Probiotic Yeasts Inhibit Virulence of Non -albicans Candida Species

Abstract

Systemic infections of Candida species pose a significant threat to public health. Toxicity associated with current therapies and emergence of resistant strains present major therapeutic challenges. Here, we report exploitation of the probiotic properties of two novel, food-derived yeasts, Saccharomyces cerevisiae (strain KTP) and Issatchenkia occidentalis (strain ApC), as an alternative approach to combat widespread opportunistic fungal infections. Both yeasts inhibit virulence traits such as adhesion, filamentation, and biofilm formation of several non-albicans Candida species, including Candida tropicalis, Candida krusei, Candida glabrata, and Candida parapsilosis as well as the recently identified multidrug-resistant species Candida auris They inhibit adhesion to abiotic surfaces as well as cultured colon epithelial cells. Furthermore, probiotic treatment blocks the formation of biofilms of individual non-albicans Candida strains as well as mixed-culture biofilms of each non-albicans Candida strain in combination with Candida albicans The probiotic yeasts attenuated non-albicans Candida infections in a live animal. In vivo studies using Caenorhabditis elegans suggest that exposure to probiotic yeasts protects nematodes from infection with non-albicans Candida strains compared to worms that were not exposed to the probiotic yeasts. Furthermore, application of probiotic yeasts postinfection with non-albicans Candida alleviated pathogenic colonization of the nematode gut. The probiotic properties of these novel yeasts are better than or comparable to those of the commercially available probiotic yeast Saccharomyces boulardii, which was used as a reference strain throughout this study. These results indicate that yeasts derived from food sources could serve as an effective alternative to antifungal therapy against emerging pathogenic Candida species.IMPORTANCE Non-albicans Candida-associated infections have emerged as a major risk factor in the hospitalized and immunecompromised patients. Besides, antifungal-associated complications occur more frequently with these non-albicans Candida species than with C. albicans Therefore, as an alternative approach to combat these widespread non-albicans Candida-associated infections, here we showed the probiotic effect of two yeasts, Saccharomyces cerevisiae (strain KTP) and Issatchenkia occidentalis (ApC), in preventing adhesion and biofilm formation of five non-albicans Candida strains, Candida tropicalis, Candida krusei, Candida glabrata, Candida parapsilosis, and Candida auris The result would influence the current trend of the conversion of conventional antimicrobial therapy into beneficial probiotic microbe-associated antimicrobial treatment.

Keywords: Caco-2 cell monolayer; Caenorhabditis elegans; Candida albicans; Candida auris; Candida glabrata; Candida krusei; Candida parapsilosis; Candida tropicalis; biofilm; mixed-species Candida biofilm; plastic adhesion assay; probiotic yeasts.

FIG 1

Probiotic treatment reduces adhesion of non-albicans Candida strains on abiotic surfaces. (A) Images show effects of potential probiotic yeasts S. cerevisiae and I. occidentalis and reference strain S. boulardii on adhesion of C. tropicalis, C. krusei, C. glabrata, and C. parapsilosis under coinoculation conditions. (B and C) Adhesion of non-albicans Candida strains to plastic surfaces was reduced in the presence of probiotic yeasts under coinoculation conditions (B) and postinoculation conditions (C). For the coinoculation conditions (indicated with a plus sign), probiotic yeasts and non-albicans Candida strains were incubated together for 3 h. For the postinoculation conditions (indicated with a vertical arrow), non-albicans Candida strains were applied to an abiotic surface for 60 min prior to seeding of probiotic isolates and were incubated for an additional 120 min. Crystal violet (0.5%) staining was used to quantify adhered non-albicans Candida cells on abiotic surfaces.

FIG 2

Probiotic yeasts S. cerevisiae, I. occidentalis, and S. boulardii prevented biofilm formation of non-albicans Candida species in both monoculture and mixed-culture biofilms with C. albicans. (A) Coinoculation of probiotics with non-albicans Candida strains for 24 h at 37°C. (B) For postinoculation of probiotics, non-albicans Candida strains were incubated for 90 min, and nonadherent cells were removed and subsequently treated with probiotic yeasts for 24 h at 37°C. Crystal violet (0.5%) staining was used to quantify the biofilm. (C) Images show the inhibitory effect of coinoculated probiotic treatment and postinoculation treatment (treatment 90 min after biofilm initiation). (D and E) Probiotic treatment inhibited mixed-culture biofilms of non-albicans Candida with C. albicans under conditions of coinoculation (D) or 90 min postinoculation, when biofilm formation had been initiated (E).

FIG 3

Probiotic yeasts S. cerevisiae, I. occidentalis, and S. boulardii inhibited morphological transition of C. tropicalis (upper row) and C. parapsilosis (bottom row). Probiotic yeasts and non-albicans Candida were coincubated for 24 h, following which unattached cells were removed and photographed using a bright-field microscope.

FIG 4

Simultaneous exposure of probiotic yeasts S. boulardii, S. cerevisiae, and I. occidentalis increased the life span of C. tropicalis (A), C. krusei (B), and C. parapsilosis (D) but not C. glabrata (C). C. elegans worms were fed a mixture of probiotic yeasts and non-albicans Candida. Live and dead worms were scored for survival each day and compared to worms fed a diet consisting only of non-albicans Candida (untreated group).

FIG 5

Effect of probiotics treatment on adhesion (A) and biofilm formation (B) of C. auris. Probiotic yeasts S. cerevisiae and I. occidentalis as well as reference strain S. boulardii and C. auris were coinoculated for 3 h and 24 h at 37°C. Adhesion and biofilm formation were quantified by crystal violet (0.5%) staining after unattached cells were removed by washing. (A) Coincubation of probiotic yeasts decreased adhesion of C. auris to abiotic surfaces. (B) Coinoculation of probiotic yeasts inhibited mixed-culture biofilms of C. auris with C. albicans.

FIG 6

(A) The effect of cell-free probiotic metabolites on adhesion of non-albicans Candida strains was quantified using crystal violet staining. (B) Graphic representation of experimental setup where probiotic yeast were inoculated into the upper chamber of the cell insertion section and non-albicans Candida strains were maintained in the lower chamber; probiotic soluble metabolites freely diffused through a 0.4-μl-pore-size membrane into and all over the media, including the lower compartment where the non-albicans Candida cells were inoculated.