Cátedra de Microbiología, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata (UNLP), La Plata, Argentina.
J Dairy Res. 2013 Feb;80(1):96-102. doi: 10.1017/S0022029912000623. Epub 2012 Dec 10.
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 lactis subps. lactis, Saccharomyces cerevisiae and Kluyveromyces marxianus) or a mixture of them (MM) to antagonise the cytopathic effect of toxins from Clostridium 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 by C. difficile spent culture supernatant (SCS), Lc. lactis CIDCA 8221 and MM supernatants were able to inhibit the cytotoxicity of SCS to Vero cells. Fraction of Lc. lactis CIDCA 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 with Lc. lactis CIDCA 8221 supernatant. However, protective effect was not affected by treatment with proteases or proteases-inhibitors tested. In conclusion, we demonstrated that kefir-isolated Lc. lactis CIDCA 8221 secreted heat-sensitive products able to protect eukaryotic cells from cytopathic effect of C. difficile toxins in vitro. Our findings provide new insights into the probiotic action of microorganisms isolated from kefir against virulence factors from intestinal pathogens.
克菲尔是一种乳制品，通过发酵牛奶获得，具有多种促进健康的特性。在这项工作中，我们测试了不同克菲尔分离的细菌和酵母菌株（Lactobacillus kefir、Lb. plantarum、Lactococcus lactis subsp. lactis、Saccharomyces cerevisiae 和 Kluyveromyces marxianus）或它们的混合物（MM）对艰难梭菌毒素（TcdA 和 TcdB）的细胞病变效应的拮抗能力。使用 Vero 细胞系进行细胞脱落测定和 F-肌动蛋白网络染色。尽管与微生物细胞孵育不能减少艰难梭菌消耗培养上清液（SCS）诱导的损伤，但 Lc. lactis CIDCA 8221 和 MM 上清液能够抑制 SCS 对 Vero 细胞的细胞毒性。Lc. lactis CIDCA 8221 上清液中含有大于 10 kDa 成分的部分负责抑制活性，并且将该部分在 100°C 下加热 15 分钟可完全消除这种能力。通过用抗 TcdA 或抗 TcdB 抗体进行斑点印迹分析，似乎在 Lc. lactis CIDCA 8221 上清液处理的 SCS 中两种毒素的浓度降低。然而，用测试的蛋白酶或蛋白酶抑制剂处理不会影响保护作用。总之，我们证明了克菲尔分离的 Lc. lactis CIDCA 8221 分泌的热敏感产物能够在体外保护真核细胞免受艰难梭菌毒素的细胞病变效应。我们的发现为从克菲尔中分离的微生物对肠道病原体毒力因子的益生菌作用提供了新的见解。
