Metabolic Plasticity and Virulence-Associated Factors of Sporothrix brasiliensis Strains Related to Familiar Outbreaks of Cat-to-Human Transmitted Sporotrichosis

Sporothrix brasiliensis is the main agent of zoonotic sporotrichosis transmitted by domestic cats in South America. In humans, sporotrichosis commonly presents with cutaneous or lymphocutaneous lesions, and in cats, with multiple ulcerated skin lesions associated with enlarged lymph nodes and respiratory signs. Fungal virulence factors may affect the clinical presentation of the mycoses. Sporothrix spp. present some virulence factors. This study aims to compare 24 S. brasiliensis strains from 12 familiar outbreaks of cat-to-human transmitted sporotrichosis. Fungal growth in different substrates, thermotolerance, resistance to oxidative stress, and production of enzymes were evaluated. An invertebrate model of experimental infection was used to compare the virulence of the strains. The strains grew well on glucose and N-acetyl-D-glucosamine but poorly on lactate. Their thermotolerance was moderate to high. All strains were susceptible to hydrogen peroxide, and the majority produced hemolysins but not phospholipase and esterase. There was no significant difference in the putative virulence-associated factors studied among the different hosts. Moreover, strains isolated from a human and a cat from four familiar outbreaks presented a very similar profile of expression of these factors, reinforcing the zoonotic transmission of S. brasiliensis in Brazil and demonstrating the plasticity of this species in the production of virulence factors.


Introduction
Species of the genus Sporothrix include dimorphic fungi, which inhabit the soil and decaying plants and can also be pathogenic to animals [1]. They belong to the Kingdom Fungi, Division Ascomycota, Class Pyrenomycetes, Order Ophiostomatales, and Family Ophiostomataceae, with about 53 species, including several species of medical and environmental relevance, such as Sporothrix schenckii, Sporothrix luriei Sporothrix albicans, S. brasiliensis, Sporothrix globosa, Sporothrix mexicana, Sporothrix pallida, Sporothrix Brunneoviolacea, and Sporothrix dimorphospora [2]. It is known that contact with decaying plant material is frequently observed in sporotrichosis cases due to S. globosa or S. schenckii, while S. brasiliensis-associated sporotrichosis is more related to zoonotic transmission by naturally infected cats [1].

Inoculum Preparation
All S. brasiliensis strains were previously cultivated at 25 • C on potato dextrose agar (PDA-Difco, Franklin Lakes, NJ, USA) to obtain conidia, and the filamentous colonies were washed with phosphate-buffered saline (PBS) pH 7.2 ± 0.1, containing 15% glycerol.
To obtain yeast cells, strains were cultivated on BHI agar (Difco, Franklin Lakes, NJ, USA) at 35 • C. Both incubations occurred for 7 days and yeasts were used in all experiments.

Evaluation of Fungal Growth 2.3.1. Growth Assay on Different Carbon Sources
The growth of strains was evaluated in the presence of glucose, lactate, and N-acetylglucosamine (GlcNAc) as carbon sources, with an initial inoculum of 1 × 10 6 yeasts/mL prepared in yeast nitrogen base medium (Difco, Franklin Lakes, NJ, USA) supplemented with 0.5% w/v lactate, 0.5% w/v GlcNAc, 0.5%, 1%, or 2% glucose (all from Sigma-Aldrich, Burlington, MA, USA). Aliquots of 100 µL were inoculated in triplicate in 96-well flat bottom plates (Jet Biofil, Guangzhou, China) and incubated at 35 • C for 7 days. The optical density of the wells at 530 nm was measured in a spectrophotometer (SpectraMax Plus 384, Molecular Devices, San Jose, CA, USA) every 24 h during the incubation period for the construction of growth curves [18].

