# A one health perspective on Diutina catenulata: Phenotypic traits, stress sensitivity, and virulence across diverse isolates

Ianca Karine Prudencio de Albuquerque, Florent Morio, Marjorie Albassier, Luc Baron, Léa Duchesne, Patrice Le Pape, Isabelle Ourliac-Garnier

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## To cite this version:

Ianca Karine Prudencio de Albuquerque, Florent Morio, Marjorie Albassier, Luc Baron, Léa Duchesne, et al.. A one health perspective on Diutina catenulata: Phenotypic traits, stress sensitivity, and virulence across diverse isolates. Journal of Medical Mycology = Journal de Mycologie Médicale, 2025, 35, pp.101583. 10.1016/j.mycmed.2025.101583. hal-05329491

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Research Paper

A one health perspective on *Diatina catenulata*: Phenotypic traits, stress sensitivity, and virulence across diverse isolates

Ianca Karine Prudencio de Albuquerque<sup>a,†</sup>, Florent Morio<sup>b,‡</sup>, Marjorie Albassier<sup>b</sup>,
Luc Baron<sup>b,§</sup>, Léa Duchesne<sup>c,‡</sup>, Patrice Le Pape<sup>b,§</sup>, Isabelle Ourliac-Garnier<sup>b,* ‖</sup>

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ARTICLE INFO

Keywords:

Diatina catenulata

One health

Virulence

Stress response

Fitness

Antifungal susceptibility

Galleria mellonella

A B S T R A C T

*Diatina catenulata*, formerly *Candida catenulata*, is found in diverse substrates, including soil, food, animals, and humans. It is recognized as an emerging human pathogen responsible for various diseases, including invasive infections in susceptible hosts, particularly in immunocompromised individuals. This study aimed to assess the phenotypic profiles, *in vitro* responses to various stressful conditions, and *in vivo* virulence of *D. catenulata* isolates from different origins (human, veterinary, and environmental). *In vitro* antifungal susceptibility to fluconazole, voriconazole, micafungin and amphotericin B was also determined for all isolates. Most *D. catenulata* isolates exhibited a light blue coloration on CHROMID® *Candida* agar. Different profiles were observed on the Spider medium, suggesting different behavior among the isolates. All isolates of *D. catenulata* were highly sensitive to oxidative and osmotic stress as demonstrated by exposure to H₂O₂ and 1.5 M NaCl, respectively whereas no unique pattern was evidenced concerning the response to cell wall stressors or membrane stressors. In addition to acquired azole resistance for some isolates, these findings provide valuable insights into the strain-dependent responses of *D. catenulata* to various stress factors. Using *Galleria mellonella*, we highlighted a wide range of virulence profiles among the isolates. Overall, this research demonstrates that the observed responses are strain-dependent rather than sample-source-dependent.

**Introduction**

*Diatina catenulata*, previously known as *Candida catenulata* [1], is an ascomycetous yeast that has been found in various settings, including food products [2–4], various hosts including humans and animals such as cows, hens, and pigeons [2,3,5–9], as well as environmental samples such as soil [10]. This species can also act as an opportunistic pathogen responsible for a wide range of diseases, including invasive infections in susceptible hosts [7]. The first isolation of *D. catenulata* from humans was reported in 1971 [11] before being described as a causative agent of onychomycosis a few years later [12]. The first reported case of fungemia involved a woman with cancer [13] and was followed by further cases of invasive infections [5–7]. Its draft genome was published in 2018 [14].

Due to its ability to colonize and infect various hosts and to survive in
the environment, deepening our knowledge about its phenotypic char-
acteristics, virulence attributes and antifungal resistance is of utmost

importance, in a One Health perspective. In line with this, our research
group recently identified critical amino acid substitutions associated
with acquired antifungal resistance in this opportunistic species[7,15].

In the present study, our objective was to use phenotypic profiling to assess whether *D. catenulata* from different sources (human, veterinary, or environmental) display the same behavior *in vitro* and *in vivo* in a model of invasive infection. These results will contribute to a more comprehensive understanding of this emerging species and its potential impact on human health.

