Cell Aggregation Capability of Clinical Isolates from Candida auris and Candida haemulonii Species Complex

The opportunistic fungal pathogens belonging to the Candida haemulonii complex and the phylogenetically related species Candida auris are well-known for causing infections that are difficult to treat due to their multidrug-resistance profiles. Candida auris is even more worrisome due to its ability to cause outbreaks in healthcare settings. These emerging yeasts produce a wide range of virulence factors that facilitate the development of the infectious process. In recent years, the aggregative phenotype has been receiving attention, as it is mainly associated with defects in cellular division and its possible involvement in helping the fungus to escape from the host immune responses. In the present study, we initially investigated the aggregation ability of 18 clinical isolates belonging to the C. haemulonii species complex (C. haemulonii sensu stricto, C. duobushaemulonii, and C. haemulonii var. vulnera) and C. auris. Subsequently, we evaluated the effects of physicochemical factors on fungal aggregation competence. The results demonstrated that cell-to-cell aggregation was a typically time-dependent event, in which almost all studied fungal isolates of both the C. haemulonii species complex and C. auris exhibited high aggregation after 2 h of incubation at 37 °C. Interestingly, the fungal cells forming the aggregates remained viable. The aggregation of all isolates was not impacted by pH, temperature, β-mercaptoethanol (a protein-denaturing agent), or EDTA (a chelator agent). Conversely, proteinase K, trypsin, and sodium dodecyl sulfate (SDS) significantly diminished the fungal aggregation. Collectively, our results demonstrated that the aggregation ability of these opportunistic yeast pathogens is time-dependent, and surface proteins and hydrophobic interactions seem to mediate cell aggregation since the presence of proteases and anionic detergents affected the aggregation capability. However, further studies are necessary to better elucidate the molecular aspects of this intriguing phenomenon.


Introduction
The Candida haemulonii species complex is classically formed by the emergent pathogens C. haemulonii sensu stricto, C. duobushaemulonii, and C. haemulonii var. vulnera [1]. The globally concerning fungus Candida auris is phylogenetically associated with this fungal complex, and together they form the C. haemulonii clade [1]. Molecular methods are required for the correct identification of these emerging opportunistic yeasts [1]. Both the A total of 18 clinical isolates of C. haemulonii species complex and C. auris were used in the present study. The isolates of the C. haemulonii complex were recovered from patients from Brazilian hospitals between 2005 and 2013 and were identified by molecular approaches as C. haemulonii (n = 5; LIPCh2 recovered from the sole of the foot, LIPCh3 from toe nail, LIPCh4 from finger nail, LIPCh7 from toe nail, and LIPCh12 from blood), C. duobushaemulonii (n = 4; LIPCh1 from finger nail, LIPCh6 from toe nail, LIPCh8 from blood, and LIPCh10 from bronchoalveolar lavage), and C. haemulonii var. vulnera (n = 3; LIPCh5 from toe nail, LIPCh9 from urine, and LIPCh11 from blood) [12]. Candida auris isolates were recovered from patients from Colombian hospitals between 2005 and 2016 (n = 6; Ca386 was recovered from a biopsy of bone tissue, Ca432 from the secretion of craniotomy, Ca485 from eye discharge, Ca446 and Ca885 from blood, and Ca881 from cerebrospinal fluid) [13]. Fungal cells were cultured in Sabouraud-dextrose broth (SDB) at 37 • C for 48 h and then used in all experiments. The yeast cells were quantified using a Neubauer chamber.

