Candida albicans Mycofilms Support Staphylococcus aureus Colonization and Enhances Miconazole Resistance in Dual-Species Interactions

Abstract

Polymicrobial inter-kingdom biofilm infections represent a clinical management conundrum. The presence of co-isolation of bacteria and fungi complicates the ability to routinely administer single antimicrobial regimens, and synergy between the microorganisms influences infection severity. We therefore investigated the nosocomial pathogens Staphylococcus aureus and Candida albicans with respect to antimicrobial intervention. We characterized the interaction using biofilm assays and evaluated the effect of miconazole treatment using in vitro and in vivo assays. Finally, we assessed the impact of biofilm extracellular matrix (ECM) on these interactions. Data indicated that the C. albicans mycofilms supported adhesion and colonization by S. aureus through close interactions with hyphal elements, significantly increasing S. aureus biofilm formation throughout biofilm maturation. Miconazole sensitivity was shown to be reduced in both mono- and dual-species biofilms compared to planktonic cells. Within a three-dimensional biofilm model sensitivity was also hindered. Galleria mellonella survival analysis showed both enhanced pathogenicity of the dual-species infection, which was concomitantly desensitized to miconazole treatment. Analysis of the ECM revealed the importance of extracellular DNA, which supported the adhesion of S. aureus and the development of the dual-species biofilm structures. Collectively, these data highlight the clinical importance of dual-species inter-kingdom biofilm infections, though also provides translational opportunities to manage them more effectively.

Keywords: Candida albicans; Staphylococcus aureus; biofilm; extracellular DNA; miconazole.

FIGURE 1

Candida albicans mycofilms facilitates Staphylococcus aureus biofilm formation. Mono- and dual-species biofilms were standardized 1 × 10⁶ CFU/mL and grown on 96-well plates for 90 min, 6 and 24 h. Biofilms were then washed with phosphate buffered saline (PBS) and biomass assessed using the crystal violet assay (A). Standardized biofilms were grown before being sonicated to remove the biomass. Live/dead PCR was then used to extract DNA and determine total and live colony forming equivalents (CFE) from mono- and dual-species biofilms (B). Biofilm morphology was then analyzed using CSLM. Biofilms were grown before being fluorescently stained using calcofluor white and SYTO9^® dyes. Resulting biofilms were then viewed on a Leica SP5 laser scanning confocal microscope and images were then processed and analyzed using Volocity 3D Image Analysis Software (C). Results represent data from three independent occasions. Statistical analysis compares dual-species biofilms to their mono-species equivalent (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001). ^##p < 0.01, compares dead cells between dual- and mono-species biofilms.

FIGURE 2

Inter-kingdom interactions decrease Staphylococcus aureus sensitivity to miconazole. Biofilms were grown for 24 h on a cellulose matrix based hydrogel model before being washed with PBS and treated with 40 mg/L of miconazole for a further 24 h. After treatment, the cellulose matrix was removed and sonicated to dislodge the biofilm biomass. Live/dead PCR was then used to extract DNA and quantify total and live CFE. Data is presented as the CFE of live cells comparing treated mono- and dual-species biofilms. Data represents duplicate samples from three independent time points with significance achieved with ^∗∗p < 0.01.

FIGURE 3

Candida albicans and Staphylococcus aureus display synergistic pathogenicity and reduced miconazole sensitivity in vivo. C. albicans (CA) and S. aureus (SA) were standardized to 5 × 10⁵ CFU/larvae and administered to larvae as a mono or co-infection (CA+SA) with percentage survival monitored across a 48 h period and data presented using a Kaplan Meier plot. Data are derived from three independent groups of 10 larvae with significance (^∗p < 0.05, ^∗∗p < 0.001) determined using the log-rank test in comparison between C. albicans and S. aureus alone and co-infection (A). After 24 h post-infection, representative larvae were snap frozen in liquid nitrogen and DNA extracted. Microbial burden was then determined using qPCR and presented as CFE (B). Data are derived from three larvae with ^∗p < 0.05. Upon 2 h post-infection of larvae with 5 × 10⁵ CFU/larvae with CA, SA, and co-infection (CA+SA), larvae were administered with 75 mg/kg of miconazole. Percentage survival was monitored across a 48 h period and represented with a Kaplan Meier plot (C). Data represents results from three independent groups of 10 larvae with significance achieved (^∗∗p < 0.01, ^∗∗∗p < 0.001) comparing C. albicans, S. aureus, and co-infection using the log-rank test.

FIGURE 4

Scanning electron micrograph of S. aureus colonizing C. albicans hyphae within dual-species biofilms. Dual-species biofilms were grown for 24 h before being fixed, processed, and imaged using a JEOL-JSM 6400 scanning electron microscope. S. aureus colonies can be seen adhering and embedded within the hyphal meshwork of C. albicans. White arrows indicate clusters of S. aureus colonies encased within extracellular matrix (ECM). Scale bar represents 5 μm at × 5000 magnification.

FIGURE 5

Extracellular DNA contributes to inter-kingdom pathogenicity. Mono- and dual-species biofilms were seeded at 1 × 10⁶ CFU/mL in black 96-well plates and eDNA release at 1.5, 6, 12, and 24 h measured using a SYBR^® Green 1 based microplate fluorescence assay (MFA) in comparison to a standard curve (A). Biofilms were washed with 0.2 M EDTA to remove the ECM and resulting eDNA quantified using the MFA described above in comparison to a standard curve (B). ECM associated DNA was then precipitated from matrix extracts and species contributions were analyzed using qPCR (C). C. albicans only biofilms were grown for 24 h in black 96-well plates. After washing biofilms were then treated with either 130 or 650 mg/L of DNase for 4 h. SYTO9^® stained S. aureus cells (1 × 10⁶ CFU/mL) were then added to the biofilm and incubated for 90 mins before being fluorescently quantified in comparison to an vehicle control treated biofilm (D). Data represents duplicate samples from three independent experiments (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001).