title: A Combination Fluorescence Assay Demonstrates Increased Efflux Pump Activity as a Resistance Mechanism in Azole-Resistant Vaginal Candida albicans Isolates.
abstract: Candida albicans is a pathogenic fungus causing vulvovaginal candidiasis (VVC). Azole drugs, such as fluconazole, are the most common treatment for these infections. Recently, azole-resistant vaginal C. albicans isolates have been detected in patients with recurring and refractory vaginal infections. However, the mechanisms of resistance in vaginal C. albicans isolates have not been studied in detail. In oral and systemic resistant isolates, overexpression of the ABC transporters Cdr1p and Cdr2p and the major facilitator transporter Mdr1p is associated with resistance. Sixteen fluconazole-susceptible and 22 fluconazole-resistant vaginal C. albicans isolates were obtained, including six matched sets containing a susceptible and a resistant isolate, from individual patients. Using quantitative real-time reverse transcriptase PCR (qRT-PCR), 16 of 22 resistant isolates showed overexpression of at least one efflux pump gene, while only 1 of 16 susceptible isolates showed such overexpression. To evaluate the pump activity associated with overexpression, an assay that combined data from two separate fluorescent assays using rhodamine 6G and alanine β-naphthylamide was developed. The qRT-PCR results and activity assay results were in good agreement. This combination of two fluorescent assays can be used to study efflux pumps as resistance mechanisms in clinical isolates. These results demonstrate that efflux pumps are a significant resistance mechanism in vaginal C. albicans isolates.
authors.affiliation: School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri, USA.
authors.forename: Somanon
authors.initials: S
authors.lastname: Bhattacharya
authors.affiliation: School of Medicine, Wayne State University, Detroit, Michigan, USA.
authors.forename: Jack D
authors.initials: JD
authors.lastname: Sobel
authors.affiliation: School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri, USA whitetc@umkc.edu.
authors.forename: Theodore C
authors.initials: TC
authors.lastname: White
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D000935:Antifungal Agents / Q000494:pharmacology; D001393:Azoles / Q000494:pharmacology*; D002176:Candida albicans / Q000187:drug effects* / Q000302:isolation & purification / Q000502:physiology; D002181:Candidiasis, Vulvovaginal / Q000382:microbiology; D025141:Drug Resistance, Fungal / Q000187:drug effects / Q000502:physiology*; D005260:Female; D005453:Fluorescence; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests / Q000379:methods; D008828:Microbiological Techniques / Q000379:methods; D014621:Vagina / Q000382:microbiology*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000935:Antifungal Agents; D001393:Azoles; C094693:CDR1 protein, Candida albicans; D005656:Fungal Proteins; D026901:Membrane Transport Proteins