Probiotic Mixture Golden Bifido Prevents Neonatal Escherichia coli K1 Translocation via Enhancing Intestinal Defense

Abstract

Escherichia coli (E. coli) K1 sepsis and meningitis is a severe infection characterized by high mortality in neonates. Successful colonization and translocation across the intestinal mucosa have been regarded as the critical steps for E. coli K1 sepsis and meningitis. We recently reported that the probiotic mixture, Golden Bifido (containing live Lactobacillus bulgaricus, Bifidobacterium, and Streptococcus thermophilus, LBS) has a preventive role against neonatal E. coli K1 bacteremia and meningitis. However, the interaction between the neonatal gut barrier, probiotics and E. coli K1 is still not elucidated. The present study aims to investigate how LBS exerts its protective effects on neonatal gut barrier during E. coli K1 infection. The beneficial effects of LBS were explored in vitro and in vivo using human colon carcinoma cell lines HT-29 and rat model of neonatal E. coli K1 infection, respectively. Our results showed that stimulation with E. coli K1 was able to cause intestinal barrier dysfunction, which were reflected by E. coli K1-induced intestinal damage and apoptosis of intestinal epithelial cells, reduction of mucin, immunoglobulin A (IgA) and tight junction proteins expression, as well as increase in intestinal permeability, all these changes facilitate E. coli K1 intestinal translocation. However, these changes were alleviated when HT-29 cells were treated with LBS before E. coli K1 infection. Furthermore, we found that LBS-treated neonatal rats (without E. coli K1 infection) have showed higher production of mucin, ZO-1, IgA, Ki67 in intestinal mucosa as well as lower intestinal permeability than that of non-treated rats, indicating that LBS could accelerate the development of neonatal intestinal defense. Taken together, our results suggest that enhancement of the neonatal intestinal defense to fight against E. coli K1 translocation could be the potential mechanism to elucidate how LBS confers a protective effect against neonatal E. coli K1 bacteremia and meningitis. This indirect mechanism makes LBS exert preventive effect on most of gut-derived pathogenic infections rather than only E. coli.

Keywords: bacterial translocation; intestinal barrier; mucin; neonatal sepsis and meningitis; probiotics; tight junction.

FIGURE 1

Lactobacillus bulgaricus, Bifidobacterium, and Streptococcus thermophilus (LBS) alleviates mucin degradation during Escherichia coli K1 infection. HT-29 cells were pre-treated with or without LBS before E. coli K1 infection. Cells treated with PBS or LBS alone were served as controls. Cellular proteins were extracted for (A) periodic acid-Schiff (PAS) assay and (C) western blot analysis. (B) HT-29 monolayers were stained with PAS and observed under light microscope (100× magnification). (D) Immunofluorescence of MUC2 was obtained by fluorescence microscopy (200× magnification), green staining represents the MUC2, the cell nuclei were stained with Hoechst 33342 (blue). Results are represented as mean ± SD. ^∗P < 0.05, ^∗∗∗P < 0.001.

FIGURE 2

Lactobacillus bulgaricus, Bifidobacterium, and Streptococcus thermophilus inhibits adhesion and invasion of E. coli K1 to HT-29 cells. HT-29 cells were cultured in 24-well plate and pre-treated with various doses of LBS for 3 h before adding E. coli K1 or co-incubated with LBS plus E. coli K1. Adhesion and invasion assays were carried out as described in section “Materials and Methods.” The numbers of associated bacteria (A,C) and intracellular bacteria (B,D) were determined. Results are represented as mean ± SD. ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 3

Pre-treatment with LBS decreases E. coli K1-induced damage and apoptosis of HT-29 cells. HT-29 cells were treated as described in Figure 1. (A) LDH activity was analyzed by microtiter plate reader and the results were expressed as a percentage of the control (untreated cells). Results are presented as mean ± SD. ^∗P < 0.05. (B) HT-29 cells were illustrated by fluorescence microcopy (100× magnification). Hoechst 33342 was used to stain nuclei of all cells (blue), PI was used to detect the dead cells (red).

FIGURE 4

Lactobacillus bulgaricus, Bifidobacterium, and Streptococcus thermophilus prevents E. coli K1-induced disruption of intestinal integrity. HT-29 cells were cultured on the upper chamber of the Transwell insert and pre-treated with or without LBS before E. coli K1 infection. Cells treated with PBS or LBS alone were served as controls. After infection, HRP was added to the upper chamber of Transwell for 1 h. Bacteria and HRP translocated from the upper chamber to the lower chamber were quantified as described in section “Materials and Methods.” (A) The OD₄₅₀ value of HRP. (B) The number of E. coli K1 CFU, which penetrated across the HT-29 monolayer. Results are represented as mean ± SD. ^∗P < 0.05, ^∗∗∗P < 0.001. (C) HT-29 cells were treated as described in Figure 1, proteins of each group were isolated for western blotting. The expressions of ZO-1 and occludin was determined, β-Actin band was used as an indicator of protein loading.

FIGURE 5

Effects of LBS on mucin expression of neonatal colon tissue with or without E. coli K1 infection. Two days old neonatal rats were orally administered with or without LBS for 3 days before E. coli K1 infection. Pups administered with PBS or LBS alone was served as controls. (A) Colon tissues received different treatment were processed with PAS stain and visualized by light microscopy (200× magnification). (B) The number of PAS-positive cells was calculated. Results are represented as mean ± SD. ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 6

Effects of LBS on ZO-1, IgA expressions and the permeability of neonatal intestinal tract with or without E. coli K1 infection. Two days old neonatal rats were treated as described in Figure 5. Paraffin-embedded small intestinal tissues were prepared for ZO-1 (A) and IgA (D) staining. Images were visualized by light microscopy (400× magnification). Semi-quantitative analysis of ZO-1 (B) and IgA (E) were performed by Image J. (C) The intestinal permeability was measured by FITIC-dextran leakage assay. The value of PBS group was assigned as 1. Data were represented as mean ± SD, n = 5. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.

FIGURE 7

Effects of LBS on proliferation of neonatal intestinal epithelium with or without E. coli K1 infection. Two days old neonatal rats were treated as described in Figure 5. (A) Small intestines were processed for Ki67 immunohistochemistry. Images were visualized by light microscopy (200× magnification). (B) Ki67-positive cells were calculated. The value of PBS group was assigned as 1. Results are represented as mean ± SD. ^∗P < 0.05, ^∗∗∗P < 0.001.

FIGURE 8

Proposed mechanisms on how LBS exert preventive effect against neonatal E. coli K1 translocation. (A) The intestinal barrier function of neonates is not fully developed, which may provide opportunities for E. coli K1 translocation across the gut barrier into the blood stream, leading to bacteremia and meningitis. (B) LBS promote the formation of neonatal intestinal barrier function, including enhancement of mucin and IgA productions and up-regulation of the expression of ZO-1 to seal the intercellular space of intestinal epithelial cells (reflected by a decrease in the intestinal permeability). This multifactorial approach enhances intestinal barrier functions and thereby limits bacterial translocation across the neonatal intestinal mucosa.