Effects of Bovine Colostrum with or without Egg on In Vitro Bacterial-Induced Intestinal Damage with Relevance for SIBO and Infectious Diarrhea

Abstract

Small intestinal bacterial overgrowth (SIBO) occurs commonly, is difficult to treat, and frequently recurs. Bovine colostrum (BC) and chicken eggs contain immunoglobulins and other components that possess antimicrobial, immunoregulatory, and growth factor activities; however, it is not known if they have the ability to reduce injury caused by the presence of bacteria associated with SIBO (Streptococcus, Escherichia coli, Staphylococcus, Bacteroides, Klebsiella, Enterococcus, and Proteus) and infectious diarrhea (enteropathogenic Escherichia coli, Salmonella). We examined the effects of BC, egg, or the combination, on bacterial growth and bacteria-induced changes in transepithelial electrical resistance (TEER) and bacterial translocation across confluent Caco-2 monolayers. BC, egg, or the combination did not affect bacterial growth. Adding bacteria to monolayers reduced TEER and (with minor variations among species) increased bacterial translocation, increased monolayer apoptosis (increased caspase-3 and Baxα, reduced Bcl2), increased intercellular adhesion molecule 1 (ICAM-1), and reduced cell adhesion molecules zonulin1 (ZO1) and claudin-1. BC, egg, or the combination reduced these effects (all p < 0.01) and caused additional increases in vascular endothelial growth factor (VEGF) and Heat Shock Protein 70 (Hsp70) expression. We conclude that BC ± egg strengthens mucosal integrity against a battery of bacteria relevant for SIBO and for infectious diarrhea. Oral BC ± egg may have clinical value for these conditions, especially SIBO where eradication of precipitating organisms may be difficult to achieve.

Keywords: antimicrobial; irritable bowel syndrome; leaky gut; nutraceuticals; repair; small intestinal bacterial overgrowth (SIBO).

Figure 1

Effect of bovine colostrum (BC), egg, or the combination on transepithelial passage of bacteria. Bacterial strains (1 × 10⁶ colony-forming units (CFU)/well) ± BC (1 mg/mL), egg (1 mg/mL), or the combination (0.6 mg/mL BC + 0.4 mg/mL egg) were added to the apical side of confluent monolayers of Caco-2 cells grown in transwell plates. The medium was collected from the basal side, 24 h later, and assessed for the number of colony-forming units (CFU)/mL. Control wells received no bacteria or test product. (A) Escherichia coli ATCC 25922 O6; (B) enteropathogenic E. coli (EPEC); (C) Salmonella; (D) Klebsiella; (E) Enterococcus; (F) Proteus; (G) Staphylococcus; (H) Streptococcus. Note that the scale of the y-axis of results for Staphylococcus and Streptococcus are lower than for other strains. Results are expressed as mean ± SEM for 6 wells. ++ signifies p < 0.01 vs. non treated (without bacteria or test product) control, * and ** signify p < 0.05 and 0.01 vs. bacteria alone. The nonpathogenic and noninvasive E. coli K12 did not result in any CFUs in this experiment, and therefore is not shown.

Figure 2

Effect of Escherichia coli ATCC 25922 O6 on Caco-2 monolayer TEER and damaging and repair pathways in the presence of BC, egg, or the combination. E. coli (1 × 10⁶ CFU/well) ± BC, egg, or the combination was added to confluent Caco-2 cells; 1 mg/mL of BC alone, 1 mg/mL egg alone or 0.6 mg/mL BC + 0.4 mg/mL egg were used. Changes in TEER (A) were determined 24 h later. Following incubation, cleared cell lysates were collected and changes in caspase-3 (B), Baxα (C), Bcl2 (D), VEGF (E), Hsp70 (F), ICAM-1 (G), ZO1 (H), and claudin-1 (I) were determined. Results expressed as mean ± SEM for 6 wells. ++ signifies p < 0.01 vs. non treated (without bacteria or test product) control, * and ** signify p < 0.05 and 0.01 vs. presence of bacteria alone. The other bacterial strains tested gave similar results (Table 1, Table 2 and Table 3).and are noted by exception in the text.

Figure 3

Effect of BC, egg, or the combination on tight junction protein, claudin-1. Bacterial strains (1 × 10⁶ CFU/well) ± BC (1 mg/mL), egg (1 mg/mL), or the combination (0.6 mg/mL BC + 0.4 mg/mL egg) were added to confluent monolayers of Caco-2 cells. Cleared lysates were prepared 24 h later. Control wells received no bacteria or test product. (A) Escherichia coli K12 (negative control); (B) EPEC; (C) Salmonella; (D) Klebsiella; (E) Enterococcus; (F) Proteus; (G) Staphylococcus; (H) Streptococcus. Results are expressed as mean ± SEM from 3 wells. ++ signifies p < 0.01 vs. non treated (without bacteria or test product) control and ** signifies p < 0.01 vs. bacteria alone.

Figure 4

Effect of BC, egg, or the combination on active caspase-3. Bacterial strains (1 × 10⁶ CFU/well) ± BC (1 mg/mL), egg (1 mg/mL), or the combination (0.6 mg/mL BC + 0.4 mg/mL egg) were added to confluent monolayers of Caco-2 cells. Cleared lysates were prepared 24 h later. Control wells received no bacteria or test product. (A) E. coli K12 (negative control); (B) EPEC; (C) Salmonella; (D) Klebsiella; (E) Enterococcus; (F) Proteus; (G) Staphylococcus; (H) Streptococcus. Results are expressed as mean ± SEM from 3 wells. ++ signifies p < 0.01 vs. non treated (without bacteria or test product) control and * & ** signify p < 0.05 & 0.01 vs. bacteria alone.

Figure 5

Effect of BC, egg, or the combination on ICAM-1. Bacterial strains (1 × 10⁶ CFU/well) ± BC (1 mg/mL), egg (1 mg/mL), or the combination (0.6 mg/mL BC + 0.4 mg/mL egg) were added to confluent monolayers of Caco-2 cells. Cleared lysates were prepared 24 h later. Control wells received no bacteria or test product. (A) E. coli K12 (negative control); (B) EPEC; (C) Salmonella; (D) Klebsiella; (E) Enterococcus; (F) Proteus; (G) Staphylococcus; (H) Streptococcus. Results are expressed as mean ± SEM from 3 wells. ++ signifies p < 0.01 vs. non treated (without bacteria or test product) control and ** signifies p < 0.01 vs. bacteria alone.