Emergence and Characterization of Mixed Candida auris Strain Infections in China

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We identified five cases of mixed C. auris -strain infections in China and investigated the genetic and biological diversity of the isolates to explore the potential causes of C. auris infection. Methods C. auris isolates from 5 infection cases were distinguished by colony morphology and verified by D1/D2-ITS alignment. Phylogenetic and genomic diversity analysis of all isolates were conducted using whole genome sequences. Comparative biological analysis of all isolates, including cellular and colony morphology, antifungal susceptibility, biofilm formation, SAP activity, and both in vitro and in vivo survival capabilities were performed. Results Five cases of mixed C. auris -strain infections in China were identified. Comparative genomic analysis revealed these infections involved strains from two distinct genetic clades (I and III) or strains from the same clade but with genetic alterations. Comparative biological analysis demonstrated the strains from mixed infections exhibit differences in several key aspects, including colony morphology, biofilm formation, SAP activity, and both in vitro and in vivo survival capabilities. Conclusion Comparative analyses revealed notable differences in biofilm formation, environmental survival, and secretion of virulence factors between the co-infecting strains of C. auris . These biological and genetic disparities may present significant challenges for the diagnosis and treatment of C. auris infections, as strains with different genetic backgrounds may exhibit varying abilities to colonize host or environmental niches. Candida auris emerging fungal pathogens antifungal resistance mixed strain infections biofilm virulence Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The emergence of Candida auris as a global health threat has raised significant concerns due to its rapid spread and multidrug resistance [ 1 – 3 ]. Since its discovery in Japan in 2009, C. auris has been reported in over 50 countries between 2009–2023 ([ 2 , 3 ] and https://www.cdc.gov/ ), promoting the World Health Organization (WHO) to categorize it as a critical fungal pathogen in 2022 ( http://www.who.int ). Since its first report in Japan in 2009 [ 4 ], Six genetic clades of C. auris (I-VI) have been identified, originating from various geographic regions, including South Asia (I), East Asia (II), South Africa (III), South America (IV), Iran (V), and Bangladesh (VI) [ 5 – 9 ]. The international spread of C. auris has been accelerated by increasing global travel and the highly transmissible nature of C. auris [ 1 , 2 , 10 ]. Multiple strains from different genetic clades have rapidly disseminated across various regions, such as the United States and China, where numerous introductions of the pathogen have been documented [ 1 , 8 , 10 ]. Unlike other pathogenic Candida species, C. auris has the unusual ability to persist for extended periods on human skin and in healthcare environments, which significantly contributes to hospital outbreaks [ 11 , 12 ]. Its capacity for long-term survival and ease of transmission makes it plausible that genetically diverse C. auris strains can co-exist within the same healthcare facility, potentially leading to co-infections. A recent study by Massic et al . (2023) provided evidence of dual-clade C. auris infections, highlighting the possibility of co-infection involving multiple strains with distinct genetic backgrounds [ 13 ]. In their research, they isolated C. auris strains from two different clades (I and III) from the same patients. However, it is worth noting that the different strains were isolated from separate anatomical locations, rather than from a single infected site on the body. Mixed-strain infections are well documented in bacterial pathogens and are associated with altered diseases dynamics, complicating diagnosis and treatment [ 14 – 17 ]. However, such cases are rarely reported in fungal pathogens. In this study, we report five cases of mixed C. auris infections in China, where phenotypically and genetically distinct strains coexisted. We performed comparative analyses to investigate the biological and genetic differences between the C. auris strains isolated from the mixed infection cases. Materials and methods Strains and culture conditions C. auris strains were routinely grown on YPD agar medium supplemented with the red dye phloxine B (2% glucose, 2% peptone, 1% yeast extract, 2% agar, and 5 µg/mL of phloxine B). All C. auris isolates were identified by sequencing the 28S D1/D2 and 18S internal transcribed spacer (ITS) regions of rDNA, and their identities were confirmed through genomic sequencing analysis [ 1 , 18 ]. Colony and cellular morphologies were examined on YPD agar containing phloxine B. Whole genome sequencing (WGS) and phylogenetic analyses Single colonies of each C. auris strains were inoculated into liquid YPD medium and grown at 30°C for 24 h. Fungal cells were collected and genomic DNA was extracted using the TIANamp Yeast DNA Kit (TianGen Biotech, Beijing, China) according to the manufacturer’s protocol. Whole genome analysis was performed according to our previous publication [ 19 ]. Briefly, the raw reads were trimmed to remove low-quality (phred score ≤ 10), ambiguous and adaptor bases using the FASTX-Toolkit v0.0.14 ( http://hannonlab.cshl.edu/fastx_toolkit/index.html ). The clean reads were mapped to the genomic assembly of C. auris strain B8441 (NCBI accession number: GCA_002759435.2) using BWA mem 0.7.17 software with default settings [ 20 ]. SAMTools v1.361 [ 20 ], Picard Tools v1.56 ( http://picard.source-forge.net ), and GATK v2.7.2 were used for SNP analysis [ 21 ]. The annotation of SNPs was analyzed using ANNOVAR [ 22 ]. The maximum likelihood phylogenetic tree was generated with whole genomic SNPs using the program RAxML-ng v1.2.1 [ 23 ] with the GTRGAMMA model (4 discrete rate categories) using 1000 bootstraps. Quantitative biofilm assays Quantitative biofilm assays were conducted in liquid YPD medium using 48-well plates. Each well received 600 µL of YPD medium to facilitate biofilm development. Fungal cells from each isolate were inoculated into the plates at a final optical density (OD 600 ) of 0.2. The plates were incubated at 30°C for 48 hours with gently shaking. After incubation, the planktonic cells were removed, and the wells were washed gently with ddH 2 O three times. The cells adhered to the bottom of the wells were treated with trypsin (T4549, Sigma-Aldrich) at 37°C for 30 min. Appropriate aliquots were diluted and plated on YPD agar medium for CFU assays. Three biological replicates were performed for each strain. Minimum inhibitory concentration (MIC) assays MICs were determined using the broth micro-dilution method according to the CLSI M27 (fourth edition) and previous studies [ 24 , 25 ]. Briefly, approximately 500 cells of each isolate were inoculated in 200 µL of RPMI 1640 medium (w/v, 1.04% RPMI 1640, 3.45% MOPs, adjusted to pH 7.0 using NaOH) in a 96-well plate. Nine antifungal agents were examined: fluconazole (FLC), voriconazole (VOC), posaconazole (POC), itraconazole (ITC), caspofungin (CAS), micafungin (MFG), anidulafungin (AFG), amphotericin B (AMB), and flucytosine (5-FC). C. auris cells were treated with antifungals and incubated at 35°C for 24 hours. Candida krusei (ATCC 6258) and Candida parapsilosis (ATCC 22019) were used as quality controls. Secreted aspartyl proteinase (Sap) activity testing Sap activity for each C. auris isolate was assessed on YCB-BSA medium as previously described [ 26 ]. In brief, 5 x 10 6 yeast cells of each isolate were resuspended in 5 µL of ddH 2 O and spotted onto the YCB-BSA plate. The plates were then incubated at 25°C, 30°C, or 37°C for 7 days. The diameter of the white precipitation zone (halo) surrounding the fungal cell spot was measured, reflecting the Sap-mediated BSA hydrolysis activity. Three biological replicates were performed for each strain. In vitro survival assays C. auris cells were initially grown on YPD medium at 30°C for three days and then suspended in ddH 2 O. Approximately 1 x 10 7 cells of each C. auris isolate in 10 µL ddH 2 O were spotted on the inner surface of polypropylene centrifuge tubes and incubated at room temperature for 0, 2, 4, 8, 16, or 30 days. The number of viable cells at each time point was determined through CFU assays. Fungal burden assays in a mouse systemic infection model All animals were housed in the P2 Laboratory at Fudan University. Mice were randomly assigned to groups for the experiments, which were conducted in accordance with the guidelines approved by the Animal Care and Use Committee of Fudan University. The isolates from the two dual-clade coinfection cases (C3-R, C3-W, C4-R, and C4-W) were used for fungal burden assays. Female BALB/c mice (15–18 g, 6–7 weeks old) were purchased from Vital River (Beijing, China). Approximately 1 x 10 7 C. auris cells in 200 µL of 1 x PBS were injected into each mouse via the tail vein. Four mice were used for each isolate. After 24 hours of infection, the mice were humanely euthanized, and the tissues (brain, liver, lung, spleen, and kidney) were harvested for fungal burden analysis. Results Identification of mixed C. auris strain infections To explore the phenotypic diversity of C. auris strains collected from hospitals in Guangdong, China (G-H1 and G-H2), between April 2023 and May 2023, we inoculated original samples on YPD agar plates containing phloxine