Aggregation Kinetic Assay
The aggregation assay was performed using a standard method previously described [14,15]. In this sense, yeasts grown in SDB were washed twice in sterile phosphate-buffered saline (PBS, pH 7.2), and then fungal suspensions containing 10 8 yeasts/mL were prepared in microcentrifuge tubes (Eppendorf, Hamburg, Germany), vortexed for 30 s and then transferred by pipetting into plastic cuvettes (1 mL/cuvette). The fungal suspensions were incubated at 37 • C without agitation for 30-, 60-, 90-, and 120-min. Aggregation was quantified as a percentage reduction in the optical density (OD) and calculated as ([OD 0 − OD f ]/OD 0 ) × 100, where OD 0 is the OD value at the start of the experiment and OD f is the value after the different incubation time periods. All measurements were performed at 530 nm using a spectrophotometer (Ultrospec 2100 Pro, Amersham Biosciences, Amersham, United Kingdom). PBS without fungal cells was used as a blank. The percentage of aggregation was calculated and used for classification of aggregation as follows: high (more than 40%), intermediate (30-40%), and low aggregation (less than 30%) [16]. One isolate of each Candida species was selected for the subsequent experiments.

Aggregation after Prolonged Periods and Assessment of Viability
The selected fungal isolates were prepared for aggregation assays as described above and incubated for 5 and 24 h at 37 • C. At each time point, the OD was read at 530 nm, and the percentage of aggregation was calculated as described above. In parallel, aliquots (10 µL) of cell suspensions were obtained at each time point, spotted on the surface of Sabouraud-dextrose agar (SDA) plates, and incubated at 37 • C for 48 h to evaluate the viability of the fungal isolates after aggregation. Fungal cells were also stained with a crystal violet solution (0.2% in water; Sigma-Aldrich, St. Louis, MO, USA) after each incubation period and observed using a light microscope to investigate cell viability. The dye is unable to enter viable (intact) cells, which remain unstained, while dead cells become blue due to the dye's ingress. In this context, control of dead cells was obtained by boiling fungal cells for 20 min, followed by staining them with the same crystal violet solution.

Light Microscope Imaging
Fungi (10 8 yeasts/mL) were incubated in PBS (pH 7.2) at 37 • C for 2 h to allow cell aggregation. Afterwards, the systems were gently mixed by pipetting, and an aliquot of 10 µL of each cell suspension was transferred to a glass slide, covered with a coverslip, and observed using a fluorescence microscope to obtain differential interference contrast images (DIC; Zeiss Fluorescence Microscope-Axio Imager D2 [Zeiss, Jena, Alemanha]). Aliquots (10 µL) of the cell suspensions taken before incubation were used as a control for negative aggregation for comparison purposes (time 0 h).

Effects of Chemicals on Aggregation
In order to investigate the influence of chemical factors on the aggregation ability of the clinical isolates of C. haemulonii species complex and C. auris, fungal suspensions were prepared as described above and then incubated for 2 h to allow aggregation in the following conditions: (i) PBS adjusted to pH 4.5, 7.2, and 8.5 to investigate the involvement of charged groups on aggregation capability [17]; (ii) 50 µg/mL of proteinase K (Invitrogen, Carlsbad, CA, USA); (iii) 0.25% trypsin (Nova Biotecnologia, São Paulo, Brazil) [9]; (iv) 0.05% to 0.25% sodium dodecyl sulfate (SDS) [18]; (v) 0.5% to 2% β-mercaptoethanol [18]; (vi) 0.5 mM to 5 mM ethylenediamine tetraacetic acid tetrasodium salt (EDTA; Sigma-Aldrich, St. Louis, MO, USA) [18]. DIC images were obtained as described above after incubation of the clinical isolates with the chemicals that affected their aggregation capability.

Effects of Temperature on Aggregation
To evaluate the influence of temperature on the aggregation capability of the isolates of the C. haemulonii species complex and C. auris, fungal cells were prepared as described above and then incubated for 2 h to allow aggregation at either 28 • C or 37 • C.

Statistics
All experiments were performed in triplicate, in three independent experimental sets. The results were analyzed statistically by Student's t-test (in comparisons between two groups) and by the Analysis of Variance One-Way ANOVA followed by Dunnett's multiple comparison test (in comparisons between three or more groups). All analyses were performed using the program GraphPad Prism 8. In all analyses, p values of 0.05 or less were considered statistically significant.

