The interplay between antifungal resistance, biocide tolerance, and virulence expression in Candidozyma (Candida) auris represents a critical challenge for infection control and patient management. In this study, we explored phenotypic patterns across clinical isolates, focusing particularly on biofilm production, esterase activity, and susceptibility to fluconazole and voriconazole. Candidozyma auris demonstrates complex resistance phenotypes to both antifungals and biocides, supported by biofilm formation, virulence factor expression, and environmental adaptability. Control of this pathogen requires a multi-pronged strategy that incorporates targeted biocide use, environmental decontamination, and careful screening of colonized patients. Research into the molecular mechanisms driving resistance, especially within dry-surface biofilms, will enhance our ability to select effective disinfectants. Infection control policies must evolve to include dynamic resistance monitoring and outbreak-tailored decolonization regimens. Only through such integrated strategies can we hope to curb the global spread of this tenacious fungal pathogen. [26, 27]
Spearman correlation analysis was conducted to assess potential associations between antifungal susceptibility, biocide tolerance, and virulence phenotypes in Candida auris. A comprehensive heatmap revealed generally weak correlations across most variables. However, several statistically significant associations emerged with moderate effect sizes (ρ > 0.3, p < 0.05). Notably, amphotericin B resistance correlated positively with isavuconazole MICs (ρ = 0.32, p = 0.028), suggesting a potential shared resistance mechanism or co-selection within this isolate set. A similar association was found between amphotericin B and TRC MIC values (ρ = 0.35, p = 0.018), indicating a possible link between polyene resistance and decreased susceptibility to certain biocides. Furthermore, amphotericin B resistance also demonstrated a statistically significant correlation with caseinase activity (ρ = 0.31, p = 0.035), suggesting a connection between antifungal tolerance and proteolytic virulence traits. Among azoles, itraconazole MICs were positively correlated with chlorhexidine (CHX) MICs (ρ = 0.32, p = 0.030), while caspofungin a representative echinocandin showed a moderate positive correlation with benzalkonium chloride (BNZ) MICs (ρ = 0.39, p = 0.007). These findings may reflect cross-resistance patterns involving alterations in the fungal cell membrane or efflux-mediated tolerance mechanisms. Interestingly, no strong correlation was found between fluconazole and CHX MICs (ρ = 0.12), or between fluconazole and combined biocide elevated MICs (mean ρ = 0.23), contradicting previously reported associations. Likewise, correlations between virulence factors such as biofilm and esterase production with fluconazole or TRC MICs were negligible (ρ < 0.3), indicating these phenotypes alone are not predictive of antifungal or biocide resistance.
In summary, the analysis suggests that while general cross-resistance trends across antifungals and biocides are limited, certain drug pairs particularly amphotericin B, TRC, and BNZ may share susceptibility profiles, potentially driven by common cellular targets or adaptive responses. The observed link between amphotericin B resistance and caseinase activity further supports a potential interplay between virulence and resistance. These relationships warrant further functional and mechanistic studies to elucidate the underlying molecular pathways and their relevance to clinical management.
Candidozyma (Candida) auris has emerged as a critical healthcare-associated pathogen, largely due to its ability to persist in the hospital environment and resist both antifungal drugs and common disinfectants. Eventhough, we could not conclude any biocide resistance, previously we showed that this species resistant to several biocides frequently used in healthcare, particularly quaternary ammonium compounds (QACs) such as BNZ, and chlorhexidine-based solutions. [16] These agents, although routinely applied for surface or skin decontamination, often fail to eradicate C. auris, especially when it exists within biofilms. Environmental surfaces in hospitals, including medical equipment and patient surroundings, can serve as reservoirs for persistent colonization. Biofilm-forming strains of C. auris further increase the difficulty of eradication by reducing the efficacy of standard disinfection practices. [28] This pathogen’s robust ability to colonize dry surfaces has been associated with hospital outbreaks, demonstrating that environmental contamination plays a central role in its transmission. [29] Therefore, understanding biocide tolerance mechanisms and optimizing disinfection strategies is crucial to reduce the spread of C. auris within clinical settings.
