Vol.: (0123456789) Mycopathologia (2026) 191:63 https://doi.org/10.1007/s11046-026-01082-5

ORIGINAL ARTICLE

Infection by Different Clades of Candidozyma auris in a Galleria mellonella Model: Determining Virulence Levels Gabriel Davi Marena · Alejandro Lopez · Gabriela Corrêa Carvalho · Javier Pemán · Jose Manuel Perez‑Royo · Victor Garcia‑Bustos · Ángel González   · Paula Muñoz Brell · María Dolores Pérez Ruiz · Lara Zaragoza Macian · Carmen Vicente Saez · Antonia Avalos Mansilla · Tais Maria Bauab · Marlus Chorilli · Alba Ruiz‑Gaitán Received: 1 April 2025 / Accepted: 8 May 2026 © The Author(s) 2026 Abstract Introduction  Candidozyma auris (formerly Candida auris) is an emerging yeast that causes bloodstream infections, especially in immunocompromised patients, and presents high resistance and virulence rates. To date, six clades have been established worldwide and the number of outbreaks caused by this microorganism has been increasing every year, causing concern in the medical community. Therefore, this study investigated the heterogeneity among clades of C. auris by evaluating the virulence profile and mechanism of infection using an in vivo model of Galleria mellonella.

Methods  G. mellonella was infected with different clades (I, II, III and IV) of C. auris, C. albicans ATCC 5341 and C. parapsilosis ATCC 22019 for virulence and histopathologic evaluation. Results  Aggregative strains of C. auris InP13 (I) and VEN C6 (IV) had a greater rate of melanization and larval mortality among the C. auris isolates, therefore, being the most aggressive strains. C. albicans caused the most melanization among all strains at the highest inoculum concentration ­(106 cells/mL). Histopathologic examination showed a greater number of granulomas in the lower and upper extremities of G. mellonella. The granulomas ranged from 0.07–

0.11  nm in diameter. All strains showed biofilms

adhering to larval tissue, which was more evident for InP13, VEN C6 and C. albicans. Infiltration of tissues by yeasts, pseudohyphae and chlamydospores (a Handling Editor: Sudha Chaturvedi.

G. D. Marena · A. Lopez · J. Pemán · J. M. Perez‑Royo ·

V. Garcia‑Bustos · A. Ruiz‑Gaitán (*)

Health Research Institute La Fe, Avenida Fernando Abril Martorell, 106 Torre A 6a Planta, 46026 Valencia, Spain e-mail: albacruiz@gmail.com G. D. Marena · G. C. Carvalho · M. Chorilli Department of Drugs and Medicines, School of Pharmaceutical Sciences, São Paulo State University (UNESP), Araraquara, São Paulo 14800‑903, Brazil G. D. Marena · G. C. Carvalho · T. M. Bauab School of Pharmaceutical Sciences, Department of Biological Sciences, São Paulo State University (UNESP), Araraquara, São Paulo 14800‑903, Brazil G. C. Carvalho Federal University of Mato Grosso Do Sul (UFMS), Faculty of Pharmaceutical Sciences, Food and Nutrition (FACFAN), Campo Grande, Brazil J. Pemán · P. M. Brell · A. Ruiz‑Gaitán Department of Medical Microbiology, University and Polytechnic La Fe Hospital, Valencia, Spain Á. González (*) Basic and Applied Research Group (MICROBA), School of Microbiology, University of Antioquia, Medellín, Colombia e-mail: angel.gonzalez@udea.edu.co M. D. P. Ruiz · L. Z. Macian · C. V. Saez · A. A. Mansilla Department of Pathological Anatomy, La Fe Hospital, Valencia, Spain

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Page 2 of 15 Vol:. (1234567890) resistance structure formed by C. albicans in stress environments) morphotypes were observed. Conclusion  The aggregative strains were more virulent and had a greater ability to form biofilms and granulomas, showing heterogeneity among the different C. auris clades.

