(Authors 1, 3, 4): Departamento de Biologia, Universidade do Minho
(Author 2): Laboratório de Citometria, IPATIMUP
In some yeasts, genes responsible of generalised resistance to a large number of structurally and functionally unrelated drugs have been described. This phenotype is known as pleiotropic drug resistance (PDR) and is related with multidrug membrane transporters which drive hydrophobic compounds out of the cell. These transporters are classified in two distinct superfamilies: the ATP-binding cassette proteins (ABC) and the major facilitator superfamily proteins (MFS). A screening for this pleiotropic resistance phenotype becomes relevant when dealing with immunocompromised patients and patients receiving broad-spectrum antibiotic therapy. Also, in the agriculture the development of pleiotropic resistance to fungicides is becoming a nowadays problem.
In the present work we tried to assess in which extension the presence of active drug efflux pumps is generalised in yeasts. For such purposes we used 16 different yeast species and calcein-AM, BCECF-AM and DIOC5, earlier described as substrates for the proteins responsible for drug pleiotropic resistance. Among the species tested some exhibited fluorescence after staining (negative response) whereas others, were fluorescent only when stained after incubation under inhibitory conditions for either ABC and MFS classes of proteins (positive response). The results suggested the presence of active efflux pumps in the positive response species and in the negative ones the absence of pumps with specificity to the probes used. The data obtained with fluorescence microscopy were validated by flow cytometry which is a more sensitive technique and allows quantification of the results. Hence the method based on fluorescence microscopy could be useful for screening of multidrug pumps in yeasts.