**Materials and methods**

Strains

A total of fourteen *D. catenulata* isolates were studied, including six of human origin, five of veterinary origin and three environmental strains, respectively[7] (Table 1). Additionally, *C. albicans* (SC5314),

<footnote><sup>a</sup> Universidade Federal de Pernambuco, Departamento de Medicina Tropical, Recife, Pernambuco, Brazil</footnote>

<footnote><sup>b</sup> Nantes Université, CHU Nantes, Cibles et Médicaments des Infections et de l'Immunité, IICiMed, UR 1155, F-44000, Nantes, France</footnote>

<footnote><sup>c</sup> Nantes Université, CHU Nantes, Public Health Department, Nantes, France</footnote>

<footnote>* Corresponding author at: UR 1155 - IICiMed, Institut de Recherche en Santé 2, 22 Boulevard Bénoni Goullin, 44200, Nantes, France.
E-mail address: isabelle.ourliac@univ-nantes.fr (I. Ourliac-Garnier).</footnote>
<table><caption>**Table 1**
List of the strains included in this study.</caption><thead><tr><th>Strain n° (internal code)</th><th>Other reference</th><th>Species</th><th>Country of isolation</th><th>Source</th></tr></thead><tbody><tr><td>DCAT1</td><td>ROC2119</td><td><i>Diutina catenulata</i></td><td>France</td><td>Human</td></tr><tr><td>DCAT7</td><td>CLE9871-1</td><td><i>Diutina catenulata</i></td><td>France</td><td>Human</td></tr><tr><td>DCAT8</td><td>CLE9871-2</td><td><i>Diutina catenulata</i></td><td>France</td><td>Human</td></tr><tr><td>DCAT25</td><td>TOU6178</td><td><i>Diutina catenulata</i></td><td>France</td><td>Human</td></tr><tr><td>DCAT27</td><td>CBS565</td><td><i>Diutina catenulata</i></td><td>Puerto Rico</td><td>Human</td></tr><tr><td>DCAT33</td><td>CBS6092</td><td><i>Diutina catenulata</i></td><td>United Kingdom</td><td>Human</td></tr><tr><td>DCAT14</td><td>1569</td><td><i>Diutina catenulata</i></td><td>Italy</td><td>Hen</td></tr><tr><td>DCAT31</td><td>CBS2743</td><td><i>Diutina catenulata</i></td><td>Netherlands</td><td>Cheese</td></tr><tr><td>DCAT35</td><td>CBS6174</td><td><i>Diutina catenulata</i></td><td>Netherlands</td><td>Milk</td></tr><tr><td>DCAT29</td><td>CBS2014</td><td><i>Diutina catenulata</i></td><td>Netherlands</td><td>Hen</td></tr><tr><td>DCAT38</td><td>CBS14346</td><td><i>Diutina catenulata</i></td><td>USA</td><td>Insect</td></tr><tr><td>DCAT24</td><td>WY3-10-4</td><td><i>Diutina catenulata</i></td><td>Ireland</td><td>Environmental</td></tr><tr><td>DCAT36</td><td>CBS7135</td><td><i>Diutina catenulata</i></td><td>Netherlands</td><td>Environmental</td></tr><tr><td>DCAT43</td><td>BES4379</td><td><i>Diutina catenulata</i></td><td>France</td><td>Environmental</td></tr><tr><td>SC5314</td><td>--</td><td><i>Candida albicans</i></td><td>–</td><td>Human</td></tr><tr><td>ATCC 22019</td><td>–</td><td><i>Candida parapsilosis</i></td><td>Puerto Rico</td><td>Human</td></tr><tr><td>ATCC 750</td><td>–</td><td><i>Candida tropicalis</i></td><td>–</td><td>Human</td></tr><tr><td>ATCC 2001</td><td>–</td><td><i>Nakaseomyces glabratus</i> (<i>Candida glabrata</i>)</td><td>–</td><td>Human</td></tr><tr><td>CAAU941</td><td>–</td><td><i>Candida auris</i> (now <i>Candidozyma auris</i>)</td><td>Colombia</td><td>Human</td></tr></tbody></table>

C. parapsilosis (ATCC 22019), C. tropicalis (ATCC 750), and Nakaseomyces glabratus (formerly Candida glabrata, ATCC 2001), and one Candidozyma auris (formerly Candida auris) from Colombia (CAAU941)[16] were used as comparators.

