Investigation into In Vitro and In Vivo Caenorhabditis elegans Models to Select Cheese Yeasts as Probiotic Candidates for their Preventive Effects against Salmonella Typhimurium

Abstract

The design of multiscale strategies integrating in vitro and in vivo models is necessary for the selection of new probiotics. In this regard, we developed a screening assay based on the investigation of the potential of yeasts from cheese as probiotics against the pathogen Salmonella Typhimurium UPsm1 (ST). Two yeasts isolated from raw-milk cheese (Saccharomyces cerevisiae 16, Sc16; Debaryomyces hansenii 25, Dh25), as well as S. cerevisiae subspecies boulardii (CNCM I-1079, Sb1079), were tested against ST by applying in vitro and in vivo tests. Adherence measurements to Caco-2 and HT29-MTX intestinal cells indicated that the two tested cheese yeasts presented a better adhesion than the probiotic Sb1079 as the control strain. Further, the Dh25 was the cheese yeast most likely to survive in the gastrointestinal tract. What is more, the modulation of the TransEpithelial Electrical Resistance (TEER) of differentiated Caco-2 cell monolayers showed the ability of Dh25 to delay the deleterious effects of ST. The influence of microorganisms on the in vivo model Caenorhabditis elegans was evaluated by measuring the longevity of the worm. This in vivo approach revealed that this yeast increased the worm's lifespan and protected it against ST infection, confirming that this in vivo model can be useful for screening probiotic cheese yeasts.

Keywords: Caenorhabditis elegans; Probiotics; Salmonella Typhimurium; cheese yeasts.

Figure 1

Change in TransEpithelial Electrical Resistance (TEER) level of yeast-exposed Caco-2 cells during apical incubation with S. boulardii 1079, D. hansenii 25, S. cerevisiae 16, and Dulbecco’s Modified Eagle’s Minimal essential medium (DMEM) control medium throughout the 24 h incubation period, expressed by reference to resistance measured at time 0. The values are mean ± SD (error bars) of three independent experiments. A high enhancement of the TEER is observed in the presence of the yeasts.

Figure 2

S. Typhimurium culture and D. hansenii 25/S. Typhimurium co-culture (24, 48, 72, and 96 h) modulations of human epithelial cell barrier function. Control with DMEM medium only. The values are mean ± SD (error bars) of three independent experiments. An increased co-culture duration delayed the deleterious effect of Salmonella on the TEER.

Figure 3

(a) Influence of D. hansenii 25 (Dh25), E. coli OP50, S. boulardii 1079 (Sb1079), S. cerevisiae 16 (Sc16), and S. Typhimurium (ST) on the longevity of the C. elegans wild-type N2 strain in a solid medium. (b) Influence of Dh25, E. coli OP50, Sb1079, Sc16, and ST on survival of the C. elegans wild-type N2 strain in a liquid medium. (c) Preventive effects of Dh25 on ST survival of the C. elegans mutant AU37 strain in a liquid medium. Mean survival, with half of the population dead, is represented on the abscissa. The asterisks indicate the p-values (log-rank test) against E. coli OP50 (***, p < 0.001). C. elegans AU37 mutant is more sensible to the pathogenic bacterium Salmonella Typhimurium and is protected by the yeasts Debaryomyces hansenii 25.