Aggregation Is a Time-Dependent Event in C. haemulonii Clade
The auto-aggregation capability of the 18 clinical isolates from the C. haemulonii species complex and C. auris studied herein was observed to be a time-dependent event ( Figure 1). When analyzing each species individually, we observed that C. auris isolates had a very similar percentage of aggregation at each time point, with mean aggregation ranging from 3.1 ± 1.8% after 30 min to 50.8 ± 3.7% after 120 min of incubation. Similar results were observed for C. duobushaemulonii isolates, with the mean percentage of aggregation varying from 6.3 ± 0.8% after 30 min to 57.7 ± 3.6% after 120 min. On the other hand, C. haemulonii and C. haemulonii var. vulnera isolates exhibited a more variable profile among the different isolates. In this sense, the mean aggregation of C. haemulonii isolates varied from 13.1 ± 8.9% after 30 min to 46.9 ± 10.8% after 120 min, while the mean percentage of aggregation of C. haemulonii var. vulnera isolates was 7.9 ± 2.4% and 54.3 ± 11.4% after 30 and 120 min of incubation, respectively.
Supporting our observations, Tomici and coworkers [17] also demonstrated that the autoaggregation percentage in isolates of C. albicans, C. krusei, C. glabrata, and Saccharomyces boulardii increases over time. Cellular aggregation is a well-known phenomenon in the microbial field, described in both bacteria and fungi, not only in natural environments but in mammalian hosts as well. For instance, nitrogen-fixing bacteria such as Azospirillum, Klebsiella, and Azotobacter can aggregate and flocculate, which contributes to their dispersal and survival in soil [18]. Microorganisms can exhibit the ability of auto-aggregation, characterized by the aggregation between cells of the same microbial strain, or coaggregation, characterized by the aggregation between different microbial strains or species, or even interkingdom interactions [19]. The formation of dental caries, for example, is highly mediated by the coaggregation process, and, for this reason, several coaggregation studies have focused on microorganisms of the human oral cavity, such as Streptococcus salivarius and Candida albicans, among others [19,20]. Additionally, the coaggregation of Lactobacillus with potential intestinal pathogens, such as Escherichia coli and Klebsiella spp., as well as some Candida species, as an anti-infection mechanism has also been investigated by research groups [17,21].
The global threat posed by C. auris depends in part on its described aggregative phenotype [4]. The impact of this phenotype on fungal cells and virulence is still being investigated, and studies are somehow contradictory, but it has been shown to influence biofilm formation, fungal virulence, and antifungal susceptibility, including tolerance to clinical concentrations of sodium hypochlorite, with some isolates persisting alive after 14 days of treatment [22].

Aggregation after Prolonged Periods and Viability
The aggregation ability of the selected fungal isolates was evaluated for prolonged periods (5 and 24 h) of incubation in PBS under inert conditions. The results revealed that aggregation remained time-dependent and, except for the isolate LIPCh5 of C. haemulonii var. vulnera, all the other isolates exhibited aggregation around 80% after 5 h and 90% after 24 h of incubation ( Figure 2A). Similar results were observed with C. albicans and C. krusei ATCC strains, but not with C. glabrata isolates, which exhibited considerably lower aggregation ability after the same incubation periods [17].
Fungal cells remained viable after aggregation, as can be observed by the fungal growth on SDA after the different incubation periods ( Figure 2B). Corroborating this finding, we stained the fungal cells with a crystal violet solution after the incubation periods and observed that the dye was unable to enter the cells, demonstrating their viability, including those forming the aggregates ( Figure 2C). On the other hand, fungal cells boiled for 20 min (control of dead cells) and then stained with the same dye turned blue, indicating that the dye entered the dead cells ( Figure 2C). Recently, Pelletier and coworkers [23] demonstrated that macrophages are unable to clear C. auris aggregates, which could benefit the fungi during a systemic infection by facilitating the persistence of infection. An elegant study conducted by Forgács and coworkers [24] showed the presence of large aggregates of C. auris, formed by single and budding yeast cells, in the kidneys, livers, and hearts of immunosuppressed mice after six days of infection. Moreover, despite exhibiting an aggregative or non-aggregative phenotype in vitro, the C. auris isolates presented the same behavior in vivo, and the authors speculate that these aggregates in tissues could protect the fungi from the host immune system [24].
All clinical isolates of the C. haemulonii species complex and C. auris that underwent testing demonstrated high aggregation after 2 h of incubation, according to the criteria established in this study (i.e., aggregation >40%), except for one C. haemulonii isolate (LIPCh7), which showed intermediate aggregation.
The following isolates were chosen for further experiments: LIPCh4 from C. haemulonii, LIPCh6 from C. duobushaemulonii, LIPCh5 from C. haemulonii var. vulnera, and Ca386 from C. auris. Candida duobushaemulonii and C. auris isolates were randomly selected for the study because very little difference was observed in their aggregation capabilities after 2 h of incubation. In contrast, for C. haemulonii and C. haemulonii var. vulnera, the isolates that showed the highest aggregation ability were chosen for the further experiments.