Yeast Growth Assay in Powdered Claw Suspension
Healthy cat claws were crushed with a mortar and pestle, sterilized by autoclavation, and subsequently mixed with a minimum medium (MM) containing 15 mM glucose, 10 mM MgSO 4 7·H 2 O, 29 mM KH 2 PO 4 , 13 mM glycine, and 3 µM thiamine (all compounds from Merck Millipore, Darmstadt, Germany) to form a suspension of 2% w/v powdered claws. An inoculum of 1 × 10 6 yeast cells/mL was prepared in MM with or without (control) powdered claws and added to the wells of a 96-well plate in a 200 µL/well volume. Plates were incubated at 35 • C for 7 days, and the growth was analyzed by the absorbance measurement at 530 nm using a microplate reader (SpectraMax M3; Molecular Devices, San Jose, CA, USA). Mitochondrial activity was determined with the CyQUANT XTT assay (Thermo Fisher, Waltham, MA, USA). A mixture of XTT reagent and electron coupling reagent was prepared at a ratio of 6 to 1. Seventy microliters of the mixture were added to each well. Then, the plate was incubated at 37 • C for 4 h in the dark in a 5% CO 2 incubator. The optical densities (ODs) were determined, in two technical replicates, using the SpectraMax ® Plus 384 Microplate Reader (SpectraMax M3; Molecular Devices, San Jose, CA, USA) at 450 nm and 660 nm. Mitochondrial activity was expressed by the decrease in OD (450-660 nm), discounting the average of the ODs of the controls [29].

Thermotolerance Evaluation
An inoculum of 1 × 10 6 yeasts/mL was prepared from all strains. Then, 5 µL aliquots of the cell suspension were inoculated in triplicate in two Petri dishes (90 mm) containing PDA medium and incubated at 25 • C and 35 • C for a total period of 7 days. After the incubation, the diameter of the colonies was measured in millimeters, and then the percentage of growth inhibition (%GI) was calculated, as described [30]. The thermotolerance of each strain was classified as %GI from 0 to 33%-high thermotolerance, %GI from 34 to 65%-moderate thermotolerance, and %GI from 66 to 100%-low thermotolerance.

Resistance to Oxidative Stress
For the analysis of oxidative stress in S. brasiliensis, 57.5 mL of BHI medium (Difco, Franklin Lakes, NJ, USA), sterilized in an autoclave and cooled to 50 • C, was supplemented with 2.5 mL of a cell suspension (1 × 10 6 yeasts/mL) in 0.45% NaCl solution. This suspension was added to a Petri dish (15 × 150 mm) and then gently shaken to obtain a homogeneous medium. After solidification, four wells of 5 mm in diameter and distributed equidistantly were made on each plate. In each well, 65 µL of 5% v/v hydrogen peroxide (Proquimios, Rio de Janeiro, Brazil) was applied. The assessment of resistance to oxidative stress was carried out by measuring the diameter of the fungal growth inhibition halo formed around each well, with the aid of a pachymeter, after 7 days of incubation at 35 • C [18].

Urease Production
In a final volume of 2 mL of Christensen urea broth (peptone 0.1%, NaCl 0.5%, KH 2 PO 4 0.2%, urea 2%, glucose 0.1%, phenol red 0.0016%), 200 µL of a cell suspension containing 1 × 10 6 yeasts/mL was added. These suspensions were incubated at 35 • C for 7 days, then centrifuged at 10,000× g for 10 min, and 100 µL of the supernatant was transferred in triplicate to a sterile 96-well flat-bottom polystyrene plate (Jet Biofil, Guangzhou, China). Candida krusei (ATCC 6258) and Candida parapsilosis (ATCC 22019) were used as negative controls and Cryptococcus neoformans (H99) as a positive control of urease production. The absorbance of the samples was measured in an Epoch Biotek spectrophotometer at 559 nm [31].

Hemolytic, Phospholipase, and Esterase Production
Enzymatic activity was measured in Petri dishes (90 mm) containing specific media for testing each enzyme. Each enzymatic activity was determined through the Pz value Pz = A B , where A is the colony diameter in millimeters (mm) and B is the colony diameter (mm) plus the halo of enzymatic activity around the fungal colony. To prepare the inoculum, a cell suspension of 1 × 10 6 yeasts/mL was used, where 5 µL aliquots were inoculated in triplicate on the surface of the undermentioned media and incubated for 7 days at 35 • C. Positive samples showed a halo around the colonies. Candida albicans (ATCC 18804) was used as a positive control to assess the quality of the culture media [22]. The enzymatic activity was classified into four categories as described: Pz: 1-Negative; Pz between 0.70 and 0.99-low activity; Pz between 0.40 and 0.69-moderate activity; and Pz lower than 0.39-high activity [32]. The hemolytic activity was determined by 5% Sheep Blood Agar (PlastLabor, Rio de Janeiro, Brazil) [22]. Egg Yolk Agar, composed of 2% glucose (Sigma-Aldrich, St Louis, MO, USA), 1% peptone, 0.5% yeast extract (Difco, Franklin Lakes, NJ, USA), 4% NaCl, 0.074% CaCl 2 (Sigma-Aldrich, Burlington, MA, USA), 1.5% Agar (Difco, Franklin Lakes, NJ, USA), plus 8% organic egg yolk emulsion at pH 7.0, was used to determine phospholipase activity [32]. Tween Agar (1% peptone; 0.5% yeast extract; 0.01% CaCl 2 ; 1.5% agar), plus 0.1% sterile Tween 80 (Sigma-Aldrich, Burlington, MA, USA), pH 7.0 was used to determine esterase activity [33].

