RESPONSE OF Candida tropicalis BIOFILMS TO OXIDATIVE STRESS: IMPLICATION OF PERSISTER CELLS.

MA DA SILVA; MARIA G PARAJE; JOSE BARONETI; PAULINA LAURA PÁEZ

San Miguel de Túcuman

Congreso; SAMIGE; 2017

UNT

Persisters cells (PCs) are defined as phenotypic variants of the wild type that display tolerance to killingby high doses of antimicrobial (ATM) drugs. Upon removal of the ATM pressure, these cells switch backto a growing state, thereby giving rise to a new population genetically identical to the original one.PCs are distinguished from resistant mutants because do not exhibit an increased minimal inhibitoryconcentration (MIC), and represents about 0.1 to 1% of the population. PCs play an important role inrecalcitrance of chronic infections. The aim of this work was to study the oxidative stress and antioxidantresponse of Candida tropicalis biofilm formed from PCs fraction upon antifungal (ATF) treatment.C. tropicalis NCPF 3111 was used. Biofilm formation was assayed by adhesion to 96-well plate and crystalviolet stain (0.1OD595nm=1BBU) and PCs fraction was determined by colony forming units counting.Biofilm was also analyzed by Scanning Confocal Laser Microscopy (SCLM) by Calcofluor White stain.Extracellular reactive oxygen species (ROS) were measuredby the reduction of the nitro-blue tetrazolium(NBT) reaction, while probe 2¢,7¢-Dichlorodihydrofluorescein diacetate was used for intracellular ROSmeasurement by SCLM. Reactive nitrogen intermediates (RNI) were measured by Griess assay. Superoxidedismutase (SOD) activitywas assayed based on the inhibition of NBT reduction and total antioxidantcapacity was measured by FRAP assay. The experimental design proposed allowed comparing oxidativestress and antioxidant response of two different biofilms. ?Biofilms 1? obtained from planktonic cellsand exposed to 200 μg/ml of AmB. A second biofilm, derived from PCs that survived drug treatment(?Biofilm 2?), was again treated with 200 μg/ml AmB. A classic biphasic killing curve indicative of PCspresence was obtained. The equal MIC confirmed that they were PCs. A greater ATF effect -higher BBUreduction- was observed in ?Biofilms 1?. Both biofilms showed similar basal ROS levels. An increasewas observed upon AmB treatment, being it greater in ?Biofilms 1?. RNI measurements showed similarprofiles as ROS after AmB treatment in both biofilms. In relation to antioxidant system, specificallySOD enzyme, a higher activation was observed in ?Biofilm 2? when treated with AmB. Same effectwas observed for total antioxidant capacity of biofilm. The last also showed significant differences ofbasal levels. Result obtained by SCLM agreed with BBU assay. These results demonstrate that ?Biofilm2? shows a major capacity to respond to the stress generated upon ATF treatment. It could due to thefact that the cells giving rise to ?Biofilm 2? had been previously exposed to AmB. The finding of a CPssubpopulation with different oxidative status would help to solve the puzzle of biofilm resistance toATFs indicating that the oxidative misbalance may be important.