Aggregation after Prolonged Periods and Viability
The aggregation ability of the selected fungal isolates was evaluated for prolonged periods (5 and 24 h) of incubation in PBS under inert conditions. The results revealed that aggregation remained time-dependent and, except for the isolate LIPCh5 of C. haemulonii var. vulnera, all the other isolates exhibited aggregation around 80% after 5 h and 90% after 24 h of incubation ( Figure 2A). Similar results were observed with C. albicans and C. krusei ATCC strains, but not with C. glabrata isolates, which exhibited considerably lower aggregation ability after the same incubation periods [17].

Morphological Analysis of Cellular Aggregation
The four clinical isolates belonging to the C. haemulonii clade selected in the present study were analyzed both before and after a 2-h incubation at 37 °C in PBS at pH 7.2. Interestingly, we observed that the members of the C. haemulonii species complex exhibited clusters of cells even after vigorous vortex mixing, which represents the time 0 h of Fungal cells remained viable after aggregation, as can be observed by the fungal growth on SDA after the different incubation periods ( Figure 2B). Corroborating this finding, we stained the fungal cells with a crystal violet solution after the incubation periods and observed that the dye was unable to enter the cells, demonstrating their viability, including those forming the aggregates ( Figure 2C). On the other hand, fungal cells boiled for 20 min (control of dead cells) and then stained with the same dye turned blue, indicating that the dye entered the dead cells ( Figure 2C).

Morphological Analysis of Cellular Aggregation
The four clinical isolates belonging to the C. haemulonii clade selected in the present study were analyzed both before and after a 2-h incubation at 37 • C in PBS at pH 7.2. Interestingly, we observed that the members of the C. haemulonii species complex exhibited clusters of cells even after vigorous vortex mixing, which represents the time 0 h of the experiment. These aggregates became noticeably larger after the incubation period, indicating the occurrence of significant cell-to-cell interactions. In contrast, C. auris also displayed clusters of cells at 0 h, but these were considerably smaller than those observed in the C. haemulonii complex isolates and remained similarly sized after incubation. However, the number of aggregates and the number of cells per aggregate visibly increased, but these were clearly smaller in comparison with the C. haemulonii complex isolates (Figure 3).  pipetting, and observed using a light microscope. DIC images represent the isolates LIPCh4 from C. haemulonii, LIPCh6 from C. duobushaemulonii, LIPCh5 from C. haemulonii var. vulnera, and Ca386 from C. auris before (0 h) and after incubation (2 h). Images were obtained at ×20 magnification. Bars = 50 µm.

Effects of Chemical Factors on Aggregation
Our clinical isolates could be seen as single and budding yeast cells before and after incubation, and the same was observed by other authors with aggregates of C. auris both in vitro [23] and in vivo, using an immunosuppressed murine model [24].