Keywords  Candida auris · Candidozyma auris · Galleria mellonella · Infection · Virulence · Granulomas · Biofilms Introduction Candidozyma auris (formerly Candida auris) is an emerging, pathogenic, and multidrug-resistant yeast known to cause systemic infections and outbreaks, with high mortality rates in patients with comorbidities or those undergoing intensive care. However, C. auris infections have undergone drastic changes, since these outbreaks are increasing every day, and the number of patients who have lost their lives is worrying. Once installed in a healthcare facility,

C. auris persists for a long time, is highly resist-

ant to antifungal therapy and disinfectants, and rapidly spreads among susceptible patients [1, 2]. In 2025, the European Centre for Disease Prevention and Control (ECDC) published a report highlighting the worrying increase in C. auris cases in European hospitals, posing a serious threat to patients and the healthcare system. The report emphasizes the importance of early diagnosis and transmission control to prevent rapid and widespread spread [3]. Currently, epidemiology suggests six distinct clades of C. auris: Clade I isolated in South Asia, Clade II from in East Asia, Clade III in South Africa, and Clade IV isolated in South America. The clade V has already been reported in Iran and is characterized by approximately 200,000 nucleotides that differ from the other clades. According to this information, there is heterogeneity among C. auris species, which can be justified by geographic location and recurrent climate changes [4]. Finally, and more recently, clade VI was detected and sequenced in Singapore in 2023 [5]. The antifungal resistance and susceptibility profile has been observed to be variable. Furthermore, difficult identification by conventional and molecular methods, resistance to common disinfectants, unclear dissemination mechanisms, and uncertain environmental niches are considered factors that hinder the control of C. auris worldwide [6, 7]. Moreover, virulence mechanisms such as the ability to form biofilms, surface adhesion or phospholipases and proteinases production are of concern to researchers worldwide [8, 9].

An in vivo model using Galleria mellonella has become one of the most widely used and effective assays in antimicrobial and infection studies. With similar characteristics to mammalian cells and their immune response, the G. mellonella model aids in antimicrobial discovery by evaluating compounds and the mechanism of pathogens infection through virulence and immunological assays [10–13]. An additional feature of this insect is melanin production: the enzyme phenol oxidase oxidises catecholamines (e.g., L-DOPA) to quinones that polymerise into melanin, a dark pigment that contributes to immune defence. Melanin encapsulates and immobilises fungi, restricting gas exchange and nutrient access, and can ultimately kill the microorganism [14].

Therefore, this study evaluated the virulence potential and infection mechanism of four clinical isolates belonging to clades I, II, III, and IV of C. auris in a G. mellonella model.

Material and Methods Fungal Strains Four clinical strains were assayed: C. auris VPCI479/ P13 (CLADE I – InP13), C. auris CBS10913 (CLADE II – JAP 1), C. auris CBS 15603 (CLADE III – SP96), C. auris VEN C6 (CLADE IV – VEN C6); in addition, two reference strains of Candida species were also included: C. albicans SC 5134 and C. parapsilosis ATCC 22019.

Aggregation Assays An aliquot of colony forming units of each strain was transferred into a 0.85% saline solution to observe the presence or absence of yeast clusters under an optical microscope.

Mycopathologia (2026) 191:63 Page 3 of 15  63 Vol.: (0123456789) Virulence Assays An in vivo model of G. mellonella (250–350 mm in height) was used for the virulence test. The larvae were first separated into groups of 20 and kept in a petri dish at 37 °C for 24 h for environmental adaptation. Before infection, a sterile toothpick containing 70% alcohol was used to clean the proleg region, the area where the yeast inoculum would be injected. Inoculum of each Candida spp. was standardised to a final concentration of ­104, ­105 and ­106 cells/larvae in a PBS solution containing 20  µg/mL ampicillin (Sigma Aldrich, Steinheim, North Rhine-Westphalia, Germany), as described by Mesa-Arango et al. [15]. Then, using a Hamilton Microliter™ syringe, 10 µL of each Candida ssp. inoculum was injected into the penultimate proleg of each larva and incubated

at 37  °C (20/group). Every 24  h of incubation, for

10 days, dead larvae were quantified. The experiment

was performed in triplicate and on different days. Evaluation of Melanization Process The melanization assay followed the infection model described above and was evaluated for 10  days. Each larva was observed individually and compared with the uninfected control group (cream colour, no changes). Melanization was quantified as a percentage based on larval colour, as shown in Fig. 1, and according to the protocol of Garcia-Bustos et al. [16]. The inoculum concentrations evaluated were ­104, ­105, and ­106 cells/larva. However, ­104 cells/larva did not cause melanization and is therefore not shown in the graph.