## Mycological identification

All strains were previously identified to the species level using the VITEK® matrix-assisted laser desorption/ionization time-of-flight mass spectrometry instrument (database v3.2.0, BioMérieux, Marcy l'Etoile, France) and further confirmed by ITS rDNA barcoding[7].

## Phenotypic profiling

### Macroscopic aspect on chromogenic medium

All *D. catenulata* isolates were grown on Sabouraud dextrose agar (SDA) (BioMérieux,) at 30 °C ± 2 °C for 24 h, before being streaked on CHROMID® Candida agar plates (CAN2) (BioMérieux). Plates were incubated at 30 °C ± 2 °C for 48 h before macroscopic examination of the colonies. *C. albicans* SC5314, *C. parapsilosis* ATCC 22,019, *C. auris* CAAU941, *C. tropicalis* ATCC 750, and *N. glabratus* ATCC 2001 were used as comparators.

### Filamentation assay

Filamentation was assessed by growing each yeast isolate on Spider

medium (2.5 g of nutrient agar, 2.5 g of mannitol, 0.5 g of KH₂PO₄, 3.4 g agar, in 250 mL distilled water, pH 7.2)[17]. Briefly, cells were pre-grown on SDA agar overnight. An inoculum containing 1 × 10³ cells/mL was then prepared in YPD broth, and 20 µL of this suspension were spotted on Spider agar plates. Plates were incubated at 30 °C ± 2 °C for seven days before macroscopic examination of the colonies. This experiment was performed in duplicate, with at least ten colonies assessed per replicate. *C. albicans* SC5314, *C. parapsilosis* ATCC 22019, *C. auris* CAAU941, *C. tropicalis* ATCC 750, and *N. glabratus* ATCC 2001 were used as comparators.

### Response to stressful conditions determined by spot assays

Inoculum suspensions were prepared with representative colonies grown at 30 °C ± 2 °C for 18–24 h from a SDA plate. Five colonies were then cultured in liquid yeast extract-peptone-dextrose (YPD) for 18 to 24 h at 30 °C ± 2 °C with shaking at 200 rpm. This suspension was then diluted 1:10, and the optical density (OD) was measured at 600 nm using a DensiCHEK Plus (BioMérieux) and adjusted to a final OD of 1 corresponding to a concentration of 10⁷ cells/mL. This suspension was then subjected to a 1:10 serial dilution, resulting in four dilutions. 5 µL of each dilution were then spotted on YPD plates containing each stressor or on a control plate (YPD only).

Different *in vitro* stressful conditions, including cell wall, oxidative, osmotic, and membrane stress, were assessed. Caffeine (12.5 mM) was employed to induce a cell wall stress. Oxidative stress was induced by the addition of hydrogen peroxide (H₂O₂) at final concentrations of 5 mM. Osmotic stress was evaluated with sodium chloride (NaCl) at a concentration of 1.5 M, along with sorbitol at 1 M and glycerol at 8 % (vol/vol). Lastly, membrane stress was induced by using sodium dodecyl sulfate (SDS) at a final concentration of 0.01 %. Growth was assessed after 48 h of incubation at 30 °C, and each experiment was performed in duplicate and included the comparator strains.

Isolates were then classified according to the extent of growth (relative to the control) in the presence of stressors. For this analysis, each plate was photographed and analyzed according to the protocol described by Petropavlovskiy et al. [18]. To minimize the potential saturation of the spot at higher concentrations or to avoid the marked decrease in intensity that occurs at lower concentrations, we chose the third spot (10⁴ cells/mL), as the ideal concentration for gray value measurement. Images were subsequently processed in ImageJ software by converting them to 8-bit format, followed by gray value densitometry, background subtraction, and grayscale measurement. The quantified data were then organized and managed in Microsoft Excel. Graphs were subsequently generated and analyzed using Prism version 8.0.1 software.