Biofilm formation is a fundamental virulence strategy of C. auris, which not only protects the cells from antifungal agents but also contributes significantly to biocide tolerance. [30, 31] Dry surface biofilms have recently been recognized as a particularly concerning phenotype, capable of persisting despite treatment with sodium hypochlorite [28] Transcriptomic analyses of these biofilms reveal upregulation of efflux pumps and iron acquisition systems, contributing to the organism's metabolic adaptation and biocide resistance. This mechanism parallels the behavior seen in bacterial biofilms and emphasizes that biofilm-mediated resistance in fungi should be evaluated more rigorously. Clinical isolates have demonstrated planktonic susceptibility but formed highly tolerant biofilms on dry abiotic surfaces, particularly under low-nutrient or organic-rich conditions. [32] The biofilm matrix is believed to reduce biocide penetration and promote the survival of embedded cells, especially during intermittent or low-level disinfection. Consequently, surface disinfection protocols must address both planktonic and biofilm forms of C. auris. The data underscore the limitations of chlorine-based disinfectants alone and suggest that alternative agents or combinations, such as povidone-iodine or hydrogen peroxide vapor, may be more effective in biofilm disruption. [33]
Virulence attributes such as phospholipase production, esterase activity, hemolysis, and aggregative phenotype may contribute to environmental persistence and biocide tolerance in C. auris. Studies have shown that isolates with high biofilm-forming potential often co-express these virulence factors, which may reinforce the integrity of the biofilm and shield cells from chemical insults. [16, 32] It is plausible that the extracellular matrix produced in strong biofilm-forming strains also retains or neutralizes biocides before they can exert lethal effects. In some models, aggregative phenotypes exhibited both enhanced biofilm robustness and decreased susceptibility to hydrogen peroxide or CHX. [32] These phenotypic traits could thus serve as biomarkers to predict disinfection outcomes. Further studies correlating specific virulence factors with biocide MIC values will be crucial in refining infection control protocols, especially in outbreak situations. Understanding these links can also assist in designing targeted surface decontamination approaches in high-risk wards.
Candida auris exhibits a wide array of virulence factors that contribute to its pathogenic potential in clinical settings. One of the most prominent virulence traits is its ability to form robust biofilms on both biotic and abiotic surfaces. These biofilms offer protection against antifungal agents and the host immune response, thus facilitating persistent infections. [34] In a study involving Turkish clinical isolates, all seven tested C. auris strains demonstrated moderate to strong biofilm-forming capacity. Interestingly, while biofilm positivity was consistent, the expression of other enzymatic virulence traits such as proteinase and hemolysis was entirely absent, indicating a strain-specific virulence phenotype. The phenotypic profile of these isolates suggests that biofilm formation may be a dominant and conserved virulence mechanism across different geographic regions. [34] Moreover, the weak activity of phospholipase and esterase among Turkish isolates contrasts with the strong enzyme production commonly seen in C. albicans, emphasizing species-specific virulence adaptations. Understanding these phenotypic patterns may aid in identifying more effective treatment approaches and infection control strategies.
The phenotypic variability in C. auris virulence traits is further supported by studies that used in vitro assays to assess enzymatic and adhesion properties. Li et al. [30] demonstrated that untreated C. auris isolates show high levels of adhesion to abiotic surfaces, elevated cell surface hydrophobicity (CSH), and considerable biofilm mass as determined by XTT and violet crystal assays. These phenotypes were significantly reduced following exposure to baicalein, a natural compound that disrupted both membrane integrity and gene expression associated with virulence. [30] In untreated conditions, flocculation tests and hydrophobicity assays revealed consistent aggregation behavior across multiple strains, supporting the high virulence potential of these isolates. Furthermore, scanning electron microscopy visualized dense, multilayered biofilms with strong surface adhesion, confirming the phenotypic positivity of these virulence traits. The transcriptomic data supported these findings by showing downregulation of adhesion and biofilm-associated genes following baicalein exposure, indicating a direct link between gene regulation and phenotypic expression. Overall, these results illustrate that adhesion, CSH, and biofilm formation are strongly positive phenotypes in most C. auris isolates.
