Dynamics of biofilm formation and the interaction between Candida albicans and methicillin-susceptible (MSSA) and -resistant Staphylococcus aureus (MRSA)

Abstract

Polymicrobial biofilms are an understudied and a clinically relevant problem. This study evaluates the interaction between C. albicans, and methicillin- susceptible (MSSA) and resistant (MRSA) S. aureus growing in single- and dual-species biofilms. Single and dual species adhesion (90 min) and biofilms (12, 24, and 48 h) were evaluated by complementary methods: counting colony-forming units (CFU mL-1), XTT-reduction, and crystal violet staining (CV). The secretion of hydrolytic enzymes by the 48 h biofilms was also evaluated using fluorimetric kits. Scanning electron microscopy (SEM) was used to assess biofilm structure. The results from quantification assays were compared using two-way ANOVAs with Tukey post-hoc tests, while data from enzymatic activities were analyzed by one-way Welch-ANOVA followed by Games-Howell post hoc test (α = 0.05). C. albicans, MSSA and MRSA were able to adhere and to form biofilm in both single or mixed cultures. In general, all microorganisms in both growth conditions showed a gradual increase in the number of cells and metabolic activity over time, reaching peak values between 12 h and 48 h (ρ<0.05). C. albicans single- and dual-biofilms had significantly higher total biomass values (ρ<0.05) than single biofilms of bacteria. Except for single MRSA biofilms, all microorganisms in both growth conditions secreted proteinase and phospholipase-C. SEM images revealed extensive adherence of bacteria to hyphal elements of C. albicans. C. albicans, MSSA, and MRSA can co-exist in biofilms without antagonism and in an apparent synergistic effect, with bacteria cells preferentially associated to C. albicans hyphal forms.

Fig 1. Mean log₁₀ CFU mL^-1 values for grown of C. albicans (A), MSSA and MRSA (B) as single- and dual-species grown in RPMI at 37°C.

Error bars represent standard deviation. The uppercase letters show differences among time periods and lowercase letters show differences among the growth conditions (Tukey post-hoc test at ρ>0.05).

Fig 2. Absorbance values of XTT reduction (Abs XTT) obtained from 90 min of adhesion and biofilms of C. albicans, MSSA, and MRSA as single- and dual-species grown in RPMI at 37°C.

Error bars represent standard deviation. The uppercase letters show differences among time periods and lowercase letters show differences among the growth conditions (Tukey post-hoc test at ρ>0.05).

Fig 3. Absorbance values of CV solutions (Abs CV) obtained from biofilms of C. albicans, MSSA, and MRSA as single- and dual-species grown in RPMI at 37°C.

Error bars represent standard deviation. The uppercase letters show differences among time periods and lowercase letters show differences among the growth conditions (Tukey post-hoc test at ρ>0.05).

Fig 4. SAP (A) and PL-C (B) activities of biofilms of C. albicans, MSSA, and MRSA, as single- and dual-species grown in RPMI at 37°C, and the positive controls.

Error bars represent standard deviation. The uppercase letters show differences among the growth conditions (Games-Howell post-hoc test at ρ>0.05).

Fig 5. Scanning electron microscopy of adhered single- and dual-species (90 min) and biofilms (12 h, 24 h, and 48 h) of C. albicans, MSSA, and MRSA in RPMI at 37°C.

The bar in images corresponds to 20 μm for C. albicans (magnification of x2500) and 20 μm for bacteria and mixed growth (magnification of x5000).