Growth levels were used to categorize the effect of the stressors as follows: no growth, very strong effect; <25 % compared to the control, strong effect; 25 % to 50 %, moderate effect; 50 % to 75 %, slight effect; and >75 %, weak effect. The Student's *t*-test was used for statistical analysis between study groups, and a p-value of <0.05 was considered statistically significant.

### *In vitro* antifungal susceptibility testing

The *in vitro* susceptibility of *D. catenulata* isolates to four systemic antifungals, fluconazole (FLC, Carbosynth®), voriconazole (VRC, Carbosynth®), amphotericin B (AMB, Biosynth®) and micafungin (MCF, Sigma-Aldrich) was determined using the EUCAST reference microdilution method, as specified in EUCAST E.DEF 7.3.2[19]. Minimal inhibitory concentrations (MIC) were established based on a reduction in turbidity of at least 50 % for FLC, VRC and MCF and at least 90 % for AMB, relative to the control well (no antifungal). MIC₉₀ were then determined for each antifungal, and represent the MIC value for which ≥90 % of the strains within the test population are inhibited

### *In vivo* virulence

Galleria mellonella larvae used in the study were bred in-house. These
larvae were housed in a dark incubator at 30 °C ± 2 °C and fed a diet of wheat flour, oat flakes, pollen, glycerol, and honey[20]. Homogeneous groups of ten larvae of the 6th instar (weight around 300 mg) with a cream-colored cuticle were randomly selected for the experiments. For each survival assay, inoculum suspensions were prepared in 1X PBS (Corning®, Tewksbury, USA) from colonies obtained after a 24-hour growth on SDA. The final inoculum concentration was adjusted to 5 × 10⁸ cells/mL. Prior to injection, the ventral surface of all larvae was disinfected with 70 % ethanol, and then 10 µL of the inoculum suspension was injected into the left penultimate proleg using a 0.3 mL 30 G insulin syringe (BD Medical, Le Pont de Claix, France). Larvae were then incubated in the dark at 37 °C without food, and survival was assessed daily for five days. Two uninfected groups were used as negative controls: one group was inoculated with PBS, while the other was not inoculated. Lack of movement in response to physical stimuli, with or without melanization, was used to determine death.

Survival curves were generated using the Graphpad Prism 8.0.1 software and analyzed using the log-rank test, Kaplan-Meier survival curves, or the Cox model with the determination of Hazard Ratios. These experiments were performed at least three times on different days.

D. catenulata isolates were categorized according to their survival rate five days after infection, using the 25th and 75th percentiles (P₂₅ and P₇₅) for calculation. Isolates with a survival rate below the 25th percentile were considered highly virulent. In contrast, those with a median survival above the 75th percentile were classified as low virulent. To go further in the description of highly virulent isolates, we calculated larval mortality at day +1 post-infection ("rapid virulence score") as described by Marcos-Zambrano et al. [21]. Furthermore, the following mortality rates were utilized as reference values: low-virulence (≤ 30 % mortality = ≥ 70 % survival), moderate-virulence (31–80 % mortality = 20–69 % survival), and high-virulence (> 80 % mortality = < 20 % survival).

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Macroscopic aspect of 14 *Diatina catenulata* and other species used as comparator strains when grown on CHROMID® *Candida* agar plates. A = human isolates; B = veterinary isolates; C = environmental isolates, and D = other species (comparators).</figcaption></figure>

# Results

## Macroscopic aspect on chromogenic medium

As depicted in Fig. 1, all but one (DCAT29) of the isolates tested in this study appeared as light blue colonies on chromogenic plates, similar to C. albicans. DCAT29 exhibited a mixed profile, displaying two distinct colors: white and light blue, the latter exhibiting the same blue as the other *D. catenulata* isolates.

## Filamentation assay on Spider medium

All isolates were also cultured on Spider agar plates, a medium that has been shown to induce yeast hyphal formation. As depicted in Fig. 2, except DCAT33 – which exhibited two distinct phenotypes (fluffy and smooth) – all the other isolates exhibited a single phenotype on this medium, although different aspects were evidenced according to the isolates.

