P2-40 Anti-apoptotic Effect of Bioactives from Lactobacillus helveticus and Lactobacillus acidophilus against Salmonella Typhimurium Infection on Epithelial Cells

Tuesday, August 5, 2014
Exhibit Hall D (Indiana Convention Center)
Jingya Peng, University of Guelph, Guelph, Canada
Sapna Sharma, University of Guelph, Guelph, Canada
Angela Tellez, University of Guelph, Guelph, Canada
Rocio Morales, University of Guelph, Guelph, Canada
Akalate Tessema, University of Guelph, Guelph, Canada
Mansel Griffiths, University of Guelph, Guelph, Canada
Introduction: Both in vitro and in vivo studies have shown that bioactive components produced by Lactobacillus helveticus and Lactobacillus acidophilus exert a protective effect against infections caused by a variety of enteric pathogens, including Salmonella.

Purpose: The goal of this study was to determine the mode of protective effect from bioactive molecules produced by L. helveticus (LH-2) and L. acidophilus (La-5) against Salmonella Typhimurium infection.

Methods: Bioactives were produced after 48 hours fermentation in milk or whey protein-based media for LH-2 and La-5, respectively. Colonic carcinoma HT-29 cells were grown in Transwell inserts for 40 days until polarized (~120 ohm×cm2). Cell membrane integrity was measured through transephithelial electrical resistance (TEER). The nontoxic doses of bioactives were determined using trypan blue exclusion/SRB assay. Bioactives were pre-incubated 24 h prior to Salmonella infection and co-incubated during Salmonella infection. Lactate dehydrogenase (LDH) activity was tested using Abcam® LDH-cytotoxicity assay kit. Apoptosis was measured by flow cytometry with BDTM APO-Direct kit. Finally, an invasion assay was carried out using chicken hepatoma LMH cells as an in vitro model for Salmonella presence in poultry.

Results: The protein concentration of 370μg/ml LH-2 and 1.5mg/ml La-5 were nontoxic for the epithelial cells. LH-2 and La-5 exhibited protective effects of TEER (P < 0.05), LDH and apoptosis (P < 0.01) against S. Typhimurium on polarized HT-29 cells. In infected polarized HT-29 cells, LDH was reduced up to 49.1% in presence of LH-2 and up to 46.8% when La-5 was present. In terms of apoptosis, compared with positive control 62.5% and 75.8% less cells underwent apoptosis with LH-2 and La-5 treatment, respectively. Salmonella invasion of LMH cells was reduced less than one log cycle. 

Significance: These data suggest that LH-2 and La-5 bioactives reduce the amount of cells undergo apoptosis following Salmonella infection.