Background

Candidiasis is a serious threat, especially to individuals with compromised immune systems and comorbidities. Hospitalized patients, particularly those who have undergone organ or bone marrow transplants or are receiving immune-suppressive therapies, are highly susceptible to infections [1]. Prolonged hospital stays further increase the risk of contracting hospital-acquired infections [2]. A critical concern is the emergence of Candida auris, a fungal pathogen first isolated in 2009 [3]. Since then, C. auris has become a global health concern, with reports of drug resistance emerging from as many as 32 countries, particularly during the COVID-19 pandemic among hospitalized patients [4]. C. auris as a drug-resistant nosocomial pathogen has a reported mortality rate of 30%–50% in infected persons [5]. This pathogen can cause various infections, including ear, systemic (fungemia), wound, lung, urinary tract, and gut infections [5–7]. Due to its high resistance to various antifungal treatments and its potential for pan-drug resistance, the World Health Organization (WHO) has listed C. auris in the critical priority group of fungal pathogen priority list (FPPL) released in October 2022 [8].

Unlike other Candida species, C. auris seldom colonizes the gut but can persist on the skin by forming resilient biofilms, which causes rapid spread of this fungus in hospitals [9]. C. auris demonstrates remarkable resilience, especially in its biofilm form, which shows substantial resistance [10]. According to the Centers for Disease Control and Prevention (CDC), C. auris biofilms are resistant to common disinfectants, including ammonium salt–based solutions, and can endure harsh conditions [11]. C. auris can persist in the cutaneous layer by dividing and forming biofilms, which can cause bloodstream infection when they reach the blood vessels [12]. This underscores the necessity of studying biofilm formation in infection scenarios to develop effective treatments. Given the widespread drug resistance, there is an urgent need for robust screening platforms to identify novel inhibitors. Drug repurposing must be validated through rigorous in vitro and in vivo screening methods. However, a major challenge is establishing a reliable method to form biofilm models that accurately replicate in vivo conditions. The phenotypes of C. auris biofilms can vary significantly based on culture conditions, complicating research outcomes. Current methods, such as those using Thermanox (polystyrene) coverslips or porcine skin surfaces [13,14], either fail to produce typical biofilm architecture or lack cost-effectiveness for large-scale screenings. A standard surface for biofilm proliferation is crucial for reproducibility, as effective drug screening requires the typical multilayered architecture of biofilms.

To address these issues, we proposed a novel in vitro method for growing C. auris biofilms on gelatin-coated coverslips derived from bovine skin. Gelatin, a derivative of collagen, mimics the matrix of mammalian cells, thereby more closely replicating natural conditions. This method successfully forms heterogeneous, multilayered biofilms of C. auris. The architecture of these biofilms has been analyzed using scanning electron microscopy (SEM) and compared with previous studies [14–16]. Furthermore, the versatility and accuracy of this method have been demonstrated using confocal scanning laser microscopy (CSLM) to measure biofilm thickness. The application of this innovative in vitro technique is not only limited to C. auris biofilms but can also be used to study biofilm formation in other Candida species, such as Candida glabrata and Candida albicans. This method is a potent, cost-effective solution for large-scale drug screenings.

Here, we describe a stepwise procedure for creating a substratum for biofilm growth and measuring biofilm thickness with confocal laser scanning microscopy and ultrastructures by scanning electron microscopy. As mentioned above, it results in reliable outcomes and is suitable for various Candida species with different media. This method has been successfully used in previously published studies, demonstrating its effectiveness and reliability [15,16].