Kefir-isolatedLactococcus lactissubsp.lactisinhibits the cytotoxic effect ofClostridium difficile in vitro

Abstract

Kefir is a dairy product obtained by fermentation of milk with a complex microbial population and several health-promoting properties have been attributed to its consumption. In this work, we tested the ability of different kefir-isolated bacterial and yeast strains (Lactobacillus kefir, Lb. plantarum, Lactococcus lactissubps.lactis, Saccharomyces cerevisiaeandKluyveromyces marxianus) or a mixture of them (MM) to antagonise the cytopathic effect of toxins fromClostridium difficile(TcdA and TcdB). Cell detachment assays and F-actin network staining using Vero cell line were performed. Although incubation with microbial cells did not reduce the damage induced byC. difficilespent culture supernatant (SCS),Lc. lactisCIDCA 8221 and MM supernatants were able to inhibit the cytotoxicity of SCS to Vero cells. Fraction ofLc. lactisCIDCA 8221 supernatant containing components higher than 10 kDa were responsible for the inhibitory activity and heating of this fraction for 15 min at 100 °C completely abrogated this ability. By dot-blot assay with anti-TcdA or anti-TcdB antibodies, concentration of both toxins seems to be reduced in SCS treated withLc. lactisCIDCA 8221 supernatant. However, protective effect was not affected by treatment with proteases or proteases-inhibitors tested. In conclusion, we demonstrated that kefir-isolatedLc. lactisCIDCA 8221 secreted heat-sensitive products able to protect eukaryotic cells from cytopathic effect ofC. difficiletoxinsin vitro. Our findings provide new insights into the probiotic action of microorganisms isolated from kefir against virulence factors from intestinal pathogens.