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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.

Candida tropicalis is a prominent non-Candida albicans Candida species involved in cases of candidemia, mainly causing infections in patients in intensive care units and (or) those presenting neutropenia. In recent years, several studies have reported an increase in the recovery rates of azole-resistant C. tropicalis isolates. Understanding C. tropicalis resistance is of great importance, since resistant strains are implicated in persistent or recurrent and breakthrough infections. In this review, we address the main mechanisms underlying C. tropicalis resistance to the major antifungal classes used to treat candidiasis. The main genetic basis involved in C. tropicalis antifungal resistance is discussed. A better understanding of the epidemiology of resistant strains and the mechanisms involved in C. tropicalis resistance can help improve diagnosis and assessment of the antifungal susceptibility of this Candida species to improve clinical management.

Introduction.Cryptococcusspecies are pathogens commonly associated with cases of meningoencephalitis in individuals who are immunosuppressed due to AIDS.Aim.The aim was to evaluate the effects of the antiretroviral darunavir alone or associated with fluconazole, 5-flucytosine and amphotericin B against planktonic cells and biofilms ofCryptococcusspecies.Methodology.Susceptibility testing of darunavir and the common antifungals against 12 members of theCryptococcus neoformans/Cryptococcus gattiispecies complex was evaluated by broth microdilution. The interaction between darunavir and antifungals against planktonic cells was tested by a checkerboard assay. The effects of darunavir against biofilm metabolic activity and biomass were evaluated by the XTT reduction assay and crystal violet staining, respectively.Results.Darunavir combined with amphotericin B showed a synergistic interaction against planktonic cells. No antagonistic interaction was observed between darunavir and the antifungals used. AllCryptococcusspecies strains were strong biofilm producers. Darunavir alone reduced biofilm metabolic activity and biomass when added during and after biofilm formation (PPConclusion.Darunavir presents antifungal activity against planktonic cells ofCryptococcusspecies and synergism with amphotericin B. In addition, darunavir led to reduced biofilm formation and showed activity against mature biofilms ofCryptococcusspecies. Activity of the antifungals against mature biofilms was enhanced in the presence of darunavir.

Introduction. Sporotrichosis, caused by species of the Sporothrix schenckii complex, is the most prevalent subcutaneous mycosis in many areas of Latin America. Statins are a class of drugs widely used for lowering high sterol levels through their action on 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme in the synthesis of sterol. Aim. In this study, the antifungal activity of statins (simvastatin, atorvastatin, pravastatin) against planktonic cells and biofilms of S. schenckii complex species was evaluated, as well as the interaction of pravastatin with classical antifungals (amphotericin B, itraconazole, terbinafine). Methodology. Eighteen strains of Sporothrix species were used. The antifungal susceptibility assay was performed using the broth microdilution method. Mature biofilms were exposed to statins and metabolic activity was measured by the XTT reduction assay. Results. MICs of statins ranged from 8 to 512 μg ml−1 and from 8 to 256 μg ml−1 for filamentous and yeast forms, respectively. Regarding mature biofilms, MICs of 50 % inhibition (SMIC50) were 128 μg ml−1 for simvastatin and atorvastatin and >2048 μg ml−1 for pravastatin. MICs of 90 % inhibition (SMIC90) were 512 μg ml−1 for simvastatin and >2048 μg ml−1 for atorvastatin and pravastatin. Conclusion. These results highlight the antifungal and antibiofilm potential of statins against S. schenckii complex species.

This study aimed to evaluate the effect of diclofenac on minimum inhibitory concentrations of antifungals against planktonic cells and biofilms of Candida tropicalis. Susceptibility testing of plan...

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.

Aim: To evaluate the inhibition of efflux pumps by using promethazine (PMZ) as a strategy to control Fusarium solani species complex (FSSC). Materials & methods: The susceptibility of FSSC strains to PMZ and the interaction between PMZ and antifungals were evaluated. The efflux pump activity was confirmed by flow cytometry with rhodamine 6G. Finally, PMZ was tested against FSSC biofilms. Results: PMZ inhibited FSSC planktonic growth and showed synergism with antifungals. PMZ reduced R6G efflux and inhibited cell adhesion, impaired the development of biofilms and disrupted mature biofilms. PMZ-challenged biofilms showed increased sensitivity to amphotericin B. Conclusion: The study provides indirect evidence of the occurrence of efflux pumps in FSSC and opens a perspective for this target in the control of fusariosis.

