Draft genome of a commonly misdiagnosed multidrug resistant pathogen Candida auris

Abstract

Background: Candida auris is a multidrug resistant, emerging agent of fungemia in humans. Its actual global distribution remains obscure as the current commercial methods of clinical diagnosis misidentify it as C. haemulonii. Here we report the first draft genome of C. auris to explore the genomic basis of virulence and unique differences that could be employed for differential diagnosis.

Results: More than 99.5 % of the C. auris genomic reads did not align to the current whole (or draft) genome sequences of Candida albicans, Candida lusitaniae, Candida glabrata and Saccharomyces cerevisiae; thereby indicating its divergence from the active Candida clade. The genome spans around 12.49 Mb with 8527 predicted genes. Functional annotation revealed that among the sequenced Candida species, it is closest to the hemiascomycete species Clavispora lusitaniae. Comparison with the well-studied species Candida albicans showed that it shares significant virulence attributes with other pathogenic Candida species such as oligopeptide transporters, mannosyl transfersases, secreted proteases and genes involved in biofilm formation. We also identified a plethora of transporters belonging to the ABC and major facilitator superfamily along with known MDR transcription factors which explained its high tolerance to antifungal drugs.

Conclusions: Our study emphasizes an urgent need for accurate fungal screening methods such as PCR and electrophoretic karyotyping to ensure proper management of fungemia. Our work highlights the potential genetic mechanisms involved in virulence and pathogenicity of an important emerging human pathogen namely C. auris. Owing to its diversity at the genomic scale; we expect the genome sequence to be a useful resource to map species specific differences that will help develop accurate diagnostic markers and better drug targets.

Fig. 1

In vitro antifungal susceptibility testing of clinical isolates of Candida: All the isolates were identified as C. haemulonii by Vitek2. Susceptibility testing was done by broth microdilution method at 37 °C for 48 h as mentioned in materials and methods. a Comparison of MIC₅₀ values of all isolates for Fcz indicates all clinical isolates have MIC range of 32–64 μg/ml. b Comparison of MIC₅₀ values of all isolates for AmB shows that all clinical isolates are resistant to AmB. Candida isolate Ci 6684 has the highest MIC₅₀ value of > = 16 μg/ml

Fig. 2

ITS phylogeny and electrophoretic karyotyping reveals that Ci 6684 belongs to C. auris clade. a Phylogenetic tree based on the partial sequence of 18 s rRNA, ITS1, 5.8 s rRNA complete sequence, ITS2 and 28 s rRNA partial sequences of species belonging to the C. haemulonii complex and related Candida species. Our isolate Ci 6684 is related to C. auris as it falls in the same clade. b Electrophoretic karyotyping shows 5 bands similar to those reported for C. auris by Oh J B et al., where they reported genotypic relatedness between C. haemulonii and closely related species. C. albicans (lane 1) shows four distinct bands. Corresponding C. auris lane (lane 2) shows five bands

Fig. 3

Evolutionary position of Candida auris isolate 6684 in the pathogenic fungal tree. a Phylogenetic tree based on orthologs of 95 conserved proteins from 11 pathogenic species under the phylum Ascomycota. b Amino acid substitution matrix by maximum likelihood estimation. c Tajima’s neutrality test indicates low level of polymorphism in the house keeping machinery wherein m = number of sequences, n = total number of sites, S = Number of segregating sites, ps = S/n, T = ps/a1, p = nucleotide diversity, and D is the Tajima test statistic. d Tajima’s relative rate test however indicates that these species have evolved at different rates. All evolutionary analyses were conducted in MEGA6

Fig. 4

Candida auris has a highly divergent genome. a, b, c, d Synonymous Codon Usage distribution of Candida auris isolate 6684 with respect to C. albicans (SC-5314 (a) and WO-1 (b)), C. lusitaniae ATCC 42720 and C. glabrata CBS 138. These plots were generated by correspondence analysis and depict the variability in the sum of synonymous codon usage and amino acid usage. These graphs depict the codon usage bias relating it to the evolution of pathogenic fungus. a, b, c, d Whole (or draft) genome dot plot alignment showing genomic synteny of Candida auris isolate 6684 with respect to other well known pathogenic Candida species. The y-axis is the largest scaffold of Candida auris 6684 and the x-axis is the largest chromosome (or scaffold) of the corresponding genome being compared

Fig. 5

Summary of functional annotation of Candida auris genome. a Annotation results against RefSeq fungal protein database shows 5.1 % (429 out of 8358 protein coding genes) were annotated functionally with predicted names. The rest of the genome remains uncharacterized. b C. auris 6684 has highest number of orthologs in C. lusitaniae ATCC 42720. However most of them were annotated as hypothetical

Fig. 6

Functional annotation of C. auris genome. a, b, c Represents Level 2 GO terms for the three main domains. The most abundant terms in Biological process (a) is cellular process, metabolic process and single-organism process and in Molecular functions (b), binding, catalytic activity and transporter activity. The common cellular component (c) termed are the cell, organelle and membrane. d Distribution of various enzymes into the six enzyme classes according to E.C numbers. e, f represents gene families predicted in C. auris that are orthologous to C. albicans

Fig. 7

Identification of mating gene MF α and mating loci in C. auris. a The amino acid sequence of the MF α gene. Regions encoding the mature α pheromone peptide is shown in blue color. Possible Kex2 cleavage sites are shown in red colour. Additional DA residues present in three of the peptides have been underlined. b Scaffold 18 shows conservation of non sex genes of MTL locus, however, MF α gene is present in a different scaffold

Fig. 8

PCR based diagnostic method to differentiate between C. auris and C. haemulonii. a Primers based on C. auris 6684 MFα gene gives a specific amplicon at 400 bp. b Amplification was not seen in the case of C. haemulonii 8176. However, four clinical isolates identified to be C. haemulonii by Manipal Hospital, showed a band at 400 bp. c The PFGE profile of these four clinical isolates was similar to that of C. auris 6684