Hydrogen Sulfide Producers Drive a Diarrhea-Like Phenotype and a Methane Producer Drives a Constipation-Like Phenotype in Animal Models.

BACKGROUND: We recently demonstrated that diarrhea-predominant irritable bowel syndrome (IBS-D) subjects have higher relative abundance (RA) of hydrogen sulfide (H2S)-producing Fusobacterium and Desulfovibrio species, and constipation-predominant IBS (IBS-C) subjects have higher RA of methanogen Methanobrevibacter smithii. AIMS: In this study, we investigate the effects of increased methanogens or H2S producers on stool phenotypes in rat models. METHODS: Adult Sprague-Dawley rats were fed high-fat diet (HFD) for 60 days to increase M. smithii levels, then gavaged for 10 days with water (controls) or methanogenesis inhibitors. To increase H2S producers, rats were gavaged with F. varium or D. piger. Stool consistency (stool wet weight (SWW)) and gas production were measured. 16S rRNA gene sequencing was performed on stool samples. RESULTS: In HFD diet-fed rats (N = 30), stool M. smithii levels were increased (P < 0.001) after 52 days, correlating with significantly decreased SWW (P < 0.0001) at 59 days (R = - 0.38, P = 0.037). Small bowel M. smithii levels decreased significantly in lovastatin lactone-treated rats (P < 0.0006), and SWW increased (normalized) in lovastatin hydroxyacid-treated rats (P = 0.0246), vs. controls (N = 10/group). SWW increased significantly in D. piger-gavaged rats (N = 16) on day 10 (P < 0.0001), and in F. varium-gavaged rats (N = 16) at all timepoints, vs. controls, with increased stool H2S production. 16S sequencing revealed stool microbiota alterations in rats gavaged with H2S producers, with higher relative abundance (RA) of other H2S producers, particularly Lachnospiraceae and Bilophila in F. varium-gavaged rats, and Sutterella in D. piger-gavaged rats. CONCLUSIONS: These findings suggest that increased M. smithii levels result in a constipation-like phenotype in a rat model that is partly reversible with methanogenesis inhibitors, whereas gavage with H2S producers D. piger or F. varium results in increased colonization with other H2S producers and diarrhea-like phenotypes. This supports roles for the increased RA of methanogens and H2S producers identified in IBS-C and IBS-D subjects, respectively, in contributing to stool phenotypes.

Rare isolation of Fusobacterium varium from a case of Fournier's gangrene.

Fusobacterium is a gram negative obligate anaerobic bacilli, a normal inhabitant of gastrointestinal tract, oropharynx and female genital tract. Here we report a case of Fourniers gangrene from which Fusobacterium varium has been isolated along with certain other pathogens. There are only a few reported cases of Fusobacterium varium in literature and it has never been reported from Fournier's gangrene. Through this report we intend to shed some light on the pathogenic potential of anaerobes which are considered as normal flora.

Draft genome sequence of two Fusobacterium varium strains isolated from patients in Kazakhstan with colorectal cancer.

Fusobacterium varium is a Gram-negative, invasive, obligate anaerobe in the gastrointestinal tract microbiota, associated with various clinical conditions, including colorectal cancer (CRC). Here, we announce the draft genome sequence of two F. varium strains Fv36kaz and Fv63kaz from patients with CRC in Kazakhstan.

Adhesion of Bacteroides vulgatus and Fusobacterium varium to the Colonic Mucosa of Healthy Beagles.

