Rapid detection of efflux pumps and their relation with drug resistance in yeast cells.
BACKGROUND
Cell drug resistance can be due to the presence of active efflux pumps (AEP). Identification of yeast cells with a resistance phenotype is important either from a clinical, agricultural or biotechnological point of view. Rapid and reliable methods to detect AEP can be therefore very useful.


METHODS
Some yeast cells change their staining by calcein-AM, BCECF-AM, rhodamine 123 and DiOC(5), when pretreated with verapamil, CCCP or ATP depletion, or when pretreated with specific antimicrobial agents. This fact may be interpreted as an indication of the presence/absence of AEP. Six yeast species were tested with a flow cytometric method (FCM) and an epifluorescence microscopic method (EFM), and ten other species were evaluated only by EFM. The minimum inhibitory concentration (MIC) of penconazol, benomyl and cycloheximide for Saccharomyces cerevisiae and Kluyveromyces marxianus, were determined by growth inhibition on solid medium and were compared to the staining 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 of at least one AEP indicated by the cytometric data.


CONCLUSIONS
The FCM revealed to be a robust assay whereas the EFM can be used as a preliminary test. It is possible to identify resistance/sensitivity patterns in yeast cells through cytometric detection methods of different efflux pumping systems.
Prudêncio
C
C
Centro de Ciências do Ambiente - Departamento de Biologia, Universidade do Minho, Braga Codex, Portugal.
Sansonetty
F
F
Sousa
M J
MJ
Côrte-Real
M
M
Leão
C
C
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D001542:Benomyl / Q000494:pharmacology; D001693:Biological Transport, Active / Q000502:physiology*; D003513:Cycloheximide / Q000494:pharmacology; D004351:Drug Resistance / Q000502:physiology; D005434:Flow Cytometry / Q000379:methods*; D005659:Fungicides, Industrial / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D008856:Microscopy, Fluorescence / Q000379:methods; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000502:physiology; D012568:Schizosaccharomyces / Q000187:drug effects / Q000502:physiology; D014230:Triazoles / Q000494:pharmacology
D018528:ATP-Binding Cassette Transporters; D005659:Fungicides, Industrial; D014230:Triazoles; C087058:penconazole; D003513:Cycloheximide; D001542:Benomyl