First two fungemia cases caused by Candida haemulonii var. vulnera in China with emerged antifungal resistance

Candida haemulonii var. vulnera is a rare variant of C. haemulonii, which has been previously reported to cause human infections. Owing to the close kinship between C. haemulonii sensu stricto and C. haemulonii var. vulnera, accurate identification of C. haemulonii var. vulnera relied on DNA sequencing assay targeting, for example, rDNA internal transcribed spacer (ITS) region. In this work, two strains of C. haemulonii var. vulnera were collected from the China Hospital Invasive Fungal Surveillance Net (CHIF-NET). The identification capacity of three matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and VITEK 2 YST ID biochemical methods were evaluated against ITS sequencing. In addition, antifungal susceptibility testing was performed using Sensititre YeastOne. Moreover, we comprehensively screened drug-resistant related genes by whole-genome sequencing. The two strains were not correctly identified to species variant level using MALDI-TOF MS and YST ID cards. Both strains were resistant to amphotericin B (minimum inhibitory concentration [MIC] > 2 μg/ml). Moreover, strain F4564 and F4584 exhibited high MIC to fluconazole (>256 μg/ml) and 5-flucytosine (>64 μg/ml), respectively, which were supposed to result from key amino acid substitutions Y132F and G307A in Erg11p and V58fs and G60K substitutions in Fur1p. The rare species C. haemulonii var. vulnera has emerged in China, and such drug-resistant fungal species that can cause invasive diseases require further close attention.

Candida haemulonii var. vulnera is a rare variant of C. haemulonii, which has been previously reported to cause human infections. Owing to the close kinship between C. haemulonii sensu stricto and C. haemulonii var. vulnera, accurate identification of C. haemulonii var. vulnera relied on DNA sequencing assay targeting, for example, rDNA internal transcribed spacer (ITS) region. In this work, two strains of C. haemulonii var. vulnera were collected from the China Hospital Invasive Fungal Surveillance Net (CHIF-NET). The identification capacity of three matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and VITEK 2 YST ID biochemical methods were evaluated against ITS sequencing. In addition, antifungal susceptibility testing was performed using Sensititre YeastOne. Moreover, we comprehensively screened drug-resistant related genes by whole-genome sequencing. The two strains were not correctly identified to species variant level using MALDI-TOF MS and YST ID cards. Both strains were resistant to amphotericin B (minimum inhibitory concentration [MIC] > 2 µg/ml). Moreover, strain F4564 and F4584 exhibited high MIC to fluconazole (>256 µg/ml) and 5-flucytosine (>64 µg/ml), respectively, which were supposed to result from key amino acid substitutions Y132F and G307A Introduction Candida haemulonii var. vulnera belongs to the C. haemulonii species complex, along with C. haemulonii sensu stricto and C. duobushaemulonii (Gade et al., 2020;Rodrigues et al., 2021;Ramos et al., 2022). Generally, C. haemulonii species complex isolates display high multidrug-resistant rates and transmission properties, which have attracted increased attention (Gade et al., 2020).
In this study, we reported two fungemia cases caused by Candida haemulonii var. vulnera found from China Hospital Invasive Fungal Surveillance Net (CHIF-NET) study. Clinical characters, identification capacity of Vitek YST card and three matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems, isolates' antifungal susceptibility phenotypes, and potential resistant mechanisms were illustrated. To our best knowledge, these were the first Candida haemulonii var. vulnera infection cases reported in China, including the first 5-flucytocine resistant strain discovered globally.

Ethics statement
This study was approved by the Human Research Ethics Committee of the Peking Union Medical College Hospital (No. S-263). Written informed consent was obtained from all patients who participated in this study, which aimed to culture and study the isolates obtained from the patients.

Microorganisms and identification
From 2010 to 2017 (Table 1), two C. haemulonii var. vulnera isolates were collected from different hospitals in two provinces from the CHIF-NET study. The colony  Phenotypic characteristics of two C. haemulonii var. vulnera isolates on Sabouraud dextrose agar. The plate contains colonies with typical appearance of circular, smooth, and moist (A,B). Microscopic examination showed budding yeast cells with Gram's staining (C,D). Scale bar = 10 µm (C).
morphology of two strains is smooth, moist, and circular, which is the typical appearance of Candida species. Gramstained microscopy showed budding yeast cells (Figure 1). The isolates were identified using Autof MS 1000 (Autobio, Zhengzhou, China), Smart MS (DL, Zhuhai, China), and Vitek MS (bioMérieux, Marcy l'Étoile, France) MALDI-TOF MS systems, in addition to Vitek 2 YST ID Card using VITEK 2 (9.02 version, bioMérieux, Marcy-l'Etoile, France) following the manufacturer's instructions. Primers ITS1 and ITS4 (Hou et al., 2016) were used for ITS amplification and sequencing, and Sanger sequencing was performed using ABI 3730XL DNA analyzer (Thermo Fisher Scientific, Cleveland, OH, USA). A phylogenetic tree of the ITS sequences was constructed using Mega X based on 1000 bootstrap replicates using the maximum likelihood method (Kumar et al., 2018).