Histopathological Analysis To carry out the histopathology analysis, larvae were inoculated with ­104 cells/larvae of each C. auris isolate and with ­105 cells/larvae for non-C. auris strains and kept at 37ºC. Two larvae were collected 120  h after infection, euthanized in 5% ethanol, and placed into tubes containing 10  mL of 4% formalin for 20 days to preserve the tissue and fixation. thereafter, Fig. 1   Melanin level scale in a model of Galleria mellonella infected with Candida spp

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Page 4 of 15 Vol:. (1234567890) larvae were cut sagittal and fixed in paraffin. Hematoxylin–eosin (HE) staining was performed for the morphological analysis of organelles, granulomas, and cellular infiltration Periodic acid-Schiff staining (PAS) was performed to assess tissue morphology, granulomas, yeast morphology, and yeast infiltration into tissues. Uninfected larvae were processed similarly for comparative purposes. One infected larva of each yeast strain was selected for analysis in a Philips IMS Scanners to observe the regions with the highest number of granulomas. The mean diameter of the granulomas was measured using a Slide Viewer program.

Ethical Statement Galleria mellonella is not classified as an animal under current European and international regulations, and therefore its use does not require ethical approval by institutional animal care and use committees. This model provides a valuable, ethical, and high throughput platform for preliminary in vivo testing, helping to reduce the use of vertebrate animals in line with the 3Rs principles (Replacement, Reduction, and refinement) in research.

Statistical Analysis For virulence/survival analysis we used One-way analysis of variance (ANOVA) was used for comparison between infected and uninfected control groups using GraphPad Prism 8.0.

Results Aggregative and Non‑aggregative Phenotype Determination of C. auris Isolates Before the biological tests, microscopic analysis was carried out to determine the aggregative and nonaggregative strains and, according to the analysis, it was possible to determine that the aggregative strains were clade I, III and IV (InP13, SP96 and VEN C6, respectively) with the JAP 1 isolate (Clade II) being the only non-aggregative one.

Candida auris Clades I and IV Appear to be More Lethal for G. mellonella Larvae As shown in Fig.  2, the aggregative isolates InP13, SP96 and VEN C6 were the ones that caused the highest mortality rate and were considered the most virulent (p < 0.0001). Among the aggregative isolates, InP13 was the most virulent and its mortality rate was directly related to an increase in yeast concentration. The mortality rate in larvae infected with InP13 was greater than 50% after 7, 9, and 9 days postinfection when inoculum ­106, ­105 and ­104 yeasts/larva were used, respectively. After 10 days of infection, mortality was 83.9, 90.5 and 100% in groups infected with inoculum ­104, ­105 and ­106 yeasts/larvae, respectively (p < 0.0001).

VEN C6 (clade IV) was the second most virulent strain among C. auris strains, causing mortality greater than 50% on days 8, 9, and 9 postinfection when inoculum of ­106, ­105, and ­104 yeasts/larva were used, respectively. After 10 days of infection, mortality was 84.3, 90.5, and 100% in groups infected with inoculum ­104, ­105, and ­106 yeasts/larvae, respectively. SP96 (clade III, aggregative) and JAP 1 (clade II, non-aggregative) were the least virulent strains, with JAP 1 having the lowest mortality. After 10 days of infection with SP96, mortality was 50.9, 85.6, and 95.3% in groups infected with inoculum ­104, ­105, and ­106 yeasts/larvae, respectively. For the group infected with JAP 1, the mortality rate after 10 days was 43.6, 43.3, and 89.2% with inoculum ­104, ­105, and ­106 yeasts/larvae, respectively.

