Mechanism of molecular interaction of sitagliptin with human DPP4 enzyme - New Insights.

PURPOSE: Dipeptidyl peptidase 4 (DPP4) inactivates a range of bioactive peptides. The cleavage of insulinotropic peptides and glucagon-like peptide 1 (GLP1) by DPP4 directly influences glucose homeostasis. This study aimed to describe the mode of interaction between sitagliptin (an antidiabetic drug) and human DPP4 using in silico approaches. MATERIALS AND METHODS: Docking studies were conducted using AutoDock Vina, 2D and 3D schematic drawings were obtained using PoseView and PLIP servers, and the DPP4-sitagliptin complex was visualized with Pymol software. RESULTS: The best affinity energy to form the DPP4-sitagliptin complex was E-value â€‹= â€‹- 8.1 â€‹kcal â€‹mol-1, as indicated by docking simulations. This result suggests a strong interaction. According to our observations, hydrophobic interactions involving the amino acids residues Tyr663 and Val712, hydrogen bonds (Glu203, Glu204, Tyr663, and Tyr667), π-Stacking interactions (Phe355 and Tyr667), and halogenic bonds (Arg123, Glu204, and Arg356) were prevalent in the DPP4-sitagliptin complex. Root Mean Square Deviation prediction also demonstrated that the global structure of the human DPP4 did not have a significant change in its topology, even after the formation of the DPP4-sitagliptin complex. CONCLUSION: The stable interaction between the sitagliptin ligand and the DPP4 enzyme was demonstrated through molecular docking simulations. The findings presented in this work enhance the understanding of the physicochemical properties of the sitagliptin interaction site, supporting the design of more efficient gliptin-like iDPP4 inhibitors.

Antifungal activity of sustainable histone deacetylase inhibitors against planktonic cells and biofilms of Candida spp. and Cryptococcusneoformans.

The limited therapeutic options for fungal infections and the increased incidence of fungal strains resistant to antifungal drugs, especially Candida spp., require the development of new antifungal drugs and strategies. Histone deacetylase inhibitors (HDACi), like vorinostat, have been studied in cancer treatment and have antifungal effects, acting alone or synergistically with classical antifungals. Here we investigated the antifungal activity of two novel sustainable HDACi (LDT compounds) based on vorinostat structure. Molecular docking simulation studies reveal that LDT compounds can bind to Class-I HDACs of Candida albicans, C. tropicalis, and Cryptococcus neoformans, which showed similar binding mode to vorinostat. LDT compounds showed moderate activity when tested alone against fungi but act synergistically with antifungal azoles against Candida spp. They reduced biofilm formation by more than 50% in C. albicans (4 µg/mL), with the main action in fungal filamentation. Cytotoxicity of the LDT compounds against RAW264.7 cells was evaluated and LDT536 demonstrated cytotoxicity only at the concentration of 200 µmol/L, while LDT537 showed IC50 values of 29.12 µmol/L. Our data indicated that these sustainable and inexpensive HDACi have potential antifungal and antibiofilm activities, with better results than vorinostat, although further studies are necessary to better understand the mechanism against fungal cells.

Fungal infections are neglected diseases that affect more than a billion people worldwide. Some histone deacetylase inhibitors can act against fungal cells. Our data reveal that HDACi LDT536 and LDT537 have potential antibiofilm and antifungal activities.

Inhibitory effect of proteinase K against dermatophyte biofilms: an alternative for increasing the antifungal effects of terbinafine and griseofulvin.

This study aimed to evaluate the effect of proteinase K on mature biofilms of dermatophytes, by assays of metabolic activity and biomass. In addition, the proteinase K-terbinafine and proteinase K-griseofulvin interactions against these biofilms were investigated by the checkerboard assay and scanning electron and confocal microscopy. The biofilms exposed to 32 µg ml-1 of proteinase K had lower metabolic activity and biomass, by 39% and 38%, respectively. Drug interactions were synergistic, with proteinase K reducing the minimum inhibitory concentration of antifungals against dermatophyte biofilms at a concentration of 32 µg ml-1 combined with 128-256 µg ml-1 of terbinafine and griseofulvin. Microscopic images showed a reduction in biofilms exposed to proteinase K, proteinase K-terbinafine and proteinase K-griseofulvin combinations. These findings demonstrate that proteinase K has activity against biofilms of dermatophytes, and the interactions of proteinase K with terbinafine and griseofulvin improve the activity of drugs against mature dermatophyte biofilms.