DNA extraction and whole-genome sequencing
The whole genomic DNA of C. haemulonii var. vulnera was extracted as previously reported (Huang et al., 2022). The 350-bp DNA library was constructed using NEBNext R Ultra TM , following the manufacturer's instructions. Library integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies). Sequencing was performed on an Illumina NovaSeq using the PE150 strategy (Beijing Novogene Bioinformatics Technology Co., Ltd.). The Illumina reads generated in this study were obtained from the National Center for Biotechnology Information (NCBI) under BioProject PRJNA890168.

Antifungal susceptibility testing
Antifungal susceptibility testing was performed using Sensititre YeastOne YO10 methodology (Thermo Scientific, Cleveland, OH, USA). Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019 were used as quality controls. As clinical breakpoints or epidemiological cutoff values against C. haemulonii var. vulnera have not been established, we used interpretation criteria for Candida species referring to CLSI M27-S3 guidelines (CLSI, 2008), including MIC value of ≥32 µg/mL as resistance to fluconazole ≥32 µg/mL as resistance to 5-flucytosine. In addition, MIC of ≥2 µg/mL was used for interpreting "resistance" to amphotericin B (Pfaller et al., 2012).

Review of Candida haemulonii var. vulnera cases reported
A literature review of previously reported C. haemulonii var. vulnera infections was done. A literature search was performed on 28 August 2022, using the following three databases: PubMed 1 , Web of Science 2 , and Embase 3 . The terms "Candida haemulonii var. vulnera" were entered in the category of "Title/Abstract" in the PubMed Advanced Search Builder, and "TS (Candida haemulonii var. vulnera)" was entered into the Web of Science database. The search in Embase was conducted in the advanced search area, including the terms "Candida haemulonii var. vulnera': ab,ti.". All hits were further screened manually to find out all infection cases with antifungal susceptibility reports.

Isolate information
The first case was an 88-year-old male patient who was admitted to the intensive care unit (ICU) with clinical diagnosis of pulmonary infection. F4564 was isolated from peripheral blood of this patient and initially misidentified as "Candida krusei" using CHROMagar chromogenic medium at local laboratory ( Table 1). The second case was a 12-year-old boy who was admitted to the general surgical ward, and clinical diagnosis was abdominal infection. F4584 was isolated from peripheral blood of this patient and identified as "Candida spp." using CHROMagar chromogenic medium initially ( Table 1).
Identification of Candida. haemulonii var. vulnera using ITS sequencing, MALDI-TOF MS, and Vitek 2 The ITS sequences of the isolates exhibited 100% identity with the corresponding ITS sequences of the reference C. haemulonii var. vulnera CBS12439 T isolates (GenBank accession number: JX459686.1). Furthermore, both clinical isolates were identified as C. haemulonii by the Autof MS 1000 and Vitek MS, but with "no identification" results by Smart MS. While using the Vitek 2 Compact system, one strain was identified with low discrimination and the other was identified as C. haemulonii (score = 94%) ( Table 1). In phylogenetic tree generated by C. haemulonii complex, C. auris,

FIGURE 2
Phylogenetic tree for rDNA ITS region of previously reported Candida haemulonii, C. duobushaemulonii, C. pseudohaemulonii, C. auris, and C. haemulonii var. vulnera, including two C. haemulonii var. vulnera clinical strains identified in this study. Phylogenetic tree was constructed using Mega X with 1000 bootstrap replicates based on the maximum likelihood method.
Frontiers in Microbiology 04 frontiersin.org and C. pseudohaemulonii ITS sequences, F4564 and F4584 were in the branch of C. haemulonii var. vulnera (Figure 2).

Genome analysis
We sequenced genomes of F4564 and F4584, and their total genome sizes were 13.51 Mb and 13.26 Mb, respectively, with an average GC content of 45% (N50 was 346,464 bp and 283,647 bp, and a number of assembled contigs were 270 and 262, comprising 95.7% and 95.6% of data according to BUSCO analysis, respectively).

Antifungal susceptibility
MIC values obtained for the nine antifungal agents against C. haemulonii var. vulnera isolates are shown in Table 1. F4564 was resistant to fluconazole with an MIC of >256 µg/ml. In addition, F4564 was classified as susceptible dose-dependent to voriconazole (2 µg/ml) and itraconazole (0.25 µg/ml). F4684 was resistant to 5-flucytosine (>64 µg/ml) and susceptible dose-dependent to itraconazole. In addition, both isolates had high MIC values of 4 µg/ml for amphotericin B, while neither strain was non-susceptible to posaconazole, caspofungin, anidulafungin, and micafungin (Table 1).

Potential genomic variations contributed to antifungal resistance
Compared with the reference genome K1, which is from an isolate susceptible to all antifungals except for amphotericin B, we found that F4564 has two previously reported key amino acid substitutions (Y132F and G307A) in Erg11p. Interestingly, we discovered two novel mutations in Fur1p (V58fs and G60K) in strain F4584 with high 5-flucytosine MIC. However, we did not find any key variation in sterol metabolismrelated genes like ERG2, ERG3, and ERG6 that may result in amphotericin B resistance. In addition, all strains were susceptible to echinocandins and did not carry substitutions in Fks1p or Fks2p.

