Pathogenic potential of Saccharomyces strains isolated from dietary supplements

Abstract

Saccharomyces cerevisiae plays a beneficial role in health because of its intrinsic nutritional value and bio-functional properties, which is why it is also used as a dietary supplement. However, the perception that S. cerevisiae is harmless has changed due to an increasing number of infections caused by this yeast. Given this scenario, we have tested whether viable strains contained in dietary supplements displayed virulence-associated phenotypic traits that could contribute to virulence in humans. We have also performed an in vivo study of the pathogenic potential of these strains using a murine model of systemic infection by intravenous inoculation. A total of 5 strains were isolated from 22 commercial products and tested. Results highlight one strain (D14) in terms of burden levels in brains and kidneys and ability to cause death, whereas the other two strains (D2 and D4) were considered of low virulence. Our results suggest a strong relationship between some of the virulence-associated phenotypic traits (ability to grow at 39°C and pseudohyphal growth) and the in vivo virulence in a mouse model of intravenous inoculation for isolates under study. The isolate displaying greatest virulence (D14) was evaluated in an experimental murine model of gastrointestinal infection with immunosuppression and disruption of mucosal integrity, which are common risk factors for developing infection in humans, and results were compared with an avirulent strain (D23). We showed that D14 was able to spread to mesenteric nodes and distant organs under these conditions. Given the widespread consumption of dietary supplements, we recommend only safe strains be used.

Figure 1. Molecular characterization of yeast strains analyzed.

HinfI mtDNA restriction patterns of yeasts isolated from commercial products (A) and of yeast strains used as controls (B); δ-PCR amplification patterns of yeasts isolated from commercial products (C). The DNA of phage λ digested with Pst I and a 100-bp DNA ladder marker (Invitrogen Life Technologies) served as the size standard respectively.

Figure 2. Growth at different temperatures of commercial and control S. cerevisiae strains.

Ten-fold serial dilutions of the indicated strains were dropped on YPD plates and incubated for 24 h at 30, 37, 39 and 42°C. +++: growth in all the dilutions; ++: growth in the first two dilutions; +: growth in the first dilution; ±: growth in the first drop without dilution; –: no growth.

Figure 3. Generation time of commercial and control S. cerevisiae strains on YPD at 30°C and 37°C.

Error bars correspond to standard deviations. ∧not determined. *p<0.05 with regard to the avirulent strains (CECT 10.431 and W303), as assessed by Students t-test.

Figure 4. Extracellular secretion of phospholipase (A) and protease (B) of commercial and control S. cerevisiae strains.

Activity is expressed as Pz value (see Material and Methods section) and the level of activity is indicated between brackets according the following code: Pz = 1 (Negative activity: –); 0,99≥Pz≥0,70 (Low activity: +); 0,69≥Pz≥0,50 (Moderate activity: ++); Pz≤0,50 (High activity: +++). ∧not determined (these strains were unable to growth at the optimum temperature to determine this activity). Results are expressed as the mean ± SD.

Figure 5. Differences in pseudohyphal growth of commercial and control S. cerevisiae strains in SLAD medium.

Examples of colony morphologies of pseudohyphal growth: (Ph −) absence of pseudohyphal growth, (Ph +) intermediate pseudohyphal development, (Ph ++) high pseudohyphal development. The images were taken with a Leica Camera associated with a microscope Leica HPS60 at 40X magnification.

Figure 6. Examples of invasive growth in commercial and control S. cerevisiae strains.

Three degrees of invasiveness were determined based on the residual growth remained after washing. Invasive growth (+): yeast cells attached to the agar surface; Invasive growth (±): yeast cells partially anchored to the agar; Invasive growth (–): yeast cells fully eliminated.

Figure 7. Differences in phosphorylation of MAPKs Slt2 and Kss1 in dietary Sacharomyces strains.

Phosphorylation of MAPKs Slt2 and Kss1 in extracts from the indicated commercial S. cerevisiae strains growing at 24°C and from the laboratory BY4741 strain, growing both at 24°C and 39°C. Phospho-Kss1 and phospho-Slt2 were detected by immunoblotting analysis with anti-phospho-p42/44 and the protein load monitored using anti-Slt2, anti-Kss1 and anti-actin antibodies.

Figure 8. Adherence of commercial and control yeast strains to plastic and catheters.

(A) Percentage of cells of the different yeast strains adhered to polystyrene Petri plates incubated 1 h at 37°C with 5% CO₂ in glucosaline solution. (B) Adherence of yeast strains to polystyrene microtiter plates incubated 1 h at 37°C with 5% CO₂ in glucosaline solution determined by absorbance. (C) Adherence of yeast strains to sections (1 cm) of polyurethane intravenous catheters incubated 1 and 24 h at 37°C in glucosaline solution and RPMI. First bar: 1 h, glucosaline solution; Second bar: 24 h, glucosaline solution; Third bar: 1 h, RPMI medium; Fourth bar: 24 h, RPMI medium).

Figure 9. Kaplan-Meier plot of the cumulative mortality of BALB/c mice intravenously challenged with Saccharomyces strains.

(A) D14 survival curve representing the combination of four experiments was compared to that of the rest of dietetic strains (D2, D4, D23) and control strains (CECT 10.431, W303, YJM128); (B) Comparison of D14 survival curves obtained in four independent trials.

Figure 10. Comparative burdens of Saccharomyces strains recovered in brains and kidneys after intravenous infection of BALB/c mice.

Day +7: D2, D4, W303 and CECT 10.431 (n = 4 each); D14 and D23 (n = 6 each); YJM118 (n = 10). Day +15: W303 (n = 3); D2, D4 and CECT 10.431 (n = 4 each); D14 and D23 (n = 7 each); YJM118 (n = 9). Day +30: W303 (n = 3); D2, D4, D23 and CECT 10.431 (n = 6 each); D14 and YJM128 (n = 11). Results are expressed as the mean ± SD.

Figure 11. Evaluation of the presence of yeasts in mouse feces during the assay of gastrointestinal infection.

Fecal counts of dietetic strains D14 (♦) and D23 (▪) after oral administration of ICR/Swiss mice receiving antibiotic supplementation in the drinking water and immunosuppressant intraperitoneally are shown. Each point represents the mean of 12 mice (day 1), 6 mice (day 3), 5 mice (day 5) and 4 mice (day 7). Results are expressed as the mean ± SD. (D): Dexamethasone injected intraperitoneally.

Figure 12. Translocation and dissemination from the gut of strains D14 and D23 after oral inoculation.

Burdens of dietetic strains D14 (A) and D23 (B) are shown after oral administration of ICR/Swiss mice receiving antibiotic supplementation in the drinking water and immunosuppressant intraperitoneally. Each bar represents polled data from 2 to 4 mice and exceptionally from 1 mouse (asterisk). Results are expressed as the mean ± SD.