A novel diterpenic compound derived from Streptomyces chrestomyceticus has also been shown to suppress key virulence factors in C. auris. Singh et al. [31] found that subinhibitory concentrations of this compound (55B3) significantly inhibited biofilm biomass, yeast-to-hyphae transition, phospholipase B secretion, and aspartyl proteinase activity. While the hyphal switch is not a predominant feature of C. auris compared to C. albicans, phenotypic tests confirmed detectable levels of phospholipase and protease activity in some isolates. These virulence markers were suppressed by 55B3 in a dose-dependent manner, with strong biofilm inhibition observed even at early developmental stages. Microscopic observations further revealed structural disruption in the biofilm matrix and a reduction in fungal metabolic activity. This phenotypic inhibition was not accompanied by cytotoxicity in mammalian cell lines, suggesting therapeutic potential. Importantly, the ability to phenotypically detect these virulence traits and monitor their modulation provides valuable insight into the multifactorial pathogenesis of C. auris and identifies targets for antifungal development.
Despite the detection of multiple virulence factors, the extent and consistency of their expression vary across C. auris strains. As reviewed by Gómez-Gaviria et al. [35], biofilm formation remains the most reliably detected positive trait, while phenotypic switching and dimorphism (yeast-to-hyphal or pink-white transitions) are less prominent but still documented under specific environmental cues. These phenotypes enable C. auris to adapt rapidly to changes in host environments and antifungal pressure. Additionally, secreted exoenzymes such as phospholipase and hemolysin have been inconsistently reported; in some isolates, these activities are undetectable, suggesting a partial or clade-specific expression. The presence or absence of these traits may affect tissue invasion potential and immune evasion. These findings underscore the importance of performing comprehensive phenotypic profiling when evaluating strain virulence. Accurate assessment of trait positivity or negativity may assist in epidemiological mapping and inform the design of targeted antifungal or anti-virulence therapies.
Several studies highlight variability in the biocidal susceptibility of C. auris, dependent on concentration, formulation, and contact time of the agent. For example, while high concentrations of chlorine-based disinfectants achieve acceptable levels of reduction, lower concentrations may not be effective, especially in the presence of organic load. [16, 36] Similarly, CHX demonstrates inconsistent activity, with some studies reporting suboptimal reduction even at concentrations typically used for hand hygiene and skin antisepsis. [33] Octenidine-based solutions appear more reliable, showing stronger fungicidal activity within shorter exposure times. The inconsistent susceptibility of C. auris strains particularly across different clades further complicates infection control strategies, suggesting that disinfectant efficacy testing must be tailored for each institutional setting. [16] This is especially important in high-risk environments such as ICUs, where the potential for nosocomial transmission is heightened by environmental persistence. Regulatory frameworks should thus mandate biocide efficacy validation not only against standard test organisms like C. albicans, but also against biofilm-forming C. auris isolates. [36]
Although the overlap between antifungal and biocide resistance is still under investigation, some evidence suggests that shared mechanisms such as efflux pump upregulation may contribute to cross-resistance. ABC transporters, which are known to mediate azole resistance, may also expel biocidal compounds like QACs or CHG from fungal cells. [28] Additionally, genomic analyses have indicated that prolonged environmental exposure to sub-inhibitory concentrations of biocides might select for strains with increased drug tolerance. [37, 38] However, this relationship is not uniform across all strains, and current data do not consistently demonstrate a direct link between antifungal resistance and biocide insensitivity. Still, the potential for cumulative adaptive responses especially in clonal strains within outbreak settings warrants cautious interpretation. Comprehensive surveillance programs should thus monitor both antifungal and biocide susceptibility profiles concurrently. [39]
Persistent environmental contamination by C. auris significantly elevates the risk of healthcare-associated infections. This pathogen’s capacity to survive on surfaces for up to several weeks, combined with its high transmissibility, positions it among the most environmentally stable fungal agents currently known. Consequently, enhanced cleaning protocols must include validated surface disinfectants active against biofilm forms, as well as proper contact time and mechanical cleaning. Additionally, routine environmental screening and decontamination of high-touch surfaces are essential in outbreak control. The implementation of no-touch disinfection methods such as UV-C light or hydrogen peroxide vapor may complement manual cleaning, particularly in patient turnover situations. [40, 41]
Another major consideration is the impact of host colonization and skin decontamination protocols on outbreak management. Unlike many Candida species that colonize mucosal sites, C. auris preferentially colonizes the skin, which may explain its higher rates of environmental dissemination. [42, 43] Standard decolonization agents like 2–4% chlorhexidine have shown limited efficacy against skin-colonizing C. auris, particularly in its biofilm form. [40] In contrast, povidone-iodine preparations may offer superior efficacy, although patient tolerance and widespread availability may limit their routine use. Newer formulations such as continuously active disinfectants or antiseptic-impregnated wash mitts with octenidine may prove beneficial. [33, 42] A tailored approach to patient decolonization, taking into account resistance profiles and colonization burden, will be critical in reducing nosocomial transmission.
