Research Article
Complete Genome Sequence of Candida mucifera from an Otitis Media Patient
https://doi.org/10.21203/rs.3.rs-4134268/v1
This work is licensed under a CC BY 4.0 License
published 02 Sep, 2024
You are reading this latest preprint version
We describe for the first time, a high-quality genome for a rare human yeast pathogen Candida mucifera, from a patient with chronic suppurative otitis media. Antifungal susceptibility profile was determined, and a potential mechanism responsible for intrinsically reduced azole susceptibility in Trichomonascus ciferrii-Candida mucifera species complex was proposed.
Candida mucifera
Fungal infection
Whole genome sequencing
Reduced azole susceptibility
Resistant mechanism
The yeast species Candida mucifera (also referred to as Blastobotrys mucifer in a recent study [1]) falls within the Trichomonascus ciferrii species complex as per the latest NCBI taxonomy database (https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=191334). This species complex is composed of ascomycetous heterothallic yeasts, formerly referred to as the Stephanoascus ciferrii complex, and currently comprises three species, namely Trichomonascus ciferrii, Candida allociferrii, and C. mucifera [2]. C. mucifera is a rare human fungal pathogen, which has only been reported in China in chronic suppurative otitis media cases [3]. Additionally, this species has been detected in environmental and animal sources (https://wi.knaw.nl/page/fungal_display/3954) [4]. Of note, all previously described animal and clinical strains exhibited notable reduced fluconazole susceptibility with minimal inhibitory concentration [MIC] values ranging from 32 to 128 mg/L) [3].
The isolate described in this study originated from an 8-year-old boy admitted to a hospital in South China (2018), suffering from chronic suppurative otitis media and repeated otorrhea (with yellow abscess) for two years. He had a moderate hearing loss but with no significant symptoms of pain, fever, or bleeding in ears. Specialized examination revealed large perforations in both eardrums, and hence tympanoplasty was carried out. Intraoperative tissue was sent for routine culture in the local mycology lab, and yeast colonies (isolate ID no. 17G1312) were observed which were initially identified as “T. ciferrii” by VITEK 2 Yeast Identification Card (bioMérieux, France).
On Sabouraud dextrose medium (Thermo, USA), the isolate exhibited pale yellow yeast colonies after incubation at 35°C for 72 hours. When cultured on Candida chromogenic medium (CHROMagar, France), the colonies appeared blue to green in color (Supplementary Figure 1). Antifungal susceptibility testing was performed on the isolate using YeastOne Sensititre (Thermo, USA). After 48 hours of incubation, the MICs of the tested isolate were as follows: fluconazole 16 mg/L, itraconazole 0.25 mg/L, voriconazole 0.12 mg/L, posaconazole 0.25 mg/L, micafungin 0.03 mg/L, anidulafungin 0.03 mg/L, caspofungin 0.03 mg/L, flucytosine 0.5 mg/L, and amphotericin B 1 mg/L.
The internal transcribed spacer (ITS) region of isolate 17G1312 was amplified and sequenced using ABI 3730xl platform (Thermo, USA). The strain’s sequence was 100% (576/576) identical to that of C. mucifera strain 80408 (GenBank accession no. OR761613.1) and 99.5% (555/558) to C. mucifera type strain CBS 7409T (GenBank accession no. NR_130678.1), but only 95.4% (554/581) similarity to T. ciferrii type strain CBS 5295T (GenBank accession no. NR_111160.1). The maximum likelihood tree constructed based on ITS region sequences, further support assigning the isolate 17G1312 as C. mucifera (Supplementary Figure 2).
As there was no publicly available genome information for C. mucifera, we conducted whole-genome sequencing of the isolate 17G1312 using short-reads on the Illumina MiSeq PE150 (Illumina, USA) and long-reads on the PacBio Sequel II (PacBio, USA). The assembly process involved converting the BAM file to a FASTA file using SAMtools v1.9 [5], assembly using Flye v2.9.3 [6], and polishing the assembly results using Pilon v1.23, with short reads filtered by fastp v0.23.4 [7, 8]. The genome completeness was assessed using BUSCO v5.2.2 and the saccharomycetes_odb10 database [9], revealing an integrity of 96.2% (single-copy: 95.5%, duplicated: 0.7%). The genome of this new strain comprises 8 scaffolds with a total length of 16,402,853 bp (15.6 Mb). The N50 is 2,475,350 bp, and the GC content is 44.9%.