Survival Tests on Molitor
For survival studies, we use the adapted methodology proposed by Lozoya-Pérez and collaborators [34], where larvae of at least 1 cm of uniform color, without dark spots or grayish marks, were selected. The animals were infected in the last left proleg with 10 µL of S. brasiliensis yeast cell suspensions (1 × 10 6 yeasts/mL), using a Hamilton syringe (701 N, 26 caliber, 10 µL capacity), into the hemocoel. Then, animals were kept in Petri dishes of 14 cm at 37 • C. Mortality changes were recorded for 10 days. A total of 40 animals were included in each experimental group: 10 infected, 10 control, 10 animals untouched, and 10 inoculated only with PBS. The animal groups were under observation for up to 10 days, and live animals at the end of this period, along with those killed by the fungal inoculum, were decapitated with a sterile scalpel. The hemolymph was used to calculate the colonyforming units (CFUs) by serial dilutions that were incubated on Mycosel plates (Difco, Franklin Lakes, NJ, USA), at 28 • C for 72 h. The experiment was performed in triplicate.

Statistical Analyses
All experiments were performed in triplicate, in three independent experimental sets, except when strictly described before, and the results were expressed as mean ± standard deviation. The results were analyzed using the GraphPad Prism 9 software (La Jolla, San Diego, CA, USA), with p-values less than 0.05 to determine the significance. Non-parametric tests were used as appropriate and necessary in the different analyses and comparisons. The overall analysis of all virulence-related phenotypes was performed through a heat map constructed with the Heatmapper tool [35]. The correlation between the expression of different phenotypes by the same strain was assessed using Spearman's correlation in GraphPad Prism 9 software.

Fungal Growth on Different Substrates
The growth profile of the isolates in the presence of glucose was similar among the 24 S. brasiliensis, regardless of glucose concentrations ( Figure 1). In general, all isolates grew poorly in the presence of lactate ( Figure 2). The growth of the S. brasiliensis strains herein studied was more abundant in the presence of GlcNAc than lactate ( Figure 3). Growth curves for human-and cat-derived S. brasiliensis strains from the familiar outbreaks were similar for all carbon sources evaluated; cases 1, 3, 4, 6, 11, and 12 presented significant growth differences on lactate and cases 1, 6, 8, and 11 on GlcNAc (p-value ≤ 0.05, Mann-Whitney test).
J. Fungi 2023, 9, x FOR PEER REVIEW 6 of 21 heat map constructed with the Heatmapper tool [35]. The correlation between the expression of different phenotypes by the same strain was assessed using Spearman's correlation in GraphPad Prism 9 software.