B. The strains from 5 cases exhibited two or more distinct colony morphologies based on coloration after 3 days of incubation at 30°C and an additional day at room temperature (Fig. 1 A). Details of the five cases are presented in Table 1 . We re-plated red (R) and white (W) colonies on phloxine B-containing medium and cultured them for 3 days at 30°C (Fig. 1 B). The white colonies (C1-W, C2-W, C3-W, C4-W, and C5-W) produced cells that formed relatively larger aggregates compared to the red colonies (C1-R, C2-R, C3-R, C4-R, and C5-R), suggesting that the white cell type may exhibit enhanced adhesion or greater potential for biofilm formation. Table 1 Information on five mixed strain infection cases of C. auris . Case # Strain # (Clades) Hospital Source Department Patient information Collection date Sex Age Clinical diagnosis 1 C1-R (III) & C1-W (III) G-H1 Sputum ICU Female 48 Guillain-Barre syndrome, thermoplegia, type 2 diabetes Apr 17th 2023 2 C2-R (III) & C2-W (III) G-H1 Urine Cardiology Female 68 Hypertension, vesico-intestinal fistula, urinary tract infection May 4th 2023 3 C3-R (I) & C3-W (III) G-H1 Sputum ICU Female 51 Basal ganglia cerebral hemorrhage, hypertension May 15th 2023 4 C4-R (I) & C4-W (III) G-H1 Catheter ICU Male 65 Mitral insufficiency,atrial fibrillation, cardiac insufficiency May 12th 2023 5 C5-R (I) & C5-W (I) G-H2 Venous blood Neurology Female 52 Pulmonary infection, chronic nephritic syndrome, autoimmune hepatitis, hypertension Apr 27th 2023 Notes: This table is associated with Fig. 1 . The five mixed strain infection cases were identified in two hospitals in Guangdong, China (G-H1 and G-H2). Strain identification was performed by analyzing the D1/D2 and 18S internal transcribed spacer (ITS) regions of the 28S rDNA sequences. As shown in Table 1 , strains C1-R, C1-W, C2-R, and C2-W from cases 1 and 2 were classified as belonging to clade III (South African); strains C5-R and C5-W from case 5 belonged to clade I (South Asian); and C3-R and C4-R, as well as C3-W and C4-W from cases 3 and 4, were identified as clades I and III, respectively. Notably, in case 3, some Candida parapsilosis colonies (labelled “P” as pink colonies, Fig. 1 A) were identified by ITS sequencing, indicating the occurrence of a co-infection with multiple Candida species. Comparative genomic analysis of C. auris strains from mixed infections To further investigate and characterize the C. auris strains from mixed infections, we conducted whole genome sequencing analysis. In cases 1, 2, and 5, we identified only 4 single nucleotide polymorphism (SNP) differences between strains C1-R and C1-W (case 1), 4 SNPs between C2-R and C2-W (case 2), and 10 SNPs between C5-R and C5-W (case 5). These nucleotide alterations impacted several genes, which may contribute to the morphological differences observed between the red (R) and White (W) colony phenotypes. In cases 3 and 4, where the infections were caused by strains from different genetic clades, we identified over 46,000 SNPs ( Table S1 ). This high number of SNPs reflects the genetic divergence between the strains C3-R and C3-W, and C4-R and C4-W, as they belong to distinct genetic clades. Using whole genome sequences, we conducted a phylogenetic analysis of the mixed C. auris strains, constructing a phylogenetic tree (Fig. 2 A). The strains from the same genetic clades identified in this study clustered closely together, suggesting their genetic relatedness. Further phylogenetic analysis of clade III (Fig. 2 B) and I (Fig. 2 C) strains isolated in China revealed that the strains from the same clades identified in this study may share common origins. For instance, clade III strains, such as C1-R, C1-W, C2-R, C2-W, C3-W, and C4-W showed greater genetic distances from other C. auris isolates from provinces like Liaoning and Beijing (Fig. 2 B). Similarly, clade I isolates like C3-R, C4-R, C5-W, and C5-R were more genetically distant from isolates from Anhui, Jiangsu and Shandong provinces (Fig. 2 C). Interestingly, the clade I strains isolated in this study appeared closely related to strain BJ004 from Beijing, suggesting that local transmissions may have occurred in Guangdong Province. These findings highlight the complex epidemiology and possible local dissemination of C. auris strains within China. Comparative biological analysis of C. auris strains from mixed infections Distinct biological characteristics of fungal pathogens can influence their infection potential and therapeutic outcomes. We next conducted antifungal susceptibility analysis on isolates from mixed infection cases. As shown in Table S2 , all strains, except those from case 5, displayed a minimum inhibitory concentration (MIC) of 128 mg/L for fluconazole. Strains from case 5 showed an MIC of 64 mg/L (C5-R) and 32 mg/L (C5-W) for the same drug. For amphotericin B, the 4 clade I strains (C3-R, C4-R, C5-R and C5-W) exhibited an MIC of 2 mg/L, while the 6 clade III strains had an MIC of 1 mg/L. For the other 7 antifungals (voriconazole, posaconazole, itraconazole, caspofungin, micafungin, anidulafungin, and flucytosine), all strains demonstrated relatively low MICs. According to the tentative antifungal breakpoints established by the CDC, all strains were resistant to fluconazole, and four clade I isolates were resistant to amphotericin B ( Table S2 ). Given the observed differences in aggregation between the strains from mixed infection (Fig. 1 B), we next evaluated their biofilm-forming ability. As shown in Fig. 3 , the white (W) isolates (C1-W, C2-W, C3-W, C4-W and C5-W) exhibited significantly stronger biofilm formation compared to the red (R) isolates (C1-R, C2-R, C3-R, C4-R and C5-R). Additionally, the clade III strains demonstrated a higher biofilm-forming capacity than the clade I strains, based on CFU analysis of the biofilms (Fig. 3 ). These results are consistent with the differences in aggregation ability on agar plates (Fig. 1 B). Secreted aspartyl proteinases (SAPs) are key virulence factors in pathogenic Candida species [ 27 ]. We compared SAP secretion among the strains from mixed infections using YCB-BSA assays at three different temperatures. As shown in Fig. 4 , the strains exhibited varying SAP activities at 37°C, the physiological temperature of the human body. With the exception of strain C2-R, all strains showed relatively low SAP activity at 25°C and 30°C. However, some strains (C1-W, C2-R, C3-R, C4-R, and C5-R) displayed higher SAP activity at 37°C than the others, indicating that C. auris strains isolated from the same patient can exhibit differences in SAP activity and virulence. C. auris strains isolated from mixed infections differ in in vitro and in vivo survival abilities A notable characteristic of C. auris is its ability to persist in the environment [ 28 , 29 ]. To assess the in vitro survival abilities of isolates from mixed infections, cells from each isolate were spotted on the inner surface of polypropylene Eppendorf tubes and incubated at room temperature. Colony-forming unit (CFU) assays were then performed to assess the viability of the C. auris cells. As shown in Fig. 5 , the white isolates (C1-W, C2-W, C3-W, C4-W and C5-W) exhibited generally higher survival rates compared to the red isolates (C1-R, C2-R, C3-R, C4-R and C5-R), mirroring the pattern observed in biofilm formation. These results suggest that isolates from mixed infections may have distinct abilities to persist in the environment. Next, we examined the susceptibility of the isolates to the human antimicrobial peptide LL-37, which plays a role in regulating skin immunity [ 30 ]. As shown in Fig. 6 , strains C1-W, C2-W, C3-R, C4-R, and C5-R exhibited relatively higher survival rates in the presence of LL-37 compared to their respective counterparts. Given the potential impact of genetic and biological differences on virulence, we conducted fungal burden assays using strains C3-W, C3-R, C4-W, and C4-R in a mouse infection model. As shown in Fig. 7 , the white (C3-W and C4-W) and red (C3-R and C4-R) isolates exhibited comparable fungal burdens in the liver, lungs, spleen, and kidneys. However, strains C3-W and C4-W demonstrated significantly higher fungal burdens in the brain compared to strains C3-R and C4-R. Taken together, isolates from mixed infections not only differ in their in vitro survival abilities but also display distinct behaviors during in vivo infections. Discussion Over the past decade, the emerging fungal pathogen C. auris has rapidly spread worldwide, frequently causing both inter-hospital and intra-hospital transmissions [ 1 , 2 , 18 ]. Since the first reported case of C. auris infection in China in 2018, hundreds of cases, including both screenings and infections, have been documented, suggesting multiple introductions into the country [ 1 ]. In this study, we report the emergence of mixed-strain infections involving C. auris in Guangdong, China. These infections involved strains from two distinct genetic clades (I and III) or strains with genetic alterations (Fig. 1 and Table 1 ). Our comparative analyses revealed notable genetic and biological diversity among isolates from the same patients. A recent study from southern Nevada, USA, also reported coinfections involving clades I and III of C. auris during a large outbreak [ 13 ]. Given that strains from clades I and III have opposite mating types (“ a ” and “α” for clades I and III, respectively), this discovery raises the possibility of mating occurring in C. auris . However, genomic analyses have shown that, aside from STE6 , which encodes the a-pheromone transporter