPB5 didn't induce significant changes of gut microbiota in healthy hosts, but expedited the structural shifts 3 days earlier toward the disease phase in BPB5-augmented than DSS-treated animals. The differential response of gut microbiota in healthy and DSS-treated mice to the same potentially beneficial bacterium with drastically different health consequences suggest that animals with dysbiotic gut microbiota should also be employed for the safety assessment of probiotic candidates.

Vaginal Microbiota.

The knowledge about the normal and abnormal vaginal microbiome has changed over the last years. Culturing techniques are not suitable any more for determination of a normal or abnormal vaginal microbiota. Non culture-based modern technologies revealed a complex and dynamic system mainly dominated by lactobacilli.The normal and the abnormal vaginal microbiota are complex ecosystems of more than 200 bacterial species influenced by genes, ethnic background and environmental and behavioral factors. Several species of lactobacilli per individuum dominate the healthy vagina. They support a defense system together with antibacterial substances, cytokines, defensins and others against dysbiosis, infections and care for an normal pregnancy without preterm birth.The numbers of Lactobacillus (L.) iners increase in the case of dysbiosis.Bacterial vaginosis (BV) - associated bacteria (BVAB), Atopobium vaginae and Clostridiales and one or two of four Gardnerella vaginalis - strains develop in different mixtures and numbers polymicrobial biofilms on the vaginal epithelium, which are not dissolved by antibiotic therapies according to guidelines and, thus, provoke recurrences.Aerobic vaginitis seems to be an immunological disorder of the vagina with influence on the microbiota, which is here dominated by aerobic bacteria (Streptococcus agalactiae, Escherichia coli). Their role in AV is unknown.Vaginal or oral application of lactobacilli is obviously able to improve therapeutic results of BV and dysbiosis.

Dysbiosis and Immune Dysregulation in Outer Space.

In space, the lifestyle, relative sterility of spaceship and extreme environmental stresses, such as microgravity and cosmic radiation, can compromise the balance between human body and human microbiome. An astronaut's body during spaceflight encounters increased risk for microbial infections and conditions because of immune dysregulation and altered microbiome, i.e. dysbiosis. This risk is further heightened by increase in virulence of pathogens in microgravity. Health status of astronauts might potentially benefit from maintaining a healthy microbiome by specifically managing their diet on space in addition to probiotic therapies. This review focuses on the current knowledge/understanding of how spaceflight affects human immunity and microbiome.