Sinergic effect of amphotericin B and gold nanoparticles combination on regular and persister cells of Candida tropicalis biofilm.

DA SILVA MARÍA ÁNGEL, BARONETTI JOSÉ LUIS, GARCÍA MARTÍNEZ JOAQUÍN, PÁEZ PAULINA LAURA, PARAJE MARÍA GABRIELA.

San Luis

Congreso; XIII Congreso Argentino de Microbiología General.; 2018

Persister cells (PCs) are a small number of cells which wereproposed to be dormant and nongrowing phenotypic variants of the isogenic cellpopulation. PCs are distinguished from resistant mutants due to donot exhibit an increased minimal inhibitory concentration (MIC), and represent about 0.1 to 1% of the population. The existence of this small cell subpopulationwas described for some fungal species of Candida, however, its existence in Candidatropicalis biofilms is controversial. The aim of this work was tostudy the antifungal (ATF) effect of amphotericin B (AmB) and gold nanoparticles(AuNPs) combination over PCs in C. tropicalis biofilm.C. tropicalis NCPF 3111 biofilm formation was assayed by adhesion to 96-well plate andcrystal violet (CV) stain (0.1 OD595nm=1BBU). The PCs experimental designproposed allowed comparing supra MIC ATF activity, oxidative stress andantioxidant response between two different biofilms, ?Biofilms 1 -B1-? obtained fromplanktonic cells and exposed to supra MIC AmB treatment; and ?Biofilm 2 -B2-? asecond biofilm, derived from PCs that survived to drug treatment. Biofilm was also analyzed byscanning electron microscope (SEM) and PCs fraction was determined by colony forming unitscounting. Extracellular reactive oxygen species (ROS) were measured by thereduction of the nitro-blue tetrazolium (NBT) reaction, and Reactive nitrogenintermediates (RNI) were measured by Griess assay. Superoxide dismutase (SOD)activity was assayed based on the inhibition of NBT reduction; total reducedglutathione (GSHt) by enzymaticdetermination and total antioxidant capacity was measured by FRAP assay.A classic biphasic killing curve,indicative of PCs presence, was obtained and the equal MIC confirmedthat they were PCs. We observed that AmB (200 μg / ml)/AuNPs (0.63mM) combination produced a greater biofilm reduction (95%) than control biofilm.SEM images show a marked decrease in the amount of exopolysaccharides of the biofilmmatrix, with predominant yeast form. These images show concordance withthe marked decrease in biomass observed by staining with CV. ROS and RNI concentrationshowed similar increase in B1and B2, whereas antioxidant defenses (SOD, GSHt and FRAP) were significantlyhigher in B2 than in B1, indicating that the oxidative misbalance may beimportant. PCs play an important role in recalcitranceof chronic infections, therefore the finding synergic ATF effect wouldhelp to solve the puzzle of biofilm resistance to ATFs.