necessary for “a” cell mating [ 31 ], multiple genes involved in the mating regulation (e.g., STE2, STE50 , and FUS1 ) were mutated in C. auris strains from one or more clades (data not shown). In our current study, we identified two dual-clade coinfection cases involving clades I and III, as well as three mixed infection cases caused by strains with genetic and phenotypic variations within a single clade (III or I). Unlike the previously reported dual-clade infections, where strains from different clades were isolated from separate body sites [ 13 ], we isolated all strains from the same samples in our mixed infection cases. Our findings indicate that strains from mixed infections exhibit differences in several key aspects, including colony morphology, biofilm formation, SAP activity, and both in vitro and in vivo survival capabilities. Coinfections with genetically and biologically distinct strains could pose new challenges for clinical diagnosis and treatment, as strains with different genetic backgrounds may exhibit varying abilities to colonize host or environmental niches. For example, C. auris strain C2-R demonstrated enhanced secretion of the SAP virulence factor (Fig. 4 ), which may contribute to improved skin colonization. These coinfections could potentially lead to increased virulence and varied treatment outcomes. We also observed that certain C. auris strains exhibited superior environmental persistence compared to others (Fig. 5 ). It remains to be determined whether the co-existence of genetically diverse strains impacts hospital transmission dynamics. Further investigation is warranted to understand how these mixed infections influence transmission, virulence, and treatment effectiveness in clinical settings. In summary, considering the ease of transmission and environmental persistence of C. auris , along with increasing international travel, the presence of genetically diverse with the same clinical setting is plausible. Consequently, coinfections are likely to occur when multiple C. auris strains of different origins are introduced into hospitals. This emerging situation could complicate the control, diagnosis, and treatment of C. auris infections, posing significant challenges for healthcare management. Declarations Acknowledgments This work was supported by the National Key Research and Development Program of China (grant no. 2021YFC2300400 to G.H., and no. 2022YFC2303000 to J.B. and H.D.), National Natural Science Foundation of China (nos. 31930005 and 82272359 to G.H., nos. 32170193 to J.B., and 82172290 to H.D.), and Shanghai Science and Technology Innovation Action Plan 2023 “Basic Research Project” (23JC1404200 to GH). The content is the sole responsibility of the authors and does not represent the views of the funders. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Author contributions DH, BJ, CHQ and HGH designed the study. DH, HYF, GPH, LWH, ZTH, and GZY carried out sample collection and experiments. BJ and DH analyzed data. DH, BJ, and HGH wrote this manuscript. All authors contributed to and reviewed the final manuscript. No potential conflict of interest was reported by the authors. Data availability The Genomic data has been deposited in the NCBI SRA database (SRR30963043-SRR30963052). References Bing J, Du H, Guo P, et al. Candida auris-associated hospitalizations and outbreaks, China, 2018-2023. Emerg Microbes Infect. 2024;13(1):2302843. De Gaetano S, Midiri A, Mancuso G, et al. Candida auris Outbreaks: Current Status and Future Perspectives. Microorganisms. 2024;12(5). Chowdhary A, Jain K, Chauhan N. Candida auris Genetics and Emergence. 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Bba-Biomembranes. 2006;1758(9):1408-1425. Magee BB, Legrand M, Alarco AM, et al. Many of the genes required for mating in Saccharomyces cerevisiae are also required for mating in Candida albicans. Mol Microbiol. 2002;46(5):1345-51. Additional Declarations No competing interests reported. Supplementary Files Supplementaryinformation1024.docx Supplementary information Table S1. SNP analysis of the C. auris strains isolated from mixed infections. Table S2. Antifungal susceptibility of C. auris strains isolated from co-infection cases. Cite Share Download PDF Status: Published Journal Publication published 14 Apr, 2025 Read the published version in Infection → Version 1 posted Editorial decision: Revision requested 03 Jan, 2025 Reviews received at journal 01 Jan, 2025 Reviewers agreed at journal 24 Dec, 2024 Reviewers agreed at journal 23 Dec, 2024 Reviews received at journal 06 Dec, 2024 Reviewers agreed at journal 16 Nov, 2024 Reviewers invited by journal 03 Nov, 2024 Editor assigned by journal 29 Oct, 2024 Submission checks completed at journal 29 Oct, 2024 First submitted to journal 28 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5346326","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":373538352,"identity":"4856a355-bc8c-4b5e-a33f-fcf05bde5338","order_by":0,"name":"Han Du","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Han","middleName":"","lastName":"Du","suffix":""},{"id":373538354,"identity":"0b855545-46ef-4c85-a95b-289cad444b96","order_by":1,"name":"Yanfeng Huang","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Yanfeng","middleName":"","lastName":"Huang","suffix":""},{"id":373538356,"identity":"f3f82f4e-5cea-49ad-9ce8-39bb40ec8404","order_by":2,"name":"Penghao Guo","email":"","orcid":"","institution":"First Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Penghao","middleName":"","lastName":"Guo","suffix":""},{"id":373538357,"identity":"f3f59d59-4017-494d-9531-1133e8988852","order_by":3,"name":"Weihong Liang","email":"","orcid":"","institution":"Shanghai Pulmonary Hospital","correspondingAuthor":false,"prefix":"","firstName":"Weihong","middleName":"","lastName":"Liang","suffix":""},{"id":373538358,"identity":"3ad9b550-35b3-4044-92ec-f45ea4393304","order_by":4,"name":"Tianhong Zheng","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Tianhong","middleName":"","lastName":"Zheng","suffix":""},{"id":373538359,"identity":"e3e6a6e1-d29a-4a16-92cd-792dc3629654","order_by":5,"name":"Zhangyue Guan","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Zhangyue","middleName":"","lastName":"Guan","suffix":""},{"id":373538361,"identity":"3bc18b54-79dc-49ec-adb6-37e5bae3f255","order_by":6,"name":"Jian Bing","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYBACA2YgIVEhx8BwAMRlI1rLGWNStIAIxjZStJiz8x5+YTnPILHv+NkDDB/KDjPwz27Ar8WymS/NQnKbQeLMM3kJjDPOHWaQuHOAgMMO85gZSG77k7jhQI4BM2/bYQYDiQRitMwxSNxw/o0B818itRg/kGwAarkBtIWRGC2WzTxmDBLHDIxn3nhjcLDnXDqPxA0CWsz5zxh/lqgxkO07n2P44EeZtRz/DAJagIBNWgLKOgDEPATVAwHzxw/EKBsFo2AUjIKRCwAHNENSwDq6dAAAAABJRU5ErkJggg==","orcid":"","institution":"Fudan University","correspondingAuthor":true,"prefix":"","firstName":"Jian","middleName":"","lastName":"Bing","suffix":""},{"id":373538364,"identity":"f97c1276-a480-4bf7-b843-e65b7c1f2b4f","order_by":7,"name":"Haiqing Chu","email":"","orcid":"","institution":"Shanghai Pulmonary Hospital","correspondingAuthor":false,"prefix":"","firstName":"Haiqing","middleName":"","lastName":"Chu","suffix":""},{"id":373538368,"identity":"3dd57c1b-95d3-4d7e-b1e8-1a5b32d2ad57","order_by":8,"name":"Guanghua Huang","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Guanghua","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2024-10-28 10:38:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5346326/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5346326/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s15010-025-02536-6","type":"published","date":"2025-04-14T15:57:24+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69362477,"identity":"a8892f1b-91d5-43f3-bb5b-916b0ccdd268","added_by":"auto","created_at":"2024-11-19 14:32:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1487267,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eColony and cellular morphologies of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. auris\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003estrains isolated from mixed infection cases. \u003c/strong\u003e(A) Colony morphologies of the original strain samples. Fungal cells were plated on YPD agar containing the red dye phloxine B and cultured at 30°C for 3 days, followed by an additional day at room temperature. R (red) and W (white) indicate colonies with different colorations. P (light pink) indicates \u003cem\u003eC. parapsilosis\u003c/em\u003e colonies. (B) Colony and cellular morphologies of \u003cem\u003eC. auris\u003c/em\u003e strains isolated from each case. Cells of purified strains were plated on YPD agar containing phloxine B and cultured at 30°C for 3 days. The “+” symbol indicates the degree of cell aggregation. Scale bar: 5 mm for colony; 10 μM for cells.\u003c/p\u003e","description":"","filename":"Figure11012.png","url":"https://assets-eu.researchsquare.com/files/rs-5346326/v1/2e328e9cb3f1df82f5b807ff.png"},{"id":69360296,"identity":"eb89ecc8-bee3-466c-9530-3ee8707e82f6","added_by":"auto","created_at":"2024-11-19 14:16:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":73873,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis of\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e C. auris\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003estrains isolated from co-infection cases.