Aim: This study investigated the effect of terpinen-4-ol against Sporothrix schenckii complex and its interactions with antifungals. Materials & methods: The antifungal activity of terpinen-4-ol was evaluated by broth microdilution. The potential effect on cellular ergosterol concentration was evaluated by spectrophotometry. The antibiofilm activity was evaluated by violet crystal staining and XTT reduction assay. The potential pharmacological interactions with antifungals were evaluated by the checkerboard assay. Results: terpinen-4-ol (T-OH) showed minimal inhibitory concentrations ranging from 4 to 32 mg/l decreasing cellular ergosterol content and presented a SMIC ranging from 64 to 1024 mg/l for Sporothrix spp. The combinations of T-OH with itraconazole or terbinafine were synergistic. Conclusion: T-OH has antifungal activity against Sporothrix spp. and acts synergistically with standard antifungals.

Aim: This study aimed to evaluate the effects of proton pump inhibitors (PPIs) on growth and melanin production by Cryptococcus spp. Materials & methods: Minimum inhibitory concentrations (MICs) of omeprazole, esomeprazole, rabeprazole, pantoprazole and lansoprazole against Cryptococcus spp. were determined and the effect of PPIs on melanin production was evaluated, in the presence or absence of copper sulfate or glutathione. Results: PPIs showed MICs ranging from 125–1000 μg/ml and decreased melanization by Cryptococcus cells. Addition of copper sulfate or gluthatione restored melanogenesis of cells grown in the presence of PPIs. The presence of PPIs and glyphosate decreased copper sulfate toxicity (1 mM). Conclusion: PPIs inhibited melanogenesis of Cryptococcus spp., possibly by chelating copper or inhibiting copper ATPase transport.

Studies on the fungal microbiota of reptiles and amphibians are necessary to better understand of host-microbe interactions and the establishment of fungal disease in these animals. However, these studies are limited. The present researchidentified yeasts from free-ranging reptiles and amphibians from the Caatinga biome andevaluated the virulence factors production, the antifungal susceptibility in planktonic and biofilm growth and the pathogenicity of Candida famata isolates. Twenty-nine isolates of the genera Candida, Cryptococcus and Rhodotorula were identified by phenotypic and/or molecular methods and production of hydrolytic enzymes in vitro by these genera of fungi was evaluated. In addition, susceptibility of planktonic cells and biofilms to azoles and amphotericin B was evaluated. The pathogenicity of C. famata, the most prevalent yeast species isolated, was evaluated using Caenorhabditis elegans model. C. famata was the most prevalent yeast in amphibian and reptilian microbiota. Phospholipase and protease production was observed in 18/29 and 11/29 of the yeast isolates, respectively, while 100% formed biofilms. Itraconazole presented high minimal inhibitory concentrations against C. famata and C. tropicalis. Amphotericin B reduced the biomass and metabolic activity of biofilms. C. famata induced the mortality of C. elegans. In conclusion, reptiles and amphibians are colonized by yeasts capable of producing important virulence factors, especially by Candida spp. that present low susceptibility to azoles which may result from imbalances in ecosystem. Finally, C. famata isolated from these animals presented high pathogenicity, showing the importance of the study of reptile and amphibians fungal microbiota. RESUMO: Estudos sobre a microbiota fúngica de répteis e anfíbios são necessários para melhor compreender as interações hospedeiro-microrganismo e o estabelecimento de doenças fúngicas nesses animais. No entanto, esses estudos são limitados. O objetivo da presente pesquisa foi identificar leveduras isoladas de répteis e anfíbios do bioma Caatinga e avaliar a produção de fatores de virulência, a sensibilidade a antifúngicos no crescimento planctônico e de biofilme e a patogenicidade de Candida famata. Vinte e nove isolados dos gêneros Candida, Cryptococcus e Rhodotorula foram identificados por métodos fenotípicos e/ou moleculares e a produção de enzimas hidrolíticas in vitro por esses gêneros de fungos foi avaliada. Além disso, foi avaliada a suscetibilidade de células planctônicas e biofilmes a azólicos e anfotericina B. A patogenicidade de C. famata, a espécie de levedura isolada mais prevalente, foi avaliada usando Caenorhabditis elegans. C. famata foi a levedura mais prevalente na microbiota de anfíbios e répteis. A produção de fosfolipase e protease foi observada em 18/29 e 11/29 dos isolados de levedura, respectivamente, enquanto 100% formaram biofilmes. O itraconazol apresentou altas concentrações inibitórias mínimas contra C. famata e C. tropicalis. A anfotericina B reduziu a biomassa e atividade metabólica dos biofilmes. C. famata induziu a mortalidade de C. elegans. Em conclusão, répteis e anfíbios são colonizados por leveduras capazes de produzir importantes fatores de virulência, especialmente por cepas de Candida spp. que apresentam baixa suscetibilidade a azólicos que podem resultar de desequilíbrio no ecossistema. Por fim, C. famata isolados desses animais apresentaram alta patogenicidade, mostrando a importância do estudo da microbiota fúngica de répteis e anfíbios.