Most strains (9 out of 14) showed smooth colonies (as exemplified with DCAT1), similar to *C. parapsilosis*, *N. glabratus* and *C. auris* isolates. Others (4 out of 14) exhibited a wrinkled phenotype (e.g. DCAT29) as observed with *C. tropicalis* ATCC 750. Finally, two isolates showed a fluffy phenotype (e.g. DCAT38 and DCAT33).

## Sensitivity to stressful conditions

To assess possible variation in stress response among isolates, each strain was grown on agar plates containing various stressors. Differences in growth were assessed visually and then quantified as described in the ‘Materials and Methods’ section. As shown, in Figure S1, Figure S2 and Fig. 3, all 14 isolates of *D. catenulata* showed high sensitivity to oxidative stress revealed by the strong inhibitory effect of H₂O₂ at 5 mM. Furthermore, among osmotic stressors, the response profile varies between isolates. Although 1.5 M NaCl significantly affected the growth of all isolates, except DCAT38, glycerol 8 % (v/v), and sorbitol 1 M only affected some strains. Notably, a single isolate was sensitive to all the stressors tested (DCAT7).

Additionally, we categorized the stress factors based on their impact on fungal growth, as shown in Fig. 3. No unique pattern was evidenced when analyzing the response of *D. catenulata* to osmotic stress, suggesting strain-dependent sensitivity. Moreover, some variations in sensitivity to specific stress factors were evidenced, with some isolates exhibiting unique profiles. A higher sensitivity to sorbitol was observed in five isolates (DCAT7, DCAT35, DCAT36, DCAT38, and DCAT43). Five isolates (DCAT7, DCAT25, DCAT29, DCAT31, and DCAT33) appeared more sensitive to glycerol. Focusing on the cell wall stressor caffeine, only DCAT7 showed a reduced growth compared to the control. Overall, the main differences between strains were observed when cells were exposed to 0.01 % SDS, which shows from no effect to strong inhibition with statistically significant differences for seven isolates, as shown in Fig. 3.

## In vitro antifungal susceptibility testing

The susceptibility profile of each *D. catenulata* isolates is given in Table 2. Overall, MICs ranged from 0.5 to 32 µg/mL for FLC, 0.015 to 0.5 µg/mL for VRC and AMB, respectively. All isolates displayed low MICs for MCF. Notably, two human *D. catenulata* clinical isolates (DCAT1 and DCAT8) showed reduced *in vitro* susceptibility to fluconazole (> 4 µg/mL), linked to the acquisition of the Y132F substitution in Erg11p, as described elsewhere[7].

## In vivo virulence in a galleria mellonella model

Overall, we globally observed a lower virulence of *D. catenulata*, when compared to the other opportunistic yeast species used as
Phenotype on Spider Medium

<figure><img src="image_5.png" /><figcaption>Fig. 2. Macromorphological aspects of colonies of *D. catenulata* isolates grown on solid Spider medium after seven days of incubation at 30 °C ± 2 °C.</figcaption></figure>

