Five azole-susceptible Candida glabrata isolates obtained before 1975 became resistant to fluconazole, itraconazole, and voriconazole within 4 days of in vitro fluconazole exposure. This cross-resistance was stable for at least 4 months after removal of fluconazole and was associated with increased CgCDR1 and CgCDR2 expression.Candida glabrata has become one of the most common causes of Candida bloodstream infections (BSI) worldwide, now accounting for 7.5 to 18.3% of cases in different countries (20). The emergence of C. glabrata is of concern because of recent reports that up to 18% of incident bloodstream isolates are resistant to fluconazole (6,8,20,21). Moreover, resistance to azole antifungal agents, including fluconazole and itraconazole, can emerge rapidly when patients with C. glabrata infection are treated with these drugs (10, 25). Resistance can also emerge when patients, such as hematopoietic stem cell transplant recipients, are given prolonged periods of azole prophylaxis (2). In these cases, both the acquisition of new strains and increased resistance in existing strains have been described (2,10,15). Multiple mechanisms of resistance have been reported, including increased expression of the ABC transporter genes CgCDR1 and CgCDR2 (PDH1), which result in decreased accumulation of intracellular fluconazole (2,11,17,23), and increased expression of the CgERG11 gene encoding the fluconazole target, 14␣-lanosterol demethylase (15,22). Recent studies have described up-regulation of these genes in C. glabrata isolates upon fluconazole exposure (2, 9, 15, 22), including some for which the previous fluconazole exposure history was unknown.We therefore sought to determine whether the rapid acquisition of fluconazole resistance by C. glabrata is an innate characteristic of this organism or whether multiple exposures to azoles over time are required. To ensure that the isolates tested had not been previously exposed to azole antifungal agents, we studied susceptible isolates of C. glabrata obtained between 1917 and 1975, a time period before the first introduction of oral or parenteral formulations of azole drugs. These C. glabrata isolates were then examined for their capacity to become resistant to azoles after in vitro exposure to fluconazole and for the length of time required for isolates to become resistant. In addition, we studied the molecular mechanisms associated with the acquired resistance and the stability over time of such resistance after the removal of fluconazole.Isolates. Three clinical C. glabrata isolates, CBS 138, CBS 860, and CBS 4692, which were isolated in 1917, 1935, and 1960, respectively, were obtained from the Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands. Two other clinical C. glabrata isolates, 73/124 and 75/015, isolated in 1973 and 1975, were obtained from the University of Aberdeen, Aberdeen, United Kingdom. Species identification was confirmed by using species-specific Candida DNA probes in a PCR-enzyme immunoassay as described previously (7).Antifunga...