Limosilactobacillus reuteri is a vertebrate symbiont that is widely appreciated as being of significant ecological importance for human health. As a unique feature, L. reuteri converts glycerol to the antimicrobial compound reuterin using enzymes encoded in its propanediol-utilization operon and evolves with host-driven diversification. Reuterin-producing L. reuteri HLRE13 was selectively isolated from poultry previously and confirmed to inhibit the growth of Staphylococcus aureus in vitro. However, it remains unclear whether L. reuteri HLRE13 retains these antagonistic properties when ingested in specific-pathogen-free mice. Here, we investigated the ameliorative effects and potential mechanisms of action of L. reuteri HLRE13 in combination with glycerol on S. aureus-induced infection phenotypes in mice. Firstly, our results confirmed that L. reuteri HLRE13 effectively inhibited the intestinal colonization of S. aureus CMCC26003; Secondly, L. reuteri HLRE13 combined with glycerol could alleviate the intestinal tissues damage caused by S. aureus through increasing the expression of ZO-1, Occludin, and MUC-2, ameliorate the intestinal systemic inflammatory response, and maintain the balance of gut microbiota by increasing the relative abundance of Lactobacillus and reducing the relative abundance of Staphylococcus. Furthermore, the colonization resistance was also found on L. reuteri HLRE13 combined with glycerol against S. aureus in pseudo germ-free mice, and they exerted the similar effects on alleviating intestinal damage and improving immune function. Combining these results, we speculate that reuterin-producing L. reuteri antagonize S. aureus in mice without the gut microbiota-dependent manner. Overall, our findings will provide a theoretical foundation for the scientific cognition of L. reiteri in maintaining intestinal health by producing reuterin.

Based on the metabolic activity and biotransformation capacity of the resting cells of Limosilactobacillus reuteri, its ability to metabolize functional oligosaccharides and antagonize foodborne pathogens was explored. A phylogenetic tree for L. reuteri was constructed based on its 16S rRNA gene sequence. The environmental tolerance of the resting cells of L. reuteri was analyzed by acid and bile salt tolerance, and antibiotic sensitivity assays. The effects of functional oligosaccharides on the metabolism, surface hydrophobicity, adhesion capacity and reuterin (a broad-spectrum antibacterial) production ability of L. reuteri were evaluated by using modified media. Then, the inhibitory effect of the resting cells of L. reuteri on the adhesion of Staphylococcus aureus to Caco-2 cells and its growth in skim milk was explored. Results showed that both L. reuteri strains tested could tolerate extreme acidic conditions and bile salts for more than 3 h and were susceptible to common antibiotics. All functional oligosaccharides tested except xylo-oligosaccharides could be metabolized by the resting cells, and its growth, surface hydrophobicity and adhesion capacity were improved significantly by raffinose (P < 0.05). Raffinose could promote the production of reuterin from L. reuteri HLRE13 by utilizing glycerol, attaining a concentration of (1.34 ± 0.03) g/L. Glycerol promoted the inhibitory effect of L. reuteri HLRE13 on a variety of foodborne pathogens significantly (P < 0.001), and inhibited the adhesion of S. aureus to Caco-2 cells by 41.67% through exclusion. Furthermore, glycerol could promote L. reuteri HLRE13 to reduce the number of S. aureus to 10³ CFU/mL during co-culture. This study will provide a scientific basis for the functional development of resting cells of L. reuteri and its synergistic efficacy with functional oligosaccharides.