\u003c/strong\u003e Whole genomic SNPs were used to conducted a maximum likelihood phylogenetic tree under the GTRGAMMA model (4 discrete rate categories) using RAxML-NG (v1.2.1) with 1000 bootstrap replicates. Color codes indicate different cases. Scale bars represent frequencies of base-pair differences. (A) Phylogenetic tree of strains from all three clades of \u003cem\u003eC. auris\u003c/em\u003e identified in China. (B) Phylogenetic tree of clade III strains identified in china. (C) Phylogenetic tree of clade I strains identified in china. Strains isolated from mixed infections in this study (strains from the same case highlighted in the same color): C1-R and C1-W from case 1; C2-R and C2-W from case 2; C3-R and C3-W from case 3; C4-R and C4-W from case 4; C5-R and C5-W from case 5. The original locations where strains were identified are shown in the brackets.\u003c/p\u003e","description":"","filename":"Figure21012.png","url":"https://assets-eu.researchsquare.com/files/rs-5346326/v1/31c849f9b28952164ad8b547.png"},{"id":69361620,"identity":"b1c55618-94f8-46d1-bc7d-b2c5ed84c019","added_by":"auto","created_at":"2024-11-19 14:24:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":61435,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative analysis of biofilm formation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. auris\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e strains isolated from mixed infection cases. \u003c/strong\u003eQuantitative biofilm assays were performed in 48-well plates. Fungal cells of each isolate were inoculated into the wells and incubated at 30°C for 48 hours with gently shaking. The cells adhered to the well bottom were treated with trypsin and subject to CFU assays. Error bars represent the standard errors of three replicates. \u003cem\u003eP \u003c/em\u003evalue are indicated (two-tailed Student’s \u003cem\u003et-\u003c/em\u003etest).\u003c/p\u003e","description":"","filename":"Figure31012.png","url":"https://assets-eu.researchsquare.com/files/rs-5346326/v1/567bb9bdf1d3f25cfd89f840.png"},{"id":69361624,"identity":"dfe05241-fb81-4a74-895f-9f820845d64e","added_by":"auto","created_at":"2024-11-19 14:24:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":719035,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative analysis of secreted aspartic protease (SAP) activity of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. auris\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e strains isolated from mixed infection cases. \u003c/strong\u003eApproximately 5 ×10\u003csup\u003e6\u003c/sup\u003e cells of each isolate in 5 μL of ddH\u003csub\u003e2\u003c/sub\u003eO were spotted onto YCB-BSA agar. After incubation at 25°C, 30°C, or 37°C for 7 days, the zone of white precipitation (halos) around the drop inoculum, which indicates SAP activity, was imaged and measured from the edge of growth outward (in mm). The mean values and standard deviations are presented in the corresponding images. N/A indicates no visible halo observed.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure41012.png","url":"https://assets-eu.researchsquare.com/files/rs-5346326/v1/e099ecb23075a2802b7c446a.png"},{"id":69360300,"identity":"3d77f649-0bde-438c-9a90-7f4d39b9448d","added_by":"auto","created_at":"2024-11-19 14:16:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":154950,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative analysis of survival ability of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. auris\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e strains isolated from mixed infection cases on polypropylene surface. \u003c/strong\u003eApproximately 1 x 10\u003csup\u003e7\u003c/sup\u003e cells of each isolate in 10 μL ddH\u003csub\u003e2\u003c/sub\u003eO were spotted on the inner surface of polypropylene centrifuge tubes (1.5 mL) and incubated at room temperature for 0, 2, 4, 8, 16, or 30 days. The number of viable cells at each time point was determined by CFU assay. The dotted line indicates no colony formation. Error bars represent standard errors of three biological replicates. ­\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure51012.png","url":"https://assets-eu.researchsquare.com/files/rs-5346326/v1/2e39564a1310c0e3e28c2c0c.png"},{"id":69360297,"identity":"580dda97-b478-44ad-80a9-d1782a1db407","added_by":"auto","created_at":"2024-11-19 14:16:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":35650,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative analysis of susceptibility of\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e C. auris\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e strains isolated from co-infection cases to human antimicrobial peptide LL-37.\u003c/strong\u003e Single isolates from each case were tested using quantitative antimicrobial killing assays (n = 3). \u003cem\u003eC. auris\u003c/em\u003e cells (1 x 10\u003csup\u003e7\u003c/sup\u003e cells/mL) were re-suspended in 1mM PPB. The cells were treated with 10 μM LL-37 for 1 h at 37 °C, and then diluted and plated onto YPD medium for CFU analysis. The survival rate (%) of each strain was calculated. \u003cem\u003eP \u003c/em\u003evalues are indicated (two-tailed Student’s \u003cem\u003et-\u003c/em\u003etest).\u003c/p\u003e","description":"","filename":"Figure61012.png","url":"https://assets-eu.researchsquare.com/files/rs-5346326/v1/750be852ef720f358de8f17e.png"},{"id":69361621,"identity":"084bdf7c-2b2c-4fd3-82e6-6bceedaf2779","added_by":"auto","created_at":"2024-11-19 14:24:33","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":99580,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFungal burden analysis of\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e C. auris\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e strains isolated from co-infection cases.\u003c/strong\u003e \u003cem\u003eC. auris\u003c/em\u003e isolates from cases 3 and 4 (C3-R and C3-W; C4-R and C4-W) were used for fungal burden assays. Approximately 1 x 10\u003csup\u003e7\u003c/sup\u003e cells were injected into each mouse via the tail vein. Four mice per\u003cem\u003e C. auris\u003c/em\u003e strain were used. At 24 h post-infection, mice were euthanized, and 5 tissues (brain, liver, lung, spleen and kidney) were used for CFU assays. The fungal burden in each tissue (CFU/g) was calculated. \u003cem\u003eP \u003c/em\u003evalues are indicated (two-tailed Student’s \u003cem\u003et-\u003c/em\u003etest).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure71012.png","url":"https://assets-eu.researchsquare.com/files/rs-5346326/v1/29c7c9186fc986a087010da2.png"},{"id":81050803,"identity":"569ab54b-3263-4139-b1cb-34db8d4d84c9","added_by":"auto","created_at":"2025-04-21 16:05:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4845224,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5346326/v1/a19c7b92-8295-454c-bdc5-6c5240b716be.pdf"},{"id":69362476,"identity":"e6ec3546-9191-4df0-89e5-1c37e3c82185","added_by":"auto","created_at":"2024-11-19 14:32:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17902,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable S1. SNP analysis of the C. auris strains isolated from mixed infections.\u003c/p\u003e\n\u003cp\u003eTable S2. Antifungal susceptibility of C. auris strains isolated from co-infection cases.\u003c/p\u003e","description":"","filename":"Supplementaryinformation1024.docx","url":"https://assets-eu.researchsquare.com/files/rs-5346326/v1/af12f684ae15b55d4c5f2bed.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Emergence and Characterization of Mixed Candida auris Strain Infections in China","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe emergence of \u003cem\u003eCandida auris\u003c/em\u003e as a global health threat has raised significant concerns due to its rapid spread and multidrug resistance [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Since its discovery in Japan in 2009, \u003cem\u003eC. auris\u003c/em\u003e has been reported in over 50 countries between 2009\u0026ndash;2023 ([\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cdc.gov/\u003c/span\u003e\u003cspan address=\"https://www.cdc.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), promoting the World Health Organization (WHO) to categorize it as a critical fungal pathogen in 2022 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.who.int\u003c/span\u003e\u003cspan address=\"http://www.who.int\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Since its first report in Japan in 2009 [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], Six genetic clades of \u003cem\u003eC. auris\u003c/em\u003e (I-VI) have been identified, originating from various geographic regions, including South Asia (I), East Asia (II), South Africa (III), South America (IV), Iran (V), and Bangladesh (VI) [\u003cspan additionalcitationids=\"CR6 CR7 CR8\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe international spread of \u003cem\u003eC. auris\u003c/em\u003e has been accelerated by increasing global travel and the highly transmissible nature of \u003cem\u003eC. auris\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Multiple strains from different genetic clades have rapidly disseminated across various regions, such as the United States and China, where numerous introductions of the pathogen have been documented [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Unlike other pathogenic \u003cem\u003eCandida\u003c/em\u003e species, \u003cem\u003eC. auris\u003c/em\u003e has the unusual ability to persist for extended periods on human skin and in healthcare environments, which significantly contributes to hospital outbreaks [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Its capacity for long-term survival and ease of transmission makes it plausible that genetically diverse \u003cem\u003eC. auris\u003c/em\u003e strains can co-exist within the same healthcare facility, potentially leading to co-infections. A recent study by Massic \u003cem\u003eet al\u003c/em\u003e. (2023) provided evidence of dual-clade \u003cem\u003eC. auris\u003c/em\u003e infections, highlighting the possibility of co-infection involving multiple strains with distinct genetic backgrounds [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In their research, they isolated \u003cem\u003eC. auris\u003c/em\u003e strains from two different clades (I and III) from the same patients. However, it is worth noting that the different strains were isolated from separate anatomical locations, rather than from a single infected site on the body.