Probiotic Activity of Enterococcus faecium and Lactococcus lactis Isolated from Thai Fermented Sausages and Their Protective Effect Against Clostridium difficile

Abstract

Lactic acid bacteria, Enterococcus faecium and Lactococcus lactis, previously isolated from Thai fermented sausages were elucidated their probiotic properties especially in the control of Clostridium difficile 630. Both isolates survived in simulated gastric solution at pH 3 followed in simulated intestinal solution at pH 8. The presence of skimmed milk also helped the bacteria to survive through acidic and alkaline in gastrointestinal conditions. The adhesion properties of both isolates were tested using a human colon adenocarcinoma cell line. The result showed that both isolates exhibited desirable probiotic properties which adhered to Caco-2 cells. The neutralized cell-free supernatant of both isolates demonstrated that no cytotoxicity toward Caco-2 cells vice versa cell-free supernatant of C. difficile 630 toward Caco-2 cell demonstrated high toxicity. The immunomodulation effect in response to bacterial neutralized cell-free supernatant and cell-free supernatant was also studied. The expression level of pro-inflammatory cytokine of Caco-2 cell which are tumor necrosis factor-α and interleukin-8 was evaluated using quantitative reverse transcriptase PCR. Both isolates were able to diminish the expression level of TNF-α and IL-8 induced by the cell-free supernatant of C. difficile 630. Hence, these isolates would be able to improve the gut health through counteracting the C. difficile-associated intestinal inflammation in human cell lines. These results may contribute to the development of the isolates using as probiotics.

Keywords: Clostridium difficile; Enterococcus faecium; LAB; Lactococcus lactis; Probiotics.

Fig. 1

Cell viability (log CFU/ml) of E. faecium (a) and L. lactis (b) incubated in simulated gastric solution at pH 2 (square) and pH 3 (circle), followed by simulated intestinal solution at pH 8; and gastric solution at pH 2 mixed with skimmed milk (triangle), followed by intestinal solution at pH 8. Bars represent standard deviation values

Fig. 2

Adhesion ability of E. faecium and L. lactis to Caco-2 cells. The bacteria added to the Caco-2 cells vs. those which adhered are represented. Bars represent standard deviation values

Fig. 3

Cell viability of Caco-2 cells incubated with the NCFS of E. faecium or L. lactis combined with the CFS of CD630 for 24 h (co-incubation: E. faecium+CD630, L. lactis+CD630). The Caco-2 cells were pre-incubated with the NCFS of E. faecium or L. lactis for 2 h, followed by the addition of CFS of CD630 for 24 h (protection: E. faecium → CD630, L. lactis → CD630) and vice versa (prevention: CD630 → E. faecium, CD630 → L. lactis). Results are expressed as means ± SEM of triplicate experiments. The statistically significant difference from the control with the p value ≤ 0.05 and ≤ 0.01 was indicated by one asterisk (*) and double asterisks (**), respectively

Fig. 4

Immunofluorescence staining of occludin in Caco-2 cells incubated with MRS (a), the NCFS of E. faecium (b), the NCFS of L. lactis (c), BHI (d), and the CFS of CD630 (e)

Fig. 5

Fold differences in pro-inflammatory cytokine expression; IL-8 (a) and TNF-α (b) after incubation with the NCFS of E. faecium, L. lactis, and the CFS of CD630. Results are expressed as means ± standard deviation for duplicate experiments. The statistically significant difference from the control with the p value ≤ 0.05 and ≤ 0.01 was indicated by one asterisk (*) and double asterisks (**), respectively