Presence and characterisation of ecotin in F. necrophorum

Fusobacterium necrophorum is a causative agent of Lemierre’s syndrome (LS) in humans. LS is characterised by thrombophlebitis of the jugular vein and bacteraemia. Disseminated intravascular coagulation is also a documented symptom. F. necrophorum is a Gram-negative, anaerobic bacterium known to possess virulence genes such as a haemolysin, filamentous haemagglutinin and leukotoxin, which target host blood components. Ecotin is a serine protease inhibitor that has not previously been characterised in F. necrophorum, but in E.coli has been shown to have a potent anticoagulant effect. Next generation and Sanger sequencing were used to confirm the presence of the ecotin gene in the genomes of a collection of F. necrophorum clinical and reference strains. When translated, it was found to be a highly conserved protein made up of159 amino acids. Enzyme/substrate inhibition assays demonstrated that F. necrophorum ecotin inhibits human plasma kallikrein and human neutrophil elastase in a dose-dependent manner. Data will also be presented on the anticoagulant effects of ecotin during activated partial thromboplastin time, thrombin time and prothrombin time tests on human donor blood. The mechanisms for how this organism reaches the bloodstream and the significance of this serine protease inhibitor during F. necrophorum infections remain to be elucidated

Comparison of virulence genes found in draft genomes of F. necrophorum

Fusobacterium necrophorum is a causative agent of persistent sore throat syndrome, tonsillar abscesses and Lemierre’s syndrome (LS) in humans. LS is characterised by thrombophlebitis of the jugular vein and bacteraemia. It is a Gram-negative, anaerobic bacterium which to date has no available reference genome. Draft genomes suggest it to be a single circular chromosome of approximately 2.2Mb. A reference strain of each of the two F. necrophorum subspecies and a clinical isolate from a LS patient were sequenced on a Roche 454 GS-FLX+. Sequence data was assembled using Roche GS Assembler and the resulting contigs annotated using xBASE, Pfam and BLAST. The annotation data was mined for gene products associated with virulence revealing a leukotoxin, haemolysin, filamentous haemagglutinnin, adhesin, hemin receptor, phage genes, CRISPR-associated proteins, ecotin and a putative type V secretion system. Data will be presented on comparative genomics of the three strains, with a focus on putative virulence genes. Tools such as Artemis Comparison Tool and ClustalO were used for sequence alignments and PhyML was used to generate phylogenetic trees. Conserved motifs associated with virulence were also located. Understanding variations at the genomic level may help to explain the increased virulence of some F. necrophorum strains.

Akkermansia muciniphila and improved metabolic health during a dietary intervention in obesity: relationship with gut microbiome richness and ecology

Objective: Individuals with obesity and type 2 diabetes differ from lean and healthy individuals in their abundance of certain gut microbial species and microbial gene richness. Abundance of Akkermansia muciniphila, a mucin-degrading bacterium, has been inversely associated with bodyfat mass and glucose intolerance in mice, but more evidence is needed in humans. The impact of diet and weight loss on this bacterial species is unknown. Our objective was to evaluate the association between fecal A. muciniphila abundance, fecal microbiome gene richness, diet, host characteristics, and their changes after calorie restriction (CR). Design: The intervention consisted of a 6-week CR period followed by a 6-week weight stabilization (WS) diet in overweight and obese adults (N=49, including 41 women). Fecal A. muciniphila abundance, fecal microbial gene richness, diet and bioclinical parameters were measured at baseline and after CR and WS. Results: At baseline A. muciniphila was inversely related to fasting glucose, waist-to-hip ratio, and subcutaneous adipocyte diameter. Subjects with higher gene richness and A. muciniphila abundance exhibited the healthiest metabolic status, particularly in fasting plasma glucose, plasma triglycerides and body fat distribution. Individuals with higher baseline A. muciniphila displayed greater improvement in insulin sensitivity markers and other clinical parameters after CR. A. muciniphila was associated with microbial species known to be related to health. Conclusion: A. muciniphila is associated with a healthier metabolic status and better clinicaloutcomes after CR in overweight/obese adults, however the interaction between gut microbiota ecology and A. muciniphila has to be taken into account.

Effects of targeted delivery of propionate to the human colon on appetite regulation, body weight maintenance and adiposity in overweight adults.

OBJECTIVE: The colonic microbiota ferment dietary fibres, producing short chain fatty acids. Recent evidence suggests that the short chain fatty acid propionate may play an important role in appetite regulation. We hypothesised that colonic delivery of propionate would increase peptide YY (PYY) and glucagon like peptide-1 (GLP-1) secretion in humans, and reduce energy intake and weight gain in overweight adults. DESIGN: To investigate whether propionate promotes PYY and GLP-1 secretion, a primary cultured human colonic cell model was developed. To deliver propionate specifically to the colon, we developed a novel inulin-propionate ester. An acute randomised, controlled cross-over study was used to assess the effects of this inulin-propionate ester on energy intake and plasma PYY and GLP-1 concentrations. The long-term effects of inulin-propionate ester on weight gain were subsequently assessed in a randomised, controlled 24-week study involving 60 overweight adults. RESULTS: Propionate significantly stimulated the release of PYY and GLP-1 from human colonic cells. Acute ingestion of 10 g inulin-propionate ester significantly increased postprandial plasma PYY and GLP-1 and reduced energy intake. Over 24 weeks, 10 g/day inulin-propionate ester supplementation significantly reduced weight gain, intra-abdominal adipose tissue distribution, intrahepatocellular lipid content and prevented the deterioration in insulin sensitivity observed in the inulin-control group. CONCLUSIONS: These data demonstrate for the first time that increasing colonic propionate prevents weight gain in overweight adult humans.