\u003c/p\u003e \u003cp\u003eMixed-strain infections are well documented in bacterial pathogens and are associated with altered diseases dynamics, complicating diagnosis and treatment [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, such cases are rarely reported in fungal pathogens. In this study, we report five cases of mixed \u003cem\u003eC. auris\u003c/em\u003e infections in China, where phenotypically and genetically distinct strains coexisted. We performed comparative analyses to investigate the biological and genetic differences between the \u003cem\u003eC. auris\u003c/em\u003e strains isolated from the mixed infection cases.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrains and culture conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eC. auris\u003c/em\u003e strains were routinely grown on YPD agar medium supplemented with the red dye phloxine B (2% glucose, 2% peptone, 1% yeast extract, 2% agar, and 5 \u0026micro;g/mL of phloxine B). All \u003cem\u003eC. auris\u003c/em\u003e isolates were identified by sequencing the 28S D1/D2 and 18S internal transcribed spacer (ITS) regions of rDNA, and their identities were confirmed through genomic sequencing analysis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Colony and cellular morphologies were examined on YPD agar containing phloxine B.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWhole genome sequencing (WGS) and phylogenetic analyses\u003c/h3\u003e\n\u003cp\u003eSingle colonies of each \u003cem\u003eC. auris\u003c/em\u003e strains were inoculated into liquid YPD medium and grown at 30\u0026deg;C for 24 h. Fungal cells were collected and genomic DNA was extracted using the TIANamp Yeast DNA Kit (TianGen Biotech, Beijing, China) according to the manufacturer\u0026rsquo;s protocol. Whole genome analysis was performed according to our previous publication [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Briefly, the raw reads were trimmed to remove low-quality (phred score\u0026thinsp;\u0026le;\u0026thinsp;10), ambiguous and adaptor bases using the FASTX-Toolkit v0.0.14 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://hannonlab.cshl.edu/fastx_toolkit/index.html\u003c/span\u003e\u003cspan address=\"http://hannonlab.cshl.edu/fastx_toolkit/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The clean reads were mapped to the genomic assembly of \u003cem\u003eC. auris\u003c/em\u003e strain B8441 (NCBI accession number: GCA_002759435.2) using BWA mem 0.7.17 software with default settings [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. SAMTools v1.361 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], Picard Tools v1.56 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://picard.source-forge.net\u003c/span\u003e\u003cspan address=\"http://picard.source-forge.net\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and GATK v2.7.2 were used for SNP analysis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The annotation of SNPs was analyzed using ANNOVAR [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The maximum likelihood phylogenetic tree was generated with whole genomic SNPs using the program RAxML-ng v1.2.1 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] with the GTRGAMMA model (4 discrete rate categories) using 1000 bootstraps.\u003c/p\u003e\n\u003ch3\u003eQuantitative biofilm assays\u003c/h3\u003e\n\u003cp\u003eQuantitative biofilm assays were conducted in liquid YPD medium using 48-well plates. Each well received 600 \u0026micro;L of YPD medium to facilitate biofilm development. Fungal cells from each isolate were inoculated into the plates at a final optical density (OD\u003csub\u003e600\u003c/sub\u003e) of 0.2. The plates were incubated at 30\u0026deg;C for 48 hours with gently shaking. After incubation, the planktonic cells were removed, and the wells were washed gently with ddH\u003csub\u003e2\u003c/sub\u003eO three times. The cells adhered to the bottom of the wells were treated with trypsin (T4549, Sigma-Aldrich) at 37\u0026deg;C for 30 min. Appropriate aliquots were diluted and plated on YPD agar medium for CFU assays. Three biological replicates were performed for each strain.\u003c/p\u003e\n\u003ch3\u003eMinimum inhibitory concentration (MIC) assays\u003c/h3\u003e\n\u003cp\u003eMICs were determined using the broth micro-dilution method according to the CLSI M27 (fourth edition) and previous studies [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Briefly, approximately 500 cells of each isolate were inoculated in 200 \u0026micro;L of RPMI 1640 medium (w/v, 1.04% RPMI 1640, 3.45% MOPs, adjusted to pH 7.0 using NaOH) in a 96-well plate. Nine antifungal agents were examined: fluconazole (FLC), voriconazole (VOC), posaconazole (POC), itraconazole (ITC), caspofungin (CAS), micafungin (MFG), anidulafungin (AFG), amphotericin B (AMB), and flucytosine (5-FC). \u003cem\u003eC. auris\u003c/em\u003e cells were treated with antifungals and incubated at 35\u0026deg;C for 24 hours. \u003cem\u003eCandida krusei\u003c/em\u003e (ATCC 6258) and \u003cem\u003eCandida parapsilosis\u003c/em\u003e (ATCC 22019) were used as quality controls.\u003c/p\u003e\n\u003ch3\u003eSecreted aspartyl proteinase (Sap) activity testing\u003c/h3\u003e\n\u003cp\u003eSap activity for each \u003cem\u003eC. auris\u003c/em\u003e isolate was assessed on YCB-BSA medium as previously described [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In brief, 5 x 10\u003csup\u003e6\u003c/sup\u003e yeast cells of each isolate were resuspended in 5 \u0026micro;L of ddH\u003csub\u003e2\u003c/sub\u003eO and spotted onto the YCB-BSA plate. The plates were then incubated at 25\u0026deg;C, 30\u0026deg;C, or 37\u0026deg;C for 7 days. The diameter of the white precipitation zone (halo) surrounding the fungal cell spot was measured, reflecting the Sap-mediated BSA hydrolysis activity. Three biological replicates were performed for each strain.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro survival assays\u003c/h2\u003e \u003cp\u003e \u003cem\u003eC. auris\u003c/em\u003e cells were initially grown on YPD medium at 30\u0026deg;C for three days and then suspended in ddH\u003csub\u003e2\u003c/sub\u003eO. Approximately 1 x 10\u003csup\u003e7\u003c/sup\u003e cells of each \u003cem\u003eC. auris\u003c/em\u003e isolate in 10 \u0026micro;L ddH\u003csub\u003e2\u003c/sub\u003eO were spotted on the inner surface of polypropylene centrifuge tubes and incubated at room temperature for 0, 2, 4, 8, 16, or 30 days. The number of viable cells at each time point was determined through CFU assays.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFungal burden assays in a mouse systemic infection model\u003c/h3\u003e\n\u003cp\u003eAll animals were housed in the P2 Laboratory at Fudan University. Mice were randomly assigned to groups for the experiments, which were conducted in accordance with the guidelines approved by the Animal Care and Use Committee of Fudan University.\u003c/p\u003e \u003cp\u003eThe isolates from the two dual-clade coinfection cases (C3-R, C3-W, C4-R, and C4-W) were used for fungal burden assays. Female BALB/c mice (15\u0026ndash;18 g, 6\u0026ndash;7 weeks old) were purchased from Vital River (Beijing, China). Approximately 1 x 10\u003csup\u003e7\u003c/sup\u003e \u003cem\u003eC. auris\u003c/em\u003e cells in 200 \u0026micro;L of 1 x PBS were injected into each mouse via the tail vein. Four mice were used for each isolate. After 24 hours of infection, the mice were humanely euthanized, and the tissues (brain, liver, lung, spleen, and kidney) were harvested for fungal burden analysis.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIdentification of mixed\u003c/strong\u003e \u003cstrong\u003eC. auris\u003c/strong\u003e \u003cstrong\u003estrain infections\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the phenotypic diversity of \u003cem\u003eC. auris\u003c/em\u003e strains collected from hospitals in Guangdong, China (G-H1 and G-H2), between April 2023 and May 2023, we inoculated original samples on YPD agar plates containing phloxine B. The strains from 5 cases exhibited two or more distinct colony morphologies based on coloration after 3 days of incubation at 30\u0026deg;C and an additional day at room temperature (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Details of the five cases are presented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. We re-plated red (R) and white (W) colonies on phloxine B-containing medium and cultured them for 3 days at 30\u0026deg;C (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). The white colonies (C1-W, C2-W, C3-W, C4-W, and C5-W) produced cells that formed relatively larger aggregates compared to the red colonies (C1-R, C2-R, C3-R, C4-R, and C5-R), suggesting that the white cell type may exhibit enhanced adhesion or greater potential for biofilm formation.