Haq et al. reported the effectiveness of a novel one-step anionic surfactant disinfectant containing dodecylbenzene sulfonic acid against multiple clades of C. auris, including isolates from clades I–IV. Compared to traditional quaternary ammonium compounds, which showed limited efficacy, this new disinfectant demonstrated promising activity and practical advantages such as rapid action and minimal residue. However, certain clades have shown reduced susceptibility to commonly used agents such as SHC and UV-C light, underscoring the role of phylogenetic diversity in resistance patterns. [44] Supporting these findings, Cadnum et al. [45] found that sporicidal and hydrogen peroxide-based products were significantly more effective against C. auris than quaternary ammonium-based disinfectants. This is particularly concerning given that many healthcare settings continue to rely on such less effective agents. The review by Omardien and Teska [46] adds that C. auris biofilms, which are commonly formed on skin and surfaces, further complicate disinfection. These biofilms can shield fungal cells from disinfectants like CHX and hydrogen peroxide, reducing their efficacy. The biofilm matrix can trap antifungal molecules such as fluconazole, rendering them ineffective. In this context, the tolerance of C. auris to environmental stressors including high salinity and desiccation further enhances its persistence and dissemination in healthcare facilities. Ku et al. [47] emphasized that although chlorine-based products remain among the most effective for surface disinfection, the addition of mechanical cleaning protocols is often necessary for thorough decontamination. Moreover, persistent colonization of patients despite body washes with CHX calls into question the long-term efficacy of biocidal strategies alone. Taken together, these findings suggest that C. auris resistance to biocides is multifactorial, influenced by clade-specific genetic characteristics, biofilm formation, and environmental adaptability. The variability in susceptibility across different biocides and clades highlights the importance of localized surveillance and clade identification in shaping effective infection control policies.
Candida auris is now recognized as a global fungal threat due to its multidrug resistance and persistence in healthcare environments. The earliest Japanese isolates, primarily derived from non-invasive ear discharge samples, belonged to clade II and showed relatively low resistance rates only 20% were resistant to fluconazole, while all were susceptible to echinocandins and amphotericin B. [48] These findings suggest that clade II strains, which are common in East Asia, may inherently carry lower antifungal resistance burdens. However, surveillance is still essential as clade shifts or horizontal transmission could introduce resistance elements. The same study emphasized the absence of resistance to echinocandins, making them a therapeutic mainstay in this region. In contrast, other global clades have demonstrated far higher resistance profiles, indicating that regional strain typing is essential for guiding therapy. Thus, antifungal susceptibility in C. auris is clade-dependent, reinforcing the need for local epidemiological data.
In Russia, a large-scale study of 112 clinical C. auris isolates revealed a markedly different antifungal resistance profile. Nearly all isolates (111 of 112) were fluconazole-resistant, and 17% exhibited resistance to amphotericin B. [49] The prevalence of azole resistance is particularly alarming given the widespread empirical use of fluconazole, especially in intensive care settings. The high resistance rates suggest that clade I strains, which were dominant in this cohort, have likely acquired multiple resistance mechanisms, possibly through antifungal pressure in nosocomial environments. Furthermore, 3.6% of the isolates were also resistant to flucytosine, and a small fraction demonstrated reduced susceptibility to echinocandins. This multidrug resistant phenotype mirrors global observations where clade I strains are associated with hospital outbreaks and treatment failure. The concurrent phospholipase activity found in many of these isolates also implies that resistance may be coupled with high virulence, exacerbating clinical outcomes. [49]
The Italian experience further underscores the challenges of treating C. auris infections with standard antifungal regimens. A case of candidemia in a critically ill ICU patient in Southern Italy revealed high clonality between isolates from two different patients, suggesting nosocomial transmission. [50] Both strains belonged to clade I and demonstrated elevated MICs to fluconazole and borderline susceptibility to amphotericin B, consistent with CDC tentative breakpoints. Interestingly, despite the administration of caspofungin during empiric therapy, the patient’s condition deteriorated rapidly, suggesting that echinocandin susceptibility may not always translate to clinical success. Environmental surveillance in the ICU also detected C. auris DNA on nearly half of high-touch surfaces, emphasizing the environmental persistence and potential for reinfection. These findings support the inclusion of antifungal resistance screening in infection control measures and suggest a need for environmental decontamination strategies to accompany clinical treatment.
