P008 Molecular mechanisms associated with fluconazole resistance and genetic diversity in clinical Candida krusei isolates from North India

Abstract

Objectives Candida krusei accounts for 2.8% of invasive candidiasis worldwide. Fluconazole resistance and its underlying mechanism in clinical isolates of C. krusei (n = 137) collected from eight hospitals in India were investigated. Also, genetic diversity of C. krusei strains among different hospitals was studied through short tandem repeat (STR) genotyping. Material and Method All the isolates were identified by MALDI-TOF MS. Antifungal susceptibility test was done by using broth microdilution method (CLSI-M27). To evaluate the genetic relatedness among the strains, STR typing was done by using 9 STR markers. To understand the fluconazole-resistant mechanisms in C. krusei, known fluconazole resistance mechanisms such as alterations in target enzyme ERG11 and drug transporters ABC1, and ABC2 were investigated in 35 C. krusei isolates [18 fluconazole-susceptible (FLU-S), and 17 fluconazole-susceptible dose-dependent (FLU-SDD)]. Furthermore, transcriptomics of one FLU-SDD (MIC 32 mg/L) and one FLU-S (MIC 4 mg/l) isolate was performed. Results Majority (77%) of C. krusei isolates were from bloodstream infections. Notably, 70% of candidemia cases occurred in neonatal intensive care units (NICUs). Remarkably, 81% (n = 110) were detected as fluconazole-SDD (MIC 16-32 mg/l), and the remaining 19% were FLU-S (MIC ≤ 8 mg/l). Marked genetic diversity with 51 diverse STR types was noticed among the 106 isolates. Interestingly, two ongoing candidemia outbreaks were observed in two geographically separated hospitals both representing NICU isolates. In addition, a large cluster containing isolates from six different hospitals was observed. ERG11 mutation analysis revealed that it did not harbor any mutation contributing to the flu-resistance. Overexpression of the ABC1 gene in 11 FLU-SDD isolates out of 17 as compared to FLU-S isolates was noted. However, no alteration was observed in the expression of ERG11 and ABC2 in both groups. Transcriptomics analysis revealed a significant number of differentially regulated genes were distributed in various gene-ontology terms including transport (10 genes), mitogen-activated protein kinase (MAPK) signaling (8 genes, MSG5, PTP3, STE50, BNR1, OPY2, STE5, SKN7, and RLM1), ergosterol biosynthesis (3 genes, ERG24, ERG25, and ERG26) and transcription factors (7 genes). In addition to the up-regulation of ergosterol pathway genes, overexpression of key transcriptional regulator of ergosterol biosynthesis genes UPC2 was observed in FLU-SDD isolates as compared with susceptible. Additionally, FLU-SDD isolate showed 2-fold increased expression of PDR12, plasma membrane ATP-binding cassette (ABC) transporter. Next, ICL1 (Isocitrate Lyase), a major glyoxylate-synthesizing enzyme was found to be 5-fold down-regulated in FLU SDD isolate compared to susceptible. The loss of ICL1 alters the expression of the FKS1, ERG11, and CDR2 genes in C. albicans. Taken together, the increased expression of PDR12 and altered MAPK singling network may partially account for the FLU resistance in C. krusei FLU-SDD isolate. Conclusion Candida krusei isolates among different hospitals showed large genetic diversity (54 different genotypes). Also, the presence of C. krusei clonal strains in six different hospitals suggests possible introduction from a widespread environmental source and human-to-human transmission. In comparison to other Candida species, the resistant mechanism in C. krusei seems to be more complex. Therefore, an in-depth study of other resistance mechanism pathways in C. krusei is further warranted.