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eInformation on five mixed strain infection cases of \u003cem\u003eC. auris\u003c/em\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"9\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eCase #\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eStrain #\u003c/p\u003e\n \u003cp\u003e(Clades)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eHospital\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSource\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eDepartment\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003ePatient information\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eCollection date\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eClinical diagnosis\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC1-R (III)\u003c/p\u003e\n \u003cp\u003e\u0026amp;\u003c/p\u003e\n \u003cp\u003eC1-W (III)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG-H1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSputum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eICU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGuillain-Barre syndrome, thermoplegia, type 2 diabetes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eApr 17th 2023\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC2-R (III)\u003c/p\u003e\n \u003cp\u003e\u0026amp;\u003c/p\u003e\n \u003cp\u003eC2-W (III)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG-H1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUrine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCardiology\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHypertension, vesico-intestinal fistula, urinary tract infection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMay 4th\u003c/p\u003e\n \u003cp\u003e2023\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC3-R (I)\u003c/p\u003e\n \u003cp\u003e\u0026amp;\u003c/p\u003e\n \u003cp\u003eC3-W (III)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG-H1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSputum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eICU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBasal ganglia cerebral hemorrhage, hypertension\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMay 15th 2023\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC4-R (I)\u003c/p\u003e\n \u003cp\u003e\u0026amp;\u003c/p\u003e\n \u003cp\u003eC4-W (III)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG-H1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCatheter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eICU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMitral insufficiency,atrial fibrillation, cardiac insufficiency\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMay 12th 2023\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC5-R (I)\u003c/p\u003e\n \u003cp\u003e\u0026amp;\u003c/p\u003e\n \u003cp\u003eC5-W (I)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG-H2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVenous blood\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNeurology\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePulmonary infection, chronic nephritic syndrome, autoimmune hepatitis, hypertension\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eApr 27th 2023\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eNotes:\u0026nbsp;\u003c/strong\u003eThis table is associated with \u003cstrong\u003eFig. 1\u003c/strong\u003e. The five mixed strain infection cases were identified in two hospitals in Guangdong, China (G-H1 and G-H2). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStrain identification was performed by analyzing the D1/D2 and 18S internal transcribed spacer (ITS) regions of the 28S rDNA sequences. As shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, strains C1-R, C1-W, C2-R, and C2-W from cases 1 and 2 were classified as belonging to clade III (South African); strains C5-R and C5-W from case 5 belonged to clade I (South Asian); and C3-R and C4-R, as well as C3-W and C4-W from cases 3 and 4, were identified as clades I and III, respectively. Notably, in case 3, some \u003cem\u003eCandida parapsilosis\u003c/em\u003e colonies (labelled \u0026ldquo;P\u0026rdquo; as pink colonies, Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA) were identified by ITS sequencing, indicating the occurrence of a co-infection with multiple \u003cem\u003eCandida\u003c/em\u003e species.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparative genomic analysis of\u003c/strong\u003e \u003cstrong\u003eC. auris\u003c/strong\u003e \u003cstrong\u003estrains from mixed infections\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate and characterize the \u003cem\u003eC. auris\u003c/em\u003e strains from mixed infections, we conducted whole genome sequencing analysis. In cases 1, 2, and 5, we identified only 4 single nucleotide polymorphism (SNP) differences between strains C1-R and C1-W (case 1), 4 SNPs between C2-R and C2-W (case 2), and 10 SNPs between C5-R and C5-W (case 5). These nucleotide alterations impacted several genes, which may contribute to the morphological differences observed between the red (R) and White (W) colony phenotypes. In cases 3 and 4, where the infections were caused by strains from different genetic clades, we identified over 46,000 SNPs (\u003cstrong\u003eTable \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e). This high number of SNPs reflects the genetic divergence between the strains C3-R and C3-W, and C4-R and C4-W, as they belong to distinct genetic clades.\u003c/p\u003e\n\u003cp\u003eUsing whole genome sequences, we conducted a phylogenetic analysis of the mixed \u003cem\u003eC. auris\u003c/em\u003e strains, constructing a phylogenetic tree (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). The strains from the same genetic clades identified in this study clustered closely together, suggesting their genetic relatedness. Further phylogenetic analysis of clade III (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB) and I (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC) strains isolated in China revealed that the strains from the same clades identified in this study may share common origins. For instance, clade III strains, such as C1-R, C1-W, C2-R, C2-W, C3-W, and C4-W showed greater genetic distances from other \u003cem\u003eC. auris\u003c/em\u003e isolates from provinces like Liaoning and Beijing (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). Similarly, clade I isolates like C3-R, C4-R, C5-W, and C5-R were more genetically distant from isolates from Anhui, Jiangsu and Shandong provinces (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). Interestingly, the clade I strains isolated in this study appeared closely related to strain BJ004 from Beijing, suggesting that local transmissions may have occurred in Guangdong Province. These findings highlight the complex epidemiology and possible local dissemination of \u003cem\u003eC. auris\u003c/em\u003e strains within China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparative biological analysis of\u003c/strong\u003e \u003cstrong\u003eC. auris\u003c/strong\u003e \u003cstrong\u003estrains from mixed infections\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDistinct biological characteristics of fungal pathogens can influence their infection potential and therapeutic outcomes. We next conducted antifungal susceptibility analysis on isolates from mixed infection cases. As shown in \u003cstrong\u003eTable S2\u003c/strong\u003e, all strains, except those from case 5, displayed a minimum inhibitory concentration (MIC) of 128 mg/L for fluconazole. Strains from case 5 showed an MIC of 64 mg/L (C5-R) and 32 mg/L (C5-W) for the same drug. For amphotericin B, the 4 clade I strains (C3-R, C4-R, C5-R and C5-W) exhibited an MIC of 2 mg/L, while the 6 clade III strains had an MIC of 1 mg/L. For the other 7 antifungals (voriconazole, posaconazole, itraconazole, caspofungin, micafungin, anidulafungin, and flucytosine), all strains demonstrated relatively low MICs. According to the tentative antifungal breakpoints established by the CDC, all strains were resistant to fluconazole, and four clade I isolates were resistant to amphotericin B (\u003cstrong\u003eTable S2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eGiven the observed differences in aggregation between the strains from mixed infection (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB), we next evaluated their biofilm-forming ability. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, the white (W) isolates (C1-W, C2-W, C3-W, C4-W and C5-W) exhibited significantly stronger biofilm formation compared to the red (R) isolates (C1-R, C2-R, C3-R, C4-R and C5-R). Additionally, the clade III strains demonstrated a higher biofilm-forming capacity than the clade I strains, based on CFU analysis of the biofilms (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). These results are consistent with the differences in aggregation ability on agar plates (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003eSecreted aspartyl proteinases (SAPs) are key virulence factors in pathogenic \u003cem\u003eCandida\u003c/em\u003e species [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. We compared SAP secretion among the strains from mixed infections using YCB-BSA assays at three different temperatures. