L. rhamnosus CNCM I-3690 survival, adaptation, and small bowel microbiome impact in human

Abstract

Fermented foods and beverages are a significant source of dietary bacteria that enter the gastrointestinal (GI) tract. However, little is known about how these microbes survive and adapt to the small intestinal environment. Colony-forming units (CFU) enumeration and viability qPCR of Lacticaseibacillus rhamnosus CNCM I-3690 in the ileal effluent of 10 ileostomy subjects during 12-h post consumption of a dairy product fermented with this strain demonstrated the high level of survival of this strain during human small intestine passage. Metatranscriptome analyses revealed the in situ transcriptome of L. rhamnosus in the small intestine, which was contrasted with transcriptome data obtained from in vitro cultivation. These comparative analyses revealed substantial metabolic adaptations of L. rhamnosus during small intestine transit, including adjustments of carbohydrate metabolism, surface-protein expression, and translation machinery. The prominent presence of L. rhamnosus in the effluent samples did not elicit an appreciable effect on the composition of the endogenous small intestine microbiome, but significantly altered the ecosystem's overall activity profile, particularly of pathways associated with carbohydrate metabolism. Strikingly, two of the previously recognized gut-brain metabolic modules expressed in situ by L. rhamnosus (inositol degradation and glutamate synthesis II) are among the most dominantly enriched activities in the ecosystem's activity profile. This study establishes the survival capacity of L. rhamnosus in the human small intestine and highlights its functional adjustment in situ, which we postulate to play a role in the probiotic effects associated with this strain.

Keywords: L. rhamnosus; human; in situ gene expression; metatranscriptome; probiotic fate; small intestine.

Figure 1.

L. rhamnosus CNCM I-3690 recovery and concentration in ileal effluent. Samples were collected prior and collectively obtained during 4-h periods over a total timespan of 12 h(4, 8, and 12) after consuming L. rhamnosus CNCM I-3690 fermented product. Serial dilutions were plated in triplicate and L. rhamnosus CNCM I-3690 colonies were enumerated and identity confirmed after 4 (black dots), 8 (magenta dots), and 12 (green dots) hours, while the total amount recovered per volunteer is indicated with a blue asterisk. L. rhamnosus CNCM I-3690 recovery peaked after 8 h in most volunteers (Panel Figure 1a, b), while it was not detected before consumption (data not shown). Ileostomy effluent volumes fluctuated considerably, affecting the L. rhamnosus concentration per sample (Panel b), averaging approximately 2*10⁸ CFU/mL.

Figure 2.

The small intestinal environment inducesMicrobiota composition analysis and L. rhamnosus recovery. a) L. rhamnosus CNCM I-3690 gene copies recovery in ileal effluent, assessed via qPCR using both PMAxx treated (black bars) and non-treated samples (pink-bars). Samples were collected prior and collectively obtained during 4-h periods over a total timespan of 12 h (4, 8, and 12) after consuming L. rhamnosus CNCM I-3690 fermented product and prior of it (0 H); b) overview of the microbiota composition throughout the study per volunteer at genus level, only the top 25 genera are displayed (comprising >95% of the total community). The genus formerly known as Lactobacillus is colored in red.

Figure 3.

Transcriptome analysis of L. rhamnosus recovered from small intestinal effluent compared with in vitro conditions. a) Principal component analysis of L. rhamnosus transcriptome samples; separating the in situ samples from the MRS samples that also separate from the milk grown samples (green dots). b) volcano plot of the differential expression analysis performed via edgeR, in blue and in red downregulated (blue) and upregulated (red) genes in situ compared to in vitro; cut-off: FDR adjusted-p-value≤0.05, Log2FC≥2. c) Gene Set Enrichment Analysis (GSEA) using the differentially expressed genes (enrichment cut-off, p-value < 0.05; fold enrichment is given as the ratio between “hits” and “expected hits” on the selected gene category) identifies 13 KEGG categories, of which 11 belong to “Metabolism” encompassing 8 belonging to “Carbohydrate metabolism” (green box). Bar colours represents an average gene expression per pathway higher (red) or lower (blue) in situ compared to in vitro condition; d) heatmap of the expression of enriched pathways belonging to the KEGG category “Metabolism”, for each pathway, the average gene expression per volunteer was used to calculate the fold change from the row mean; hierarchical clustering: complete linkage based on Euclidian distances. Among the overrepresented gene sets, only two (Fatty acid biosynthesis and Pyruvate metabolism) had a higher expression in vitro than in situ, while the remaining 9 were higher expressed in situ.

Figure 4.

L. rhamnosus response to the small intestinal environment. Panel a: The expression values are displayed as edgeR derived, FDR < 0.05, logFC ≥ 2, displayed as a heat map representing fold change relative to the mean expression per row; blue, and red intensities reflect relative down- and up-regulation, white reflects mean-level expression. The variable environmental conditions encountered by L. rhamnosus in each individual is reflected by diverse in situ gene expression of nutrient acquisition genes (e.g., sugar import functions, panel a, parts I and II). Conversely, a ribosomal protein gene cluster is consistently induced in situ, while a few ribosomal protein transcripts are induced in MRS-cultures (panel a, part III), suggesting adjustment of translation machinery associated functions in these contrasting conditions. The degree of variation of in situ gene expression is illustrated by the coefficient of variation color scale at the left end of the heatmaps (white, red). The adjacent color scale (green, white, violet) indicates the average Log2FC in intestinal samples compared to in vitro samples. At the left end of the heatmap display a per category hierarchical clustering is displayed using linkage based on Euclidian distances. Panel b: expression of the global regulator Catabolite Control Protein A gene (ccpA) is induced in situ relative to in vitro, but variable in in the intestinal samples (panels aI and aII). Panel c: variable in situ induction of the pilus encoding operon (spaD-F), which plays a role in adhesion and immune-related effects of L. rhamnosus. Significance of the differences in expression was assessed by FDR adjusted p-value <0.05; ****: FDR adjusted p-value <0.0001.

Figure 5.

Ecosystem contribution by L. rhamnosus on the overall activity profile of the microbiota. The “contribution enrichment factor”, calculated as log₂ of the fraction of all L. rhamnosus reads mapped to a category divided by the fraction of all other reads mapped to that category, was assessed for the main KEGG metabolism classes (panel a) and for the carbohydrate metabolism major categories (panel b). The analyses showed that L. rhamnosus dedicates more of its transcriptome compared with the endogenous microbiota to “Carbohydrate metabolism”, “Galactose metabolism”, “Fructose and mannose metabolism”, and “Inositol phosphate metabolism” (Wilcoxon tests, Holm-Sidak Post hoc test * p-value < 0.05, ** p-value < 0.01, ***p-value < 0.005). The “Inositol phosphate metabolism” and the “Glutamate biosynthesis II” are identified as two of the nine Gut-Brain Modules encoded by L. rhamnosus genome and they are significantly more expressed by L. rhamnosus than by the small intestinal endogenous microbiota (c, Mann–Whitney test, Holm-Sidak Post hoc test ***p-value <0.005, **** p-value <0.001). L. rhamnosus is responsible for most of the Inositol degradation expression (d, red represent in L. rhamnosus contribution, in blue the endogenous one) and that most of the 13-genes-long operon (e), encoding the entire Inositol degradation pathway, is highly expressed in situ compared with in vitro conditions.