The relative abundances of Bacteroidetes and Fusobacteria phyla have been reported to be decreased in dogs with chronic enteropathies. In colitis, obligate anaerobes (e.g., Bacteroides and Fusobacterium) are likely to vanish in response to the heightened oxidative stress in the colon's inflammatory environment. The ability to adhere to the colonic mucosa is viewed as an essential step for obligate anaerobic bacteria to colonize and subsequently interact with the host's epithelium and immune system. The reintroduction of a balanced community of obligate anaerobic bacteria using probiotics can restore the microbial function in the intestine. We found no studies on dogs regarding the adhesion properties of Bacteriodes vulgatus and Fusobacterium varium on paraffin-embedded canine colonic mucosa. Thus, the objective of this study is to investigate the adhesion capacities of these two bacterial species to paraffin-embedded colonic mucosa from healthy dogs. Additionally, we investigated their hydrophobicity properties to determine whether differences in adhesion capability can be explained by this factor. The results of our study showed that B. vulgatus adhered significantly lower than F. varium to the canine colonic mucosa (p = 0.002); however, B. vulgatus showed higher hydrophobicity (46.1%) than F. varium (12.6%). In conclusion, both bacteria have potential as probiotics, but further studies will be required to determine the efficacy and safety of the strains to be used, which strains to use, and the reasons other than hydrophobicity for attachment.

Gut microbes exacerbate systemic inflammation and behavior disorders in neurologic disease CADASIL.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a cerebral small vessel disease that carries mutations in NOTCH3. The clinical manifestations are influenced by genetic and environmental factors that may include gut microbiome. RESULTS: We investigated the fecal metagenome, fecal metabolome, serum metabolome, neurotransmitters, and cytokines in a cohort of 24 CADASIL patients with 28 healthy household controls. The integrated-omics study showed CADASIL patients harbored an altered microbiota composition and functions. The abundance of bacterial coenzyme A, thiamin, and flavin-synthesizing pathways was depleted in patients. Neurotransmitter balance, represented by the glutamate/GABA (4-aminobutanoate) ratio, was disrupted in patients, which was consistent with the increased abundance of two major GABA-consuming bacteria, Megasphaera elsdenii and Eubacterium siraeum. Essential inflammatory cytokines were significantly elevated in patients, accompanied by an increased abundance of bacterial virulence gene homologs. The abundance of patient-enriched Fusobacterium varium positively correlated with the levels of IL-1ß and IL-6. Random forest classification based on gut microbial species, serum cytokines, and neurotransmitters showed high predictivity for CADASIL with AUC = 0.89. Targeted culturomics and mechanisms study further showed that patient-derived F. varium infection caused systemic inflammation and behavior disorder in Notch3R170C/+ mice potentially via induction of caspase-8-dependent noncanonical inflammasome activation in macrophages. CONCLUSION: These findings suggested the potential linkage among the brain-gut-microbe axis in CADASIL. Video Abstract.

Unexpected finding of Fusobacterium varium as the dominant Fusobacterium species in cattle rumen: potential implications for liver abscess etiology and interventions.

Fusobacterium varium has been generally overlooked in cattle rumen microbiome studies relative to the presumably more abundant liver abscess-causing Fusobacterium necrophorum. However, F. varium was found to be more abundant in the rumen fluid of cattle and under culture conditions tailored to enrich F. necrophorum. Using near-full length 16S ribosomal ribonucleic acid sequencing, we demonstrate that F. varium grows under restrictive conditions commonly used to enumerate F. necrophorum, suggesting that previous F. necrophorum abundance assessment may have been inaccurate and that F. varium may be an underestimated member of the ruminal bacterial community. Fusobacterium varium were not as susceptible as F. necrophorum to in-feed antibiotics conventionally used in feedlots. Exposure to tylosin, the current gold standard for liver abscess reduction strategies in cattle, consistently hindered growth of the F. necrophorum strains tested by over 67% (P < 0.05) relative to the unexposed control. In contrast, F. varium strains were totally or highly resistant (0%-13% reduction in maximum yield, P < 0.05). Monensin, an ionophore antibiotic, had greater inhibitory activity against F. necrophorum than F. varium. Finally, preliminary genomic analysis of two F. varium isolates from the rumen revealed the presence of virulence genes related to those of pathogenic human F. varium isolates associated with active invasion of mammalian cells. The data presented here encourage further investigation into the ecological role of F. varium within the bovine rumen and potential role in liver abscess development, and proactive interventions.