Effects of Chemical Factors on Aggregation
The adaptation of opportunistic pathogens to different pH values favors their survival in the hostile environment of the human body, for example, facing basic pH in the mouth, acidic pH in the stomach, and neutral pH in the large intestine. In order to investigate the potential involvement of charged groups in the aggregation of C. haemulonii complex and C. auris isolates, we tested their ability to aggregate at different pH values. Since pH values ranging from 4.5 to 8.5 are relevant for biological systems, we evaluated the impact of PBS adjusted at three different pHs (4.5, 7.2, and 8.5) on the aggregation capability of the isolates and observed that none of the Candida species tested were affected by pH variation under the conditions used in our study (p > 0.05; Figure 4). On the other hand, Tomicic and coworkers [17] reported that the auto-aggregation of C. albicans, C. krusei, C. glabrata, and S. boulardii varied depending on pH values after 5 h of incubation, with the highest auto-aggregation observed at acidic pH (pH 4.5) and the lowest at basic pH (pH 8.5); however, after 24 h of incubation, no differences were observed in the auto-aggregation ability of these different Candida species.  Subsequently, the fungi were treated with two broad-spectrum proteases, proteinas K and trypsin, to investigate whether surface proteins, such as adhesins, play a role in th aggregation process of C. haemulonii species complex and C. auris. Our results showed tha both proteinase K and trypsin led to a significant reduction in the percentage of aggrega tion (p < 0.05), indicating that surface proteins are indeed important in cell-cell interaction that lead to aggregation in these emerging Candida species ( Figure 5). Subsequently, the fungi were treated with two broad-spectrum proteases, proteinase K and trypsin, to investigate whether surface proteins, such as adhesins, play a role in the aggregation process of C. haemulonii species complex and C. auris. Our results showed that both proteinase K and trypsin led to a significant reduction in the percentage of aggregation (p < 0.05), indicating that surface proteins are indeed important in cell-cell interactions that lead to aggregation in these emerging Candida species ( Figure 5).
Subsequently, the fungi were treated with two broad-spectrum proteases, proteina K and trypsin, to investigate whether surface proteins, such as adhesins, play a role in t aggregation process of C. haemulonii species complex and C. auris. Our results showed th both proteinase K and trypsin led to a significant reduction in the percentage of aggreg tion (p < 0.05), indicating that surface proteins are indeed important in cell-cell interactio that lead to aggregation in these emerging Candida species ( Figure 5). Bing and colleagues [7] demonstrated that treatment of C. auris with proteinase and trypsin led to the separation of cell clumps into individual yeast cells in an isolate C. auris that did not exhibit the typical aggregative phenotype; however, the enzym were not able to disrupt the aggregates of a typical aggregative isolate of C. auris. Furthe more, quantitative transcriptional expression assays demonstrated that the relative e pression levels of the ALS4 gene in the typical aggregative isolate were comparable those of a non-aggregative strain of C. auris, whereas the isolate whose aggregates we disrupted by the action of proteinase K and trypsin exhibited 400 times higher relati expression levels of the ALS4 gene [9]. Therefore, those authors suggested the existence two aggregative phenotypes in C. auris: the typical aggregative phenotype resulting fro a defect in cell division and release of the budding daughter cells, and the new aggregati phenotype caused by the expansion of the ALS4 gene adhesin [9]. Additionally, t Figure 5. Effects of proteinase K (PK) and trypsin on the aggregation capability of the clinical isolates of the C. haemulonii species complex and C. auris. The aggregation was evaluated by the reduction in the optical density (at 530 nm) of fungal suspensions in PBS containing the fungi (10 8 yeasts/mL), PK (50 µg/mL), and trypsin (0.25%) after 2 h of incubation at 37 • C. The results were expressed as percentages of aggregation of isolates LIPCh4 from C. haemulonii, LIPCh6 from C. duobushaemulonii, LIPCh5 from C. haemulonii var. vulnera, and Ca386 from C. auris. The values represent the mean ± standard deviation of three independent experiments. The symbols represent the significant difference in aggregation capability between PBS and the PK or Trypsin systems. The asterisks mean the following: (****) p < 0.0001; (***) p < 0.001; (**) p < 0.01.