Furthermore, C. albicans 5314 caused the highest number of larval deaths, with an inoculum of ­106 yeast/larva. After 10 days of infection, mortality was 28.8, 48.5, and 100% in groups infected with inoculums ­104, ­105, and ­106 yeast/larvae, respectively. C. parapsilosis causes lower mortality at ­106 yeasts/ larva, with approximately 30% lethality after 10 days of infection.

Melanization Process in G. mellonella did not Differ Among the Different Strains (Belonging to Clades I‑IV) of C. auris Figure 3 shows the results of the melanization assay and as observed, there was no evidence of melanin levels for the inoculum of ­104 yeasts/larva with any of the clinical isolates. However, the injection of

Mycopathologia (2026) 191:63 Page 5 of 15  63 Vol.: (0123456789) Fig. 2   Survival analysis of G. mellonella larvae infected with Candida spp. A G. mellonella larvae inoculated with ­104; B ­105; and C ­106 yeasts/larva. InP13 (clade I): C. auris VPCI479/P13; JAP 1 (clade II): C. auris CBS10913; SP96 (clade III): C. auris

CBS 15603; VEN C6

(clade IV): C. auris VEN C6; Ca 5314: C. albicans ATCC 5314; Cp 22019: C. parapsilosis ATCC 22019; PBS + AmP: Phosphate Buffered Saline + Ampicillin at 20 µg/mL. (*): p < 0.05; (**): p < 0.005; (***): p < 0.001; (****): p < 0.0001

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Page 6 of 15 Vol:. (1234567890) ­105 yeasts/larva caused a total of 13% (C. parapsilosis) 15.2% (C. albicans), 18.5% (VEN C6), 19.5% (InP13), 21% (JAP 1) and 21% (SP96) of melanization in the larval body (Fig. 3A). However, there was no statistical difference between the infected groups, only compared to the group treated with PBS + AmP, with a p < 0.05 (Fig.  3A). Noteworthy, it was observed that the ­106 yeasts/larva inoculum, as shown in Fig. 3B, the group infected with

C. albicans showed 49.5% of melanization at the

end of the period evaluated, being the species that caused the most melanization process (p < 0.0001). On the other hand, JAP 1 was the strain with least melanization, with 31% (p = 0.0121). Finally, larvae infected with the higher inoculum ­(106 cells) of VEN C6, C. parapsilosis, InP13, and SP96 showed 36.5, 37, 37.5, and 42% of melanization, respectively, with a significant difference compared to the uninfected group (p < 0.0001).

Histological Analysis of Galleria mellonella Figures  4 and 5 show the histological analysis of G. mellonella infected with Candida spp. Fig. 3   Melanin production in G. mellonella infected with Candida spp. A: ­105 yeasts/larvae; B: ­106 yeasts/ larvae; InP13: C. auris

VPCI479/P13; JAP 1: C.

auris CBS10913; SP96: C.

auris CBS 15603; VEN C6:

C. auris VEN C6; Ca 5314:

C. albicans ATCC 5314; Cp 22019: C. parapsilosis ATCC 22019; PBS + AmP:

Phosphate Buffered Saline + Ampicillin at 20 µg/mL. (*): p < 0.05; (****): p < 0.0001

Mycopathologia (2026) 191:63 Page 7 of 15  63 Vol.: (0123456789) Tissue sections were stained with HE and PAS, respectively.