The conventional method of liver abscess prevention in feedlot cattle is in-feed use of tylosin to target Fusobacterium necrophorum, which has been presumed to be the most common Fusobacterium species within the ruminal compartment. Our investigation into ruminal Fusobacterium, however, revealed a different species, Fusobacterium varium, to be abundant and ubiquitous in ruminal content samples. Furthermore, growth conditions tailored to enrich F. necrophorum consistently promoted growth of F. varium, and the bovine isolates tested had much lower susceptibilities to the commonly fed antibiotics tylosin and monensin compared to F. necrophorum. Fusobacterium varium is an emerging pathogen in humans and preliminary genome sequencing of two ruminal F. varium isolates revealed genes linked to pathogenicity. While the ecological role of F. varium in the rumen is still not fully understood, our findings draw attention to this pathogen and its potential implication in liver abscesses.

Bacterial Species Associated With Human Inflammatory Bowel Disease and Their Pathogenic Mechanisms.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract with unknown etiology. The pathogenesis of IBD results from immune responses to microbes in the gastrointestinal tract. Various bacterial species that are associated with human IBD have been identified. However, the microbes that trigger the development of human IBD are still not clear. Here we review bacterial species that are associated with human IBD and their pathogenic mechanisms to provide an updated broad understanding of this research field. IBD is an inflammatory syndrome rather than a single disease. We propose a three-stage pathogenesis model to illustrate the roles of different IBD-associated bacterial species and gut commensal bacteria in the development of human IBD. Finally, we recommend microbe-targeted therapeutic strategies based on the three-stage pathogenesis model.

Reduction of Fumarate to Succinate Mediated by Fusobacterium varium.

Accumulation of succinate as a fermentation product of Fusobacterium varium was enhanced when the anaerobic bacterium was grown on complex peptone medium supplemented with fumarate. Residual substrates and fermentation products were determined by proton NMR spectroscopy. Cells collected from the fumarate-supplemented medium (8-10 h after inoculation) supported the conversion of fumarate to succinate when suspended with fumarate and a co-substrate (glucose, sorbitol, or glycerol). Succinate production was limited by the availability of fumarate or reducing equivalents supplied by catabolism of a co-substrate via the Embden-Meyerhof-Parnas (EMP) pathway. The choice of reducing co-substrate influenced the yield of acetate and lactate as side products. High conversions of fumarate to succinate were achieved over pH 6.6-8.2 and initial fumarate concentrations up to 300 mM. However, at high substrate concentrations, intracellular retention of succinate reduced extracellular yields. Overall, the efficient utilization of fumarate (≤ 400 mM) combined with the significant extracellular accumulation of succinate (corresponding to ≥ 70% conversion) indicated the effective utilization of fumarate as a terminal electron acceptor by F. varium and the potential of the methodology for the bioproduction of succinate.

Characterization of Fusobacterium isolates from the respiratory tract of white-tailed deer (Odocoileus virginianus).

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

Corticotropin-releasing factor secretion from dendritic cells stimulated by commensal bacteria.

AIM: To study the production and secretion of corticotropin-releasing factor (CRF) by dendritic cells and the influence of commensal bacteria. METHODS: JAWSII cells (ATCC CRL-11904), a mouse dendritic cell line, were seeded into 24-well culture plates and grown for 3 d. Commensal bacterial strains of Clostridium clostrodiiforme (JCM1291), Bacteroides vulgatus (B. vulgatus) (JCM5856), Escherichia coli (JCM1649), or Fusobacterium varium (F. varium) (ATCC8501) were added to the cells except for the control well, and incubated for 2 h. After incubation, we performed enzyme-linked immunosorbent assay for the cultured medium and reverse transcription polymerase chain reaction for the dendritic cells, and compared these values with controls. RESULTS: The level of CRF secretion by control dendritic cells was 40.4 ± 6.2 pg/mL. The CRF levels for cells incubated with F. varium and B. vulgatus were significantly higher than that of the control (P < 0.0001). CRF mRNA was present in the control sample without bacteria, and CRF mRNA levels in all samples treated with bacteria were above that of the control sample. F. varium caused the greatest increase in CRF mRNA expression. CONCLUSION: Our results suggest that dendritic cells produce CRF, a process augmented by commensal bacteria.