<table><thead><tr><th rowspan="2"></th><th rowspan="2">Isolates</th><th colspan="3">Osmotic stress</th><th>Cell wall stress</th><th>Oxidative stress</th><th>Membrane stress</th><th rowspan="2"></th></tr><tr><th>NaCl - 1.5 M</th><th>Sorbitol - 1 M</th><th>Glycerol - 8% v/v</th><th>Caffeine - 12.5 mM</th><th>H<sub>2</sub>O<sub>2</sub> - 5 mM</th><th>SDS - 0.01%</th></tr></thead><tbody><tr><td rowspan="5">Comparator strains</td><td>SC5314</td><td>*</td><td>*</td><td></td><td>*</td><td></td><td></td><td rowspan="5">No growth</td></tr><tr><td>ATCC 22019</td><td>*</td><td>*</td><td></td><td></td><td></td><td></td></tr><tr><td>CAAU941</td><td>*</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>ATCC 750</td><td>*</td><td>*</td><td>*</td><td></td><td>*</td><td>*</td></tr><tr><td>ATCC 2001</td><td>*</td><td>*</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="6">Human origin</td><td>DCAT1</td><td>*</td><td></td><td></td><td></td><td>*</td><td></td><td rowspan="6">Strong effect (&gt;0% to ≤25%)</td></tr><tr><td>DCAT7</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td></tr><tr><td>DCAT8</td><td>*</td><td></td><td></td><td></td><td>*</td><td></td></tr><tr><td>DCAT25</td><td>*</td><td></td><td></td><td></td><td>*</td><td>*</td></tr><tr><td>DCAT27</td><td>*</td><td></td><td></td><td></td><td>*</td><td></td></tr><tr><td>DCAT33</td><td>*</td><td></td><td>*</td><td></td><td>*</td><td></td></tr><tr><td rowspan="5">Veterinary origin</td><td>DCAT14</td><td>*</td><td></td><td></td><td></td><td>*</td><td></td><td rowspan="5">Moderate effect (&gt;25% to ≤50%)</td></tr><tr><td>DCAT29</td><td>*</td><td></td><td>*</td><td></td><td>*</td><td>*</td></tr><tr><td>DCAT31</td><td>*</td><td></td><td>*</td><td></td><td>*</td><td></td></tr><tr><td>DCAT35</td><td>*</td><td>*</td><td></td><td></td><td>*</td><td>*</td></tr><tr><td>DCAT38</td><td>*</td><td></td><td></td><td></td><td>*</td><td>*</td></tr><tr><td rowspan="3">Environmental origin</td><td>DCAT24</td><td>*</td><td></td><td></td><td></td><td>*</td><td></td><td rowspan="3">Slight effect (&gt;50% to ≤75%)</td></tr><tr><td>DCAT36</td><td>*</td><td>*</td><td></td><td></td><td>*</td><td></td></tr><tr><td>DCAT43</td><td>*</td><td>*</td><td></td><td></td><td>*</td><td>*</td></tr></tbody></table>

Note: SDS = Sodium Dodecyl Sulfate; SC5314 = *Candida albicans*; ATCC 22019 = *Candida parapsilosis*; CAAU941 = *Candida auris* (now *Candidozyma auris*); ATCC 750 = *Candida tropicalis*, and ATCC 2001 = *Nakaseomyces glabratus*.

<caption>Fig. 3. Comparison of *Diutina catenulata* strains growth under normal conditions or exposure to various stressors. The percentage of growth in the presence of the stress factors relative to the control condition is color-coded. Asterisks indicate statistically significant differences (p-value < 0.05) (supplementary Figure S1 and S2).</caption>

comparators (Fig. 4A). The survival curves of *D. catenulata* strains from human, veterinary, and environmental sources are shown in Figure S3. Looking at day 5 survival, three isolates (DCAT7, DCAT25, and DCAT33), were of low virulence, seven isolates showed moderate virulence, and four isolates were considered highly virulent, but differences in virulence did not correlate with the origins of the strains. Of note, human clinical strains showed a wider range of virulence, from low (DCAT7, DCAT25, and DCAT33) to highly virulent (DCAT27).

Additionally, to gain further insights, the rapid virulence score (i.e. 1-day post-infection) was determined for *D. catenulata* isolates formerly classified as highly virulent (i.e. DCAT27, DCAT29, DCAT31, DCAT35) and all comparator strains. As shown in Fig. 4B, 1-day post infection, the

comparator strains ATCC 750, CAAU941, and SC5314 were classified as highly virulent, ATCC 22,019 as moderately virulent, and ATCC 2001, DCAT27, DCAT29, DCAT31, and DCAT35 as low virulent.

Discussion

In this study, we compared the phenotypes, stress responses, and virulence of *D. catenulata* isolates from human, veterinary, and environmental sources to better understand the species and identify potential origin-related differences in behavior. Furthermore, *in vitro* antifungal susceptibility to fluconazole (Diflucan), voriconazole, micafungin and amphotericin B was evaluated.
<caption>**Table 2**</caption>

*In vitro* susceptibility of 14 *Diatina catenulata* strains to four systemic antifungals, determined by the EUCAST method.

