S8.5d Cryptococcus neoformans and Cryptococcus gattii clinical isolates from Colombia develop heteroresistance to fluconazole at high concentrations

Abstract

S8.5 Genotyping of Cryptococcus neoformans and C. gattii, September 23, 2022, 3:00 PM - 4:30 PMIntroductionCryptococcosis is a worldwide mycosis caused by Cryptococcus neoformans and Cryptococcus gattii. Although resistance to antifungals is infrequent, isolates with decreased susceptibility to fluconazole have been reported globally, including Colombia, which may be due to 1) heteroresistance, defined as the ability to adapt to increasing concentrations of this azolic antifungal, and 2) point mutations in the EGR11 gene encoding the fluconazole target enzyme, lanosterol 14-α-demethylase.ObjectiveTo determine the development of heteroresistance to fluconazole in C. neoformans and C. gattii clinical isolates from Colombia and to amplify and sequence the ERG11 gene of the isolates to seek for mutations that might characterize resistant or heteroresistant phenotypes.MethodsThe minimum inhibitory concentration (MIC) to fluconazole was determined in 31 and 24 isolates of C. neoformans and C. gattii, respectively, using broth microdilution. Heteroresistance was evaluated by plating each isolate on YPD agar that contained fluconazole at concentrations equal to the MIC of each isolate. Heteroresistant colonies were then replated at increasing concentrations of fluconazole, as high as 128 μg/ml.ResultsAll isolates were susceptible to fluconazole with MICs of 1 μg/ml (n = 3), 2 μg/ml (n = 6), 4 μg/ml (n = 17), 8 μg/ml (n = 23), 16 μg/ml (n = 5), and 32 μg/ml (n = 1). However, all isolates developed heteroresistant colonies, with increments in the MIC from 2 to 6 dilutions. Notably, 5 (16.1%) isolates of C. neoformans and 8 (33.3%) of C. gattii, grew up to 64 μg/ml of fluconazole, which is the MIC that defines resistance to this azole, and 1 (3,2%) isolate of C. neoformans and 4 (16.7%) of C. gattii grew up to 128 μg/ml of fluconazole. Currently, the ERG11 gene is being amplified for further sequencing. Conclusion: clinical isolates of C. neoformans and C. gattii that develop heteroresistance to fluconazole in high concentrations circulate in Colombia, which is important since this characteristic contributes to the relapse of cryptococcosis during therapy with this triazole.