Fungal Growth on Different Substrates
The growth profile of the isolates in the presence of glucose was similar among the 24 S. brasiliensis, regardless of glucose concentrations ( Figure 1). In general, all isolates grew poorly in the presence of lactate ( Figure 2). The growth of the S. brasiliensis strains herein studied was more abundant in the presence of GlcNAc than lactate ( Figure 3). Growth curves for human-and cat-derived S. brasiliensis strains from the familiar outbreaks were similar for all carbon sources evaluated; cases 1, 3, 4, 6, 11, and 12 presented significant growth differences on lactate and cases 1, 6, 8, and 11 on GlcNAc (p-value ≤ 0.05, Mann-Whitney test).    The majority of S. brasiliensis strains exhibited better growth in the MM supplemented with powdered claws than in MM without any supplementation after 7 days of incubation ( Figure 4A). Besides growth, metabolic activity after this incubation time was quantified by XTT-reaction assay ( Figure 4B). The human S. brasiliensis strain from outbreak 7 did not have metabolic activity after this incubation period. The metabolic activity of humanand cat-derived strains was similar on both MM and MM supplemented with cat claw powder. In addition, outbreaks 3, 4, and 6 presented differences between growth in MM of human and cat strains (p-values = 0.0059, =0.0042, and <0.0001, respectively, Tukey's multiple comparisons test). Results are expressed as the mean and standard deviation of readings obtained from three independent experiments performed in technical triplicates. Asterisks indicate significant differences between growth curves of human-and cat-derived strains from the familiar outbreak. The asterisk symbol indicates a statistically significant difference between the growth curves of paired cases, pvalue (Mann-Whitney test) ≤ 0.05.
The majority of S. brasiliensis strains exhibited better growth in the MM supplemented with powdered claws than in MM without any supplementation after 7 days of incubation ( Figure 4A). Besides growth, metabolic activity after this incubation time was quantified by XTT-reaction assay ( Figure 4B). The human S. brasiliensis strain from outbreak 7 did not have metabolic activity after this incubation period. The metabolic activity of human-and cat-derived strains was similar on both MM and MM supplemented with cat claw powder. In addition, outbreaks 3, 4, and 6 presented differences between growth in MM of human and cat strains (p-values = 0.0059, =0.0042, and <0.0001, respectively, Tukey's multiple comparisons test). The optical density (530 nm) of cultured isolates was measured daily for 7 days. Results are expressed as the mean and standard deviation of readings obtained from three independent experiments performed in technical triplicates. Asterisks indicate significant differences between growth curves of human-and cat-derived strains from the familiar outbreak. The asterisk symbol indicates a statistically significant difference between the growth curves of paired cases, p-value (Mann-Whitney test) ≤ 0.05.

Susceptibility to Physical and Chemical Stressors
All strains analyzed showed thermotolerance, ranging from high (n = 1) (%GI 0 to 33%) to moderate thermotolerance (n = 23) (%GI 34 to 65%). Cases 6 and 8 showed significant differences between human and animal isolates ( Figure 5A) (p-values = 0.0313 and <0.0001, respectively, Mann-Whitney test). Figure 5B presents data about the oxidative stress of the clinical S. brasiliensis isolates herein studied. The oxygen-derived oxidant susceptibility profile of strains isolated from cats and humans varied (p-value ≤ 0.0001, Mann-Whitney test). Moreover, the paired strains from outbreak 6 showed a significant difference between them (p-value = 0.0370, Mann-Whitney test).

Susceptibility to Physical and Chemical Stressors
All strains analyzed showed thermotolerance, ranging from high (n = 1) (%GI 0 to 33%) to moderate thermotolerance (n = 23) (%GI 34 to 65%). Cases 6 and 8 showed significant differences between human and animal isolates ( Figure 5A) (p-values = 0.0313 and <0.0001, respectively, Mann-Whitney test). Figure 5B presents data about the oxidative stress of the clinical S. brasiliensis isolates herein studied. The oxygen-derived oxidant sus- Whitney test). Moreover, the paired strains from outbreak 6 showed a significant difference between them (p-value = 0.0370, Mann-Whitney test).

Production of Extracellular Enzymes
All samples produced urease, as visually determined by the change of the medium color to pink, like that observed for the C. neoformans strain and contrasting with that observed for the C. parapsilosis and C. krusei control strains that did not change the color of the Christensen's urea broth. Outbreaks 1, 4, 5, 8, and 9 showed significant differences between human-and cat-derived strains ( Figure 6A