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.

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 β-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.

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.

This study evaluated the effect of the protease inhibitor ritonavir (RIT) on Trichosporon asahii and Trichosporon inkin. Susceptibility to RIT was assessed by the broth microdilution assay and the effect of RIT on protease activity was evaluated using azoalbumin as substrate. RIT was tested for its anti-biofilm properties and RIT-treated biofilms were assessed regarding protease activity, ultrastructure and matrix composition. In addition, antifungal susceptibility, surface hydrophobicity and biofilm formation were evaluated after pre-incubation of planktonic cells with RIT for 15 days. RIT (200 μg ml−1) inhibited Trichosporon growth. RIT (100 μg ml−1) also reduced protease activity of planktonic and biofilm cells, decreased cell adhesion and biofilm formation, and altered the structure of the biofilm and the protein composition of the biofilm matrix. Pre-incubation with RIT (100 μg ml−1) increased the susceptibility to amphotericin B, and reduced surface hydrophobicity and cell adhesion. These re...

Purpose. The aim of this study was to evaluate the in vitro and ex vivo biofilm-forming ability of dermatophytes on a nail fragment. Methodology. Initially, four isolates of Trichophyton rubrum, six of Trichophyton tonsurans, three of Trichophyton mentagrophytes, ten of Microsporum canis and three of Microsporum gypseum were tested for production biomass by crystal violet assay. Then, one strain per species presenting the best biofilm production was chosen for further studies by optical microscopy (Congo red staining), confocal laser scanning (LIVE/DEAD staining) and scanning electron (secondary electron) microscopy. Results. Biomass quantification by crystal violet assay, optical microscope images of Congo red staining, confocal microscope and scanning electron microscope images revealed that all species studied are able to form biofilms both in vitro and ex vivo, with variable density and architecture. M. gypseum, T. rubrum and T. tonsurans produced robust biofilms, with abundant matrix and biomass, while M. canis produced the weakest biofilms compared to other species. Conclusion. This study sheds light on biofilms of different dermatophyte species, which will contribute to a better understanding of the pathophysiology of dermatophytosis. Further studies of this type are necessary to investigate the processes involved in the formation and composition of dermatophyte biofilms.

Cryptococcus neoformans/Cryptococcus gattii complex species are etiological agents of cryptococcosis, a systemic mycosis that cause respiratory infection and meningoencephalitis. To establish the infection, these yeasts produce virulence factors, such as melanin, which contribute to pathogenicity and antifungal tolerance. The aim of this study was to investigate melanin production by the C. neoformans/C. gattii complex in the presence of different precursors of melanogenesis and evaluate the effect of melanization on the antifungal susceptibility of these species to fluconazole, flucytosine and amphotericin B. Epinephrine, norepinephrine, dopamine and caffeic acid were used as substrates for melanin production, and l -dopa was used as positive control. The susceptibility of melanized strains (n = 6), after exposure to epinephrine or l -dopa, was evaluated by broth microdilution assay, and non-melanized strains were used as control. The antifungal activity of amphotericin B against melanized strains was also investigated by time kill assay. All Cryptococcus spp. strains produced melanin after exposure to the tested substrates. After exposure to epinephrine, minimum inhibitory concentration (MIC) ranges were 1–8 μg/mL for fluconazole, 2–8 μg/mL for flucytosine and 0.125–1 μg/mL for amphotericin B, while, after exposure to l -dopa, MIC ranges were 2–8 μg/mL for fluconazole, 4–8 μg/mL for flucytosine, and 0.125–0.5 μg/mL for amphotericin B. Similar results were observed for non–melanized strains. The production of melanin after exposure to epinephrine was higher than that induced by l -dopa. Melanized cells of both species were more tolerant to amphotericin B than the non-melanized control, emphasizing the importance of melanin production for fungal virulence.

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 β-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.