<table><thead><tr><th rowspan="2">STRAIN</th><th colspan="4">MIC determination according to the EUCAST methode (µg/mL)</th></tr><tr><th>FLC</th><th>VRC</th><th>AMB</th><th>MCF</th></tr></thead><tbody><tr><td>DCAT1</td><td>16</td><td>0.125</td><td>0.250</td><td>0.031</td></tr><tr><td>DCAT7</td><td>1</td><td>0.015</td><td>0.250</td><td>0.031</td></tr><tr><td>DCAT8</td><td>32</td><td>0.500</td><td>0.500</td><td>0.015</td></tr><tr><td>DCAT25</td><td>1</td><td>0.015</td><td>0.250</td><td>0.015</td></tr><tr><td>DCAT27</td><td>2</td><td>0.062</td><td>0.250</td><td>0.015</td></tr><tr><td>DCAT33</td><td>2</td><td>0.062</td><td>0.250</td><td>0.031</td></tr><tr><td>DCAT14</td><td>1</td><td>0.031</td><td>0.500</td><td>0.031</td></tr><tr><td>DCAT29</td><td>2</td><td>0.015</td><td>0.125</td><td>0.015</td></tr><tr><td>DCAT31</td><td>4</td><td>0.031</td><td>0.250</td><td>0.015</td></tr><tr><td>DCAT35</td><td>0.5</td><td>0.015</td><td>0.500</td><td>0.015</td></tr><tr><td>DCAT38</td><td>2</td><td>0.062</td><td>0.500</td><td>0.031</td></tr><tr><td>DCAT24</td><td>4</td><td>0.062</td><td>0.250</td><td>0.031</td></tr><tr><td>DCAT36</td><td>1</td><td>0.031</td><td>0.500</td><td>0.031</td></tr><tr><td>DCAT43</td><td>4</td><td>0.062</td><td>0.500</td><td>0.031</td></tr><tr><td>Median</td><td>2</td><td>0.047</td><td>0.250</td><td>0.031</td></tr><tr><td>MIC<sub>90</sub></td><td>16</td><td>0.125</td><td>0.500</td><td>0.031</td></tr></tbody></table>

Note: FLC = Fluconazole; VRC = Voriconazole; AMB = Amphotericin B; MCF = Micafungin. MIC<sub>90</sub> was calculated as the concentration that inhibits the growth of 90 % of the tested isolates (here 13 strains) and Median was calculated as the concentration that inhibits the growth of 50 % of the tested isolates.

Firstly, it should be noted that the blue coloration of *D. catenulata* on the CHROMID® chromogenic medium and the presence of mixed profiles, can lead to misinterpretations for untrained eyes, because of its similarity to that of *C. albicans* on this medium. However, on closer observation, the predominant light blue coloration seen in *D. catenulata* isolates differs from the darker blue typically observed with *C. albicans*. Furthermore, *D. catenulata* harbors distinctive traits on Spider agar which were not related to their origin. A comparable within species diversity was previously documented by Zuza-Alves *et al.* [22] in environmental isolates of *C. tropicalis*, wherein three phenotypes (fluffy, wrinkled, and smooth) were observed on Spider agar. The findings of Anderson *et al.* [23] also show that *C. albicans* isolates can also exhibit remarkable plasticity.

Here we performed the first assessment of the sensitivity of *D. catenulata* to various stressful conditions and observed that *D. catenulata* isolates demonstrated greater sensitivity to oxidative stress induced by H₂O₂ than more common clinically relevant species, used

here as comparators. Results showed that half of *D. catenulata* isolates also demonstrated increased sensitivity to 0.01 % SDS. Among the control strains, only *C. tropicalis* ATCC 750 demonstrated similar sensitivity to SDS, which is consistent with the findings of Lyon *et al.* [24].