A significant factor complicating antifungal therapy in C. auris infections is the variability in susceptibility testing methods and interpretation. Arendrup et al. [51] compared CLSI, EUCAST, and commercial strip-based methods (Etest, MTS) and found significant discrepancies in amphotericin B resistance categorization. While CLSI and EUCAST broth microdilution showed consistent modal MICs near 1 mg/L, strip-based methods often yielded higher MICs, sometimes classifying isolates as resistant when broth methods did not. This inconsistency reflects methodological artifacts and emphasizes the need for standardized interpretive criteria specific to C. auris. Moreover, some isolates demonstrated bimodal MIC distributions with certain testing methods, suggesting underlying heterogeneity in drug susceptibility even within clades. As no official clinical breakpoints exist for many antifungals against C. auris, clinicians must rely on tentative thresholds and contextual data to guide therapy. These differences in susceptibility interpretation may lead to either overestimation or underestimation of resistance rates, affecting treatment outcomes. Taken together, the antifungal susceptibility profile of C. auris is complex and shaped by geographic, clade-related, and methodological variables. While some clades like clade II retain broad susceptibility to echinocandins and amphotericin B, others such as clade I show multidrug resistance, including to azoles and occasionally amphotericin B. [52] The presence of biofilm formation and hydrolytic enzyme activity in resistant strains may further complicate eradication and treatment. Testing method discrepancies must also be resolved to ensure accurate resistance detection. Overall, integrated surveillance, standardized testing, and molecular typing are indispensable for guiding therapy and outbreak control strategies. Future antifungal development and stewardship programs must account for the evolving resistance landscape of C. auris, particularly in high-burden settings. [53]
Studies from Türkiye reporting C. auris infections are rising in recent years. [16, 34, 54–57] However biocide susceptibility was evaluated in just two of them. [16, 34] Our current study representing Turkish isolates, however has several limitations that should be acknowledged. First, all 47 isolates analyzed belonged exclusively to Clade 1, which restricts the generalizability of the findings across other C. auris clades. However, Clade 1 remains the most globally prevalent and clinically relevant clade, particularly in outbreak scenarios, and our findings contribute to filling the gap in region-specific data from Turkey, where such clade-based susceptibility data are limited. The phenotypic diversity observed in virulence and susceptibility traits may differ in isolates from Clades II–V and the recently described African genotype. Second, biocide resistance interpretations were based on epidemiological cutoff values (ECOFFs) extrapolated from C. albicans, as standardized biocide breakpoints for C. auris are currently lacking. This may lead to potential misclassification of resistance. But it enables standardized comparison and has been adopted in other peer-reviewed studies in the absence of formal CLSI or EUCAST guidelines for biocides. Third, virulence assessment relied primarily on in vitro assays, which may not fully replicate host-pathogen interactions in vivo. Additionally, all analyses were based on phenotypic methods without integrating genomic resistance or virulence markers, which limits the mechanistic insight into observed correlations. Nonetheless, standardized phenotypic assays such as esterase, phospholipase, and biofilm production remain valuable and widely accepted surrogates for virulence screening in initial pathogenesis studies. Future research integrating in vivo models and genomic analyses is warranted and already planned as a follow-up to this work. Lastly, although correlations between antifungal and biocide MICs were explored using non-parametric methods, the study design was observational and hypothesis-generating; causal relationships cannot be inferred. While our correlation analysis revealed statistically significant associations between antifungal/biocide resistance and virulence factors, we clearly stated that the design was exploratory and does not support causal inference. We hope that by openly discussing these limitations, we have provided sufficient transparency while reinforcing the relevance and applicability of our findings in understanding the evolving threat of Clade 1 C. auris in clinical settings.