The herbicide paraquat alters growth and melanin production on the Cryptococcus neoformans/Cryptococcus gattii species complex.

Paraquat (1,10-dimethyl-4,4-bipyridinium dichloride; PQ) is a free-radical producing herbicide that affects cell membranes and can upset the environmental balance of microorganisms present in soil, such as Cryptococcus spp. This study aimed to evaluate the in vitro activity of PQ against Cryptococcus spp. in planktonic and biofilm forms, as well as the protective effect of antioxidant agents against the antifungal effect of PQ and the kinetics of melanin production in response to PQ. Susceptibility to PQ was evaluated by microdilution. Cryptococcus sp. strains exposed to PQ were grown in media with ascorbic acid (AA) and glutathione (GSH). Melanin production was assessed in the presence of l-3,4-dihydroxyphenylalanine (l-DOPA) + PQ. The minimum inhibitory concentration of PQ against Cryptococcus spp. ranged from 8 to 256 µg/mL. Furthermore, PQ reduced biofilm formation. AA and GSH restored the fungal growth of Cryptococcus spp. exposed to PQ. In addition, l-DOPA + PQ delayed melanin production by 24 and 48 h for C. deuterogattii and C. neoformans sensu lato, respectively, suggesting that PQ induces a fitness trade-off in melanin production. Taken together, our data suggest that the antifungal effect of PQ against Cryptococcus spp. possibly exerts selective pressures interfering with biofilm formation and melanin production by these yeasts.

Azole-Resilient Biofilms and Non-wild Type C. albicans Among Candida Species Isolated from Agricultural Soils Cultivated with Azole Fungicides: an Environmental Issue?

This study aimed to identify Candida spp. from agricultural soils cultivated with azole fungicides and investigate their susceptibility to clinical (fluconazole, itraconazole, voriconazole, and amphotericin B) and agricultural (tetraconazole and tebuconazole) antifungals in planktonic form. Additionally, Candida biofilm-forming ability and biofilm susceptibility to agricultural antifungals and voriconazole were analyzed. Species identification was performed by phenotypic and molecular assays. The susceptibility of planktonic cells was evaluated by the broth microdilution method. The biofilm metabolic activity was evaluated by the XTT reduction assay. The recovered Candida spp. were identified as C. parapsilosis sensu stricto (n = 14), C. albicans (n = 5), C. tropicalis (n = 2), C. fermentati (n = 1), and C. metapsilosis (n = 2). Minimum inhibitory concentration ranges for clinical and agricultural antifungals were ≤ 0.03-4 µg/mL and 1-128 µg/mL, respectively. Two and one C. albicans strains were considered non-wild type for voriconazole and fluconazole, respectively. All strains were biofilm producers. The minimum biofilm inhibitory concentration ranges for tetraconazole and tebuconazole were 128-> 1024 µg/mL, while for voriconazole was 512-> 1024 µg/mL. In summary, this study shows that non-wild type and azole-resilient biofilm-producing Candida species colonize agricultural soils cultivated with azole fungicides.

Essential oils encapsulated in chitosan microparticles against Candida albicans biofilms.