Elevated MIC values for fluconazole observed in human isolates align with the findings of Borman *et al.* [25], Chen *et al.* [6] and Alves *et al.* [26]. Currently, neither breakpoints nor epidemiological cut-off value (ECV/ECOFF) have been determined for *D. catenulata*. Recently, Astvad *et al.* [27] proposed recommendations to interpret MIC data for rare yeast species for which no breakpoints are available. Thus, in line with this study all *D. catenulata* strains included in the present study can be considered as susceptible to both amphotericin B and micafungin. On the contrary, a wider range of MICs was noted for azoles, showing evidence for acquired resistance which is also supported by the presence of the Y132H mutation in the 2 isolates showing the highest MICs. The finding that micafungin MICs are low for all strains including those with higher fluconazole MICs suggests that echinocandins may be a good option for treating invasive infections caused by *D. catenulata*.

In addition to antifungal susceptibility and *in vitro* response to stressful conditions, we then assessed the virulence of *D. catenulata* using the *G. mellonella* larvae model. A few years ago, Montoya *et al.* [28] demonstrated significant differences in survival rates among *Diatina mesorugosa* isolates, with a range from high virulence (100 % mortality within two days) to low virulence (average survival times of 8 to 9 days). Despite using a different methodology, our study similarly documented a wide range of virulence levels across *D. catenulata* isolates that did not correlate with the origins of the strains. Of note, the 3 out of the 4 highly virulent strains exhibited a wrinkled phenotype (DCAT27, DCAT29, and DCAT31) which suggest a possible link between phenotype and virulence in this species. Overall, human strains showed a larger range of virulence. A couple of studies of *C. auris*, also demonstrate differences in virulence across clinical isolates [29–30]. Romera *et al.* [29], observed mortality rates between 20 % and 33 % after a five-day observation period for the tested isolates, using a fungal charge of 10⁵ cells/larvae. Similarly, the study by Arias *et al.* [30], which used a fungal charge of 2.5 × 10⁵ cells/larvae, reported survival rates ranging from 55.56 % to 74.87 % after four days of analysis.

## Conclusions

Due to the strain-dependent responses rather than sample-source-

<figure><img src="image_6.png" /></figure>

Note: SC5314 = Candida albicans; ATCC 22019 = Candida parapsilosis; CAAU941 = Candida auris (now Candidozyma auris); ATCC 750 = Candida tropicalis, and ATCC 2001 = Nakaseomyces glabratus.

**Fig. 4.** Survival of *Galleria mellonella* larvae: five-day survival after infection with 14 *D. catenulata* isolates and comparators strains using an inoculum of 5 × 10⁶ cells/larvae (A) and one-day survival after infection for only highly virulent *Candida* spp. and *D. catenulata* isolates (B).
dependent responses, the results obtained in this study point to exciting opportunities for future research from a One Health perspective. Recent studies show not all strains of a given species can exhibit substantial differences in virulence, and other pathogenic factors [31]. Such variability poses challenges for treatment, as therapies effective against one strain may not be equally effective against others, as already known for antifungal resistance.

Extending the current research with a larger number of *D. catenulata* isolates would increase the generalizability of our findings. Moreover, testing additional oxidative, membrane, cell wall, and osmotic stress conditions could also provide a broader view of *D. catenulata* adaptive responses to stressful conditions. Furthermore, studies examining the interaction between different stressors are crucial to elucidate the metabolic and structural adaptations that allow *D. catenulata* cells to thrive in challenging environments, as has been done in previous studies focusing on other opportunistic yeast species[32–34].

In conclusion, continuing research from a One Health perspective is crucial to deepening our understanding of emerging fungal species, which can circulate or appear in various reservoirs.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

The authors acknowledge Prof. Geraldine Butler, Dr. Sophie Cassading, Prof. Frédéric Grenouillet, Prof. Ferry Hagen, Dr. Céline Nourrisson, and Prof. Philippe Poirier for providing some *D. catenulata* strains used in the present study and Prof. JY Rodriguez from Centro de Investigaciones Microbiológicas del Cesar (CIMCE), Valledupar 200002, Colombia for the *C. auris* strain.

## Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.mycmed.2025.101583.

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