Whole-Genome and Targeted-Amplicon Sequencing of Fluconazole-Susceptible and -Resistant Candida parapsilosis Isolates from Kuwait Reveals a Previously Undescribed N1132D Polymorphism in CDR1

LETTER

Candida parapsilosis is an opportunistic yeast pathogen causing invasive candidiasis in susceptible patients, surpassing even Candida albicans in frequency in some centers/geographical areas, and causing ∼35% of all candidemia cases in neonates (1–5). Fluconazole resistance among clinical C. parapsilosis isolates is an emerging problem and typically involves Y132F mutation in ERG11, occurring in 31% to 100% of azole-resistant isolates (5, 6). Additional mechanisms include overexpression of efflux pumps CDR1 and MDR1, possibly due to activating mutations in their transcriptional regulators, TAC1 and MRR1, respectively. However, missense mutations in transcriptional regulators TAC1 and MRR1 do not always correlate with overexpression of CDR1 and MDR1 (7, 8). This study performed next-generation sequencing (NGS) to identify novel mutations in target genes involved in conferring resistance to fluconazole in clinical C. parapsilosis isolates in Kuwait.

Initially, one fluconazole-susceptible (Kw3259/15) and two fluconazole-resistant (Kw2006/15 and Kw1590/18) C. parapsilosis isolates with known ERG11 alterations (6) were subjected to NGS by using the Illumina HiSeq platform, paired-end reads were mapped to the C. parapsilosis CDC317 genome with Burrows wheeler aligner, and data were annotated with Ensemble Variants Effect Predictor and the Candida Genome Database (methodological details are provided in the supplemental material). Subsequently, clinical C. parapsilosis isolates (n = 43) were tested for susceptibility against fluconazole by Etest and for mutations in target genes identified by NGS by PCR-sequencing/multiplex-allele-specific PCR using gene-specific primers, as described previously for other gene targets (6, 9, 10).

The genomic analysis metrics, including coverage depth, percentage of mapped reads, and total number of single-nucleotide variants detected compared with those of the reference C. parapsilosis CDC317 genome, for each of the three isolates are presented in Table S1 in the supplemental material. Compared to the reference CDC317 genome, NGS (average coverage, 175×) identified mutations N283Y in ERG11, S208G and S304G in ERG6, and I1287V in CDR1 in isolate Kw3259/15, which likely represent genetic polymorphisms. Isolate Kw2006/15 contained resistance-conferring Y132F in addition to another previously known mutation (R398I) in ERG11, S208G and S304G in ERG6, and I396V in MDR1. Isolate Kw1590/18 contained a novel mutation (N1132D) in CDR1, which was not described previously, in addition to S208G and S304G in ERG6. No mutations were detected in other relevant genes, such as MRR1 or TAC1. To confirm the association of the N1132D mutation in CDR1 with fluconazole resistance, 43 clinical C. parapsilosis isolates were tested. Twenty-one of these C. parapsilosis isolates were susceptible to fluconazole (MIC range, 0.064 to 3.0 μg/ml) and contained wild-type sequences of ERG11 and CDR1, and 22 isolates were resistant to fluconazole. Of the fluconazole-resistant isolates, 4 (MIC range, 8.0 to 48.0 μg/ml) contained wild-type ERG11 and CDR1 sequences, 6 (MIC range, 8.0 to 256.0 μg/ml) contained the Y132F mutation in ERG11 only, and 12 (MIC range, 12.0 to 256.0 μg/ml) contained the N1132D mutation in CDR1 only (Table 1).

TABLE 1.

Susceptibility to fluconazole and mutations detected in ERG11 and CDR1 among 43 clinical C. parapsilosis isolates from Kuwait

No. of isolates	Susceptibility to fluconazole	MIC range (μg/ml)	Mutation detected in:
			ERG11a	CDR1b
21	Susceptible	0.064–3.0	None	Nonec
4	Resistant	8.0–48.0	None	None
12	Resistant	12.0–256.0	None	N1132D
6	Resistant	8.0–256.0	Y132F	None

^aERG11 gene fragment of 323 bp around codon Y132 was sequenced for most isolates.

^bCDR1 gene fragment of 333 bp around codon N1132 was sequenced for all isolates.

^cOne susceptible isolate contained the T1184P mutation.

Our data show that NGS identified all genetic alterations previously detected by PCR sequencing of ERG11 in three C. parapsilosis isolates. NGS data additionally identified a novel (N1132D) mutation in CDR1 in one fluconazole-resistant isolate that was not described previously. Further screening of 43 clinical isolates showed that 12 (75%) of 16 fluconazole-resistant isolates lacking the Y132F mutation in ERG11 contained the N1132D mutation in CDR1, implicating this alteration as a possible mechanism conferring resistance to fluconazole in C. parapsilosis. Sequence comparison of C. parapsilosis CDR1 with corresponding sequences from several other Candida spp. (see Fig. S1 in the supplemental material) showed that aspartate is naturally found at the corresponding position in CDR1 in fluconazole-susceptible reference strains of several Candida and yeast species. We also carried out multiple sequence alignment of the CDR1 gene fragment from all of the C. parapsilosis isolates from Kuwait analyzed in this study. The data showed that isolates with the N1132D mutation were genetically distinct from other fluconazole-susceptible and -resistant C. parapsilosis isolates (see Fig. S2 in the supplemental material). Taken together, our data show that the N1132D mutation in CDR1 detected in some C. parapsilosis isolates in Kuwait represents a polymorphism that has not been described previously.

Data availability.

The CDR1 sequence data reported in this study were submitted to GenBank/ENA/DDBJ databases under accession no. LR814050 to LR814063. The complete genome sequences of C. parapsilosis isolates Kw3259/15, Kw2006/15, and Kw1590/18 have been deposited in NCBI Sequence Read Archive (SRA) under accession no. PRJNA661583.