Biology subjects

Pascal, G.

Publications and source records attributed to Pascal, G..

5 recordsLinked to original sources

bioRxiv2025

The fecal microbiota of lactating Holstein dairy cows: a meta-analysis highlighting key microbial profiles and methodological challenges

In cattle, the digestive microbiota has been poorly studied outside the rumen, despite its recognized role in animal physiology, health, and production. This study aimed to provide a comprehensive meta-analysis of the fecal microbiota of lactating Holstein dairy cows. A systematic online search of PubMed was performed in June 2023 to identify studies on the fecal microbiota of lactating Holstein dairy cows. Of the 526 articles retrieved from the systematic online search, only 28 met our required inclusion criteria. Raw sequencing data targeting the 16S rRNA gene (V3-V4 region) were obtained either from public repositories or directly from the authors. Two recently published articles were included as they met the inclusion criteria. A total of 2,136 samples were included in the analysis. The number of sequences per sample varied considerably between studies, and the metadata were sparse. The core microbiota was identified as the most prevalent and shared taxa, comprising 21 microbiota families that accounted for 82% of the relative abundance. The observed clustering, which depended on study, highlighted the significant impact of environmental factors and experimental conditions on the microbial communities of cattle. A multi-group analysis was performed to correct for the study effect and successfully identified three microbiota profiles. The meta-analysis approach is a rigorous and systematic method of analyzing research findings that allows for the generation of reliable and reproducible results. This approach ensures the independence of results from a single experimental facility or condition, thereby enhancing the reliability and generalizability of the findings. ImportanceThis meta-analysis provides the most comprehensive and up-to-date overview of the fecal microbiota of lactating Holstein dairy cows, based on a rigorous selection of over 2,000 samples. By controlling for inter-study variation, it successfully identified consistent microbial patterns and establishes a robust core microbiota. This work not only highlighted the microbial taxa most likely essential to bovine physiology and health, but also emphasized the need for standardized methodologies and improved data-sharing practices across studies. Ultimately, the findings pave the way for strategies that target the modulation of microbiota with potential benefits for animal health and environmental impacts.

systems biology↗

bioRxiv2025

Nested PCR to optimize rpoB metabarcoding for low-concentration and host-associated bacterial DNA

BackgroundHousekeeping genes have proved effective taxonomic markers for characterizing bacterial microbiota in short-read amplicon metabarcoding studies. A region of the rpoB gene, in particular, has been shown to minimize OTU overestimation bias with a high degree of accuracy, providing better species-level taxonomic resolution (Ogier et al. BMC Microbiol. 19:171). However, the primers for rpoB are highly degenerate, leading to potential problems in the amplification of bacterial DNA present at low concentration in the sample or embedded within eukaryotic matrices, as for the host-associated microbiota. We addressed these limitations by using a two-step PCR approach to optimize the rpoB procedure. The first PCR amplifies a 906-nucleotide region of the rpoB gene with the classical primers, referred to here as outer primers, and the second PCR then uses primers incorporating Illumina adapters, referred to here as inner primers, to amplify a 435-nucleotide subregion, the taxonomic marker for metabarcoding. ResultsWe first used in silico approaches to evaluate the universality of the outer and inner rpoB primers. We then tested the nested rpoB PCR method on commercial mock samples of known composition. The nested PCR approach increased amplification efficiency for dilute samples without biasing the bacterial composition of the mock sample revealed by metabarcoding relative to single-step PCR. We also tested the nested rpoB PCR method on field-collected samples of the lepidopteran Spodoptera frugiperda. The nested PCR outperformed single-step PCR, increasing amplification efficiency for bacterial DNA present at low concentrations (oral secretions from S. frugiperda) or embedded in eukaryotic DNA matrices (S. frugiperda larvae). ConclusionsThis method provides a promising new strategy for characterizing insect-associated microbiota that can also be applied to other host microbiomes.

microbiology↗

bioRxiv2024

metagWGS, a comprehensive workflow to analyze metagenomic data using Illumina or PacBio HiFi reads

