Lactobacillus casei DN-114 001 inhibits the ability of adherent-invasive Escherichia coli isolated from Crohn's disease patients to adhere to and to invade intestinal epithelial cells

Abstract

Ileal lesions in 36.4% of patients with Crohn's disease are colonized by pathogenic adherent-invasive Escherichia coli. The aim of this study was to determine the in vitro inhibitory effects of the probiotic strain, Lactobacillus casei DN-114 001, on adhesion to and invasion of human intestinal epithelial cells by adherent-invasive E. coli isolated from Crohn's disease patients. The experiments were performed with undifferentiated Intestine-407 cells and with undifferentiated or differentiated Caco-2 intestinal epithelial cells. Bacterial adhesion to and invasion of intestinal epithelial cells were assessed by counting CFU. The inhibitory effects of L. casei were determined after coincubation with adherent-invasive E. coli or after preincubation of intestinal cells with L. casei prior to infection with adherent-invasive E. coli. Inhibitory effects of L. casei on adherent-invasive E. coli adhesion to differentiated and undifferentiated intestinal epithelial cells reached 75% to 84% in coincubation and 43% to 62% in preincubation experiments, according to the cell lines used. Addition of L. casei culture supernatant to the incubation medium increased L. casei adhesion to intestinal epithelial cells and enhanced the inhibitory effects of L. casei. The inhibitory effects on E. coli invasion paralleled those on adhesion. This effect was not due to a bactericidal effect on adherent-invasive E. coli or to a cytotoxic effect on epithelial intestinal cells. As Lactobacillus casei DN-114 001 strongly inhibits interaction of adherent-invasive E. coli with intestinal epithelial cells, this finding suggests that the probiotic strain could be of therapeutic value in Crohn's disease.

FIG. 1.

Adhesion of L. casei DN-114 001 to intestinal epithelial cells according to multiplicity of infection (A) or to time of incubation (B). Adhesion of L. casei DN-114 001 was tested with undifferentiated Intestine-407 or Caco-2 cells cultured for 2 days and with differentiated Caco-2 cells cultured for 15 days. (A) Cultured cells were incubated at an MOI of 10 (black bars), 100 (white bars), and 500 (grey bars) for 6 h. (B) Cultured cells were incubated at an MOI of 500 for 1 h (black bars), 3 h (white bars), and 6 h (grey bars). Adhesion levels are expressed as the number of CFU per well. Data are given as means ± the standard error of the mean (SEM) of at least three separate experiments.

FIG. 2.

Increased adhesion of L. casei DN-114 001 in the presence of its spent culture supernatant. Cultured cells were incubated with L. casei DN-114 001 at an MOI of 500 for 6 h in cell culture medium alone (black bars), or supplemented with 10% (vol/vol) of its spent culture supernatant (white bars) and with 10% of neutralized supernatant (grey bars). Adhesion levels were determined as described in the legend to Fig. 1.

FIG. 3.

Inhibitory effects of L. casei DN-114 001 on the abilities of AIEC LF82 to adhere to and to invade intestinal epithelial cells in preincubation experiments. Adhesion (A) and invasion (B) of AIEC LF82 with intestinal epithelial cells preincubated with L. casei DN-114 001 alone (black bars) or supplemented with 10% (vol/vol) of its spent culture supernatant (grey bars), compared with adhesion and invasion levels of AIEC LF82 to untreated epithelial cells (white bars), taken as 100%. Preincubation of cultured cells was performed for 6 h with L. casei DN-114 001 at an MOI of 500. Infection with AIEC LF82 was performed for 3 h with an MOI of 100. Invasion was determined after gentamicin treatment for an additional hour. Results are expressed as cell-associated bacteria (adherent plus intracellular bacteria) or intracellular bacteria relative to those obtained for strain LF82 with untreated cells. Each value is the mean ± SEM of three to four separate experiments.

FIG. 4.

Adhesion (A) and invasion (B) abilities of AIEC LF82 with respect to intestinal epithelial cells in coincubation experiments with L. casei DN-114 001 alone (black bars) or supplemented with 10% (vol/vol) of L. casei DN-114 001 spent culture supernatant (grey bars), compared with monoinfection experiments with LF82 alone (white bars). A multiplicity of infection of 500 was used for L. casei DN-114 001 and an MOI of 10 was used for AIEC LF82. Cell-associated bacteria were quantified after a 6-h incubation period. Invasion was determined after gentamicin treatment for an additional hour. Results are expressed as the percentage of cell-associated bacteria (adherent plus intracellular bacteria) or intracellular bacteria relative to those obtained in monoinfection with strain LF82, taken as 100%. Each value is the mean ± SEM of three to five separate experiments.