The present study aimed at evaluating the role of captive scarlet ibises (Eudocimus ruber) and their environment as reservoirs of Aeromonas spp. and Plesiomonas spp., and analyzing the in vitro antimicrobial susceptibility and virulence of the recovered bacterial isolates. Thus, non-lactose and weak-lactose fermenting, oxidase positive Gram-negative bacilli were recovered from cloacal samples (n = 30) of scarlet ibises kept in a conservational facility and from water samples (n = 30) from their environment. Then, the antimicrobial susceptibility, hemolytic activity and biofilm production of the recovered Aeromonas spp. and Plesiomonas shigelloides strains were assessed. In addition, the virulence-associated genes of Aeromonas spp. were detected. Ten Aeromonas veronii bv. sobria, 2 Aeromonas hydrophila complex and 10 P. shigelloides were recovered. Intermediate susceptibility to piperacillin-tazobactam and cefepime was observed in 2 Aeromonas spp. and 1 P. shigelloides, respectively, and resistance to gentamicin was observed in 4 P. shigelloides. The automated susceptibility analysis revealed resistance to piperacillin-tazobactam and meropenem among Aeromonas spp. and intermediate susceptibility to gentamicin among P. shigelloides. All Aeromonas isolates presented hemolytic activity, while 3 P. shigelloides were non-hemolytic. All Aeromonas spp. and 3/10 P. shigelloides were biofilm-producers, at 28 °C, while 10 Aeromonas spp. and 6/10 P. shigelloides produced biofilms, at 37 °C. The most prevalent virulence genes of Aeromonas spp. were asa1 and ascV. Scarlet ibises and their environment harbour potentially pathogenic bacteria, thus requiring monitoring and measures to prevent contamination of humans and other animals.

The aim of this study was to evaluate the in vitro hemolytic activity and biofilm antifungal susceptibility of veterinary and human Candida tropicalis strains, as well as their pathogenesis against Caenorhabditis elegans. Twenty veterinary isolates and 20 human clinical isolates of C. tropicalis were used. The strains were evaluated for their hemolytic activity and biofilm production. Biofilm susceptibility to itraconazole, fluconazole, voriconazole, amphotericin B and caspofungin was assessed using broth microdilution assay. The in vivo evaluation of strain pathogenicity was investigated using the nematode C. elegans. Hemolytic factor was observed in 95% of the strains and 97.5% of the isolates showed ability to form biofilm. Caspofungin and amphotericin B showed better results than azole antifungals against mature biofilms. Paradoxical effect on mature biofilm metabolic activity was observed at elevated concentrations of caspofungin (8-64μg/mL). Azole antifungals were not able to inhibit mature C. tropicalis biofilms, even at the higher tested concentrations. High mortality rates of C. elegans were observed when the worms were exposed to with C. tropicalis strains, reaching up to 96%, 96h after exposure of the worms to C. tropicalis strains. These results reinforce the high pathogenicity of C. tropicalis from veterinary and human sources and show the effectiveness of caspofungin and amphotericin B against mature biofilms of this species.

Fungal infections of the respiratory tract of horses are not as frequent as those of bacterial and viral origin, often leading to worsening of clinical conditions due to misdiagnosis and incorrect treatment. Coccidioidomycosis and histoplasmosis are systemic mycoses caused by the dimorphic fungi Coccidioides spp. and Histoplasma capsulatum, respectively, which affect humans and a variety of other animals, including equines. These systemic mycoses of chronic and progressive nature can exhibit clinical manifestations similar to other microbial infections. Thus, this article broadly discusses the epidemiology, etiology, virulence, pathogenesis, clinical presentation, treatment, and diagnostic strategies of coccidioidomycosis and histoplasmosis, to support accurate diagnosis.

Aim: The purpose of this study was to evaluate the effects of the antileishmanials meglumine antimoniate and pentamidine against Sporothrix schenckii complex. Materials & methods: The antifungal activity of the two antileishmanials was assessed by broth microdilution. The interaction between the antileishmanials and antifungal drugs (amphotericin B, itraconazole and terbinafine) was evaluated by the checkerboard assay. The effect of prior exposure of Sporothrix spp. yeast cells to antileishmanials was evaluated by broth microdilution. Results: Only pentamidine showed antifungal activity against Sporothrix spp. Synergistic interactions were observed between pentamidine and the antifungals. Also, the pre-exposure to meglumine antimoniate reduced the susceptibility of Sardinella brasiliensis and S. schenckii sensu stricto to amphotericin B and itraconazole. Conclusion: Pentamidine showed antifungal activity against Sporothrix spp., indicating it is a possible therapeutic alternative for the treatment of sporotrichosis.

