Multitechnique characterisation of gut microbiota dynamics in piglets supplemented with Limosilactobacillus reuteri DSPV002C

Technical Summary: Multitechnique Characterisation of Gut Microbiota Dynamics in Piglets Supplemented with Limosilactobacillus reuteri DSPV002C

Problem Statement
The weaning process in piglets represents a critical developmental stage characterized by abrupt dietary and environmental changes that disrupt gut microbial balance and integrity. This disruption often leads to growth delays and increased susceptibility to diseases, particularly post-weaning diarrhea syndrome associated with enterotoxigenic Escherichia coli (ETEC). With the restriction of antibiotics due to antimicrobial resistance, there is an urgent need for alternative strategies, such as probiotics, to restore microbial homeostasis. However, significant gaps remain regarding the optimal strains, dosages, and administration timings, as well as a comprehensive understanding of how specific probiotic strains modulate microbial diversity and function during this transitional period.

Methodology
This study employed a multitechnique approach to characterize temporal changes in the fecal microbiota of weaned piglets supplemented with Limosilactobacillus reuteri DSPV002C.

• Experimental Design: Twenty piglets (28 ± 1 days old) were randomly assigned to a control group (CG) or a probiotic group (PG). The PG received daily macrocapsules containing a rifampicin-resistant variant of L. reuteri DSPV002C (≥9.80 log CFU/animal) from day 1 to day 42.
• Sampling: Fecal samples were collected on days 0, 7, 14, 21, 28, 35, and 42.
• Analytical Techniques:
  1. Culture-Based Methods: Plate counts were performed to quantify Lactic Acid Bacteria (LAB), the supplemented L. reuteri strain, Campylobacter spp., E. coli, and yeasts.
  2. Denaturing Gradient Gel Electrophoresis (DGGE): Used to profile microbial community structure and diversity (Shannon, Pielou, Margalef indices) on days 0, 21, and 42. Representative bands were sequenced to identify bacterial groups.
  3. 16S rRNA Gene Sequencing: Performed on a subset of animals (n=3 per group per time point) using Ion Torrent™ technology. Data analysis included alpha and beta diversity assessments (PCA, PCoA, UniFrac), relative abundance analysis at phylum and genus levels, Linear Discriminant Analysis Effect Size (LEfSe) for enriched taxa, and functional prediction of metabolic pathways using Tax4Fun2.

Key Results

• Probiotic Persistence: The supplemented L. reuteri DSPV002C strain was successfully recovered in the PG from day 7 onward throughout the trial, confirming its gastrointestinal transit and persistence. It was not detected in the CG.
• Culturable Populations: No significant differences were observed in total LAB counts between groups. Similarly, populations of Campylobacter spp., E. coli, and yeasts did not differ significantly between the CG and PG, though temporal fluctuations occurred in both groups during the post-weaning period.
• DGGE Analysis: Profiles revealed temporal clustering, with samples grouping more by sampling time than by treatment group. While beta diversity showed a trend toward separation between groups on day 42 (p < 0.10), no significant differences in diversity, richness, or homogeneity indices were found between groups. DGGE sequencing identified Firmicutes, Synergistetes, Bacteroidetes, and Proteobacteria as dominant phyla, with community restructuring occurring over time.
• 16S rRNA Sequencing:
  • Diversity: No significant differences in alpha diversity (Observed ASV, Shannon, Simpson) were detected between groups at any time point. Beta diversity analysis indicated a trend toward separation between groups on day 42 (p < 0.10), while significant temporal shifts were observed within both groups throughout the study.
  • Taxonomic Composition: Firmicutes and Bacteroidota were the dominant phyla. At the genus level, several taxa showed trends toward differential abundance (p < 0.10). For instance, Ligilactobacillus and Campylobacter were enriched in the PG on day 21, while the Eubacterium siraeum group and Angelakisella showed enrichment in the PG on day 42. Conversely, Streptococcus and Sphaerochaeta tended to be more abundant in the CG on day 42.
  • Functional Prediction: No statistically significant differences were observed in predicted metabolic pathways between the groups at any sampling time.

Significance and Claims
The authors claim that the study provides a broader characterization of temporal microbiota dynamics by integrating culture-dependent and culture-independent methodologies. The primary finding is that L. reuteri DSPV002C supplementation is associated with temporal shifts in gut microbial composition during the post-weaning period, evidenced by the persistence of the strain and subtle trends in specific genera.

However, the paper maintains a modest stance regarding its conclusions. The authors explicitly state that the results should be considered exploratory due to the limited number of animals included in the sequencing analyses (n=3), which reduces statistical power. They note that while the multitechnique approach revealed temporal restructuring and specific taxonomic trends, the absence of clear functional differences and significant beta diversity separation suggests that the microbial changes associated with this specific supplementation under these conditions were relatively subtle. The study concludes that future research with larger cohorts and complementary approaches (e.g., metabolomics, physiological parameters) is necessary to clarify the biological relevance of these microbial modulations.