Next-generation sequencing identifies a novel CDR1 mutation in fluconazole-resistant Candida parapsilosis in Kuwait. Mechanism of Fluconazole resistance

Resistance to fluconazole (FLU), mediated by mutations in ERG11 and/or overexpression of efflux pumps MDR1 and CDR1, among clinical Candida parapsilosis isolates is increasing in many countries including Kuwait. This study performed next-generation sequencing (NGS) to identify novel mutations in target genes involved in conferring resistance to FLU in C. parapsilosis. One FLU-susceptible (Kw3259/15) and 2 FLU-resistant (Kw2006/15 and Kw1590/18) C. parapsilosis isolates with known ERG11 alterations were used. NGS was performed on llumina Hiseq X platform and raw paired-end reads were mapped to the C. parapsilosis CDC317 genome with Burrows wheeler aligner. Data were compressed using SAMtools and duplicated reads were removed by Picard tools. Variants calling was done by Genome Analysis and data were annotated with Ensemble Variants Effect Predictor and Candida Genome Database. Additionally, 40 C. parapsilosis isolates including 19 FLU-susceptible and 21 FLU-resistant isolates were screened by Sanger sequencing for mutation in ERG11 and CDR1 region.Compared to reference CDC317 genome, NGS small variants of 6003, 5177 and 1915 were identified in Kw3259/15, Kw2006/15 and Kw1590/18, respectively, with an average coverage of 175X. Kw3259/15 contained N283Y in ERG11, S208G & S304G in ERG6 and I1287V in CDR1 which represent genetic polymorphisms. Kw2006/15 contained resistance-conferring Y132F in addition to previously known mutation, R398I, in ERG11, S208G & S304G in ERG6 and I396V in MDR1. Kw1590/18 contained a novel mutation (N1132D) in CDR1 which has not been described previously in addition to S208G & S304G in ERG6. Furthermore, MAS-PCR of ERG11 and PCR sequencing of CDR1 identified Y132F and N1132D mutations in 6 of 21 and 11 of 21 fluconazole-resistant isolates, respectively. These mutations were absent in 20 fluconazole-susceptible isolates. Our data show that NGS identified all genetic alterations previously detected by PCR-sequencing of ERG11 in three C. parapsilosis isolates. The N1132D mutation in ERG11 was found in 11 of 21 (52%) fluconazole-resistant isolates only. N1132D mutation in CDR1 was detected in 11 FLU-resistant isolate and absent in all which has not been described previously. Further studies are warranted to validate the role of N1132D mutation in CDR1 in conferring resistance to FLU in C. parapsilosis.