Pathogenic potential of Saccharomyces strains isolated from dietary supplements.

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

Intestinal translocation and dissemination of isolates D14 and D23 based on the number of mice that showed yeast burdens in target organs.

(a)<p>Day of sacrifice after initiation of oral administration of the yeast.</p>(b)<p>MLNs: mesenteric lymph nodes.</p>(c)<p>Other organs: brain, kidneys, liver.</p

Generation time of commercial and control <i>S. cerevisiae</i> strains on YPD at 30°C and 37°C.

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

Commercial products analyzed in this study.

(a)<p>Brewer’s yeast and wheat germ;</p>(b)<p>Brewer’s yeast;</p>(c)<p>Yeast;</p>(d)<p>Yeast and germ;</p>(e)<p>Yeast flakes;</p>(f)<p>Yeast alive;</p>(g)<p>Biological beer.</p

Growth at different temperatures of commercial and control <i>S. cerevisiae</i> strains.

<p>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.</p

Virulence-associated phenotypes observed in strains isolated from commercial products.

<p>Phenotype traits shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098094#pone-0098094-g002" target="_blank">Figures (2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098094#pone-0098094-g004" target="_blank">4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098094#pone-0098094-g005" target="_blank">5</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098094#pone-0098094-g006" target="_blank">6</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098094#pone-0098094-g007" target="_blank">7</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098094#pone-0098094-g008" target="_blank">8</a>) and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098094#pone-0098094-t004" target="_blank">Table 4</a> were simplified as follows to calculate the Spearman’s Rho correlation coefficients between strains and apply the Jaccard proximity test (excluding joint absences for considerations): growth at 37, 39 and 42°C, +++, ++ and + were simplified to +, and ± and − were simplified to −; phospholipase activity (PLA), ++ and + were simplified to +; pseudohyphal (Ph) growth,++ and + were simplified to +; invasive growth, ± and − were simplified to −; switching, multiple colony phenotypes with frequencies between 10⁻²−10⁻⁴ were considered +; MAPK activation, high Slt2 and Kss1 phosphorylation was considered + and low or non phosphorylation were considered −; Plastics adherence (ADH), Petri plates adherence values >30% and microtiter plates absorbance values >1 were considered +, Petri plates adherence values ≤30% and microtiter plates absorbance values <1 were considered −. n.d. not determined.</p

<i>S. cerevisiae</i> strains used as control and assays for which they were used.

(a)<p>(named D5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098094#pone-0098094-t001" target="_blank">Table 1</a>);</p>(b)<p>Molecular characterization of these strains have been described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098094#pone.0098094-deLlanos5" target="_blank">[44]</a>;</p>(c)<p>Growth at different temperatures,</p>(d)<p>protease and phospholipase secretion, Ph (pseudohyphal) and invasive growth;</p>(e)<p>MAPK activation;</p>(f)<p>Adherence to plastics;</p>(g)<p><i>In vivo</i> study by intravenous inoculation of strains in BALB/c mice;</p>(h)<p>(<i>MAT</i><b>a</b><i>; ura3-52; trp1D2; leu2-3_112; his3-11; ade2-1; can1-100</i>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098094#pone.0098094-Wheeler1" target="_blank">[64]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098094#pone.0098094-Thomas1" target="_blank">[76]</a>;</p>(i)<p>(<i>MAT</i><b>a</b><i>his3</i>Δ<i>1 leu2</i>Δ<i>0 met15</i>Δ<i>0 ura3</i>Δ<i>0)</i> (EUROSCARF); n.r.: Non reported.</p

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

<p>(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).</p

Differences in phosphorylation of MAPKs Slt2 and Kss1 in dietary <i>Sacharomyces</i> strains.

<p>Phosphorylation of MAPKs Slt2 and Kss1 in extracts from the indicated commercial <i>S. cerevisiae</i> 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.</p

Colony phenotype switching frequencies of commercial <i>Saccharomyces</i> strains.

<p>Colony phenotype descriptions: D2-A (5–7 mm, light pink, strong pink centre, white edge, creamy texture); D2-B (5–7 mm, light pink, strong pink centre, white edge, strong pink sector, creamy texture); D2-C (5–7 mm, strong pink, white edge, rough texture, striated); D2-D (3 mm, light pink, strong pink centre, creamy texture); D2-E (5–7 mm, light pink, strong pink centre, white edge, creamy texture, star shape); D2-F (5–7 mm, light pink, strong pink centre, white edge, white sector, creamy texture); D2-G (3 mm, strong pink, creamy texture, irregular); D2-H (4 mm, light pink, strong pink centre, white edge, strong pink sector, rough texture); D2-I (5–7 mm, light pink, strong pink centre, rough texture, striated); D4-A (5 mm, light pink, strong pink centre, creamy texture); D4-B (2 mm, light pink, strong pink centre, creamy texture, irregular); D4-C (1 mm, light pink, creamy texture, irregular); D4-D (3 mm, strong pink, creamy texture); D4-E (5 mm, light pink, strong pink centre, creamy texture, white sector); D4-F (5 mm, light pink, strong pink centre, creamy texture, irregular); D14-A (5 mm, light pink, pale pink centre, strong pink edge, creamy texture); D14-B (2–3 mm, pale pink, creamy texture); D14-C (2–3 mm, light pink, pale pink centre, creamy texture); D14-D (5 mm, light pink, pale pink centre, strong pink edge, creamy texture, white sector); D14-E (5 mm, light pink, pale pink centre, strong pink edge, creamy texture, strong pink sector); D14-F (5 mm, medium pink, pale pink centre, strong pink edge, creamy texture); D14-G (5 mm, light pink, pale pink centre, strong pink edge, creamy texture, striated); D14-H (2 mm, pale pink, dark pink edge, creamy texture); D23-A (4–5 mm, light pink, strong pink centre, creamy texture); D23-B (1–2 mm, strong pink, creamy texture); D23-C (4–5 mm, light pink, strong pink centre, creamy texture, irregular); D23-D (4 mm, strong pink, creamy texture); D23-E (7–8 mm, light pink, strong pink centre, creamy texture); D23-F (4–5 mm, light pink, strong pink centre, rough texture, striated); D23-G (5 mm, light pink, strong pink centre, strong pink sector, creamy texture); D23-H (1–2 mm, light pink, strong pink centre, creamy texture).</p