Resistance to azole antifungals continues to be a significant problem in the common fungal pathogen Candida albicans. Many of the molecular mechanisms of resistance have been defined with matched sets of susceptible and resistant clinical isolates from the same strain. Mechanisms that have been identified include alterations in the gene encoding the target enzyme ERG11 or overexpression of efflux pump genes including CDR1, CDR2, and MDR1. In the present study, a collection of unmatched clinical isolates of C. albicans was analyzed for the known molecular mechanisms of resistance by standard methods. The collection was assembled so that approximately half of the isolates were resistant to azole drugs. Extensive cross-resistance was observed for fluconazole, clotrimazole, itraconazole, and ketoconazole. Northern blotting analyses indicated that overexpression of CDR1 and CDR2 correlates with resistance, suggesting that the two genes may be coregulated. MDR1 overexpression was observed infrequently in some resistant isolates. Overexpression of FLU1, an efflux pump gene related to MDR1, did not correlate with resistance, nor did overexpression of ERG11. Limited analysis of the ERG11 gene sequence identified several point mutations in resistant isolates; these mutations have been described previously. Two of the most common point mutations in ERG11 associated with resistance, D116E and E266D, were tested by restriction fragment length polymorphism analysis of the isolates from this collection. The results indicated that the two mutations occur frequently in different isolates of C. albicans and are not reliably associated with resistance. These analyses emphasize the diversity of mechanisms that result in a phenotype of azole resistance. They suggest that the resistance mechanisms identified in matched sets of susceptible and resistant isolates are not sufficient to explain resistance in a collection of unmatched clinical isolates and that additional mechanisms have yet to be discovered.

The sterol pathway in Candida albicans is the target for several classes of antifungal drugs. Intermediates in the sterol pathway are involved in ergosterol synthesis, prenylation and dolichol synthesis. This study examines gene expression of the sterol pathway in response to lovastatin, an inhibitor of HMG-CoA reductase (Hmg1p), and fluconazole, an inhibitor of 14 alpha-lanosterol demethylase (Erg11p). Minimum inhibitory concentration (MIC) studies indicated that lovastatin acts synergistically with fluconazole in vitro. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) results indicated that genes in the early part of the sterol pathway, such as HMG1 and ERG20, did not alter expression in the presence of both lovastatin and fluconazole, whereas genes in the later part of the sterol pathway, such as ERG9 and ERG11, had increased expression in response to these drugs in mid-logarithmic growth. Genes involved in prenylation, such as RAM1 and RAM2, also respond to these drugs in mid-logarithmic growth, although another prenylation gene, CDC43, was not affected. After 24 h of growth, the relative expression of ERG20, ERG9, and ERG11 remained unchanged or increased in the presence of both drugs, while all other genes decreased in expression under all drug treatments.