Production of Extracellular Enzymes
All samples produced urease, as visually determined by the change of the medium color to pink, like that observed for the C. neoformans strain and contrasting with that observed for the C. parapsilosis and C. krusei control strains that did not change the color of the Christensen's urea broth. Outbreaks 1, 4, 5, 8, and 9 showed significant differences between human-and cat-derived strains ( Figure 6A) (cases 1, 8, and 9 p-value ≤ 0.0001, 4 = 0.0238, and 5 = 0.0001, Tukey's multiple comparisons test).
was no enzymatic production of phospholipase and esterase by the 24 S. brasiliensis strains of this study. Among these strains, four (16.7%), three isolated from humans and one isolated from a cat, from four distinct outbreaks (6H, 8H, 10C, and 11H) were unable to produce hemolysin ( Figure 6B). The production of hemolysin varied from low enzymatic activity (n= 17) (Pz between 0.70 and 0.99) to moderate (n = 3) (Pz between 0.40 and 0.699). Differences between human-and cat-derived strains were not significant (p-value < 0.05, Tukey's multiple comparisons test).   For the enzymes evaluated on agar plates, the control C. albicans strain yielded Pz values of 0.4, 0.6, and 0.6 for phospholipase, esterase, and hemolysin, respectively. There was no enzymatic production of phospholipase and esterase by the 24 S. brasiliensis strains of this study. Among these strains, four (16.7%), three isolated from humans and one isolated from a cat, from four distinct outbreaks (6H, 8H, 10C, and 11H) were unable to produce hemolysin ( Figure 6B). The production of hemolysin varied from low enzymatic activity (n= 17) (Pz between 0.70 and 0.99) to moderate (n = 3) (Pz between 0.40 and 0.699). Differences between human-and cat-derived strains were not significant (p-value < 0.05, Tukey's multiple comparisons test).   . Survival curves of Tenebrio molitor larvae infected with Sporothrix brasiliensis strains from 12 familiar outbreaks of sporotrichosis. Larvae were infected with a 10 µL suspension containing 1 × 10 6 yeast-like cells and incubated at 37 • C and observed until 10 days after inoculation. A total of 40 animals were included in each experimental group, and these were monitored daily to assess mortality, which was defined as a lack of irritability and the presence of extensive body melanization. The asterisk symbol indicates a statistically significant difference between the growth curves of paired cases, p-value (Log-rank (Mantel-Cox) test) ≤ 0.05.

Survival Tests on Tenebrio Molitor
To demonstrate fungal growth in inoculated animals, the colony-forming units (CFUs) were quantified in infected animals ( Figure 8). Cases 2, 6, and 8 show differences in CFU compared to corresponding paired cases (p = 0.0022, Mann-Whitney test).
x FOR PEER REVIEW 13 of 21 mortality, which was defined as a lack of irritability and the presence of extensive body melanization. The asterisk symbol indicates a statistically significant difference between the growth curves of paired cases, p-value (Log-rank (Mantel-Cox) test) ≤ 0.05.
To demonstrate fungal growth in inoculated animals, the colony-forming units (CFUs) were quantified in infected animals ( Figure 8). Cases 2, 6, and 8 show differences in CFU compared to corresponding paired cases (p = 0.0022, Mann-Whitney test).

Comparative Analysis of Virulence-Related Factors
As depicted in Figure 9, none of the 24 S. brasiliensis strains presented a high level of all herein-studied putative virulence-associated factors. In general, the major phenotypic features potentially related to fungal virulence among the studied S. brasiliensis strains were urease production and susceptibility to hydrogen peroxide. The dendrogram clustered the strains according to their global similarity in the production of those studied factors. It is interesting to note that human-and cat-derived strains from outbreaks 2 and 4 clustered together, while strains from outbreaks 7 and 9 clustered very closely.

Comparative Analysis of Virulence-Related Factors
As depicted in Figure 9, none of the 24 S. brasiliensis strains presented a high level of all herein-studied putative virulence-associated factors. In general, the major phenotypic features potentially related to fungal virulence among the studied S. brasiliensis strains were urease production and susceptibility to hydrogen peroxide. The dendrogram clustered the strains according to their global similarity in the production of those studied factors. It is interesting to note that human-and cat-derived strains from outbreaks 2 and 4 clustered together, while strains from outbreaks 7 and 9 clustered very closely.
Possible correlations between the different fungal phenotypes were also investigated. Figure 10 shows a heatmap representing the Spearman's correlation coefficients of the associations between different phenotypes of the studied isolates. The red color represents negative associations, whereas the blue color represents positive associations. Growth on glucose was negatively monotonically related to growth on GlcNAc (p-value = 0.039) and lactate (p-value = 0.030). Hemolysin production was positively monotonically related to growth on glucose and thermotolerance (p-values = 0.014 and =0.030, respectively), while urease production was positively monotonically related to growth on lactate and GlcNAc (p-values = 0.031 and =0.030, respectively). Possible correlations between the different fungal phenotypes were also investigated. Figure 10 shows a heatmap representing the Spearman's correlation coefficients of the associations between different phenotypes of the studied isolates. The red color represents negative associations, whereas the blue color represents positive associations. Growth on glucose was negatively monotonically related to growth on GlcNAc (p-value = 0.039) and lactate (p-value = 0.030). Hemolysin production was positively monotonically related to growth on glucose and thermotolerance (p-values = 0.014 and =0.030, respectively), while urease production was positively monotonically related to growth on lactate and GlcNAc (p-values = 0.031 and =0.030, respectively). Finally, a regression analysis was conducted to estimate the relative contributions of the studied virulence factors to the survival of T. molitor. As shown in Table 2, none of the herein-studied virulence-associated factors presented a significant association with larvae survival.