The images show the development of yeast clusters (Y) surrounded by haemocytes (H), forming granuloma structures throughout the larval tissue. During infectious and inflammatory processes, large amount of melanin is produced, creating orange/ brown areas (M). Granulomas are found throughout the larva body and are more abundant in the lower extremities (tail) and the upper extremities (head). Larvae infected with InP13 showed granulomas in their tissues with an intense immunological response; moreover, these structures were surrounded by adipose tissue in the area between the upper and lower intestines, known as the transitory region (Fig.  4A–B). InP13 isolate produced yeast infiltration in the organelles, which was caused by an intense inflammatory response and adipose tissue in the lower intestine (Fig. 5A–B). Furthermore, yeasts with defined morphologies stood out, with intense budding and hyphae/pseudohyphae formation were observed (Fig.  5B). JAP 1 isolate caused granulomas in different regions of the larval body, particularly in the head (Fig. 4C–D) and intermediate regions (Fig. 5C–D). A high level of melanization and tissue necrosis was observed (Fig. 4D), and it can also be noted that yeast-like structures were internalized by haemocytes and surrounded by adipose tissue Fig. 4   Histological analysis of G. mellonella infected with C. auris and non-C. auris and stained with HE. A: 10 × InP13; B: InP13 100x; C: JAP 1 10x; D: JAP 1 100x; E: SP96 10x; F: SP96 100x; G: VEN C6 10x; H: VEN C6 100x; I: C. albicans

5134 10x; J: C. albicans 5134 100x; K: C. parapsilosis 22019

10x; L: C. parapsilosis 22019 × 100.(h): hemolymph; (at): adipose tissue; (H): haemocytes; (Y): yeast; (M): melanin

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Page 8 of 15 Vol:. (1234567890) (Fig.  5C). Granulomas induced by the SP96 isolate were found in the tail of G. mellonella, close to the cuticle (Figs.  4 and 5 E–F); moreover, granuloma showed haemocytes with clusters of yeast surrounded by melanin (Figs. 4F and 5F). Granuloma formed by VEN C6 isolate surrounded by haemocytes and adipose tissue were found in the upper intestine (Figs. 4 G-H) and tail (Figs. 5 G-H). Invasive infection with a high degree of inflammation, melanization, and tissue necrosis was observed by C. albicans (Figs. 4 and 5, I–J). A well-defined yeast structures were observed with PAS staining, which allowed to observe budding cells (Figs. 5J) or yeasts associated with melanin formation and hyphae/pseudohyphae formation process (Figs. 4J). A cluster of small melanized regions surrounded by haemocytes was observed between the upper and lower intestines in larvae infected with C. parapsilosis (Fig. 4K–L). Another highlight was that granulomas surrounded by adipose tissue and haemocytes were found dispersed in the haemolymph of the lower intestine (Fig. 5 K–L).

Figure 6 shows the anatomy of G. mellonella without infection. The head and thorax are in the upper extremity regions (A and B). The intermediate region contains most of the organs, such as the trachea, intestine, muscle tissue, adipose tissue, and most of the haemolymph (E). The junction between the intermediate and lower ends was in the transitional region (F). Finally, the tail (E) of the lower region consisted of adipose tissue (C) and other organelles responsible for excretion. The prolegs (G) are located on the posterior surface of the larva and are composed of spore Fig. 5   Histological analysis of G. mellonella infected with C. auris and non-C. auris and stained with PAS. A: 10 × InP13; B: InP13 100x; C: JAP 1 10x; D: JAP 1 100x; E: SP96 10x; F: SP96 100x; G: VEN C6 10x; H: VEN C6 100x; I: C. albicans 5134 × 10x; J: C. albicans 5134 100x; K: C. parapsilosis 22019 10x; L: C. parapsilosis 22019 100x. (h): hemolymph; (at): adipose tissue; (H): haemocytes; (Y): yeast; (M): melanin

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Figures 7–11 show granulomas, yeast aggregates, and infiltration located in different regions of G. mellonella. As shown in Fig.  7, several granulomas were observed in the larvae’s tissue infected with the InP13 isolate. In addition, yeast infiltration in the intestinal wall located in the transitional region (the region between the lower and upper extremities) was observed. This infiltration was followed by the Fig. 6   Histological anatomy of G. mellonella without infection and stained with PAS. A: Head; B: Chest; C: adipose tissue; D: Lower region–tail; E: Intestinal wall; F: Transitory region; G: Proleg of the cuticle Fig. 7   Distribution of yeast aggregates and granulomas in G. mellonella tissue infected with C. auris InP13. Images analyzed by SlideViewer software; A–C: 20x; D and F:10x; E: 30x; Black arrows indicate granulomas or yeast aggregates; PAS staining

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Page 10 of 15 Vol:. (1234567890) presence of yeast aggregates with budding formation (Fig. 7 B, C, and E). Yeast aggregates forming a uniform biofilm-like community attached to the intestinal wall and surrounded by haemocytes throughout the outer cavity in the transitional region (Fig. 7 A) and lower end (Figs. 7 D and F) were observed. Granulomas with diameters of 0.083 ± 0.047  mm were observed throughout the tissue.

