Abstract
The aim of this study was to evaluate the antifungal activity of Terpinen-4-ol associated with nystatin, on single and mixed species biofilms formed by Candida albicans and Candida tropicalis, as well as the effect of terpinen-4-ol on adhesion in oral cells and the enzymatic activity. The minimum inhibitory concentrations and minimum fungicide concentrations of terpinen-4-ol and nystatin on Candida albicans and Candida tropicalis were determined using the microdilution broth method, along with their synergistic activity ("checkerboard" method). Single and mixed species biofilms were prepared using the static microtiter plate model and quantified by colony forming units (CFU/mL). The effect of Terpinen-4-ol in adhesion of Candida albicans and Candida tropicalis in coculture with oral keratinocytes (NOK Si) was evaluated, as well as the enzymatic activity by measuring the size of the precipitation zone, after the growth agar to phospholipase, protease and hemolysin. Terpinen-4-ol (4.53 mg mL-1) and nystatin (0.008 mg mL-1) were able to inhibit biofilms growth, and a synergistic antifungal effect was showed with the drug association, reducing the inhibitory concentration of nystatin up to 8 times in single biofilm of Candida albicans, and 2 times in mixed species biofilm. A small decrease in the adhesion of Candida tropicalis in NOK Si cells was showed after treatment with terpinen-4-ol, and nystatin had a greater effect for both species. For enzymatic activity, the drugs showed no action. The effect potentiated by the combination of terpinen-4-ol and nystatin and the reduction of adhesion provide evidence of its potential as an anti-fungal agent.
Highlights
In the oral cavity, Candida species adhere to many sites, such as mucosal cells, the tongue, surfaces of teeth and dentures, and other microorganisms
An increasing number of infections caused by non-albicans Candida species has been reported, and have been identified as the infecting pathogens isolated from various clinical specimens, which demonstrated the production of virulence factors once attributed to Candida albicans [2,3]
One of the simplest and best known protocols to investigate the antimicrobial activity of synthetic products or natural agents is the “checkerboard” test, which provides a two-dimensional arrangement of different concentrations of substances and allows the required Fractional Inhibitory Concentration Index (FICI) to be calculated to evaluate the combination of synergism, additive effect, indifference or antagonism [17]
Summary
Candida species adhere to many sites, such as mucosal cells, the tongue, surfaces of teeth and dentures, and other microorganisms To induce disease, they must adhere to epithelial cells of the oral mucosa and subsequently invade and destroy these cells. To improve its ability to colonize and establish infections, the fungus produces exoenzymes, such as aspartyl protease and phospholipase, which degrade the extracellular matrix and inhibit phagocytosis by neutrophils to induce inflammatory reactions, and hemolysin. They acquire iron, which facilitates the invasion and hyphal development of disseminated candidiasis [1,2]. An increasing number of infections caused by non-albicans Candida species has been reported, and have been identified as the infecting pathogens isolated from various clinical specimens, which demonstrated the production of virulence factors once attributed to Candida albicans [2,3]
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