Isolation and antibacterial characterization of L. rhamnosus P118. (A) Screening flowchart of P118 in vitro. (B) Candidate probiotic strains. (C) Bile salt tolerance ability of strains. (D) Biofilm forming ability of strains. (E) Screening using C. elegans infection model. (F) Interacted screening strategy. (G) Antibacterial ability of P118 under aerobic or anaerobic culture conditions. (H) Broad-spectrum pH tolerance of P118 supernatants (pH = 3.4) that adjusted to pH < 3.4 by 1M HCl or pH > 3.4 by 1M NaOH. Antibacterial effects of P118 supernatant under (I) 20 mg/mL catalase (CAT), (J) 100μg/mL proteinases (trypsin, proteinase K, pepsin), and (K) different temperatures (37, 50, 70, 90, 100, 120℃) boiled for 30 min treatments. (L) Antibacterial effects of components of P118 (boiled at 120 ℃ for 30 min or was lysed by ultrasonication at 240 W for 2 h). (M) Active ingredients of P118 protect C. elegans against S. Typhimurium infection. (N) Taxonomic classification of P118 draft genome. (O) The nearest subspecies phylogenetic neighbor of P118 draft genome was determined by percentage shared genomic content graphed as ANI versus AAI. (J, H) Prediction of secondary metabolites and bacteriocin protein-encoding gene clusters of P118. (G-M) S. Typhimurium SL1344 was selected as an indicator pathogen. Significant differences * P < 0.05, ** P < 0.01, *** P < 0.001.

A list of probiotic strains recovered from examined samples

L. rhamnosus P118 enhances mice tolerance to S. Typhimurium infection. (A) Experimental design. (B) Survival curve of mice infected with S. Typhimurium. (C) Body weight. (D) Spleen index. (E) Representative images of spleen and intestine. (F) Liver index. (G) S. Typhimurium burden in tissues and shedding in feces. (H) Representative images of H&E staining, TEM, and immunostaining (DAPI, Ki67, lysozyme, Muc2, F4/80, iNOS) in the ileum. Different lowercase letters indicate a significant difference (P < 0.05). Significant differences * P < 0.05, ** P < 0.01.

L. rhamnosus P118 improves S. Typhimurium infection-induced dysbacteriosis. (A) Principal coordinates analysis (PCoA) based on Bray-Curtis distance. (B) UpSetR plot based on bacterial absolute OTU abundances. (C & D) The fold changes of bacterial absolute OTU abundances between two groups. (E & F) Comparison of intestinal microbes by STAMP. The prefix “g_” represent the annotated level of the genus.

Microbe derived tryptophan metabolites are involved in mice tolerance to S. Typhimurium. (A) Principal coordinates analysis (PCoA) based on Bray-Curtis distance. (B & C) UpSetR plot based on fecal metabolites. (D) Metabolomics pathway enrichment. (E) Comparison of tryptophan metabolism enriched pathway. (F) Pearson correlation analysis among S. Typhimurium burden, organ indices, body weight and fecal metabolites. (G) Bio-Sankey network analysis between intestinal microbes and fecal metabolites. (H) Pathway schematic of abbreviated mammalian and microbial tryptophan metabolism. Different lowercase letters indicate a significant difference (P < 0.05). Significant differences *P < 0.05; **P < 0.01; ***P < 0.001.

Indole-3-acrylic acid enhances mice tolerance to S. Typhimurium infection. (A) Experimental design. (B) Body weight change. (C) Spleen index. (D) Liver index. (E) Colon length. (F) S. Typhimurium burden in tissues and shedding in feces. (G) Representative images of spleen and liver. (H) Villus height of ileum. (I) Muc2-positive cells in ileum. (J-N) mRNA expression levels in ileum. (O) Representative images of H&E staining and immunostaining in the ileum. Different lowercase letters indicate a significant difference (P < 0.05). Significant differences ** P < 0.01.

Macrophage depletion abrogates the protective effect of L. rhamnosus P118 and indole-3-acrylic acid against S. Typhimurium infection. (A) Experimental design. (B) Body weight change. (C) Spleen index. (D) Liver index. (E) Colon length. (F) Representative images of spleen and liver. (G) S. Typhimurium burden in tissues and shedding in feces. (H) Villus height of ileum. (I) Muc2-positive cells in ileum. (J-N) mRNA expression levels in ileum. (O) Representative images of H&E staining and immunostaining in the ileum. Different lowercase letters indicate a significant difference (P < 0.05). Significant differences ** P < 0.01.

Lacticaseibacillus rhamnosus P118 strain with great probiotic properties was screened from 290 purified isolates through two distinctive screen approaches, and P118 strain was underscored for superficial protective effects on a murine infection model. Further, multi-omics analysis pinpointed the microbe-derived tryptophan/indole could be the importance of P118 probiotic properties. Nevertheless, the newly found P118 enhances host tolerance to Salmonella infections via various pathways, including direct antibacterial actions, inhibiting Salmonella colonization and invasion, attenuating pro-inflammatory responses of intestinal macrophages, and modulating gut microbiota mediated by microbe-derived indole metabolites.

(A) Drug resistance and (B) broad-spectrum antibacterial activities of candidate probiotic strains.

(A) Circular genome map of L. rhamnosus P118. (B) phylogenetic trees of L. rhamnosus P118 and nearest phylogenetic neighbors constructed by average amino acid identify (AAI). Color gradients indicate the percentage of conserved average nucleotide identity (ANI) between each isolate and respective nearest subspecies phylogenetic neighbors.

Clinical and Histopathological observation. (A) Representative images of H&E staining tissues (liver, spleen). Scale bar with 100 μm. (B) Clinical symptom score. Histopathological quantification of H&E staining (C & D) and TEM (E & F) images observed in the ileum. Different lowercase letters indicate a significant difference (P < 0.05).

Gene and protein expression level of tight junction protein (ZO-1, Claudin-1) and inflammatory cytokines (IL-1β, IL-18) in ileum and serum of mice. Different lowercase letters indicate a significant difference (P < 0.05).

Comparison of intestinal microbes by STAMP. The prefixes “g_” and “s_” represent the annotated levels of genus and species.

L. rhamnosus P118 alters gut microbial community assembly. (A) Ecological processes of gut microbes. The inner circle indicates the percentage of deterministic and stochastic assembly processes. The outer circle indicates the contribution of detailed ecological processes. (B) The fit of neutral community model (NCM) of community assembly for gut microbes. The best fit to NCM and 95% Cis around the best fit to NCM were indicated by solid and dashed blue lines, respectively. Light blue and dark red dots indicate OTUs that occur more (above prediction) or less (below prediction) frequently than predicted by NCM, respectively, while black dots indicate the OTUs that occur within neutral prediction (neutral distribution). Donut charts shows the distinct fitting proportions of OTUs predicted by NCM.

Analysis of differential fecal metabolites. Venn diagram based on differential fecal metabolites (A) down-regulated in “S vs C” and up-regulated in “P+S vs S”, (B) up-regulated in “S vs C” and down-regulated in “P+S vs S”. (C) Significant differential fecal metabolites in “S vs C” and “P+S vs S”.

Antibacterial activities of indole-3-acrylic acid.

Indole-3-acrylic acid enhances bactericidal capacity and exerts anti-inflammatory activity in RAW 264.7 cells. Different lowercase letters indicate a significant difference (P < 0.05).