The aim of the study was to produce and characterize chitosan microparticles loaded with essential oils (CMEOs), evaluate the essential oil (EO) release profile and the CMEOs' anti-Candida activity. The chitosan microparticles (CMs) loaded with lemongrass essential oil (LEO) and geranium essential oil (GEO) were produced by the spray-drying method and characterized regarding CMEO morphological and physicochemical parameters and EO encapsulation efficiency (EE) and release profile. The planktonic activity was quantified by broth microdilution, and the activity against biofilm was quantified by biomass formation measurement. The LEO and GEO compositions were analyzed by gas chromatography combined with mass spectrometry (GC/MS), finding the main components citral (83.17%) and citronellol (24.53%). The CMs and CMEOs showed regular distribution and spherical shape (1 to 15 µm), without any morphological and physical modifications after EO incorporation. EE% ranged from 12 to 39%. In vitro release tests demonstrated the EO release rates, after 144 h, were 33% and 55% in PBS and HCl media, respectively. The minimum inhibitory concentration (MIC) values for CMEOs were lower than for CMs and pure EOs (P < 0.05). The higher CMEO biofilm inhibition percentage demonstrates the efficiency of microparticles against Candida biofilm. These results indicate that CMEOs are promising compounds that have antibiofilm activity against C. albicans.

Antifungal activity of deferiprone and EDTA against Sporothrix spp.: Effect on planktonic growth and biofilm formation.

The present study evaluated the antifungal activity of the chelators deferiprone (DFP) and ethylenediaminetetraacetic acid (EDTA) and their effect on biofilm formation of the S. schenckii complex. Eighteen strains of Sporothrix spp. (seven S. brasiliensis, three S. globosa, three S. mexicana and five Sporothrix schenckii sensu stricto) were used. Minimum inhibitory concentration (MIC) values for EDTA and DFP against filamentous forms of Sporothrix spp. ranged from 32 to 128 µg/ml. For antifungal drugs, MIC values ranged from 0.25 to 4 µg/ml for amphotericin B, from 0.25 to 4 µg/ml for itraconazole, and from 0.03 to 0.25 µg/ml for terbinafine. The chelators caused inhibition of Sporothrix spp. in yeast form at concentrations ranging from 16 to 64 µg/ml (for EDTA) and 8 to 32 µg/ml (for DFP). For antifungal drugs, MIC values observed against the yeast varied from 0.03 to 0.5 µg/ml for AMB, 0.03 to 1 µg/ml for ITC, and 0.03 to 0.13 µg/ml for TRB. Both DFP and EDTA presented synergistic interaction with antifungals against Sporothrix spp. in both filamentous and yeast form. Biofilms formed in the presence of the chelators (512 µg/ml) showed a reduction of 47% in biomass and 45% in metabolic activity. Our data reveal that DFP and EDTA reduced the growth of planktonic cells of Sporothrix spp., had synergistic interaction with antifungal drugs against this pathogen, and reduced biofilm formation of Sporothrix spp. LAY SUMMARY: Our data reveal that iron chelators deferiprone and ethylenediaminetetraacetic acid reduced the growth of planktonic cells of Sporothrix spp. as well as had synergistic interaction with antifungal drugs against this pathogen and reduced biofilm formation of Sporothrix spp.

Antifungal activity of promethazine and chlorpromazine against planktonic cells and biofilms of Cryptococcus neoformans/Cryptococcus gattii complex species.

Cryptococcus neoformans/Cryptococcus gattii are fungal pathogens that affect the central nervous system, mainly in immunocompromised individuals. Due to the limited pharmacological arsenal available for the treatment of cryptococcosis associated with cases of antifungal resistance of Cryptococcus spp. reported in some studies, the search for new compounds with antifungal potential becomes relevant. Thus, the objective of this study was to evaluate the inhibitory effect of phenothiazines (promethazine and chlorpromazine) on C. neoformans/C. gattii planktonic cells and biofilms. In vitro planktonic susceptibility testing was performed using the broth microdilution assay. The effect of phenothiazines was evaluated against biofilm formation and mature Cryptococcus biofilms. Biofilm morphology and ultrastructure were also evaluated by scanning electron microscopy. Promethazine and chlorpromazine showed antifungal activity against planktonic cells, with minimum inhibitory concentrations of 8-32 µg/ml and 4-16 µg/ml, respectively. As for biofilm formation, phenothiazines reduced biomass by 60% and metabolic activity by 90% at 64 µg/ml; while in mature biofilms, reductions of 85% and 90% in biomass and metabolic activity, respectively, were observed at 1024 µg/ml. Promethazine and chlorpromazine were also able to disrupt and fragment biofilms. In conclusion, promethazine and chlorpromazine have antifungal activity against planktonic cells and biofilms of Cryptococcus spp. These data show the potential of promethazine and chlorpromazine as antibiofilm drugs.