JAP1 infection induced formation of biofilm-like yeast aggregates located between the outer intestinal wall and surrounded by haemocytes in the transition region (Fig. 8 C). Granulomas caused by JAP 1 were found in the lower and upper extremities, formed between adipose tissue (Fig.  8 F), close to the cuticle lower proleg (Figs. 8 D and E) and head (Fig.  8 A and B), with diameters of approximately 0.11 ± 0.036 mm.

Granulomas formed by SP96 infection were observed between the lower and upper regions, reaching a diameter of 0.11 ± 0.087 mm, which were dispersed in the haemolymph (Fig. 9 A), or adhered to tissues or organelle walls (Figs. 9 B–F). Most of these granulomas presented yeast aggregates with a high degree of melanization and tissue necrosis (Figs. 9 E and F).

The larvae infected with the VEN C6 isolate presented granulomas with diameters of up to 0.072 ± 0.027  mm in the upper and lower regions. Biofilm-like aggregates of yeast were found adhered to tissue (Fig. 10 A) or inside of organelles (Figs. 10 D and E).

A different infection pattern was observed with C. albicans, which showed a greater number of yeast aggregates, tissue invasion and biofilm-like formation. Yeasts and hyphae/pseudohyphae were found invading the tissue (Fig. 11 A) or invading the intestinal wall in the transitional region (Figs. 11 D and E). Additionally, presence of tissue infiltration was observed (Fig.  11 B) together with the biofilm-like formed structures in the intestinal wall and invading other tissue/organs and forming a yeast displacement channel (Fig. 11 D). Granulomas with diameters of up to 0.1 ± 0.038 mm were observed; furthermore, dispersed yeasts or hyphae/pseudohyphae were observed within granulomas with a high degree of melanization (Fig. 11).

Discussion In this study, C. auris InP13 (clade I) and C. albicans were identified as the most virulent strains, exhibiting high melanin production and granuloma formation, which contributed to invasive infection. Fig. 8   Distribution of yeast aggregates and granulomas in G. mellonella tissue infected with C. auris JAP 1. Images analyzed by SlideViewer software; A–C: 20x; D:5x; E–F: 10x; Black arrows indicate granulomas or yeast aggregates; PAS staining

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C. albicans showed a pronounced capacity for

aggregation, biofilm, and tissue invasion, which may explain the high melanization and mortality observed. Moreover, the InP13 and VEN C6 isolates (classified in the Clade I and Clade IV, respectively) formed pronounced biofilm-like structures in G. Fig. 9   Distribution of yeast aggregates and granulomas in G. mellonella tissue infected with C. auris SP96. Images analyzed by SlideViewer software; A–C and E–F: 20x; D:10x; Black arrows indicate granulomas or yeast aggregates; PAS staining Fig. 10   Distribution of yeast aggregates and granulomas in G. mellonella tissue infected with C. auris VEN C6. Images analyzed by SlideViewer software; A: 10x; B–F:20x; Black arrows indicate granulomas or yeast aggregates; PAS staining

