Paradoxical antifungal activity and structural observations in biofilms formed by echinocandin-resistant Candida albicans clinical isolates

Echinocandin-resistant clinical isolates of Candida albicans have been reported, and keyhot spot mutations in the FKS1 gene, which encodes a major glucan synthase subunit, have been identified in these (caspofungin-resistant [CAS-R]) strains. Although these mutations result in phenotypic resistance to echinocandins in planktonic cells, there is little data on antifungal susceptibilities of CAS-R C. albicans strains within biofilms. Thus, we analyzed biofilms formed by 12 C. albicans CAS-R clinical strains in which we previously identified FKS1 hot-spot mutations and compared the sessile antifungal and paradoxical activity of anidulafungin (ANID), caspofungin (CAS), and micafungin (MICA). Biofilms were formed in a 96-well static microplate model and assayed using both tetrazoliumsalt reduction and crystal violet assays, as well as examination by scanning electron microscopy. We first sought to assess biofilm formation and structure in these fks1 mutants and found that the biofilm mass and metabolic activities were reduced in most of the fks1 mutants as compared with reference strain SC5314. Structural analyses revealed that the fks1 mutant biofilms were generally less dense and had a clear predominance of yeast and pseudohyphae, with unusual "pit"-like cell surface structures. We also noted that sessile minimum inhibitory concentrations (MICs) to ANID, CAS, and MICA were higher than planktonic MICs of all but one strain. The majority of strains demonstrated a paradoxical effect (PE) to particular echinocandins, in either planktonic or sessile forms. Overall, biofilms formed by echinocandin-resistant clinical isolates demonstrated varied PEs to echinocandins and were structurally characterized by a preponderance of yeast, pseudohyphae, and pit-like structures.