Rapid detection of efflux pumps and their relation with drug resistance inyeast cells

Background: Cell drug resistance can be due to the presence of active efflu x pumps (AEP). Identification of yeast cells with a resistance phenotype is important either from a clinical, agricultural or biotechnological point o f view. Rapid and reliable methods to detect AEP can be therefore very usef ul. Methods: Some yeast cells change their staining by calcein-AM, BCECF-AM, rh odamine 123 and DiOC(5), when pretreated with verapamil, CCCP or ATP deplet ion, or when pretreated with specific antimicrobial agents. This fact may b e interpreted as an indication of the presence/absence of AEP. Sis yeast sp ecies were tested with a flow cytometric method (FCM) and an epifluorescenc e microscopic method (EFM), and ten other species were evaluated only by EF M. The minimum inhibitory concentration (MIC) of penconazol. benomyl and cy cloheximide for Saccharomyces cerevisiae and Kluyveromyces marxianus, were determined by growth inhibition on solid medium and were compared to the st aining changes detected by FCM. Results: The FCM and the EFM allowed the detection of AEP in all the yeast species tested. High MIC values for a drug were related with the presence o f at least one AEP indicated by the cytometric data. Conclusions: The FCM revealed to be a robust assay whereas the EFM can be u sed as a preliminary test. It is possible to identify resistance/sensitivit y patterns in yeast cells through cytometric detection methods of different efflux pumping systems. (C) 2000 Wiley-Liss, Inc.