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Page 12 of 15 Vol:. (1234567890) mellonella tissues, supporting the findings of Hernando-Ortiz et al. [17] who reported that less virulent aggregative strains often developed biofilms. Similarly, Sherry et al. [18] observed increased biofilm formation in aggregative isolates. This underscores the high virulence potential of some C. auris strains, which could correlate with pathogenicity. Indeed, the aggregating phenotype has been associated with altered interactions with the immune response, modulation of biofilm production profiles as well as antifungal resistance profile [19]. The virulence characteristics of C. auris can be largely attributed to its enzymes arsenal, which includes proteinases and phospholipases, as well as its capability to form biofilm, haemolytic capacity, resistance to environmental stress, and pronounced antifungal resistance [17]. These mechanisms are differentially expressed among the different strains and clades, where independent studies have been confirmed the heterogenicity in enzyme production. As an example, phospholipase activity was detected in 37% of the isolates analysed by Larkin et al. [20] while proteinase secretion was observed in 67% of the isolates depending on the strain and clade analysed. Furthermore, C. auris exhibited virulence characteristics associated with its cell wall, such as polymorphism, resistance to abiotic surfaces, adaptation, immune system evasion, and tolerance to external conditions. The cell wall of this species is composed of an outer mannan layer that plays an important role in protecting the internal 1,3-β glucan, which hinders recognition by the host’s immune system and the ability to attack it with antifungal agents, resulting in greater resistance to antifungal agents [21].

The discovery of a new clade (IV) suggests that the genetic population exhibit significant diversity and that a non-recent speciation event or events led to the genesis of each of these separate clades [22]. According to Fayed et  al. [23], the diversity of C. auris occurs not only between clades but also within the same clade. Whole genome sequencing of different isolates from the same clade (III) showed that they are genetically distinct with a significant high number of single nucleotide polymorphisms. Furthermore, the response to antifungal agents also showed diversity; thus, of the 17 clinical isolates from clades I and III exposed to low and high concentrations of fluconazole, it was possible to observe genomic modifications, including aneuploidy, karyotype alterations and point mutations. Finally, 19 clinical isolates showed genomic differences in genes related to resistance. Fig. 11   Distribution of yeast aggregates and granulomas in G. mellonella tissue infected with C. albicans. Images analyzed by SlideViewer software; A and E: 20x; B–C and E–F: 30x; D: 10x; Black arrows indicate granulomas or yeast aggregates; PAS staining

Mycopathologia (2026) 191:63 Page 13 of 15  63 Vol.: (0123456789) One of the differences between the clades is related to distinct mutations and/or mechanisms. For example, clades I, III, and IV present point mutations in the ERG11 gene related to azole resistance, while copy number variations are seen in Clade II [24]. Regarding the virulence analysis, Hernando-Ortiz et al. [17] used a G. mellonella model where larvae were inoculated with a yeast suspension of different isolates of C. auris (phylogenetically close to the isolates of clade III) ranging from ­105 to ­107 yeast/ larva; they found a dose-dependent mortality, which is consistent with the results of the present study with the higher inoculum ­(106) reducing larval survival. The comparative study of virulence among Candida species, as examined by Garcia-Bustos et al. [16] and using G. mellonella as a model, reported to C. auris as significantly virulent, resulting in 83% of lethality within three days post-infection. C. albicans, however, demonstrated superior pathogenicity, achieving a 95% mortality rate in the same studied period. Notably, no-aggregative C. auris strains exhibited greater virulence in comparison with those aggregative strains. In the study of Romera et al. [25] C. auris strains from clinical samples caused higher larval mortality and higher melanization scores, the authors tested a C. albicans strain, finding it more virulent than C. auris. Furthermore, the authors reported that although, no differences in larval mortality were observed between aggregative and non-aggregative

C. auris strains, the later strains exhibited higher mel-

anization scores compared to aggregative strains. Melanization has been recognized as important virulence mechanism and was also investigated in this study. Interestingly, no differences in the melanization process were observed among C. auris isolates. These results suggest that, while melanization is a relevant virulence factor, its expression may vary significantly across different clades; nonetheless, the non-significant difference in the melanization of infected larvae in this study may be related to the limited number of isolates used per clade (only one isolate). Thus, Garcia-Bustos et al. [16] reported that C. albicans achieved 100% melanization in the G. mellonella larvae within 24 h of infection, whereas the non-aggregative C. auris strains Cj198 and Cj175 showed significantly lower melanization capacity; contrarywise, Romera et  al. [25] found that clinical isolates of C. auris with aggregative capacity showed increased melanization. It is important to note that melanin could be produced in response to the stress caused by the infection; thus, this compound exhibits an antimicrobial potential and is considered an innate defence mechanism in response to pathogens during the initial infection [26]. The immune response in G. mellonella is mediated by prophenoloxidase, which is predominantly found in haemocytes and enocytoids. Once the pathogens are detected, a cascade is activated that results in melanin synthesis [27]. This melanization serves as a mechanism against pathogenic invasion. More recently, Anower et al. observed that three resistant clade I isolates caused high mortality of G. mellonella larvae, while a drug-susceptible clade II isolate caused low larval mortality. Finally, melanization was more significant in groups infected with more pathogenic isolates [28].

