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

Abstract

Echinocandin-resistant clinical isolates of Candida albicans have been reported, and key-hot 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 tetrazolium-salt 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.

Garcinia xanthochymus Benzophenones Promote Hyphal Apoptosis and Potentiate Activity of Fluconazole against Candida albicans Biofilms

“…Therefore, the compounds may reinforce each other's activities. In contrast, caspofungin increases the expression of hypha-specific proteins (26,46,47), an antagonistic result. Therefore, it is not surprising that our assays have shown antagonism in efficacy assays of xanthochymol with caspofungin (data not shown).…”

In Vitro Analysis of Finasteride Activity against Candida albicans Urinary Biofilm Formation and Filamentation

“…For the studies on the treatment of mature biofilms, the cells were incubated in urine medium in a 96-well microplate to allow the formation of mature biofilms for 24 h of incubation at 37°C. Next, the wells were gently washed and the biofilms were incubated for another 24 h at 37°C with increasing log 2 concentrations of FIN (2,4,8,16,32,64,128, and 256 mg/liter in urine medium). For both the prevention and treatment of biofilm studies, the metabolic activity within the biofilms was measured using the XTT reduction assay.…”

“…(C) Representative light microscopy images of C. albicans strain SC5314 biofilms. Visual differences in the biofilm architectures are apparent at four different FIN concentrations (32,64,128, and 256 mg/liter). The pictures were taken using a ϫ40 power field.…”

“…The combination is considered synergistic when the FICI is Յ0.5, indifferent when the FICI is Ͼ0.5 to Յ4, and antagonistic when the FICI is Ͼ4. When the highest concentration of drug tested did not produce a 90 or 50% decrease in metabolic activity, the highest concentration of the drug was used in place of the MIC 90 or MIC 50 to calculate the FICI, according to published methods (29)(30)(31)(32). These concentrations are preceded by "Ͼ".…”

“…Filamentation was assayed using solid and liquid media. Briefly, an overnight culture of C. albicans SC5314 was spotted onto YPD supplemented with 10% fetal calf serum (FCS) plates with increasing concentrations of FIN (8,32,64, and 128 mg/liter). The same FIN concentrations were used in hypha-inducing Spider plates (1% nutrient broth, 1% D-mannitol, 0.2% dipotassium phosphate, 2% agar [pH 7.2]).…”