Prostaglandin E2 from Candida albicans Stimulates the Growth of Staphylococcus aureus in Mixed Biofilms

Abstract

Background: Previous studies showed that Staphylococcus aureus and Candida albicans interact synergistically in dual species biofilms resulting in enhanced mortality in animal models.

Methodology/principal findings: The aim of the current study was to test possible candidate molecules which might mediate this synergistic interaction in an in vitro model of mixed biofilms, such as farnesol, tyrosol and prostaglandin (PG) E2. In mono-microbial and dual biofilms of C.albicans wild type strains PGE2 levels between 25 and 250 pg/mL were measured. Similar concentrations of purified PGE2 significantly enhanced S.aureus biofilm formation in a mode comparable to that observed in dual species biofilms. Supernatants of the null mutant deficient in PGE2 production did not stimulate the proliferation of S.aureus and the addition of the cyclooxygenase inhibitor indomethacin blocked the S.aureus biofilm formation in a dose-dependent manner. Additionally, S. aureus biofilm formation was boosted by low and inhibited by high farnesol concentrations. Supernatants of the farnesol-deficient C. albicans ATCC10231 strain significantly enhanced the biofilm formation of S. aureus but at a lower level than the farnesol producer SC5314. However, C. albicans ATCC10231 also produced PGE2 but amounts were significantly lower compared to SC5314.

Conclusion/significance: In conclision, we identified C. albicans PGE2 as a key molecule stimulating the growth and biofilm formation of S. aureus in dual S. aureus/C. albicans biofilms, although C. albicans derived farnesol, but not tyrosol, may also contribute to this effect but to a lesser extent.

Fig 1. Growth kinetics of S.aureus in mono-microbial and in dual S. aureus/C. albicans biofilms.

(a) Biofilm thickness of S. aureus 19552 single (open bars) and mixed biofilms with C. albicans 31883 wt (clinical strain; striped) or C. albicans SC5314 wt (laboratory strain; solid bars) at various time points determined by staining with crystal violet. Total biofilm thickness is expressed as mean of the optical density (OD) measured at a wave length of 570 nm. Shown are the means and standard deviations of two independent experiments with 24 replicates each. (b) Quantification of S. aureus 19552 in mono-microbial (circle) and in dual species biofilms together with C. albicans clinical isolate 31883 (triangle) or C. albicans SC5314 laboratory strain (square). Shown are the mean cfu`s of S. aureus derived from two representative experiments with three intra-assay replicates. Asterisks indicate significant (p<0.001) differences between mono-microbial and dual species biofilms.

Fig 2. Growth-promoting properties of cell-free C. albicans supernatants.

(a) Supernatants removed from biofilms of two C. albicans wild type strains SC5314 and 31883, respectively, after the indicated time of incubation were added to 24h old biofilms of S. aureus 19552. (b) Heat-treated (striped) and untreated supernatants (open bars) were collected as described above and added to 24h-old S. aureus biofilms. S. aureus grown as mono-microbial biofilm was used as control (solid bars). Shown are the mean cfu and the standard deviations of two independent experiments each with three replicates. Asterisks indicate statistically significant differences (p<0.001).

Fig 3. Effect of farnesol on the growth of S. aureus 19552 in mono-microbial biofilms.

(a) Pre-cultered 24h old S. aureus biofilms were supplemented with different concentrations of purified farnesol and the number of cfu`s was determined. S. aureus biofilms incubated in farnesol-free medium were used as control. (b) Growth kinetics of S. aureus in dual biofilms with the farnesol-deficient C. albicans strain ATCC10231 (striped) and the farnesol producer C. albicans SC5314 (open bars). Colony forming units (cfu`s) of S. aureus derived from single species biofilms were function as control (solid bars). Shown are the mean cfu`s and the standard deviations of two representative experiments with three replicates each. Asterisks indicate significant (p<0.05) differences compared to controls.

Fig 4. PGE₂ stimulates the growth of S. aureus biofilms.

(a) S. aureus 19552 was grown in single species biofilms in the presence of various concentrations of purified PGE_2. The mean number of colony forming units (cfu`s) from untreated S. aureus biofilms were used as control. Asteriks indicate statistically significant (p< 0.05) differences between PGE<sub>2</sub>-treated and untreated biofilms. (b) Cultures of S. aureus 19552 biofilms were supplemented with heat-treated (solid bars) and untreated (open bars) PGE<sub>2</sub> supplemented medium. Shown are the mean number of S. aureus cfu`s normalized to the non-supplemented control as well as the standard deviations derived from two independent experiments and three replicates per experiment. Asterisks indicate significant (p< 0.05) differences between the groups.

Fig 5. PGE₂ synthesis in C.albicans wild type and mutant strains and its effect on S. aureus biofilms.

(a) Time-dependent accumulation of PGE₂ in culture supernatants of the C. albicans SC5314 wt (dashed), the reference strain ura3-/- (open bars), and the null mutant ura3-/-fet31-/- (solid bars) measured by EIA. (b) Effect of cell-free supernatants derived from C. albicans SC5314 wt (striped), from the reference (grey bars) and from the null mutant (solid bars) to the growth of established S. aureus 19552 biofilms (control; open bars) after further 24h of incubation. (c) PGE₂ levels in supernatants of C. albicans ATCC10231 (farnesol nonproducer; open bars) and SC5314 (farnesol producer; solid bars) were compared. Mean and standard deviations derived from two representative experiments with three replicates per test are demonstrated. Significant differences (p<0.001) between the two groups were identified by asterisks.

Fig 6. Indomethacin suppresses the stimulatory effect of C. albicans on S. aureus biofilms.

(a) Indomethacin was added to mixed biofilms of S. aureus 19552 and C. albicans SC5314 pre-cultured for different periods. PGE₂ synthesis was measured in supernatants from indomethacin-treated and untreated (control) dual biofilms by monoclonal and (b) metabolite EIA. Asterisks indicate significant differences between indomethacin-treated cultures and untreated controls. (c) The number of S. aureus cfu`s was quantified in mixed S. aureus/C. albicans biofilms after addition of graded doses of indomethacin at the indicated time points. Untreated dual species biofilms were used as control. Shown are the results of two representative experiments with three intra-assay replicates.