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, the strains exhibited varying SAP activities at 37\u0026deg;C, the physiological temperature of the human body. With the exception of strain C2-R, all strains showed relatively low SAP activity at 25\u0026deg;C and 30\u0026deg;C. However, some strains (C1-W, C2-R, C3-R, C4-R, and C5-R) displayed higher SAP activity at 37\u0026deg;C than the others, indicating that \u003cem\u003eC. auris\u003c/em\u003e strains isolated from the same patient can exhibit differences in SAP activity and virulence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. auris\u003c/strong\u003e \u003cstrong\u003estrains isolated from mixed infections differ in in vitro and in vivo survival abilities\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA notable characteristic of \u003cem\u003eC. auris\u003c/em\u003e is its ability to persist in the environment [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. To assess the \u003cem\u003ein vitro\u003c/em\u003e survival abilities of isolates from mixed infections, cells from each isolate were spotted on the inner surface of polypropylene Eppendorf tubes and incubated at room temperature. Colony-forming unit (CFU) assays were then performed to assess the viability of the \u003cem\u003eC. auris\u003c/em\u003e cells. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, the white isolates (C1-W, C2-W, C3-W, C4-W and C5-W) exhibited generally higher survival rates compared to the red isolates (C1-R, C2-R, C3-R, C4-R and C5-R), mirroring the pattern observed in biofilm formation. These results suggest that isolates from mixed infections may have distinct abilities to persist in the environment.\u003c/p\u003e\n\u003cp\u003eNext, we examined the susceptibility of the isolates to the human antimicrobial peptide LL-37, which plays a role in regulating skin immunity [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, strains C1-W, C2-W, C3-R, C4-R, and C5-R exhibited relatively higher survival rates in the presence of LL-37 compared to their respective counterparts.\u003c/p\u003e\n\u003cp\u003eGiven the potential impact of genetic and biological differences on virulence, we conducted fungal burden assays using strains C3-W, C3-R, C4-W, and C4-R in a mouse infection model. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e, the white (C3-W and C4-W) and red (C3-R and C4-R) isolates exhibited comparable fungal burdens in the liver, lungs, spleen, and kidneys. However, strains C3-W and C4-W demonstrated significantly higher fungal burdens in the brain compared to strains C3-R and C4-R. Taken together, isolates from mixed infections not only differ in their \u003cem\u003ein vitro\u003c/em\u003e survival abilities but also display distinct behaviors during \u003cem\u003ein vivo\u003c/em\u003e infections.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOver the past decade, the emerging fungal pathogen \u003cem\u003eC. auris\u003c/em\u003e has rapidly spread worldwide, frequently causing both inter-hospital and intra-hospital transmissions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Since the first reported case of \u003cem\u003eC. auris\u003c/em\u003e infection in China in 2018, hundreds of cases, including both screenings and infections, have been documented, suggesting multiple introductions into the country [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In this study, we report the emergence of mixed-strain infections involving \u003cem\u003eC. auris\u003c/em\u003e in Guangdong, China. These infections involved strains from two distinct genetic clades (I and III) or strains with genetic alterations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Our comparative analyses revealed notable genetic and biological diversity among isolates from the same patients.\u003c/p\u003e \u003cp\u003eA recent study from southern Nevada, USA, also reported coinfections involving clades I and III of \u003cem\u003eC. auris\u003c/em\u003e during a large outbreak [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Given that strains from clades I and III have opposite mating types (\u0026ldquo;\u003cb\u003ea\u003c/b\u003e\u0026rdquo; and \u0026ldquo;α\u0026rdquo; for clades I and III, respectively), this discovery raises the possibility of mating occurring in \u003cem\u003eC. auris\u003c/em\u003e. However, genomic analyses have shown that, aside from \u003cem\u003eSTE6\u003c/em\u003e, which encodes the a-pheromone transporter necessary for \u0026ldquo;a\u0026rdquo; cell mating [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], multiple genes involved in the mating regulation (e.g., \u003cem\u003eSTE2, STE50\u003c/em\u003e, and \u003cem\u003eFUS1\u003c/em\u003e) were mutated in \u003cem\u003eC. auris\u003c/em\u003e strains from one or more clades (data not shown). In our current study, we identified two dual-clade coinfection cases involving clades I and III, as well as three mixed infection cases caused by strains with genetic and phenotypic variations within a single clade (III or I). Unlike the previously reported dual-clade infections, where strains from different clades were isolated from separate body sites [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], we isolated all strains from the same samples in our mixed infection cases.\u003c/p\u003e \u003cp\u003eOur findings indicate that strains from mixed infections exhibit differences in several key aspects, including colony morphology, biofilm formation, SAP activity, and both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e survival capabilities. Coinfections with genetically and biologically distinct strains could pose new challenges for clinical diagnosis and treatment, as strains with different genetic backgrounds may exhibit varying abilities to colonize host or environmental niches. For example, \u003cem\u003eC. auris\u003c/em\u003e strain C2-R demonstrated enhanced secretion of the SAP virulence factor (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), which may contribute to improved skin colonization. These coinfections could potentially lead to increased virulence and varied treatment outcomes. We also observed that certain \u003cem\u003eC. auris\u003c/em\u003e strains exhibited superior environmental persistence compared to others (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). It remains to be determined whether the co-existence of genetically diverse strains impacts hospital transmission dynamics. Further investigation is warranted to understand how these mixed infections influence transmission, virulence, and treatment effectiveness in clinical settings.\u003c/p\u003e \u003cp\u003eIn summary, considering the ease of transmission and environmental persistence of \u003cem\u003eC. auris\u003c/em\u003e, along with increasing international travel, the presence of genetically diverse with the same clinical setting is plausible. Consequently, coinfections are likely to occur when multiple \u003cem\u003eC. auris\u003c/em\u003e strains of different origins are introduced into hospitals. This emerging situation could complicate the control, diagnosis, and treatment of \u003cem\u003eC. auris\u003c/em\u003e infections, posing significant challenges for healthcare management.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003eThis work was supported by the National Key Research and Development Program of China (grant no. 2021YFC2300400 to G.H., and no. 2022YFC2303000 to J.B. and H.D.), National Natural Science Foundation of China (nos. 31930005 and 82272359 to G.H., nos. 32170193 to J.B., and 82172290 to H.D.), and\u0026nbsp;Shanghai Science and Technology Innovation Action Plan 2023 \u0026ldquo;Basic Research Project\u0026rdquo; (23JC1404200 to GH). The content is the sole responsibility of the authors and does not represent the views of the funders. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e DH, BJ, CHQ and HGH designed the study. DH, HYF, GPH, LWH, ZTH, and GZY carried out sample collection and experiments. BJ and DH analyzed data. DH, BJ, and HGH wrote this manuscript. All authors contributed to and reviewed the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eNo potential conflict of interest was reported by the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eThe Genomic data has been deposited in the NCBI SRA database (SRR30963043-SRR30963052).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBing J, Du H, Guo P, et al. Candida auris-associated hospitalizations and outbreaks, China, 2018-2023. Emerg Microbes Infect. 2024;13(1):2302843.\u003c/li\u003e\n\u003cli\u003eDe Gaetano S, Midiri A, Mancuso G, et al. Candida auris Outbreaks: Current Status and Future Perspectives. Microorganisms. 2024;12(5).\u003c/li\u003e\n\u003cli\u003eChowdhary A, Jain K, Chauhan N. Candida auris Genetics and Emergence. Annu Rev Microbiol. 2023;77:583-602.\u003c/li\u003e\n\u003cli\u003eSatoh K, Makimura K, Hasumi Y, et al. Candida auris sp. nov., a novel ascomycetous yeast isolated from the external ear canal of an inpatient in a Japanese hospital. Microbiol Immunol. 2009;53(1):41-4.\u003c/li\u003e\n\u003cli\u003eSuphavilai C, Ko KKK, Lim KM, et al. Detection and characterisation of a sixth Candida auris clade in Singapore: a genomic and phenotypic study. Lancet Microbe. 2024;5(9):100878.\u003c/li\u003e\n\u003cli\u003eKhan T, Faysal NI, Hossain MM, et al. Emergence of the novel sixth Candida auris Clade VI in Bangladesh. Microbiol Spectr. 2024;12(7):e0354023.\u003c/li\u003e\n\u003cli\u003eDu H, Bing J, Hu T, et al. Candida auris: Epidemiology, biology, antifungal resistance, and virulence. PLoS Pathog. 2020;16(10):e1008921.\u003c/li\u003e\n\u003cli\u003eLockhart SR, Etienne KA, Vallabhaneni S, et al. Simultaneous Emergence of Multidrug-Resistant Candida auris on 3 Continents Confirmed by Whole-Genome Sequencing and Epidemiological Analyses. