Distinct patterns of gene expression associated with development of fluconazole resistance in serial candida albicans isolates from human immunodeficiency virus-infected patients with oropharyngeal candidiasis

Abstract

Resistance to fluconazole is becoming an increasing problem in the management of oropharyngeal candidiasis in human immunodeficiency virus-infected patients. Strains obtained from five patients developed decreased fluconazole susceptibility over time. DNA strain typing confirmed the high degree of relatedness among isolates from one patient and the variability among isolates from different patients. Expression of genes involved in development of fluconazole resistance was monitored in each isolate using probes specific for ERG11 (lanosterol 14alpha-demethylase), MDR1 (a major facilitator), and CDR (ATP-binding cassette or ABC transporter) genes. Increased expression of CDR genes was detected in the series of isolates from two patients. Isolates from one of the two patients also demonstrated increased ERG11 expression, whereas isolates from the other patient did not. Increased levels of MDR1 mRNA correlated with increased resistance in sequential isolates from another patient. Initial overexpression of MDR1 with subsequent overexpression of CDR genes and a final isolate again overexpressing MDR1 were detected in serial isolates from another patient. In another patient, overexpression of these genes was not detected despite an eightfold increase in fluconazole MIC. In this patient, sequence data of the ERG11 gene revealed no point mutations associated with decreased susceptibility. Five different patterns of gene expression were observed in isolates recovered from five patients who developed resistance. Therefore, these experiments demonstrate that a variety of mechanisms or combinations of mechanisms are associated with the development of fluconazole drug resistance. Additional studies are needed to estimate the frequency and clinical impact of these mechanisms of resistance.

FIG. 1

RFLP patterns generated by digestion with SfiI of genomic DNA from the different clinical C. albicans isolates recovered from successive episodes of OPC. The image is a composite of image scans from two different gels run under the same electrophoretic conditions.

FIG. 2

Northern blots of total RNA from clinical C. albicans isolates probed with ERG11, MDR1, and CDR probes. Hybridizations were performed as described in Materials and Methods. The bottom series show the amounts of 18S rRNA used to standardize signal levels according to lane loading parameters.

FIG. 3

Northern blot analysis of total RNA from clinical C. albicans isolates using probes specific for CDR1 and CDR2 (see Materials and Methods). The bottom series show the amounts of 18S rRNA used as a control for lane loading in order to normalize signal levels.