Discussion
The S. brasiliensis strains herein studied showed differences in some phenotypi characteristics that have been described as related to virulence in other human pathoge fungi [36][37][38][39][40]. These aspects should be studied from a "One Health" perspective since t species continuously passes through different mammalian hosts and environmen sources [41]. This was the reason that motivated us to compare these phenotypes

Discussion
The S. brasiliensis strains herein studied showed differences in some phenotypical characteristics that have been described as related to virulence in other human pathogenic fungi [36][37][38][39][40]. These aspects should be studied from a "One Health" perspective since this species continuously passes through different mammalian hosts and environmental sources [41]. This was the reason that motivated us to compare these phenotypes on strains obtained from humans and the cats that have infected them. In general, no differences were observed on human-and cat-derived S. brasiliensis strains, and for some outbreaks, phenotypes were extremely close for the related strains, suggesting that one passage through a mammalian host does not significantly affect the studied phenotypes.
Adaptations to environmental stressors may compel some fungi to acquire abilities that can increase their virulence and survival, driving the emergence of new threats to human, animal, and environmental health [42]. To survive the adverse conditions during parasitism, pathogenic fungi must adapt to the harsh microenvironments of the host. They may display altered phenotypic characteristics due to the selection pressure, in a process known as microevolution, as demonstrated in fungi of medical interest, such as Aspergillus fumigatus [43], C. neoformans [44], and C. albicans [45]. The present study also shows that S. brasiliensis has metabolic plasticity and may also suffer microevolution depending on the host. This plasticity to adapt to various stress conditions imposed during protracted infections on different patients was also observed previously for patients with cryptococcosis [46] and sporotrichosis [18].
Pathogenic fungi could use various carbon sources for development and survival [47,48]. Disturbances in host homeostasis can cause differences in glucose concentrations in the body [48], impacting the pathogenesis of the mycoses. For instance, colonization by Candida spp. is associated with glycemic control in patients with diabetes because hyperglycemia increases salivary glucose levels, promoting pathogen growth in the oral cavity [48]. Studies indicate that C. albicans can colonize immunocompetent individuals, and its cells are able to quickly adapt to available carbon sources in the host. Lactate, glucose, and GlcNAc are some of the carbon sources available for pathogens in the human body [49,50]. The S. brasiliensis growth studied here was noticeable in glucose since this is the main carbon source used by fungi. Concerning alternative carbon sources, the growth was slightly greater in the presence of GlcNAc than lactate, like that observed previously by our group in a group of S. brasiliensis strains isolated from human patients with chronic disseminated disease [18]. The skin tropism of S. brasiliensis may explain its preferential use of GlcNAc over lactate. GlcNAc is a glucosamine derivative that is naturally produced by humans and, together with D-glucuronate, forms hyaluronic acid, which is present in several body tissues, such as joints and skin. On the other hand, lactate is a widely abundant compound in most host niches, and it is a carboxylic acid that, in the body, is mainly found in its ionic form. Lactate is a product of glycolysis and plays fundamental roles in skeletal muscle tissue [48].
The production of extracellular enzymes allows the hydrolysis of skin components and promotes the invasion of the pathogen into the host tissues [51]. Moreover, when fungi are phagocytosed, they induce the release of oxidative chemical mediators, such as hydrogen peroxide, that cause oxidative stress in the microbe. Fungi, as an escape mechanism, produce catalases promoting fungal multiplication within macrophages [52]. The enzyme converts H 2 O 2 into water and oxygen, allowing the fungus to remain viable after the host's immune response, triggering its multiplication process [53]. The S. brasiliensis strains studied here were inhibited by hydrogen peroxide. Our results do not allow quantifying the catalase amount produced by the strains, but they show that they produce high amounts of catalase, due to the halo of growth inhibition lower than that described for A. fumigatus [54] and S. brasiliensis strains from chronic disease [18]. S. brasiliensis is an extracellular pathogen; therefore, its survival does not strongly depend on resistance to phagocytosis.