Additionally, G. mellonella larvae display other strategies against infection, such as the recruitment of immune cells to the site of infection and the production of cytokine-like proteins. The interaction of C-type lectins and imulectin-3 on haemocyte membranes with fungal wall antigens facilitates phagocytosis and phagosome formation within haemocytes leading to the destruction of the microbes. Following phagosome formation, the yeast is exposed to reactive oxygen species, acid degradation and enzymatic hydrolysis within phagolysosomes, which effectively neutralize the fungal threat. The inflammatory response, characterized by the formation of granulomas containing melanized or necrotic tissue, further indicates an active immune process [29, 30]. Histopathological examination of G. mellonella revealed granulomas with yeast aggregates adhering to organ tissues, the tracheal region and the haemolymph. Galleria, in contrast to mammals with a closed circulatory system, has an open one that permeates the entire larval body, especially the abdomen, facilitating the transport of lipids and proteins, the neutralization of endotoxins and the distribution of antimicrobial molecules, essential for larval survival [11, 31]. Both C. auris and non-C. auris species demonstrated the ability to form melanized granulomas, with a higher prevalence on the extremities. This observation needs further investigation to clarify why a greater number of granulomas were found between the head and tail. Thus, Vasquez-Muñoz et  al. [32] observed that C. auris aggregates are found dispersed in the tissue of G. mellonella, characterizing the pattern of infection and invasion. Bravo-Ruiz et al. [33]

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Page 14 of 15 Vol:. (1234567890) suggested that C. auris could develop rudimentary pseudohyphae under stressful conditions as a defence mechanism against immune responses. Smith et  al. [14] documented melanin-encapsulated C. auris pseudohyphae in the tracheal region of G. mellonella and the presence of melanin in the haemolymph adjacent to invasive pseudohyphae. In our study, conventional light microscopy revealed enlarged cells approximately 8 μm in diameter. The significant presence of granulomas was also reported in a recent study. Overall, over a 24-h period, 5 to 10 granulomas per profile were observed in a group of larvae infected with C. auris 16-1 and more than 50 granulomas per profile in groups infected with C. auris 17-1 and 18-1 [28]. Therefore, the results indicate a considerable difference in the levels of pathogenicity and virulence among the C. auris clades, which is an important factor for further investigation and the development of alternative methods with therapeutic potential that can more effectively encompass all clades of the species. Furthermore, the results may contribute to a better understanding of the pathogenesis of C. auris and its behaviour in response to a host, even if it is an alternative invertebrate in vivo model. Conclusions Our study indicates that C. auris presents heterogeneous virulence among isolates from different clades or regions. This variation may be related to the aggregative phenotype or clinical origin, as isolates from clade I (InP13) and clade IV (VEN C6) demonstrate higher virulence and a greater ability to form biofilm-like structures in G. mellonella compared to clade II (JAP 1) and clade III (SP96). Additionally, our findings highlight differences in the infection mechanisms of C. auris and C. albicans. While C. auris strains form a greater number of agglomerated yeasts and are involved in haemocytes, C. albicans and the InP13 isolate (clade I) show greater invasiveness, with the presence of filaments or pseudohyphae (C. albicans).

Acknowledgements  We would like to thank the group from the Pathology Department of La Fe Hospital for their help with the histopathological processing.

Funding  Open Access funding provided by Colombia Consortium. Fapesp, 2021/11120-3, Gabriel Davi Marena,Instituto de Salud Carlos III, JR21/00061, Alba Ruiz-Gaitán Declarations Conflicts of interest  The authors declare no conflict of interest.

Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. References

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