Whole-genome sequencing of the first Candida auris clinical isolate from a patient with sepsis in Palestine

TL;DR

Scientists sequenced a drug-resistant yeast from a sepsis patient in Palestine and found genetic markers linked to resistance.

Contribution

The study provides the first whole-genome sequence of Candida auris from Palestine and identifies resistance-related SNPs.

Findings

The isolate C. auris Pal1 belongs to geographical Clade I.

Two SNPs (Erg11Y132F and Mrr1T647N) are associated with azole resistance.

Abstract

An isolate of the multidrug-resistant yeast Candida auris (Pal1) was identified in the urine of a patient with sepsis and sequenced using both Oxford Nanopore and Illumina sequencing technologies. Phylogenetic analysis placed C. auris Pal1 in the geographical Clade I, with distinct SNPs linked to azole resistance (Erg11Y132F and Mrr1T647N).

Figures

Average nucleotide identity (ANI) analysis showing genetic relatedness of the C. auris Pal1 isolate with the genomes of 22 C. auris isolates representative of the six geographical clades (B11205: SRX1939460, B11207: SRX1939463, L1537_2020: SRX10155110, B8441: SRX7522312, B11112: SRX1939497, B11808: SRX7155656, B14308: SRX7155771, B13463: SRX7155759, B11229: SRX1939486, B11230: SRX1939487, B11225: SRX1939487, B12037: SRX7155666, B12388: SRX4745454, B12177: SRX7155717, B12336: SRX4060912, B12098: SRX7155671, IFRC2087: SRX5786024, TMML616: SRX14462689, SRR24877249: SRX20641053, Isolate A: SRX21188005, Isolate B: SRX21188006, and Isolate C: SRX21188007). The scale bar indicates the mean number of nucleotide substitutions per site.

Full text

ANNOUNCEMENT

Candida auris has emerged as a significant healthcare-associated pathogen due to its multidrug-resistant nature, and it is listed in the WHO critical priority group due to its serious global health threat. In the first prevalence study of Candida infections in Palestine, many isolates of Candida spp. were not identified at the species level (1). As many of these isolates were resistant to fluconazole, we decided to specifically screen for C. auris. C. auris Pal1 was isolated from the urine of a patient with severe sepsis admitted at Ibn Sina Hospital (Jenin, Palestine) using CHROMagar Candida medium (CHROMagar, Paris, France). Pal1 isolate was initially identified as C. auris by the VITEK2 Compact system. A single colony of this isolate was inoculated into YPD (1% Yeast extract, 2% Peptone, 2% Dextrose, and 50 µg/mL uridine) medium and incubated at 30°C overnight. The cells were then harvested by centrifugation, and genomic DNA was extracted using the YeaStar kit (Zymo Research). Genomic DNA was first subjected to Sanger sequencing of the ITS2 region (2) (PQ578863) that confirmed this isolate as a C. auris species. Whole-genome sequencing was performed at Plasmidsaurus (Eugene, OR, USA) using both Oxford Nanopore Technology (ONT) long-read and Illumina NextSeq2000 short-read sequencing technologies. The Nanopore library was prepared using v14 library prep chemistry without fragmentation or size selection and sequenced on the PromethION P24 platform (Oxford Nanopore Technologies), equipped with R.10.4.1 flow cells. The Illumina library was made using the SeqWell ExpressPlex 96 library prep kit and sequenced on an Illumina NextSeq2000 (2 × 150 bp).

Illumina sequencing reads were filtered with Fastp version 0.23.2 (3) and those from Nanopore were filtered using Filtlong version 0.2.1 (https://github.com/rrwick/Filtlong) by keeping the best 90% of reads above 1,000 bp. Read quality was verified with FastQC version 0.12.1 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). After filtration, 2.25 million Illumina reads were retained, totaling 354 Mb (96% > Q20, 85% > Q30). For Nanopore reads, 286,335 reads (N50 ~11 kb) were retained, totaling 2.4 Gb. Nanopore reads were assembled with Flye version 2.9.5-b1801 (4) at an average coverage of 200×. The assembly was then polished with Polypolish version 0.6.0 (5). The final assembly resulted in eight contigs, corresponding to the seven chromosomes and the mitochondrial genome. Annotation was performed with Funannotate version 1.8.17 (https://github.com/nextgenusfs/funannotate/) using default parameters and the Candida albicans SC5314 model (http://www.candidagenome.org), which is the closest available species.

C. auris Pal1 relatedness to different isolates that are representative of the six known geographical clades (6) was evaluated using the average nucleotide identity method (7). This analysis showed that Pal1 is closely related to isolate B11207 (8) that belongs to the geographical Clade I (9) (Fig. 1). Pal1 isolate harbors different SNPs associated with azole resistance, including Erg11^Y132F^ and Mrr1^T647N^.

Average nucleotide identity (ANI) analysis showing genetic relatedness of the C. auris Pal1 isolate with the genomes of 22 C. auris isolates representative of the six geographical clades (B11205: SRX1939460, B11207: SRX1939463, L1537_2020: SRX10155110, B8441: SRX7522312, B11112: SRX1939497, B11808: SRX7155656, B14308: SRX7155771, B13463: SRX7155759, B11229: SRX1939486, B11230: SRX1939487, B11225: SRX1939487, B12037: SRX7155666, B12388: SRX4745454, B12177: SRX7155717, B12336: SRX4060912, B12098: SRX7155671, IFRC2087: SRX5786024, TMML616: SRX14462689, SRR24877249: SRX20641053, Isolate A: SRX21188005, Isolate B: SRX21188006, and Isolate C: SRX21188007). The scale bar indicates the mean number of nucleotide substitutions per site.