Bing and colleagues [7] demonstrated that treatment of C. auris with proteinase K and trypsin led to the separation of cell clumps into individual yeast cells in an isolate of C. auris that did not exhibit the typical aggregative phenotype; however, the enzymes were not able to disrupt the aggregates of a typical aggregative isolate of C. auris. Furthermore, quantitative transcriptional expression assays demonstrated that the relative expression levels of the ALS4 gene in the typical aggregative isolate were comparable to those of a non-aggregative strain of C. auris, whereas the isolate whose aggregates were disrupted by the action of proteinase K and trypsin exhibited 400 times higher relative expression levels of the ALS4 gene [9]. Therefore, those authors suggested the existence of two aggregative phenotypes in C. auris: the typical aggregative phenotype resulting from a defect in cell division and release of the budding daughter cells, and the new aggregative phenotype caused by the expansion of the ALS4 gene adhesin [9]. Additionally, the authors demonstrated that the C. auris isolate with the new aggregative phenotype developed more robust biofilms on both polystyrene and silicone surfaces compared to the typical aggregative isolate and non-aggregative isolates of C. auris, which formed weaker biofilms [9]. Based on our findings, the clinical isolate of C. auris used in this study fits this newly described aggregative phenotype.
In order to investigate the role of proteins in aggregation and other features related to cell adhesion, we utilized chemicals, such as the protein-denaturing agent βmercaptoethanol, the chelator agent EDTA, and the anionic detergent SDS, to assess cellto-cell interactions. In this sense, treatments with 0.5 to 2% β-mercaptoethanol and 0.5 to 5 mM EDTA had no effect on the aggregation ability of the clinical isolates tested, indicating that disulfide bonds and divalent cations did not appear to mediate aggregation in the C. haemulonii complex and C. auris ( Figure 6A,B). Conversely, treatment with SDS significantly reduced the aggregation ability of all clinical isolates studied at concentrations varying from 0.10 to 0.25%, which suggests that hydrophobic interactions may play a role in cell-to-cell aggregation of isolates of the C. haemulonii complex and C. auris ( Figure 6C).
It has been reported that the use of detergents is not capable of disrupting C. auris aggregates in isolates exhibiting the typical aggregative phenotype [4]. However, in this study, we demonstrated that SDS significantly reduced the aggregation of our isolate of C. auris, which did not present the typical aggregative phenotype, and the same effect was observed for the isolates of the C. haemulonii complex. To our knowledge, until now, no other studies have evaluated the impact of protein-denaturing and chelator agents on the aggregation ability of Candida spp. or other yeasts. Nevertheless, a study conducted with the bacterium Azospirillum brasilense also demonstrated that β-mercaptoethanol and EDTA did not affect bacterial aggregation [18].
In order to investigate the role of proteins in aggregation and other features related to cell adhesion, we utilized chemicals, such as the protein-denaturing agent β-mercaptoethanol, the chelator agent EDTA, and the anionic detergent SDS, to assess cell-to-cell interactions. In this sense, treatments with 0.5 to 2% β-mercaptoethanol and 0.5 to 5 mM EDTA had no effect on the aggregation ability of the clinical isolates tested, indicating that disulfide bonds and divalent cations did not appear to mediate aggregation in the C. haemulonii complex and C. auris ( Figure 6A,B). Conversely, treatment with SDS significantly reduced the aggregation ability of all clinical isolates studied at concentrations varying from 0.10 to 0.25%, which suggests that hydrophobic interactions may play a role in cellto-cell aggregation of isolates of the C. haemulonii complex and C. auris ( Figure 6C). It has been reported that the use of detergents is not capable of disrupting C. auris aggregates in isolates exhibiting the typical aggregative phenotype [4]. However, in this Microscopic analyses confirmed the spectrometric results, revealing that the incubation of the clinical isolates of the four species tested herein with trypsin, proteinase K, and SDS drastically reduced their aggregation ability. As a result, the aggregates observed were considerably smaller than those observed in PBS systems (Figure 7). aggregation ability of Candida spp. or other yeasts. Nevertheless, a study conducted with the bacterium Azospirillum brasilense also demonstrated that β-mercaptoethanol and EDTA did not affect bacterial aggregation [18].
Microscopic analyses confirmed the spectrometric results, revealing that the incubation of the clinical isolates of the four species tested herein with trypsin, proteinase K, and SDS drastically reduced their aggregation ability. As a result, the aggregates observed were considerably smaller than those observed in PBS systems (Figure 7). Figure 7. Effects of trypsin, proteinase K, and SDS on the aggregation capability of the clinical isolates belonging to the C. haemulonii species complex and C. auris. The fungi (10 8 yeasts/mL) were incubated under inert conditions at 37 °C for 2 h in PBS (pH 7.2), trypsin (0.25%), proteinase K (50 μg/mL), and SDS (0.25%), gently homogenized by pipetting, and observed using a light microscope. DIC images represent the isolates LIPCh4 from C. haemulonii, LIPCh6 from C. duobushaemulonii, LIPCh5 from C. haemulonii var. vulnera, and Ca386 from C. auris after incubation. Images were obtained at 20 magnification. Bars = 50 μm.