Phospholipase and esterase activities were not detected on the S. brasiliensis strains under the current study conditions. It is interesting to note that we previously detected esterase production in patients with chronic sporotrichosis [18], indicating that this enzyme may have a role in fungal maintenance in hosts with compromised immunity over extended periods of time. All isolates were able to produce urease. This enzyme is responsible for the use of urea as a source of nitrogen, causing the release of ammonia, resulting in a partial increase in the acidic pH [55]. In C. neoformans, urease is important for the invasion of the central nervous system since it facilitates the crossing of the bloodbrain barrier [56]. It is known that S. brasiliensis produces higher levels of urease when compared to S. schenckii [31], and that S. brasiliensis could cause meningitis in severely immunosuppressed patients [57], which may be linked to the production of this enzyme. Interestingly, the urease levels produced by our strains were similar to that produced by the control C. neoformans strain, a traditional agent of fungal meningitis. Fungi can also secrete hemolysins responsible for the lysis of nucleated and red blood cells [58]. Four strains of the present study were unable to produce hemolysin. The production of this enzyme by S. brasiliensis isolates from human disseminated disease is useful during hematogenic dissemination [18]. Sporotrichosis in cats is progressive and blood dissemination will likely occur without proper management [59]. These proteins are important for the survival of microorganisms and are related to iron acquisition; therefore, their production is essential for the establishment of an infectious process [60].
Sporothrix spp. can be isolated from claw surfaces of naturally infected cats [61] and in an ex vivo biofilm model using the filamentous form of Sporothrix species and cat claws [26]. Our results show that the yeast-like form of S. brasiliensis can also use nutrients from cat claws to improve fungal growth, like that described in the literature [29]. This is important information, supporting that this species can survive on cat claws in its parasitic form, facilitating the zoonotic transmission of sporotrichosis. It is not possible to conclude that the added keratin facilitated the formation of biofilms, as the nail powder used may contain various nutrients for fungi. Furthermore, the low metabolic activity observed in certain Sporothrix strains may be related to this. To explore this further, future experiments may focus on identifying components present in cat nails that may promote the growth of S. brasiliensis.
The mealworm, also known as T. molitor, is an invertebrate animal model used to study fungal virulence [62]. T. molitor has been used to assess the virulence of entomopathogenic fungi such as Metarhizium robertsii [63], Metarhizium brunneum [64], and Beauveria bassiana [65] and human pathogens such as C. albicans [62], C. neoformans [62], and Fonsecaea spp. [66]. This study demonstrated the virulence of S. brasiliensis in these animals, with the median survival ranging from 2 to 7 days at 37 • C. The results that we have obtained for the yeast form are like that previously described for this species [34]. The observed outcomes concerning the yeast form align with previous reports on this particular species' behavior.
The present study suggests that S. brasiliensis has a complex phenotype expression profile to adapt to the conditions found in the hosts. The data obtained in this study demonstrated that the S. brasiliensis isolates could grow in different nutrient sources available in hosts, present thermotolerance, susceptibility to the stress generated by hydrogen peroxide, and produce hemolysin. These metabolic factors may be associated with the installation/infection of S. brasiliensis in humans and other animals, demonstrating the plasticity of S. brasiliensis strains. The clinical signs of human and cat patients infected by this fungus are most probably linked to the host immunity or to other enzymes and virulence factors not evaluated in this study because they did not demonstrate significance with the survival of the experimental model herein used. Funding: This work was supported by the Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES), Finance Code 001, and Fundação de Amparo à Pesquisa do Rio de Janeiro (FAPERJ). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data in this study are available in the presented manuscript.