Deep longitudinal multi-omics analysis of Bordetella pertussis cultivated in bioreactors highlights medium starvations and transitory metabolisms, associated to vaccine antigen biosynthesis variations and global virulence regulation.

Bordetella pertussis is the bacterial causative agent of whooping cough, a serious respiratory illness. An extensive knowledge on its virulence regulation and metabolism is a key factor to ensure pertussis vaccine manufacturing process robustness. The aim of this study was to refine our comprehension of B. pertussis physiology during in vitro cultures in bioreactors. A longitudinal multi-omics analysis was carried out over 26 h small-scale cultures of B. pertussis. Cultures were performed in batch mode and under culture conditions intending to mimic industrial processes. Putative cysteine and proline starvations were, respectively, observed at the beginning of the exponential phase (from 4 to 8 h) and during the exponential phase (18 h 45 min). As revealed by multi-omics analyses, the proline starvation induced major molecular changes, including a transient metabolism with internal stock consumption. In the meantime, growth and specific total PT, PRN, and Fim2 antigen productions were negatively affected. Interestingly, the master virulence-regulating two-component system of B. pertussis (BvgASR) was not evidenced as the sole virulence regulator in this in vitro growth condition. Indeed, novel intermediate regulators were identified as putatively involved in the expression of some virulence-activated genes (vags). Such longitudinal multi-omics analysis applied to B. pertussis culture process emerges as a powerful tool for characterization and incremental optimization of vaccine antigen production.

Lack of anti-TNF drugs levels in fistula tissue - a reason for nonresponse in Crohn's perianal fistulating disease?

INTRODUCTION: Anti-TNF therapy is recommended as treatment for patients with Crohn´s perianal fistulas. However, a significant proportion of patients have a sub-optimal response to anti-TNF therapy. Higher serum levels of anti-TNF agents have been associated with improved outcomes in perianal Crohn's disease. Currently, it is unknown whether anti-TNF agent levels can be detected in tissue from fistula tracts themselves and whether this is associated with response. AIMS AND METHODS: We undertook a pilot study to measure fistula tissue levels of anti-TNF medication (infliximab and adalimumab). We used a previously validated targeted proteomic technique, employing ultraperformance liquid chromatography-mass spectrometry, to detect/quantify anti-TNF drugs. Biopsies were obtained from fistula tracts of patients with Crohn's disease on maintenance treatment; with idiopathic (cryptoglandular) fistula tissues used as negative controls as well as positive controls (by spiking the latter tissues with anti-TNF drugs). RESULTS: Tissue was sampled from the fistula tracts of seven patients with Crohn's perianal disease (five patients were on adalimumab and two patients were on infliximab). The anti-TNF drugs, infliximab and adalimumab, were not detected in fistula samples from any of the Crohn's patients despite detection in 'spiked' positive control samples. CONCLUSION: Absence of detection of the anti-TNF drugs in fistula tissue raises the question on the role of tissue penetrance of anti-TNF drugs in response to therapy. Further work is required in a larger number of patients to validate the findings observed and investigate if any correlation exists between tissue and serum levels of anti-TNF and clinical outcome. SUMMARY: Predicting response in Crohn's fistula patients on biologic therapy is difficult with no reliable biomarkers. This pilot study uses targeted proteomics to investigate the potential role of tissue drug levels in acting as a biomarker of treatment response.

Roux-en-Y gastric bypass-induced bacterial perturbation contributes to altered host-bacterial co-metabolic phenotype.

BACKGROUND: Bariatric surgery, used to achieve effective weight loss in individuals with severe obesity, modifies the gut microbiota and systemic metabolism in both humans and animal models. The aim of the current study was to understand better the metabolic functions of the altered gut microbiome by conducting deep phenotyping of bariatric surgery patients and bacterial culturing to investigate causality of the metabolic observations. METHODS: Three bariatric cohorts (n = 84, n = 14 and n = 9) with patients who had undergone Roux-en-Y gastric bypass (RYGB), sleeve gastrectomy (SG) or laparoscopic gastric banding (LGB), respectively, were enrolled. Metabolic and 16S rRNA bacterial profiles were compared between pre- and post-surgery. Faeces from RYGB patients and bacterial isolates were cultured to experimentally associate the observed metabolic changes in biofluids with the altered gut microbiome. RESULTS: Compared to SG and LGB, RYGB induced the greatest weight loss and most profound metabolic and bacterial changes. RYGB patients showed increased aromatic amino acids-based host-bacterial co-metabolism, resulting in increased urinary excretion of 4-hydroxyphenylacetate, phenylacetylglutamine, 4-cresyl sulphate and indoxyl sulphate, and increased faecal excretion of tyramine and phenylacetate. Bacterial degradation of choline was increased as evidenced by altered urinary trimethylamine-N-oxide and dimethylamine excretion and faecal concentrations of dimethylamine. RYGB patients' bacteria had a greater capacity to produce tyramine from tyrosine, phenylalanine to phenylacetate and tryptophan to indole and tryptamine, compared to the microbiota from non-surgery, normal weight individuals. 3-Hydroxydicarboxylic acid metabolism and urinary excretion of primary bile acids, serum BCAAs and dimethyl sulfone were also perturbed following bariatric surgery. CONCLUSION: Altered bacterial composition and metabolism contribute to metabolic observations in biofluids of patients following RYGB surgery. The impact of these changes on the functional clinical outcomes requires further investigation. Video abstract.

Differences in amino acid and lipid metabolism distinguish Crohn's from idiopathic/cryptoglandular perianal fistulas by tissue metabonomic profiling and may offer clues to underlying pathogenesis.

INTRODUCTION: Few studies have investigated perianal fistula etiopathogenesis, and although the cryptoglandular theory is widely accepted in idiopathic cases, in Crohn's disease, it is thought to involve the interplay between microbiological, immunological and genetic factors. A pilot study was conducted to assess for metabolic variations in Crohn's perianal fistula tissue that might differ from that of idiopathic (cryptoglandular) perianal fistula tissue as a comparator. The goal was to identify any potential biomarkers of disease, which may improve the understanding of pathogenesis. AIMS AND METHODS: Fistula tract biopsies were obtained from 30 patients with idiopathic perianal fistula and 20 patients with Crohn's anal fistula. Two different assays were used in an ultra-high-performance liquid chromatography system coupled with a mass spectrometric detector to achieve broad metabolome coverage. Univariate and multivariate statistical data analyses were used to identify differentiating metabolic features corresponding to the perianal fistula phenotype (i.e. Crohn's disease vs. idiopathic). RESULTS: Significant orthogonal partial least squares discriminant analysis predictive models (validated with cross-validated-analysis of variance P value <0.05) differentiated metabolites from tissue samples from Crohn's vs. idiopathic anal fistula patients using both metabolic profiling platforms. A total of 41 metabolites were identified, suggesting alterations in pathways, including amino acid, carnitine and lipid metabolism. CONCLUSION: Metabonomics may reveal biomarkers of Crohn's perianal fistula. Further work in larger numbers is required to validate the findings of these studies as well as cross-correlation with microbiome work to better understand the impact of host-gut/environment interactions in the pathophysiology of Crohn's and idiopathic perianal fistulas and identify novel therapeutic targets.