Literature review
We found eight articles in all reported antifungal susceptibility of C. haemulonii var. vulnera isolates. A number of strains exhibit high MICs to fluconazole or even all azoles (Cendejas-Bueno et al., 2012), but there was not any investigation on related resistance mechanisms. In addition, one study reported the emergence of pan-echinocandin-resistant C. haemulonii var. vulnera strains (Cendejas-Bueno et al., 2012). To date, there have not been any reports describing 5-flucytosine-resistant C. haemulonii var. vulnera strain ( Table 2).

Discussion
Candida haemulonii var. vulnera was identified ever-first by ITS sequencing (Cendejas-Bueno et al., 2012). Although it has been involved in identification database of latest Vitek 2 YST ID system, the results from the current study and Rodrigues et al. (2020) still found YST ID card was not able to achieve a reliable identification between C. haemulonii sensu stricto and C. haemulonii var. vulnera. Furthermore, none of the current available MALDI-TOF MS systems could accurately identify C. haemulonii var. vulnera (Kathuria et al., 2015;Grenfell et al., 2016;Rodrigues et al., 2020), though Grenfell et al. described that some discriminatory protein peaks may be able to differentiate C. haemulonii sensu stricto and C. haemulonii var. vulnera in using FlexAnalysis and ClinProTools to analysis MALDI-TOF MS results (Grenfell et al., 2016). Therefore, DNA sequencing remained the only applicable methods for the identification of C. haemulonii var. vulnera. Interestingly, Cendejas-Bueno reported that C. haemulonii sensu stricto and C. haemulonii var. vulnera can be separated by ITS sequences, but these two species have identical RPB1, RPB2, and D1/D2 sequences. Therefore, these strategies could not be used for the identification of these two species (Cendejas-Bueno et al., 2012). To date, isolation of C. haemulonii var. vulnera has only been reported in Brazil, Argentina, and Peru. Due to the abovementioned limitations of commercial identification systems, we reidentified all C. haemulonii complex isolates collected in CHIF-NET study by ITS sequencing and finally found two C. haemulonii var. vulnera strains among >80 cases.
Previously, fluconazole-and amphotericin B-resistant C. haemulonii var. vulnera have been discovered (Cendejas-Bueno et al., 2012;Ramos et al., 2015;de Almeida et al., 2016). Like findings in previous reports, one of the isolates we discovered was also resistant to fluconazole and amphotericin B. However, we also found a 5-flucytosine-resistant strain, and it is also cross-resistant to amphotericin B. Although 5-flucytosine resistance has been recognized in C. haemulonii sensu stricto and C. duobushaemulonii in China and other regions (Kathuria et al., 2015;Hou et al., 2016), this is the first 5-flucytosineresistant C. haemulonii var. vulnera case characterized to date. Of note, both strains we discovered remained susceptible to all echinocandins.
Compared with C. haemulonii sensu stricto and C. duobushaemulonii that have been well characterized, there are very few studies on resistant mechanisms of C. haemulonii var. vulnera. Key amino acid substitutions in Erg11p and Fur1p have been noted as major reasons contributing to azole and 5-flucytosine resistance, respectively, in other C. haemulonii complex species (Gade et al., 2020) and Chen et al. (2022). In this study, it was found that the fluconazole-resistant C. haemulonii var. vulnera isolate carried Y132F and G307A mutations in Erg11p. Erg11p Y132F substitutions have been reported in a broad range of fluconazole-resistant Candida species including C. albicans, C. tropicalis, and C. haemulonii (Fan et al., 2019;Warrilow et al., 2019;Gade et al., 2020), while G307A has been reported in C. parapsilosis Binder et al., 2020). While in our 5-flucytosine-resistant C. haemulonii var. vulnera strain, a frameshift V58fs and a mutation G60K were found in Fur1p, which has not been recovered in any other Candida strains to our best knowledge. Literature review showed a high proportion of C. haemulonii var. vulnera isolates (13/17, 76.5%) were with reduced susceptibility to amphotericin B (≥2 µg/ml). However, resistant mechanisms to amphotericin B were not well understood, and in this study, we also failed to found any mutations in key genes may potentially contributed to amphotericin B resistance. As a major limitation, our study did not collect detailed medical records of these two cases; therefore, we were not able to further analyze the source of infections or assessing clinical risk factors of this species.
In conclusion, there is a potential threat posed by C. haemulonii var. vulnera, a highly antifungal-resistant fungal species. Resistance to fluconazole and 5-flucytosine in C. haemulonii var. vulnera was supposed to be resulted from variations in Erg11p and Fur1p, respectively. Although invasive infection with C. haemulonii var. vulnera remained rare, further monitoring of this specie is still warranted.

Data availability statement
The datasets presented in this study can be found in the online repositories. The names of the repository/repositories and accession numbers(s) can be found in the manuscript.