Lactobacillus agilis SNF7 Presents Excellent Antibacteria and Anti-Inflammation Properties in Mouse Diarrhea Induced by Escherichia coli

Abstract

Escherichia coli (E. coli) is a common pathogen that causes diarrhea in newborns and animals. Antibiotics are typically used to treat bacterial diarrhea, a global intestinal health issue. Probiotics have gained interest as a potential substitute for antibiotics in the management of E. coli-induced diarrhea and present novel therapeutic options. In this study, the probiotic properties of Lactobacillus agilis SNF7 (L. agilis SNF7) isolated from feces were investigated, and whole genome sequencing was performed to evaluate the properties of the strain. Furthermore, we investigated the protective effects of L. agilis SNF7 in a mouse model of E. coli K99 infection. L. agilis SNF7 exhibits a high survival rate in artificial gastroenteric fluid and bile salt environments, along with an antagonistic effect against E. coli O₁₁₁:K₅₈ (B₄), Staphylococcus aureus (S. aureus), and E. coli K99. Multiple genes with probiotic properties, including bacteriostasis, anti-inflammation, antioxidant, CAZyme, and the utilization of carbohydrate compounds, were identified in genome. L. agilis SNF7 prevented the gut barrier from being damaged by E. coli K99, reducing the clinical manifestations of the infection. Furthermore, L. agilis SNF7 reduced the expression of inflammatory cytokines (IL-6, IL-1β, and TNF-α) by inhibiting the phosphorylation of proteins linked to the NF-κB and MAPK signaling pathways. L. agilis SNF7 improved the intestinal microbial barrier, controlled the balance of the intestinal microecology, and reduced the entry of harmful microbes into the intestine. By controlling gut flora and reducing the inflammatory response, L. agilis SNF7 may be able to prevent and treat E. coli K99 infections. The application of L. agilis SNF7 in the creation of probiotic formulations to stop intestinal illnesses brought on by E. coli infections is clarified by this work.

Keywords: Escherichia coli; Lactobacillus agilis SNF7; diarrhea; probiotic properties; whole genome.

Figure 1

Experimental design and the characteristics of L. agilis SNF7 in vitro. (a) Experimental design; (b) colony morphology; (c) survival of L. agilis SNF7 at different pH levels; (d) survival of L. agilis SNF7 at different bile salt concentrations; (e) survival of L. agilis SNF7 in artificial gastrointestinal fluid; (f) inhibitory effect of L. agilis SNF7 on three strains of pathogenic bacteria; (g) inhibitory effect of L. agilis SNF7 on three strains of pathogenic bacteria after different treatments; (h) hemolysis of L. agilis SNF7 (the picture above is S. aureus and the one below is L. agilis SNF7); (i) growth curve of L. agilis SNF7; (j) acid production curve of L. agilis SNF7; (k) sensitivity of L. agile SNF7 to antibiotics. Compared with the CK group, ns means has no significant difference (p > 0.05), * means significant difference (p < 0.05).

Figure 2

Genomic predictive function of L. agilis SNF7. (a) Annotation of the genome of L. agilis SNF7 in the GO database; (b) annotation of the genome of L. agilis SNF7 in the KEGG database; (c) annotation of the genome of L. agilis SNF7 in the eggNOG database; (d) annotation of the genome of L. agilis SNF7 in the CAZy database.

Figure 3

Effects of L. agilis SNF7 on the physiological indexes of E. coli K99-induced diarrhea mice. (a) Diarrhea rate of mice during the test period; (b) fecal scores of mice during the test period; (c) body weight of mice in each group on day 8 of the experiment; (d) body weight of mice in each group on day 15 of the experiment; (e) changes in body weight of mice during the experiment; (f) spleen index; (g) liver index; (h) intestinal index. Same letters on columns means the difference is not significant (p > 0.05), ns means has no significant difference (p > 0.05). The difference is significant (p < 0.05) when there are no identical letters between the columns.

Figure 4

Effects of L. agilis SNF7 on the intestinal physical barrier of E. coli K99-induced diarrhea mice. (a) H&E staining of mouse jejunal tissue (bar = 100 µm); (b) expression of Claudin-1 in mouse jejunal tissue (bar = 100 µm); (c) expression of occludin in mouse jejunal tissue (bar = 100 µm); (d) expression of ZO-1 in mouse jejunal tissue (bar = 100 µm); (e) expression of MUC2 in mouse jejunal tissue (bar = 100 µm); (f) area fraction of Claudin-1; (g) area fraction of occludin; (h) area fraction of ZO-1; (i) area fraction of MUC2. The same letters on the column show the difference is not significant (p > 0.05). The difference is significant (p < 0.05) when there are no identical letters between the columns.

Figure 5

Effects of L. agilis SNF7 on E. coli-induced inflammatory factor secretion in mice jejunum tissues. (a) Relative expression of IL-6 mRNA; (b) relative expression of TNF-α mRNA; (c) relative expression of IL-1β mRNA; (d) IL-6 content; (e) TNF-α content; (f) IL-1β content. The same letters on the column show the difference is not significant (p > 0.05). The difference is significant (p < 0.05) when there are no identical letters between the columns.

Figure 6

Effect of L. agilis SNF7 on NF-κB and MAPK signaling pathways in E. coli K99-induced diarrhea mice. (a) Protein bands of NF-κB signaling pathways; (b) p-IκBα/IκBα; (c) p-p65/p-65; (d) protein bands of MAPK signaling pathways; (e) p-p38/p38; (f) p-ERK/ERK; (g) p-JNK/JNK. The same letters on the column show the difference is not significant (p > 0.05). The difference is significant (p < 0.05) when there are no identical letters between the columns.

Figure 7

Venn diagram, α diversity analysis, and β diversity analysis of cecum microorganisms in each group. (a) OTU Wayne diagram; (b) α diversity analysis; (c) beta-diversity analysis: PCoA analysis; (d) beta-diversity analysis: NMDS analysis. * Compared with the CK group, * means significant difference (p < 0.05).

Figure 8

Modulation effect of L. agilis SNF7 on the gut microbiota in E. coli K99-induced diarrhea mice. (a) Phyla-level species distribution histogram; (b) phylum-level species composition heat map; (c) genus-level species distribution histogram; (d) generic-level species composition heat map; (e–k) relative abundance of genera with genus-level differences between groups. The same letters on the columns show the difference is not significant (p > 0.05). The difference is significant (p < 0.05) when there are no identical letters between the columns.