SINERGIC EFFECT OF AMPHOTERICIN B AND GOLD NANOPARTICLES COMBINATION ON REGULAR AND PERSISTER CELLS OF Candida tropicalis BIOFILM

BARONETTI JL; PAEZ P; GALERA I; PARAJE, M. GABRIELA; DA SILVA M; GARCIA MARTINEZ, JOAQUIN

San luis

Congreso; XIII CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL - SAMIGE 2018; 2018

Persister cells (PCs) are a small number of cells which were proposed to be dormant and nongrowingphenotypic variants of the isogenic cell population. PCs are distinguished from resistant mutants dueto do not exhibit an increased minimal inhibitory concentration (MIC), and represent about 0.1 to 1%of the population. The existence of this small cell subpopulation was described for some fungal speciesof Candida, however, its existence in Candida tropicalis biofilms is controversial. The aim of thiswork was to study the antifungal (ATF) effect of amphotericin B (AmB) and gold nanoparticles(AuNPs) combination over PCs in C. tropicalis biofilm. C. tropicalis NCPF 3111 biofilm formationwas assayed by adhesion to 96-well plate and crystal violet (CV) stain (0.1 OD595nm=1BBU). ThePCs experimental design proposed allowed comparing supra MIC ATF activity, oxidative stress andantioxidant response between two different biofilms, ?Biofilms 1 -B1-? obtained from planktonic cellsand exposed to supra MIC AmB treatment; and ?Biofilm 2 -B2-? a second biofilm, derived from PCsthat survived to drug treatment. Biofilm was also analyzed by scanning electron microscope (SEM)and PCs fraction was determined by colony forming units counting. Extracellular reactive oxygenspecies (ROS) were measured by the reduction of the nitro-blue tetrazolium (NBT) reaction, andReactive nitrogen intermediates (RNI) were measured by Griess assay. Superoxide dismutase (SOD)activity was assayed based on the inhibition of NBT reduction; total reduced glutathione (GSHt) byenzymatic determination and total antioxidant capacity was measured by FRAP assay.A classic biphasic killing curve, indicative of PCs presence, was obtained and the equal MICconfirmed that they were PCs. We observed that AmB (200 g / ml)/AuNPs (0.63 mM) combinationproduced a greater biofilm reduction (95%) than control biofilm. SEM images show a markeddecrease in the amount of exopolysaccharides of the biofilm matrix, with predominant yeast form.These images show concordance with the marked decrease in biomass observed by staining with CV.ROS and RNI concentration showed similar increase in B1 and B2, whereas antioxidant defenses(SOD, GSHt and FRAP) were significantly higher in B2 than in B1, indicating that the oxidativemisbalance may be important. PCs play an important role in recalcitrance of chronic infections,therefore the finding synergic ATF effect would help to solve the puzzle of biofilm resistance to ATF