Cefepime and Amoxicillin Increase Metabolism and Enhance Caspofungin Tolerance of Candida albicans Biofilms.

It is well known that prolonged antibiotic therapy alters the mucosal microbiota composition, increasing the risk of invasive fungal infection (IFI) in immunocompromised patients. The present study investigated the direct effect of ß-lactam antibiotics cefepime (CEF) and amoxicillin (AMOX) on biofilm production by Candida albicans ATCC 10231. Antibacterials at the peak plasmatic concentration of each drug were tested against biofilms grown on polystyrene surfaces. Biofilms were evaluated for biomass production, metabolic activity, carbohydrate and protein contents, proteolytic activity, ultrastructure, and tolerance to antifungals. CEF and AMOX enhanced biofilm production by C. albicans ATCC 10231, stimulating biomass production, metabolic activity, viable cell counts, and proteolytic activity, as well as increased biovolume and thickness of these structures. Nevertheless, AMOX induced more significant changes in C. albicans biofilms than CEF. In addition, it was shown that AMOX increased the amount of chitin in these biofilms, making them more tolerant to caspofungin. Finally, it was seen that, in response to AMOX, C. albicans biofilms produce Hsp70 - a protein with chaperone function related to stressful conditions. These results may have a direct impact on the pathophysiology of opportunistic IFIs in patients at risk.

Potassium iodide and miltefosine inhibit biofilms of Sporothrix schenckii species complex in yeast and filamentous forms.

This study aimed to evaluate the yeast biofilm growth kinetics and ultrastructure of Sporothrix schenckii complex and assess their mature biofilm susceptibility in filamentous and yeast forms to potassium iodide (KI) and miltefosine (MIL). Yeast biofilms were evaluated by crystal violet staining, XTT reduction assay and microscopic techniques. Susceptibility of planktonic and sessile cells was analyzed by broth microdilution. S. schenckii complex in yeast form produced biofilms, with an optimum maturation at 96 h, showing multilayered blastoconidia embedded in extracellular matrix. KI and MIL minimum inhibitory concentration (MIC) ranges against planktonic cells were 62,500-250,000 µg/ml and 0.125-4 µg/ml, respectively. KI and MIL reduced biofilm metabolic activity by 75.4% and 67.7% for filamentous form and 55.1% and 51.6% for yeast form, respectively. This study demonstrated that S. schenckii complex forms biofilms in vitro, and potassium iodide and miltefosine inhibit Sporothrix spp. biofilms in both filamentous and yeast forms.

ß-lactam antibiotics & vancomycin increase the growth & virulence of Candida spp.

AIM: To investigate the direct effect of antibiotics on growth and virulence of the major Candida species associated with invasive infections. MATERIALS & METHODS: Cefepime, imipenem, meropenem, amoxicillin and vancomycin were tested at twofold the peak plasma concentration (2× PP) and the peak plasma concentration (PP). The effects of antibiotics on Candida albicans, Candida parapsilosis, Candida krusei and Candida tropicalis were investigated by colony counting, flow cytometry, proteolytic activity and virulence in Caenorhabditis elegans. RESULTS: Antibiotics increase growth and proteolytic activity of Candida spp; In addition, amoxicillin potentiates virulence of C. krusei and C. tropicalis against Caenorhabditis elegans. CONCLUSION: These results suggest that antimicrobial therapy may have a direct effect on the pathophysiology of invasive fungal infections in patients at risk.

Antifungal susceptibility and virulence of Candida parapsilosis species complex: an overview of their pathogenic potential.