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 2017;64(2):134-140.\u003c/li\u003e\n\u003cli\u003eChowdhary A, Sharma C, Meis JF. Candida auris: A rapidly emerging cause of hospital-acquired multidrug-resistant fungal infections globally. PLoS Pathog. 2017;13(5):e1006290.\u003c/li\u003e\n\u003cli\u003eLyman M, Forsberg K, Sexton DJ, et al. Worsening Spread of Candida auris in the United States, 2019 to 2021. Annals of internal medicine. 2023;176(4):489-495.\u003c/li\u003e\n\u003cli\u003eJeffery-Smith A, Taori SK, Schelenz S, et al. Candida auris: a Review of the Literature. Clinical microbiology reviews. 2018;31(1) :e00029-17.\u003c/li\u003e\n\u003cli\u003eWelsh RM, Bentz ML, Shams A, et al. Survival, Persistence, and Isolation of the Emerging Multidrug-Resistant Pathogenic Yeast Candida auris on a Plastic Health Care Surface. J Clin Microbiol. 2017;55(10):2996-3005.\u003c/li\u003e\n\u003cli\u003eMassic L, Gorzalski A, Siao DD, et al. Detection of five instances of dual-clade infections of Candida auris with opposite mating types in southern Nevada, USA. Lancet Infect Dis. 2023;23(9):e328-e329.\u003c/li\u003e\n\u003cli\u003eDiaz Caballero J, Wheatley RM, Kapel N, et al. Mixed strain pathogen populations accelerate the evolution of antibiotic resistance in patients. Nature communications. 2023;14(1):4083.\u003c/li\u003e\n\u003cli\u003eCohen T, Wilson D, Wallengren K, et al. Mixed-strain Mycobacterium tuberculosis infections among patients dying in a hospital in KwaZulu-Natal, South Africa. J Clin Microbiol. 2011;49(1):385-8.\u003c/li\u003e\n\u003cli\u003eCohen T, van Helden PD, Wilson D, et al. Mixed-strain mycobacterium tuberculosis infections and the implications for tuberculosis treatment and control. Clinical microbiology reviews. 2012;25(4):708-19.\u003c/li\u003e\n\u003cli\u003eAsare-Baah M, Seraphin MN, Salmon LAT, et al. Effect of mixed strain infections on clinical and epidemiological features of tuberculosis in Florida. Infect Genet Evol. 2021;87:104659.\u003c/li\u003e\n\u003cli\u003eBing J, Wang SJ, Xu HP, et al. A case of Candida auris candidemia in Xiamen, China, and a comparative analysis of clinical isolates in China. Mycology-Uk. 2021;13(1):68-75.\u003c/li\u003e\n\u003cli\u003eBing J, Guan Z, Zheng T, et al. Rapid evolution of an adaptive multicellular morphology of Candida auris during systemic infection. Nature communications. 2024;15(1):2381.\u003c/li\u003e\n\u003cli\u003eLi H, Handsaker B, Wysoker A, et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25(16):2078-9.\u003c/li\u003e\n\u003cli\u003eMcKenna A, Hanna M, Banks E, et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome research. 2010;20(9):1297-303.\u003c/li\u003e\n\u003cli\u003eWang K, Li M, Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic acids research. 2010;38(16):e164.\u003c/li\u003e\n\u003cli\u003eKozlov AM, Darriba D, Flouri T, et al. RAxML-NG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics. 2019;35(21):4453-4455.\u003c/li\u003e\n\u003cli\u003eCLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. 4th ed CLSI standard M27 (Clinical and Laboratory Standards Institute, 2017). 2017.\u003c/li\u003e\n\u003cli\u003eHu T, Wang S, Bing J, et al. Hotspot mutations and genomic expansion of ERG11 are major mechanisms of azole resistance in environmental and human commensal isolates of Candida tropicalis. Int J Antimicrob Agents. 2023:107010.\u003c/li\u003e\n\u003cli\u003eTao L, Du H, Guan G, et al. Discovery of a \u0026quot;white-gray-opaque\u0026quot; tristable phenotypic switching system in candida albicans: roles of non-genetic diversity in host adaptation. PLoS Biol. 2014;12(4):e1001830.\u003c/li\u003e\n\u003cli\u003eNaglik JR, Challacombe SJ, Hube B. Candida albicans secreted aspartyl proteinases in virulence and pathogenesis. Microbiol Mol Biol Rev. 2003;67(3):400-28, table of contents.\u003c/li\u003e\n\u003cli\u003eRossato L, Colombo AL. Candida auris: What Have We Learned About Its Mechanisms of Pathogenicity? Frontiers in Microbiology. 2018;9:3081.\u003c/li\u003e\n\u003cli\u003eSpivak ES, Hanson KE. Candida auris: an Emerging Fungal Pathogen. J Clin Microbiol. 2018;56(2):e01588-17.\u003c/li\u003e\n\u003cli\u003eDurr UHN, Sudheendra US, Ramamoorthy A. LL-37, the only human member of the cathelicidin family of antimicrobial peptides. Bba-Biomembranes. 2006;1758(9):1408-1425.\u003c/li\u003e\n\u003cli\u003eMagee BB, Legrand M, Alarco AM, et al. Many of the genes required for mating in Saccharomyces cerevisiae are also required for mating in Candida albicans. Mol Microbiol. 2002;46(5):1345-51.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"infection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infe","sideBox":"Learn more about [Infection](http://link.springer.com/journal/15010)","snPcode":"15010","submissionUrl":"https://submission.nature.com/new-submission/15010/3","title":"Infection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Candida auris, emerging fungal pathogens, antifungal resistance, mixed strain infections, biofilm, virulence ","lastPublishedDoi":"10.21203/rs.3.rs-5346326/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5346326/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose\u003c/strong\u003e The multidrug-resistant fungal pathogen \u003cem\u003eCandida auris\u003c/em\u003e poses an increasing global health threat due to its high transmissibility and persistence in healthcare environments. We identified five cases of mixed\u003cem\u003e C. auris\u003c/em\u003e-strain infections in China and investigated the genetic and biological diversity of the isolates to explore the potential causes of \u003cem\u003eC. auris\u003c/em\u003e infection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e \u003cem\u003eC. auris\u003c/em\u003e isolates from 5 infection cases were distinguished by colony morphology and verified by D1/D2-ITS alignment. Phylogenetic and genomic diversity analysis of all isolates were conducted using whole genome sequences. Comparative biological analysis of all isolates, including cellular and colony morphology, antifungal susceptibility, biofilm formation, SAP activity, and both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e survival capabilities were performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eFive cases of mixed\u003cem\u003e C. auris\u003c/em\u003e-strain infections in China were identified. Comparative genomic analysis revealed these infections involved strains from two distinct genetic clades (I and III) or strains from the same clade but with genetic alterations. Comparative biological analysis demonstrated the strains from mixed infections exhibit differences in several key aspects, including colony morphology, biofilm formation, SAP activity, and both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e survival capabilities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e Comparative analyses revealed notable differences in biofilm formation, environmental survival, and secretion of virulence factors between the co-infecting strains of \u003cem\u003eC. auris\u003c/em\u003e. These biological and genetic disparities may present significant challenges for the diagnosis and treatment of \u003cem\u003eC. auris\u003c/em\u003e infections, as strains with different genetic backgrounds may exhibit varying abilities to colonize host or environmental niches.\u003c/p\u003e","manuscriptTitle":"Emergence and Characterization of Mixed Candida auris Strain Infections in China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-19 14:16:28","doi":"10.21203/rs.3.rs-5346326/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-03T15:00:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-02T04:48:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"188142197385699493370723873597073185242","date":"2024-12-24T17:39:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"318521564568484713159607217230456149559","date":"2024-12-23T09:32:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-06T19:27:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"150160742510014483164420239146254724115","date":"2024-11-16T21:46:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-03T23:08:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-29T07:30:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-29T06:06:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Infection","date":"2024-10-28T10:26:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"infection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infe","sideBox":"Learn more about [Infection](http://link.springer.com/journal/15010)","snPcode":"15010","submissionUrl":"https://submission.nature.com/new-submission/15010/3","title":"Infection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ab5f0bba-bb7c-4dda-83c6-06fa41963e4a","owner":[],"postedDate":"November 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-21T16:00:06+00:00","versionOfRecord":{"articleIdentity":"rs-5346326","link":"https://doi.org/10.1007/s15010-025-02536-6","journal":{"identity":"infection","isVorOnly":false,"title":"Infection"},"publishedOn":"2025-04-14 15:57:24","publishedOnDateReadable":"April 14th, 2025"},"versionCreatedAt":"2024-11-19 14:16:28","video":"","vorDoi":"10.1007/s15010-025-02536-6","vorDoiUrl":"https://doi.org/10.1007/s15010-025-02536-6","workflowStages":[]},"version":"v1","identity":"rs-5346326","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5346326","identity":"rs-5346326","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}