Agoutin, G. | bioRxiv Science

bioRxiv2026

Late lactation represents the main window for sow-to-piglet transmission of persistent gut strains

The gut microbiota plays a key role in piglet health, and maternal microbial transmission may represent a promising lever to shape early-life microbiota and prevent post-weaning digestive disorders. This study aimed to better characterize sow-to-piglet microbiota transmission and persistence using a long-read metabarcoding approach targeting the 16S-ITS-23S region. Fecal samples (n = 204) were collected from 17 families, a family being as sow and three of her piglets, at multiple stages: late gestation (G110), early (L6) and late lactation (L28) for sows; early lactation (L6), late lactation (L28), and 5 days post-weaning for piglets. To approximate strain-level resolution, a putative strain (PS) approach was developed by clustering ASVs (n = 6064) affiliated with the same species based on abundance covariance (r > 0.9), resulting in 4857 PS. Piglet microbiota progressively diversified during lactation and converged toward that of sow. In sows, 27 {+/-} 6% of PS were persistent from late gestation to late lactation. In piglets, only 4.2 {+/-} 2.5% of PS persisted from d6 to 5 days post-weaning. Persistent PS in piglets were mainly affiliated with Limosilactobacillus reuteri and Lactobacillus amylovorus followed with Holdemanella porci and H. biformis, Lentihominibacter hominis and Dorea formicigenerans. Shared PS were significantly higher within families than between unrelated pairs (p < 0.05). Maternal transmission peaked at the end of lactation (35 {+/-} 7% at L28). Persistent transmitted PS represented 2.7 {+/-} 1.6% (d6-post-weaning) and 15.4 {+/-} 5.6% (d28-post-weaning). Early-transmitted persistent PS were mainly affiliated with Limosilactobacillus reuteri, Lactobacillus amylovorus, and Paraeggerthella hominis, whereas late-transmitted persistent PS were associated with Prevotella spp., Sphaerochaeta globosa, and Bariatricus comes. These findings highlight the significance of maternal transmission in shaping the post-weaning microbiota and identify late lactation as a critical window for microbiota transfer.

zoology↗

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↗