PURPOSE: Antifungal resistance and several putative virulence factors have been associated with the pathogenicity of the Candida parapsilosis species complex. The objective of this study was to evaluate the antifungal susceptibility, the production of virulence factors and the pathogenicity of the C. parapsilosis complex. METHODOLOGY: Overall, 49 isolates of C. parapsilosis sensu stricto, 19 C. orthopsilosis and nine C. metapsilosis were used. The planktonic and biofilm susceptibility to fluconazole, itraconazole, voriconazole, amphotericin B and caspofungin was assessed using a broth microdilution assay. Finally, the production of biofilm and hydrolytic enzymes and the fungal pathogenicity against Caenorhabditis elegans were investigated.Results/Key findings. Overall, one C. orthopsilosis was resistant to caspofungin and susceptible-dose-dependent to itraconazole, the other two C. orthopsilosis were susceptible-dose-dependent to fluconazole and itraconazole, and one C. metapsilosis was susceptible-dose-dependent to azoles. A total of 67.5 % of the isolates were biofilm producers. Amphotericin B and caspofungin caused the greatest reduction in the metabolic activity and biomass of mature biofilms. Phospholipase and protease production was observed in 55.1 % of C. parapsilosis sensu stricto, 42.1 % of C. orthopsilosis and 33.3 % of C. metapsilosis isolates. Moreover, 57.9 % of C. orthopsilosis and 20.4 % of C. parapsilosis sensu stricto isolates were ß-haemolytic, and all C. metapsilosis were α-haemolytic. Finally, the C. parapsilosis complex caused high mortality of C. elegans after 96 h of exposure. CONCLUSION: These results reinforce the heterogeneity of these cryptic species for their antifungal susceptibility, virulence and pathogenic potential, emphasizing the relevance of monitoring these emerging pathogens.

In vitro activity of azole derivatives and griseofulvin against planktonic and biofilm growth of clinical isolates of dermatophytes.

As shown by recent research, most of the clinically relevant fungi, including dermatophytes, form biofilms in vitro and in vivo, which may exhibit antimicrobial tolerance that favour recurrent infections. The aim of this study was to determine the minimum inhibitory concentrations (MICs) of itraconazole (ITC), voriconazole (VCZ) and griseofulvin (GRI) against Trichophyton rubrum, Trichophyton tonsurans, Trichophyton mentagrophytes, Microsporum canis and Microsporum gypseum in planktonic and biofilm growth. For the planktonic form, susceptibility testing was performed according to the Clinical and Laboratory Standards Institute (CLSI), document M38-A2, while biofilm susceptibility was evaluated using the XTT colorimetric essay. The planktonic growth of all strains was inhibited, with MIC values ranging from 0.00195 to 0.1225 µg/mL for VRC, 0.00195 to 0.25 µg/mL for ITC and <0.0039 to 4 µg/mL for GRI, while a 50-fold increase in the MIC was required to significantly reduce the metabolic activity (P < .05) of dermatophyte biofilms. In brief, the ability of dermatophytes to form biofilms may be a contributing factor for the recalcitrance of dermatophytoses or the dissemination of the disease.

In vitro effects of promethazine on cell morphology and structure and mitochondrial activity of azole-resistant Candida tropicalis.

The aim of this study was to evaluate the effect of promethazine on the antifungal minimum inhibitory concentrations against planktonic cells and mature biofilms of Candida tropicalis, as well as investigate its potential mechanisms of cell damage against this yeast species. Three C. tropicalis isolates (two azole-resistant and one azole-susceptible) were evaluated for their planktonic and biofilm susceptibility to promethazine alone and in combination with itraconazole, fluconazole, voriconazole, amphotericin B, and caspofungin. The antifungal activity of promethazine against C. tropicalis was investigated by performing time-kill curve assays and assessing rhodamine 6G efflux, cell size/granularity, membrane integrity, and mitochondrial transmembrane potential, through flow cytometry. Promethazine showed antifungal activity against planktonic cells and biofilms at concentrations of 64 and 128 µg/ml, respectively. The addition of two subinhibitory concentrations of promethazine reduced the antifungal MICs for all tested azole drugs against planktonic growth, reversing the resistance phenotype to all azoles. Promethazine decreased the efflux of rhodamine 6G in an azole-resistant strain. Moreover, promethazine decreased cell size/granularity and caused membrane damage, and mitochondrial membrane depolarization. In conclusion, promethazine presented synergy with azole antifungals against resistant C. tropicalis and exhibited in vitro cytotoxicity against C. tropicalis, altering cell size/granularity, membrane integrity, and mitochondrial function, demonstrating potential mechanisms of cell damage against this yeast species.

