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

MARIA ANGEL DA SILVA, JOSE BARONETTI, PAULINA LAURA PAEZ, MARIA GABRIELA PARAJE.

Tucumán

Congreso; Congreso Argentino de Microbiología General (SAMIGe) 2017; 2017

Asociación Civil de Microbiología General

Persisterscells (PCs) are defined as phenotypic variants of the wild type that displaytolerance to killing by high doses of antimicrobial (ATM) drugs. Upon removalof the ATM pressure, these cells switch back to a growing state, thereby givingrise to a new population genetically identical to the original one. PCs are distinguished from resistant mutantsbecause do not exhibit an increased minimalinhibitory concentration (MIC), and represents about 0.1 to 1% of thepopulation. PCs play an important role inrecalcitrance of chronic infections.Theaim of this work was to study the oxidative stress and antioxidant response of Candida tropicalis biofilm formed from PCsfraction upon antifungal (ATF) treatment. C. tropicalisNCPF 3111 was used. Biofilm formation was assayed by adhesion to 96-well plate and crystalviolet stain (0.1 OD595nm=1BBU) and PCs fractionwas determined by colony forming units counting. Biofilm was also analyzed byScanning Confocal Laser Microscopy (SCLM) by Calcofluor White stain. Extracellular reactiveoxygen species (ROS) were measured by the reduction of the nitro-blue tetrazolium (NBT)reaction, while probe 2′,7′-Dichlorodihydrofluorescein diacetate was used for intracellularROS measurement by SCLM. Reactive nitrogen intermediates (RNI) were measured by Griess assay. Superoxidedismutase (SOD) activity was assayedbased on the inhibition of NBT reduction and total antioxidant capacity was measuredby FRAP assay.Theexperimental design proposed allowed comparing oxidative stress and antioxidantresponse of two different biofilms. ?Biofilms 1? obtained from planktonic cellsand exposed to 200 µg/ml of AmB. A second biofilm, derived from PCs that surviveddrug treatment (?Biofilm 2?), was againtreated with 200 µg/ml AmB. A classic biphasickilling curve indicative of PCs presence was obtained. The equal MIC confirmed that they werePCs.A greater ATF effect -higher BBU reduction- was observed in ?Biofilms 1?. Both biofilms showedsimilar basal ROS levels. An increase was observed upon AmB treatment, being itgreater in ?Biofilms 1?. RNI measurements showed similar profiles as ROS after AmBtreatment in both biofilms. In relation toantioxidant system, specifically SOD enzyme, a higher activation was observed in ?Biofilm 2? when treated with AmB. Same effect was observed fortotal antioxidant capacity of biofilm. The last also showed significant differencesof basal levels. Result obtained by SCLMagreed with BBU assay. These resultsdemonstrate that ?Biofilm 2? shows amajor capacity to respond to the stress generated upon ATF treatment. It coulddue to the fact that the cells giving rise to ?Biofilm2? had been previously exposed to AmB. The finding of a CPssubpopulation with different oxidative status would help to solve the puzzle ofbiofilm resistance to ATFs indicating that the oxidative misbalance may beimportant.