Unveiling antifungal resistance and biocide tolerance in clinical isolates of Candida spp

Abstract

Candidiasis is an opportunistic infection that impacts public health. In recent years, emerging Candida species have contributed to the increase in the disease burden exacerbated by antifungal resistance. This study aimed to evaluate the antifungal susceptibility profile of Candida clinical isolates, investigate resistance mechanisms, evaluate the efflux pump activity, and assess biocide tolerance. 100 Candida isolates from hospitalized and outpatient individuals were analyzed. Antifungal susceptibility and biocide tolerance (sodium hypochlorite, hydrogen peroxide, and benzalkonium chloride) were assessed using a disk diffusion test, and the molecular resistance mechanisms were explored through polymerase chain reaction (PCR). Efflux pump activity was performed using the Cartwheel method. A high frequency of resistance (n=87/87%) to at least one antifungal was observed, with 21% (n=21) of isolates showing simultaneous resistance to multiple antifungal agents. The fungal isolates evaluated showed the highest frequency of resistance to fluconazole (n=70), followed by voriconazole (n=68), itraconazole (n=47), and caspofungin (n=20), and the highest susceptibility to amphotericin B (n=1). The most common mutation was detected in the ERG11 gene (n=38/43.7%), followed by CDR2 (n=17/19.5%), CDR1 (n=7/8%) and MDR1 (n=6/6.9%). The ERG2 and FKS1 genes were not detected in any of the isolates in this study. Besides the intrinsic efflux pump activity observed in all Candida isolates, 14 Candida albicans isolates exhibited a higher MCEtBr compared to the reference strain, with 1.0 μg/l (n=10) and 1.5 μg/l (n=4). For the non-albicans group, 6 isolates of Candida tropicalis, 3 of Candida parapsilosis, and 1 each of Meyerozyma guilliermondii (syn. Candida guilliermondii) and Pichia kudriavzevii (syn. Candida krusei) presented an MCEtBr greater than the reference at 1.0 μg/l, those findings point towards an overexpression of the mechanism. Sodium hypochlorite was the most effective biocide tested with an inhibition zone ranging from 18.3mm (±4.4) to 34.0mm (±4.0), and the least effective was hydrogen peroxide at 4.25% and benzalkonium chloride 5% with 6.00mm (±0.0). This study highlights considerable antifungal resistance in Candida spp., particularly to azoles, in association with tolerance to routinely used biocides, emphasizing the need to develop new therapeutic strategies and infection control measures for healthcare-associated infections. Keywords: Candida; Infections; Antifungal sus