Antifungal susceptibility of Sporothrix schenckii complex biofilms.

Sporotrichosis, caused by species of Sporothrix schenckii complex, is the most prevalent subcutaneous mycosis in many areas of Latin America. The aim of this study was to evaluate the ability of Sporothrix spp. to form biofilms in vitro and to characterize the growth kinetics, morphology, and antifungal susceptibility of biofilms against classical antifungals. We investigated the ability of strains to produce biofilms in vitro and determined the effects of exposure to amphotericin B, itraconazole, caspofungin, ketoconazole, voriconazole, and fluconazole at minimum inhibitory concentration (MIC) against planktonic form and at 10× MIC and 50× MIC on the biomass and metabolic activity of these biofilms. Biofilm structure was analyzed by optical microscopy using Congo-red staining, confocal and scanning electron microscopy. Strains were classified for biofilm-forming ability, through the analysis of absorbance of crystal violet retained by biomass of mature biofilms. We found that all S. brasiliensis (n = 10), S. schenckii sensu stricto (n = 2), S. globosa (n = 2), and S. mexicana (n = 4) strains were strong biofilm-producers. The analyzed biofilms had dense network of hyphae and conidia immersed in extracellular matrix, with presence of water channels. Antifungal drugs at the three tested concentrations showed different effects on biomass and metabolic activity of biofilms. However, the best inhibitory response was observed with 50× MIC of amphotericin B and caspofungin, which reduced these parameters. Furthermore, high drug concentrations, especially amphotericin B and caspofungin, showed antifungal activity against these biofilms, probably because they damaged the architecture and extracellular matrix, allowing diffusion of the drugs.

Biofilms of Candida spp. from the ocular conjunctiva of horses with reduced azole susceptibility: a complicating factor for the treatment of keratomycosis?

OBJECTIVES: This study aimed to assess the biofilm-forming ability of Candida spp. from the ocular conjunctiva of horses and to investigate the antifungal susceptibility of these biofilms. PROCEDURES: Initially, the biofilm-forming ability of 15 strains was assessed by crystal violet staining, which reveals the fungal biomass adhered to the polystyrene plates, and scanning electron microscopy. Then, the minimum inhibitory concentrations (MICs) of amphotericin B, fluconazole, itraconazole, and caspofungin were initially determined against strains in planktonic form. Afterward, antifungal susceptibility of mature biofilms was evaluated by exposing them to 10 × MIC and 50 × MIC of the tested drugs, followed by the assessment of their metabolic activity, using the oxidoreduction indicator XTT. Results were analyzed through ANOVA and Tukey's post-test, and P-values below 5% led to significant conclusions. RESULTS: Eight strains produced biofilms and were classified as strong (1/15), moderate (3/15) and weak (4/15) producers, according to the amount of crystal violet retained by the adhered fungal biomass. Biofilm metabolic activity of one C. tropicalis did not decrease after exposure to the tested antifungals, while biofilm metabolic activity of five strains was reduced by amphotericin B, but not the other drugs. One C. parapsilosis sensu stricto and one C. glabrata showed significant reduction in biofilm metabolic activity after exposure to fluconazole, itraconazole, and caspofungin, but not amphotericin B. CONCLUSIONS: The results demonstrate that Candida from the ocular conjunctiva of horses can pose as a risk to animal health as they are capable of forming biofilms, which are commonly involved in fungal keratitis.

Trends in antifungal susceptibility and virulence of Candida spp. from the nasolacrimal duct of horses.

