A Novel Robust Method Mimicking Human Substratum To Dissect the Heterogeneity of Candida auris Biofilm Formation

Candida auris is a pathogen of urgent threat level as marked by the CDC. The formation of biofilms is an essential property of this fungus to establish infection and escape drug treatment. However, our understanding of pathogenesis through biofilm is hampered by heterogeneity in C. auris biofilms observed in different studies. It is imperative to replicate in vivo conditions for studying C. auris biofilm formation in vitro. Different methods are standardized, but the surface used to form biofilms lacks consistency as well as the architecture of a typical biofilm. Here, we report an in vitro technique to grow C. auris biofilms on gelatin-coated coverslips. Interestingly, C. auris cells grown on gelatin-coated coverslips either on modified synthetic sweat media or RPMI 1640 resulted in similar multilayer biofilm formation with extracellular polymeric substances (EPS). This method is also consistent with the biofilm formation of other Candida species, such as Candida glabrata and Candida albicans. Biofilms of C. glabrata developed through this method show pseudohyphae and EPS. This method can be used to understand the molecular basis of biofilm formation, associated pathogenesis, and drug tolerance. The technique is cost-effective and would thus serve in rightful screening and repurposing drug libraries for designing new therapeutics against the less-studied high-alarm pathogen C. auris. IMPORTANCE Heterogeneity is seen when multidrug-resistant C. auris biofilm is cultured using different reported methods. Biofilm formed on the gelatin surface mimics the condition of a host environment that has multilayers and EPS. This method has feasibility for drug screening and analyzing biofilms through three-dimensional (3D) reconstruction. This in vitro biofilm formation technique is also exploited to study the formation of biofilm of other Candida species. The biofilms of C. glabrata and C. albicans can also be correctly mimicked using gelatin in the biofilm-forming environment. Thus, the novel in vitro method for biofilm formation reported here can be widely used to understand the mechanism of biofilm formation, related virulence properties, and drug tolerance of C. auris and other Candida species. This simple and low-cost technique is highly suitable for screening novel inhibitors and repurposed libraries and to design new therapeutics against Candida species.