The genome of isolate 17G1312 (GenBank assembly accession: GCA_036871495.1) was compared with two genomes of its closely-related species T. ciferrii (strains NRRL Y-10943 [GenBank assembly accession: GCA_030573635.1], and CBS 4856 [GenBank assembly accession: GCA_008704605.1]) using fastANI v1.33 [10], and the average nucleotide identity (ANI) between isolate 17G1312 and two T. ciferrii strains, was only 80.6%. Gene annotation results of T. ciferrii strain CBS 4856 were used to train a new de novo prediction model for AUGUSTUS v3.3.2 and then utilized for structural annotation of our C. mucifera genome [11], and a total of 6533 genes were predicted.
Due to the “intrinsically” reduced fluconazole susceptibility in C. mucifera and other species of the T. ciferrii complex [3], we conducted an mutation analysis of the azole target Erg11p. We observed that position 499 of Erg11p amino acid sequence in C. mucifera isolate 17G1312 (GenBank accession no. PP204302.1), and the homologous position (position 501) in T. ciferrii (GenBank accession no. PP213130.1), had a valine (V) residue. In contrast to this finding, in wild-type azole-susceptible Candida albicans strains (e.g. strain no. SC5314, GenBank accession no. XP_716761.1), the homologous position of Erg11p at position 488 has an isoleucine (I) residue. Of note, the V488I amino acid substitution in C. albicans has been reported to be responsible for fluconazole resistance [12]. Consequently, we hypothesize that the V499I substitution in C. mucifera Erg11p (corresponding to V501I substitution in T. ciferrii) introduced reduced fluconazole susceptibility within this species complex.
In summary, we described (and deposited) the complete genome of a rare human yeast pathogen, C. mucifera. In addition, our analysis provides a potential mechanism explanation for “intrinsically” reduced azole susceptibility in Trichomonascus ciferrii-Candida mucifera species complex. The methodology used, as well as the availability of data and stock cultures, adheres to the quality assurance checklist of MycopathologiaGENOMES [13].
Data availability
The isolate 17G1312 has been deposited at China General Microbiological Culture Collection Center under the accession number CGMCC2.8257. The ERG11, ITS, D1/D2 region of the isolate 17G1312 have been released in GenBank (accession nos. PP204302.1, PP204071.1, PP230918.1). All sequencing data have been deposited in the NCBI database (GenBank accession nos. CP145483.1-CP145490.1, assembly accession no. GCA_036871495.1, Biosample no. SAMN39602682, BioProject no. PRJNA1068766).
Funding
This work was financially supported by the National Key Research and Development Program of China (2022YFC2303002), and the National High Level Hospital Clinical Research Funding (2022-PUMCH-C-052).
Author Contributions
M. X. and Y.-C. X. conceived and planned the experiments. R.-C. D. and M. X. performed the genomic analysis. J. G., Y.-T. N., W. Z., X.-F. C. and G. Z. collected the isolate and clinical data. R.-C. D., Y.-T. N. and M. X. contributed to the first draft of the manuscript. J. G., T. K., and Y.-C. X. contributed to the review and editing.
Ethics approval
This study was approved by the Human Research Ethics Committee of Peking Union Medical College Hospital (No. S-263).
Conflict of interest
The authors have no relevant financial or non-financial interests to disclose.
published 02 Sep, 2024
Editorial decision: Minor revisions
17 Jun, 2024
Reviewers agreed at journal
28 May, 2024
Reviewers invited by journal
24 Apr, 2024
Editor invited by journal
02 Apr, 2024
Editor assigned by journal
01 Apr, 2024
First submitted to journal
31 Mar, 2024
You are reading this latest preprint version