This was a cross-sectional study to investigate the antifungal susceptibility and production of virulence factors in strains of Candida isolated from the outlet and the lumen of the nasolacrimal duct of horses in the state of Ceará, Brazil. The samples were obtained from 103 horses. Sterile cotton swabs were used to collect the material from the outlet of the nasolacrimal duct and urethral probes, for the instillation of 2 ml of saline solution, were used to collect samples from the lumen of the nasolacrimal duct. A total of 77 Candida isolates were obtained, with C. famata, C. tropicalis, C. guilliermondii, and C. parapsilosis sensu lato as the most prevalent species. One isolate (C. glabrata) was resistant to caspofungin. One isolate was resistant only to fluconazole (C. parapsilosis sensu lato), 11 were resistant only to itraconazole (7 C. tropicalis, 2 C. guilliermondii, 1 C. famata, 1 C. parapsilosis sensu lato), while eight C. tropicalis showed resistance to both azoles. Overall, 28 isolates produced phospholipases and 12 produced proteases. These results highlight the importance of investigating the antifungal susceptibility and virulence trends of Candida spp. from the microbiota of the nasolacrimal duct of horses.

Evidence of Fluconazole-Resistant Candida Species in Tortoises and Sea Turtles.

The aim of this study was to evaluate the antifungal susceptibility of Candida spp. recovered from tortoises (Chelonoidis spp.) and sea turtles (Chelonia mydas, Caretta caretta, Lepidochelys olivacea, Eretmochelys imbricata). For this purpose, material from the oral cavity and cloaca of 77 animals (60 tortoises and 17 sea turtles) was collected. The collected specimens were seeded on 2% Sabouraud dextrose agar with chloramphenicol, and the identification was carried out by morphological and biochemical methods. Sixty-six isolates were recovered from tortoises, out of which 27 were C. tropicalis, 27 C. famata, 7 C. albicans, 4 C. guilliermondii and 1 C. intermedia, whereas 12 strains were obtained from sea turtles, which were identified as Candida parapsilosis (n = 4), Candida guilliermondii (n = 4), Candida tropicalis (n = 2), Candida albicans (n = 1) and Candida intermedia (n = 1). The minimum inhibitory concentrations for amphotericin B, itraconazole and fluconazole ranged from 0.03125 to 0.5, 0.03125 to >16 and 0.125 to >64, respectively. Overall, 19 azole-resistant strains (14 C. tropicalis and 5 C. albicans) were found. Thus, this study shows that Testudines carry azole-resistant Candida spp.

Candida tropicalis isolates obtained from veterinary sources show resistance to azoles and produce virulence factors.

Candida tropicalis has been associated with invasive candidiasis, being the first or second most common non-Candida albicans Candida species isolated in humans with candidemia and candiduria, as well as being frequently isolated from healthy animals. This study aimed to characterize C. tropicalis isolates (n = 64) obtained from several animal species regarding antifungal susceptibility and production of virulence factors. The isolates were obtained from the microbiota of healthy animals (goats, n = 25; sheep, n = 6; psittacines, n = 14; rheas, n = 6; horses, n = 2; sirenians, n = 5; shrimp, n = 1), as well as from aquatic mammals found dead in the environment (cetaceans, n = 5). The isolates were subjected to in vitro susceptibility testing by broth microdilution according to the CLSI M27-A3 protocol against amphotericin B, caspofungin, itraconazole, and fluconazole. We also evaluated the virulence attributes, such as proteases and phospholipases, as well as biofilm formation. Resistance to itraconazole (n = 29) and fluconazole (n = 30) was detected among isolates from every source; resistance to both azoles was detected in 24 isolates, but none of them were resistant to amphotericin B and caspofungin. Protease production was detected in the majority of the isolates (n = 59), but phospholipase was produced by only a few of them (n = 6). The isolates showed different patterns in biofilm production, being considered strong producers (n = 41), moderate producers (n = 11), weak producers (n = 9) or non-producers (n = 3). In summary, C. tropicalis isolated from animals showed high rate of resistance to azoles, expressed virulence factors and therefore may represent a potential threat to human and animal health.