BackgroundTo study communities of micro-organisms taxonomically and functionally, metagenomic analyses are now often used. If there is no reference gene catalogue, a de novo approach is required. Because genomes are easier to interpret than contigs, the recovery of metagenome-assembled genomes (MAGs) by binning of contigs from metagenomic data has recently become a common task for microbial studies. However, during this process, there is a significant loss of information between the assembly and the binning of contigs. This is why it is important to produce taxonomic and functional matrices for all contigs and not just those included in correct bins. In addition, Pacbio HiFi reads (long and of good quality) are now a possible, albeit more expensive, alternative to short Illumina reads. We therefore developed a workflow that is easy to install with dependencies fixed using singularity images and easy to use on a computing cluster, that is capable of analyzing either short or long reads, and that should allow analysis at the contig and/or bin level, depending on the users choice. Following is a presentation of metagWGS, a fully automated workflow for metagenomic data analysis. It uses a new tool for refining bins (called Binette) that we will demonstrate is more efficient than competing tools. MethodsmetagWGS is a Nextflow workflow distributed with two singularity images and complete documentation to facilitate its installation and use. Because the main original features of metagWGS concern binning (short and long reads) and the analysis of HiFi reads, we compared metagWGS with the MAG construction workflow proposed by PacBio to a public dataset used by Pacbio to promote its workflow. ResultsmetagWGS differs from existing workflows by (i) offering flexible approaches for the assembly; (ii) supporting short reads (Illumina) or PacBio HiFi reads; (iii) combining multiple binning algorithms with a new bin refinement tool, referred to as "Binette", to achieve high-quality genome bins; and (iv) providing taxonomic and functional annotation for all genes, all contigs built and bins. metagWGS produces more medium (708) and high-quality (255) bins on 11 public metagenomic samples from human gut data than the Pacbio HiFi dedicated workflow, referred to as the HiFi-MAGS-pipeline (659 medium quality bins and 231 high quality bins), primarily due to the better performance of Binette.

bioinformatics↗

bioRxiv2020

Functional maturation of the gut microbiota at weaning is influenced by maternal environment in piglets

The objective of this study was to analyze in piglets the impact of weaning on the production of metabolites by gut bacteria and to determine whether early life environment influences the functional maturation of the gut microbiota. Fecal metabolome and microbiome were analyzed in piglets raised in two separate maternity farms and mixed at weaning. In piglets from both maternity farms, the relative abundance of Lactobacillus and of the predicted function "Fucose degradation" decreased after weaning while the relative abundance of Ruminococcus 2 and of the predicted function "Starch degradation" increased. In piglets from the first maternity farm, the relative concentration of biogenic amines and the relative abundance of Escherichi-Shigella decreased after weaning while the relative concentration of short chain fatty acids and the relative abundance of Christensenellaceae R-7 group and Ruminococcaceae UCG-002 increased. These changes were not observed at weaning in piglets from the second maternity farm probably because they already had high relative concentration of short chain fatty acids and higher relative abundance of Christensenellaceae R-7 group and Ruminococcaceae UCG-002 during the suckling period. In conclusion, the functional maturation of the microbiota at weaning is highly dependent on the maternal environment in piglets. ORIGINALITY - SIGNIFICANCE STATEMENTBacterial metabolites are key molecular intermediates between the gut microbiota and host cells. Our study in piglets reveals that the metabolic activity of the gut microbiota shifts at weaning, a key developmental period for intestinal and immune health. We also show that this functional maturation of the gut microbiota is strongly influenced by maternal environment. Thus, targeting early life environmental factors is a promising strategy to program health trough the production of beneficial bacterial metabolites at the suckling-to-weaning transition.

microbiology↗

bioRxiv2020

Reconstitution of intestinal stem cell niche in vitro with pharmacological inhibitors or L-WRN conditioned medium differentially regulates epithelial proliferation, differentiation and barrier function in rabbit caecum organoids

Intestinal organoids are self-organized 3-dimensional (3D) structures formed by a single layer of polarized epithelial cells. This innovative in vitro model is highly relevant to study physiology of the intestinal epithelium and its role in nutrition and barrier function. However, this model has never been developed in rabbits, while it would have potential applications for biomedical and veterinary research. Here, we cultured rabbit caecum organoids with either pharmacological inhibitors (2Ki medium) or L-WRN cells conditioned medium (L-WRN CM) to reconstitute the intestinal stem cell niche in vitro. Large spherical organoids were obtained with the 2Ki medium and this morphology was associated with a high level of proliferation and stem cells markers gene expression. In contrast, organoids cultured with L-WRN CM had a smaller diameter; a greater cell height and part of them were not spherical. When the L-WRN CM was used at low concentration (5%) for two days, the gene expression of stem cells and proliferation markers were very low, while absorptive and secretory cells markers and antimicrobial peptides were elevated. Epithelial cells within organoids were polarized in 3D cultures with 2Ki medium or L-WRN CM (apical side towards the lumen). We cultured dissociated organoid cells in 2D monolayers, which allowed accessibility to the apical compartment. Under these conditions, actin stress fibers were observed with the 2Ki medium, while perijonctionnal localization of actin was observed with the L-WRN CM suggesting, in 2D cultures as well, a higher differentiation level in the presence of L-WRN CM. In conclusion, rabbit caecum organoids cultured with the 2Ki medium were more proliferative and less differentiated than organoids cultured with L-WRN CM. We propose that organoids cultured with the 2Ki medium could be used to rapidly generate in vitro a large number of rabbit intestinal epithelial stem cells while organoids cultured with the L-WRN CM represent a suitable model to study differentiated rabbit epithelium.

cell biology↗