Response of Candida tropicalis biofilms to oxidative stress: implication of persister cells

DA SILVA M; BARONETTI JL; PAEZ P; PARAJE, MG

Congreso; XII Congreso de Microbiologìa general (SAMIGE); 2017

Persisters cells (PCs) are defined as phenotypic variants of the wild type that display tolerance to killing by high doses of antimicrobial (ATM) drugs. Upon removal of the ATM pressure, these cells switch back to 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 inhibitory concentration (MIC), and represents about 0.1 to 1% of the population. PCs play an important role in recalcitrance of chronic infections. The aim of this work was to study the oxidative stress and antioxidant response 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 crystal violet 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 ROS measurement by SCLM. Reactive nitrogen intermediates (RNI) were measured by Griess assay. Superoxide dismutase (SOD) activitywas assayed based on the inhibition of NBT reduction and total antioxidant capacity was measured by FRAP assay. The experimental design proposed allowed comparing oxidative stress and antioxidant response of two different biofilms. ?Biofilms 1? obtained from planktonic cells and 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 PCs presence was obtained. The equal MIC confirmed that they were PCs. A greater ATF effect -higher BBU reduction- was observed in ?Biofilms 1?. Both biofilms showed similar basal ROS levels. An increase was observed upon AmB treatment, being it greater in ?Biofilms 1?. RNI measurements showed similar profiles as ROS after AmB treatment in both biofilms. In relation to antioxidant system, specifically SOD enzyme, a higher activation was observed in ?Biofilm 2? when treated with AmB. Same effect was observed for total antioxidant capacity of biofilm. The last also showed significant differences of basal levels. Result obtained by SCLM agreed with BBU assay. These results demonstrate that ?Biofilm 2? shows a major capacity to respond to the stress generated upon ATF treatment. It could due to the fact that the cells giving rise to ?Biofilm 2? had been previously exposed to AmB. The finding of a CPs subpopulation with different oxidative status would help to solve the puzzle of biofilm resistance to ATFs indicating that the oxidative misbalance may be important