DOI: 10.5061/dryad.1rn8pk138
title: In vitro assessment of Bacillus subtilis DSM29784 secreted metabolites on gut chicken microbiota

description: In poultry, the probiotic Bacillus subtilis 29784 (Bs29784) sustains
 intestinal health, enhancing animal resilience and performance through the
 production of the bioactive metabolites hypoxanthine (HPX), Niacin (NA),
 and pantothenate (PTH). Here, using enterocyte in vitro models, we
 determine the functional link between these metabolites and the three
 pillars of intestinal resilience: immune response, intestinal barrier, and
 microbiota. To this end, we evaluated in vitro Bs29784 vegetative cells,
 spores, and metabolites capacity to modulate global immune regulators
 (using HT-29-NF-κB and HT-29-AP1 reporter cells), intestinal integrity
 (HT-29-MUC-2 reporter cells and Caco-2 cells), and cytokine production
 (Caco-2 cells). Finally, we simulated chickens’ intestinal fermentations
 to determine the effect of Bs29784 metabolites on the microbiota and their
 fermentation profile. Bs29784 vegetative cells reduce the inflammatory
 response more effectively than spores, indicating that their benefit is
 linked to metabolic activity. To assess this hypothesis, we studied
 individually Bs29784 metabolites. The results show that each metabolite
 had different beneficial effects. PTH and NIA reduced the activation of
 the proinflammatory pathways AP1 and NF-κB. HPX upregulated mucin
 production by enhancing MUC2 expression. HPX, NA, and PTH increased cell
 proliferation. PTH and HPX increased epithelial resilience to an
 inflammatory challenge by limiting permeability increase. In cecal
 fermentations NA increased acetate, HPX increased butyrate while PTH
 increased acetate, butyrate, and propionate. In ileal fermentations, PTH
 increased butyrate. All molecules lead changes to microbiota explaining
 the different fermentation patterns. Altogether, we show that Bs29784
 modulates intestinal health by acting on the three lines of resilience via
 its secreted metabolites.
description: All experiments were conducted according to the European Union
 Guidelines of Animal Care and legislation governing the ethical treatment
 of animals, and investigators were certified by the French government to
 conduct animal experiments. The Center for Expertise and Research in
 Nutrition facilities (Malicorne, France) are in accordance with the
 agreement no. C 03 159 4 of 6 November 2008, relative to experimentation
 on vertebrate living animals (European regulation 24/11/86 86/609 CEE;
 Ministerial decree of 19 April 1988). Twelve 28-days old broilers chickens
 were euthanized and their cecal and ileal contents were collected and
 pooled and immediately stored at -80°C. Cecal and ileal
 chicken microbial fermentation was performed in Hungate tubes in anaerobic
 buffer prepared according to Davies et al. (2000). Four technical
 replicates were performed by condition. The anaerobic buffer was composed
 of 5 solutions (A, B, C, D and E) prepared individually: solution A (per
 Liter: 5.7 g Na<sub>2</sub>HPO<sub>4</sub>, 6,2g
 KH<sub>2</sub>PO<sub>4</sub> and 0,6g
 MgSO<sub>4</sub>-7H<sub>2</sub>O), solution B (per
 Liter: 4 g NH<sub>4</sub>HCO<sub>3</sub> and 35 g
 NaHCO<sub>3</sub>), solution C (per 10mL: 132 mg
 CaCl<sub>2</sub>-2H<sub>2</sub>O, 100 mg
 MnCl<sub>2</sub>-4H<sub>2</sub>O, and 80 mg
 FeCl<sub>3</sub>-6H<sub>2</sub>O), solution D (per
 Liter: 0.1 % resazurin) and solution E (per 100mL: 4ml de NaOH 1M, 625mg
 de Na<sub>2</sub>S). The anaerobic buffer was assembled in
 anaerobic condition the day of the experiment (gazed with a mixture of
 CO<sub>2</sub> and N<sub>2</sub>): 0,01 % of
 solution A, 25,3% of solution B, 25,3% of solution C, 0,1 % of solution C,
 49,29% of ultra-pure water (18,2mΩ) and autoclaved in the presence of
 0,5g/L of L-cysteine (reducing agent). On the day of the experiment, the
 buffer was further reduced by adding 4% (<em>v/v</em>) of
 solution E before adding the cecal inoculum (final pH: 7.5 and Eh:
 -150mV). The cecal content was mixed at 5% (<em>w/v</em>) with
 the anaerobic buffer and 10 mL of the slurry was transferred in Hungate
 tubes. All treatments were added at 1mM. The fermentation was then
 performed in water bath under constant agitation (200 rpm) at 39°C for
 24h. At the end of the fermentation, samples were collected to quantify
 microbiota composition (Illumina sequencing, Genoscreen,
 France).  Sequencing of 16s was done on Illumina
 platform at GenoScreen using the Metabiote kit on the V3-V4 16s
 hypervariable region. Briefly, DNA was extracted, normalized and the
 multiplex library was prepared for Illumina Miseq Paired-end 2x300 bases.
 Quality control of the sequencing was performed using a mock community (15
 bacterial and 2 archaeal strains) including in the sequencing run. The
 primer and index were identified (100% homology) and removed to create
 demultiplexed fastq files. The fastq files were quality trimmed at Q30 at
 the end of the read, the reads were then paired assembled with minimum
 30bp alignment at 97% homology using Qiime. The demultiplexed, quality
 trimmed and assembled read were then clustered using DADA2
 software. 
description: # In vitro assessment of Bacillus subtilis DSM29784 secreted metabolites
 on gut chicken microbiota [https://doi.org/10.5061/dryad.1rn8pk138
 ](https://doi.org/10.5061/dryad.1rn8pk138) In this study, we aimed to
 investigate the mechanisms by molecules released by Bacillus subtilis
 DSM29784 modulate chicken gut microbiota, focusing on hypoxanthine (HPX),
 niacin (NIA) and panthotenate (PTH). We conducted in vitro assay targeting
 the modulation of microbiota by mimicking ileal and cecal chicken
 compartment thanks to the inocula fermentation of 28 days-old broiler
 chickens. There are 4 repetitions by condition tested.  ## Description of
 the data and file structure This README file describes the data package
 accompanying the above publication:  * *vieco-saiz_et_al_2024_Sequences
 folder* present[s](javascript:) the reads in fastq.gz format *
 *vieco-saiz_et_al_2024_metadata.txt* identifies the sequences and the
 conditions tested ## Code/Software The primer and index were identified
 (100% homology) and removed to create demultiplexed fastq files. The fastq
 files were quality trimmed at Q30 at the end of the read, the reads were
 then paired assembled with minimum 30bp alignment at 97% homology using
 Qiime. The demultiplexed, quality trimmed and assembled read were then
 clustered using DADA2 software. The DADA2 package infers exact amplicon
 sequence variants (ASVs) from high-throughput amplicon sequencing data.
 The DADA2 pipeline takes as input demultiplexed fastq files, and outputs
 the sequence variants and their sample-wise abundances after removing
 substitution and chimera errors. Taxonomic classification is done via a
 native implementation of the RDP naive Bayesian classifier. The normalized
 ASV table (normalized to the lower number of sequence/sample) is then
 analyzed using Phyloseq (phyloseq object containing ASV table, taxonomic
 assignment, and environmental data) and Vegan package for diversity under
 R environment.