qPCR analysis of C. albicans virulence genes

Real‐time qPCR was performed as described previously (Cavalcanti et al. 2015; Morse et al. 2018). Putative virulence genes of C. albicans included adhesins involved in adhesion to biotic and abiotic surfaces and hyphal formation/promotion (agglutinin‐like sequence 3 (ALS3), epithelial adhesin 1 (EPA1), hyphal wall protein 1 (HWP1)); and production of secreted hydrolytic enzymes involved in nutrient acquisition, hyphal formation, tissue invasion and subsequent damage (phospholipase D1 (PLD1) and secreted aspartyl proteinases 4 and 6 (SAP4, SAP6)). The primer sequences were as detailed in Table S1. The analysis of relative gene expression was performed according to the ^ΔΔCt method (Bustin et al. 2009) normalised to the ACT1 housekeeping gene expression of C. albicans‐only biofilms. Statistical analysis of gene expression was performed using the ^ΔΔCt values using one‐way analysis of variance, with Tukey's multiple comparisons test, at 95% confidence.