title: Heteroresistance to Fluconazole Is a Continuously Distributed Phenotype among Candida glabrata Clinical Strains Associated with In Vivo Persistence.
abstract: Candida glabrata causes persistent infections in patients treated with fluconazole and often acquires resistance following exposure to the drug. Here we found that clinical strains of C. glabrata exhibit cell-to-cell variation in drug response (heteroresistance). We used population analysis profiling (PAP) to assess fluconazole heteroresistance (FLC(HR)) and to ask if it is a binary trait or a continuous phenotype. Thirty (57.6%) of 52 fluconazole-sensitive clinical C. glabrata isolates met accepted dichotomous criteria for FLC(HR) However, quantitative grading of FLC(HR) by using the area under the PAP curve (AUC) revealed a continuous distribution across a wide range of values, suggesting that all isolates exhibit some degree of heteroresistance. The AUC correlated with rhodamine 6G efflux and was associated with upregulation of the CDR1 and PDH1 genes, encoding ATP-binding cassette (ABC) transmembrane transporters, implying that HetR populations exhibit higher levels of drug efflux. Highly FLC(HR) C. glabrata was recovered more frequently than nonheteroresistant C. glabrata from hematogenously infected immunocompetent mice following treatment with high-dose fluconazole (45.8% versus 15%, P = 0.029). Phylogenetic analysis revealed some phenotypic clustering but also variations in FLC(HR) within clonal groups, suggesting both genetic and epigenetic determinants of heteroresistance. Collectively, these results establish heteroresistance to fluconazole as a graded phenotype associated with ABC transporter upregulation and fluconazole efflux. Heteroresistance may explain the propensity of C. glabrata for persistent infection and the emergence of breakthrough resistance to fluconazole.
  
  
  
  Heteroresistance refers to variability in the response to a drug within a clonal cell population. This phenomenon may have crucial importance for the way we look at antimicrobial resistance, as heteroresistant strains are not detected by standard laboratory susceptibility testing and may be associated with failure of antimicrobial therapy. We describe for the first time heteroresistance to fluconazole in C. glabrata, a finding that may explain the propensity of this pathogen to acquire resistance following exposure to fluconazole and to persist despite treatment. We found that, rather than being a binary all-or-none trait, heteroresistance was a continuously distributed phenotype associated with increased expression of genes that encode energy-dependent drug efflux transporters. Moreover, we show that heteroresistance is associated with failure of fluconazole to clear infection with C. glabrata Together, these findings provide an empirical framework for determining and quantifying heteroresistance in C. glabrata.
authors.affiliation: Infectious Diseases Unit, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel Sackler School of Medicine, Tel Aviv, Israel.
authors.forename: Ronen
authors.initials: R
authors.lastname: Ben-Ami
authors.affiliation: Infectious Diseases Unit, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel.
authors.forename: Offer
authors.initials: O
authors.lastname: Zimmerman
authors.affiliation: Infectious Diseases Unit, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel.
authors.forename: Talya
authors.initials: T
authors.lastname: Finn
authors.affiliation: Infectious Diseases Unit, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel.
authors.forename: Sharon
authors.initials: S
authors.lastname: Amit
authors.affiliation: Infectious Diseases Unit, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel.
authors.forename: Anna
authors.initials: A
authors.lastname: Novikov
authors.affiliation: Department of Molecular Microbiology and Biotechnology, George Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
authors.forename: Noa
authors.initials: N
authors.lastname: Wertheimer
authors.affiliation: Department of Molecular Microbiology and Biotechnology, George Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
authors.forename: Mor
authors.initials: M
authors.lastname: Lurie-Weinberger
authors.affiliation: Department of Molecular Microbiology and Biotechnology, George Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Jberman@post.tau.ac.il.
authors.forename: Judith
authors.initials: J
authors.lastname: Berman
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000935:Antifungal Agents / Q000378:metabolism / Q000494:pharmacology*; D001692:Biological Transport; D041221:Candida glabrata / Q000187:drug effects* / Q000302:isolation & purification; D002177:Candidiasis / Q000382:microbiology*; D004195:Disease Models, Animal; D025141:Drug Resistance, Fungal; D044127:Epigenesis, Genetic; D015725:Fluconazole / Q000378:metabolism / Q000494:pharmacology*; D014644:Genetic Variation; D005828:Genetics, Population; D006801:Humans; D051379:Mice; D010641:Phenotype
chemical_list: D018528:ATP-Binding Cassette Transporters; D000935:Antifungal Agents; D015725:Fluconazole