Lacticaseibacillus rhamnosus Reduces the Pathogenicity of Escherichia coli in Chickens

Abstract

Lacticaseibacillus rhamnosus is a recognized probiotic that is widely used in scientific research and clinical applications. This study found that the Lacticaseibacillus rhamnosus GG (LGG) strain can reduce the adhesion of Escherichia coli (E. coli) to primary chicken intestinal epithelial cells by 75.7% and inhibit 41.7% of the E. coli that adhere to intestinal epithelial cells. Additionally, LGG showed strong inhibitory ability on the growth of E. coli, Staphylococcus aureus, Salmonella Paratyphi B, and Salmonella Enteritidis in vitro. Furthermore, the influence of LGG on the growth performance, intestinal flora, immunity, and disease resistance of chickens was explored. Chickens fed with LGG exhibited increased average daily weight gain and concentrations of sIgA, IgG, and IgM than did controls. After 21 days of feeding, a diet with LGG increased the diversity of intestinal microbiota and maintained intestinal health. Moreover, LGG promoted immunologic barriers by upregulating cytokines and chemokines via the Toll-like receptor. The major pro-inflammatory factors, including Myd88, NF-κB, Il6, and Il8, were upregulated compared to controls. After being challenged with E. coli, the survival rate of chickens fed with LGG was significantly higher than those in the control group, and decreased numbers of E. coli were detected in the heart and lungs of the LGG group. In summary, oral administration of LGG to chickens could improve growth performance, maintain intestinal homeostasis, and enhance innate immune response and disease resistance.

Keywords: Lacticaseibacillus rhamnosus; adhesion; disease resistance; innate immune response; intestinal microbiota.

FIGURE 1

Antibacterial activity of LGG against Staphylococcus aureus, Salmonella Paratyphi B, O1 E. coli, and Salmonella Enteritidis. Four pathogenic bacteria were spread onto the LB agar plates. Then (A) LGG culture supernatant, (B) lysate, (C) LGG culture, and (D) LGG cells were transferred to holes (5-mm diameter) punched into the agar palates. The antimicrobial activity was determined by the size of the inhibition zone. Bars represent the means ± standard deviations of three independent repetitions. Nd, not detected.

FIGURE 2

Effects of LGG on E. coli adhesion to primary chicken intestinal epithelial cells. (A) The LGG and E. coli cultures were added to primary chicken intestinal epithelial cells (MOI = 100:1), followed by 1-h incubation at 37°C and 5% CO₂. Adhesion percentage of LGG and E. coli to primary chicken intestinal epithelial cells was expressed as the number of adherent bacteria relative to the total number of bacteria. (B) Competition, inhibition, and displacement of E. coli adhesion in the presence of LGG. LGG and E. coli (1:1) were simultaneously added to primary chicken intestinal epithelial cells for competition assay. In the inhibition assay, LGG was first added to primary chicken intestinal epithelial cells and incubated for 1 h, after which unbound LGG was removed and E. coli was added to the wells. On the contrary, in the displacement assay, E. coli was added first. Bars represent the means ± standard deviations of three independent repetitions.

FIGURE 3

Effects of dietary LGG on cecal microbial diversity in chickens. The cecal contents of chickens fed with LGG and the controls were collected for high-throughput sequence analysis after 21 days of feeding. (A) α-diversity of OTU rank abundance. (B) OTU Venn diagram between the LGG group and the control group. (C) Principal component analysis (PCA) plot based on the distribution of bacterial community. (D) Principal coordinates analysis (PCoA) plot using Brary–Curtis distances. The percentage represents the contribution of the principal component to the sample difference.

FIGURE 4

Comparison of identified relative abundance in cecal microbes. Relative abundance of bacterial (A) phylum level and (B) family level. Relative abundance of (C) Ruminococcaceae, (D) Lachnospiraceae, and (E) Lactobacillaceae. Bars represent the means ± standard deviations of three independent repetitions. *p < 0.05. (F) The differential abundance between LGG and the control group based on metastats difference analysis. The abundance distribution of the five species with the greatest difference between the two groups was presented.

FIGURE 5

The expression of Tlr4, Mhc II-α, Myd88, NF-κB, Ifn-α, Il1β, Il6, and Il8 in the spleens and livers of chickens after 21 days of feeding. The fold change represents the target gene expression in the diet with LGG compared to the diet of the control group. The relative gene expression levels were normalized to β-actin. Bars represent the means ± standard deviations (n = 5). *p < 0.05.

FIGURE 6

Disease resistance of chickens fed with LGG diet post-challenge with E. coli. Chickens were challenged with 10⁶ CFU E. coli after 21 days of feeding. (A) The survival rate of chickens after infection with E. coli. (B) E. coli content in hearts, livers, spleens, lungs, and kidneys of infected chickens at 1 and 3 dpi (log₁₀ CFU g^{– 1}). Bars represent the means ± standard deviations (n = 5). *p < 0.05.

FIGURE 7

The expression of immune-related genes in the spleens and livers of infected chickens at 1 and 3 dpi. Expression of (A) Tlr4, (B) Mhc II-α, (C) Myd88, (D) NF-κB, (E) Ifn-α, (F) Il1β, (G) Il6, and (H) Il8. The fold change represents the target gene expression in the diet with LGG compared to the diet of the control group. The relative gene expression levels were normalized to β-actin. Bars represent the means ± standard deviations (n = 5). *p < 0.05.