Modulation of virulence factors and wall proteins on Candida spp. in the presence of Carvacrol

Objectives: The objective of the study was to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of carvacrol against Candida spp. The data were employed to evaluate the impact of carvacrol at the MIC on virulence factors and to examine the effect of sub-inhibitory concentrations on wall protein expression. Methods: The MIC and MFC of carvacrol was determined. Subsequently, the ability to form biofilms in the presence of carvacrol, XTT-menadione assay and crystal violet assay (CVA) was assessed using a spectrophotometer at wavelengths of 490 nm (XTT-menadione) and 560 (CVA). The alteration in the capacity of microorganisms to adhere to abiotic surfaces was evaluated following a three-hour incubation period in the presence of carvacrol, employing the Cristalviolet assay. The protein components were also characterised by densitometry and mass spectrometry (MALDI-TOF). The efficacy of the treatment was tested in vivo using Galleria mellonella larvae within five days of infection. Results: The MIC and MFC for the species tested was: C. glabrata MIC=0,39 MFC=0,78 ; C. krusei MIC=0,39 MFC=0,78. The presence of carvacrol induced a significant inhibition on both an early (3h) and a pre-formed (24h) biofilm in all species tested except C. krusei biomass in the biofilm at 3h. There were no significant differences between metabolic activity and total biomass. In the presence of carvacrol, at a concentration equal to the MIC, there is a 52% inhibition of adherence in C. glabrata. For C. krusei, the presence of carvacrol does not impact adherence. Densitometric analysis showed an alteration in proteins involved in the metabolism and adhesion process to human cells in the presence of carvacrol. The presence of carvacrol increased larval survival in all species, 10% C. glabrata, 30% C. krusei. Conclusions: The presence of carvacrol modifies the virulence factors tested in both of the species, reducing the ability to form biofilms and reducing the ability to adhere to plastic surfaces in all species tested. The presence of carvacrol also affects the expression of proteins involved in glycolysis, such as phosphoglycerate mutase, and in human cell adhesion and invasion, such as enolase. Finally, the presence of carvacrol increases the survival of infected larvae, although there are differences between species.