Spilanthol as a promising antifungal alkylamide for the treatment of vulvovaginal candidiasis.

Spilanthol is a bioactive alkylamide from the native Amazon plant species, Acmella oleracea. However, antifungal activities of spilanthol and its application to the therapeutic treatment of candidiasis remain to be explored. This study sought to evaluate the in vitro and in vivo antifungal activity of spilanthol previously isolated from A. oleracea (spilanthol(AcO)) against Candida albicans ATCC® 10231™, a multidrug-resistant fungal strain. Microdilution methods were used to determine inhibitory and fungicidal concentrations of spilanthol(AcO). In planktonic cultures, the fungal growth kinetics, yeast cell metabolic activity, cell membrane permeability and cell wall integrity were investigated. The effect of spilanthol(AcO) on the proliferation and adhesion of fungal biofilms was evaluated by whole slide imaging and scanning electron microscopy. The biochemical composition of the biofilm matrix was also analyzed. In parallel, spilanthol(AcO) was tested in vivo in an experimental vulvovaginal candidiasis model. Our in vitro analyses in C. albicans planktonic cultures detected a significant inhibitory effect of spilanthol(AcO), which affects both yeast cell membrane and cell wall integrity, interfering with the fungus growth. C. albicans biofilm proliferation and adhesion, as well as, carbohydrates and DNA in biofilm matrix were reduced after spilanthol(AcO) treatment. Moreover, infected rats treated with spilanthol(AcO) showed consistent reduction of both fungal burden and inflammatory processes compared to the untreated animals. Altogether, our findings demonstrated that spilanthol(AcO) is an bioactive compound against planktonic and biofilm forms of a multidrug resistant C. albicans strain. Furthermore, spilanthol(AcO) can be potentially considered for therapeutical treatment of vulvovaginal candidiasis caused by C. albicans. LAY SUMMARY: This study sought to evaluate the antifungal activity of spilanthol against Candida albicans ATCC® 10 231™, a multidrug-resistant fungal strain. Our findings demonstrated that spilanthol(AcO) can be potentially considered for therapeutical treatment of vulvovaginal candidiasis caused by C. albicans.

Whole slide imaging is a high-throughput method to assess Candida biofilm formation.

New strategies that enable fast and accurate visualization of Candida biofilms are necessary to better study their structure and response to antifungals agents. Here, we applied whole slide imaging (WSI) to study biofilm formation of Candida species. Three relevant biofilm-forming Candida species (C. albicans ATCC 10231, C. glabrata ATCC 2001, and C. tropicalis ATCC 750) were cultivated on glass coverslips both in presence and absence of widely used antifungals. Accumulated biofilms were stained with fluorescent markers and scanned in both bright-field and fluorescence modes using a WSI digital scanner. WSI enabled clear assessment of both size and structural features of Candida biofilms. Quantitative analyses readily detected reductions in biofilm-covered surface area upon antifungal exposure. Furthermore, we show that the overall biofilm growth can be adequately assessed across both bright-field and fluorescence modes. At the single-cell level, WSI proved adequate, as morphometric parameters evaluated with WSI did not differ significantly from those obtained with scanning electron microscopy, considered as golden standard at single-cell resolution. Thus, WSI allows for reliable visualization of Candida biofilms enabling both large-scale growth assessment and morphometric characterization of single-cell features, making it an important addition to the available microscopic toolset to image and analyse fungal biofilm growth.