Effects of Temperature on Aggregation
All experiments were conducted at 37 °C to mimic the normal temperature of the human body. However, we also evaluated the aggregation ability of the isolates at room temperature (28 °C). The results indicated that the clinical isolates of C. haemulonii complex and C. auris tended to form fewer aggregates at 28 °C, but no significant differences were Figure 7. Effects of trypsin, proteinase K, and SDS on the aggregation capability of the clinical isolates belonging to the C. haemulonii species complex and C. auris. The fungi (10 8 yeasts/mL) were incubated under inert conditions at 37 • C for 2 h in PBS (pH 7.2), trypsin (0.25%), proteinase K (50 µg/mL), and SDS (0.25%), gently homogenized by pipetting, and observed using a light microscope. DIC images represent the isolates LIPCh4 from C. haemulonii, LIPCh6 from C. duobushaemulonii, LIPCh5 from C. haemulonii var. vulnera, and Ca386 from C. auris after incubation. Images were obtained at ×20 magnification. Bars = 50 µm.

Effects of Temperature on Aggregation
All experiments were conducted at 37 • C to mimic the normal temperature of the human body. However, we also evaluated the aggregation ability of the isolates at room temperature (28 • C). The results indicated that the clinical isolates of C. haemulonii complex and C. auris tended to form fewer aggregates at 28 • C, but no significant differences were observed (p > 0.05; Figure 8). Tomicic and coworkers [9] reported that C. krusei and C. glabrata isolates exhibited a higher percentage of auto-aggregation at 37 • C compared to the same process at 28 • C and 42 • C; conversely, for C. albicans, a higher percentage of auto-aggregation was observed at 42 • C. observed (p > 0.05; Figure 8). Tomicic and coworkers [9] reported that C. krusei and C. glabrata isolates exhibited a higher percentage of auto-aggregation at 37 °C compared to the same process at 28 °C and 42 °C; conversely, for C. albicans, a higher percentage of auto-aggregation was observed at 42 °C.

Conclusions
This study collectively demonstrated the ability of the C. haemulonii species complex and C. auris to form cell aggregates in a typically time-dependent manner. The presence of proteinase K, trypsin, and SDS significantly impacted the auto-aggregation process, suggesting that surface proteins and hydrophobic interactions play a crucial role in mediating the cell-to-cell adhesion of these Candida species. Nevertheless, further studies are necessary to better elucidate the molecular aspects of this intriguing phenomenon.

Conclusions
This study collectively demonstrated the ability of the C. haemulonii species complex and C. auris to form cell aggregates in a typically time-dependent manner. The presence of proteinase K, trypsin, and SDS significantly impacted the auto-aggregation process, suggesting that surface proteins and hydrophobic interactions play a crucial role in mediating the cell-to-cell adhesion of these Candida species. Nevertheless, further studies are necessary to better elucidate the molecular aspects of this intriguing phenomenon.