COMPARISON BETWEEN CLSI AND EUCAST METHODS FOR TESTING SUSCEPTIBILITY OF CANDIDA SPP TO MELALEUCA ALTERNIFOLIA ESSENTIAL OIL

Tatiana Maria Souza Moreira (UNESP); Rosemeire Cristina Linhari Rodrigues Pietro (UNESP)

Tea tree oil is an antimicrobial essential oil from Melaleuca alternifolia (Cheel.) active against several bacteria, fungus and Candida species. In order to analyze the susceptibility profile of different species of Candida treated with tea tree oil, the present study compared CLSI (Clinical and Laboratory Standards Institute; formerly NCCLS, National Committee for Clinical Laboratory Standards) and EUCAST (European Committee on Antibiotic Susceptibility Testing) susceptibility methods. Strains tested: Candida albicans ATCC 64548, Candida krusei ATCC 6258, Candida parapsilosis ATCC 22019, Candida tropicalis ATCC 750. CLSI method used standard RPMI 1640 medium (0.2% glucose) and an inoculum containing 0.5x10³-2.5x10³ CFU/mL. EUCAST method used double-strength RPMI 1640 medium (2% glucose) and 0.25x10⁵-1.25x10⁵ inoculum. Both tests were done in flat-bottom microdilution plates. Tea tree oil dilutions in aqueous 1% Tween 80 ranged from 1% to 0.002%. Plates were incubated at 35° C and readings were done at 24 and 48 h. Values of 48 h CLSI and 24 h EUCAST readings were considered in agreement when discrepancies were no more than 2log₂ dilutions. MIC (minimum inhibitory concentration) values ranged from 0.125 to 0.5% of essential oil by CLSI and EUCAST methods and were in agreement with values obtained in previous studies that used CLSI or other MIC determination methods. MIC values obtained according CLSI at 48 h and EUCAST at 24 h were no more than 2 log₂ dilutions to C. parapsilosis and C. tropicalis. There was no agreement for MIC values obtained with the other species, showing that there is still a disagreement between the two methods when analyzing Candida susceptibility to this essential oil sample. EUCAST proposed a methodology of supplementation of RPMI 1640 with 2% glucose for increasing the turbidity and then shorting MIC determination to 24 h, but CLSI is an extensive standard method used in fungal susceptibility to plant samples. It is up to the researcher validate the more useful method meanwhile there is no agreement between those references.