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.

The antifungal activity of some statins against different fungal species has been reported. Thus, at the first moment, the in vitro antifungal activity of simvastatin, atorvastatin and pravastatin was tested against Candida spp. and Cryptococcus spp. Then, in a second approach, considering that the best results were obtained for simvastatin, this drug was evaluated in combination with antifungal drugs against planktonic growth and tested against biofilms of Candida spp. and Cryptococcus spp. Drug susceptibility testing was performed using the microdilution broth method, as described by the Clinical and Laboratory Standards Institute. The interaction between simvastatin and antifungals against planktonic cells was analyzed by calculating the fractional inhibitory concentration index. Regarding biofilm susceptibility, simvastatin was tested against growing biofilm and mature biofilm of one strain of each tested yeast species. Simvastatin showed inhibitory effect against Candida spp. and Cryptococcus spp. with minimum inhibitory concentration values ranging from 15.6 to 1000 mg L−1 and from 62.5 to 1000 mg L−1, respectively. The combination of simvastatin with itraconazole and fluconazole showed synergism against Candida spp. and Cryptococcus spp., while the combination of simvastatin with amphotericin B was synergistic only against Cryptococcus spp. Concerning the biofilm assays, simvastatin was able to inhibit both growing biofilm and mature biofilm of Candida spp. and Cryptococcus spp. The present study showed that simvastatin inhibits planktonic cells and biofilms of Candida and Cryptococcus species. Keywords: Candida, Cryptococcus, Simvastatin, Antifungal activity, Biofilm

The aim of this study was to evaluate the effects of cefepime, meropenem, piperacillin/tazobactam (TZP) and vancomycin on strains of Candida albicans and Candida tropicalis in planktonic and biofilm forms. Twenty azole-derivative-resistant strains of C. albicans ( n =10) and C. tropicalis ( n =10) were tested. The susceptibility of planktonic Candida spp. to the antibacterial agents was investigated by broth microdilution. The XTT reduction assay was performed to evaluate the viability of growing and mature biofilms following exposure to these drugs. Minimum inhibitory concentrations (MICs) ranged from 0.5mg/mL to 2mg/mL for cefepime, TZP and vancomycin and from 0.5mg/mL to 1mg/mL for meropenem and the drugs also caused statistically significant reductions in biofilm cellular activity both in growing and mature biofilm. Since all of the tested drugs are commonly used in patients with hospital-acquired infections and in those with catheter-related infections under antibiotic-lock therapy, it may be possible to obtain an additional benefit from antibiotic-lock therapy with these drugs, namely the control of Candida biofilm formation.

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

The yeast Malassezia pachydermatis is a component of the microbiota of dogs and cats, however it can cause otitis and seborrheic dermatitis in these animals. The objective of this study was to determine the antifungal susceptibility, and evaluate virulence and pathogenicity of 25 M. pachydermatis strains from animals. Susceptibility to ketoconazole, fluconazole, itraconazole, voriconazole, terbinafine, and amphotericin B was evaluated by broth microdilution assay. In addition, biofilm-forming ability, protease, phospholipase, hemolysin and melanin production and adhesion to epithelial cells by this yeast species were assessed. Finally, strain pathogenicity was investigated using the nematode Caenorhabditis elegans. Concerning the planktonic susceptibility, minimum inhibitory concentrations varied from 64 μg/mL for azole derivatives, 1 to >16 μg/mL for amphotericin B and 0.03 to 0.25 μg/mL for terbinafine. All strains were classified as strong biofilm producers, and ketoconazole, fluconazole and amphotericin B presented the best inhibitory effect against mature biofilms. All fungal isolates produced proteases, whereas 14/25 strains were positive for phospholipase production. Hemolytic activity was not observed and 18/25 strains showed dark pigmentation in the presence of L-DOPA. Regarding adhesion to epithelial cells, a low adhesion rate was observed in 10/12 evaluated strains. C. elegans mortality rate reached 95.9% after 96 h of exposure of the worms to M. pachydermatis. This yeast species produces important virulence factors and presents high pathogenicity, corroborating its clinical importance.

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.

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.

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.

Objective: To report atypical morphological features of Trichophyton (T.) tonsurans strains associated with tinea capitis. Methods: Eighty-two T. tonsurans strains isolated in Ceará, Brazil, were analyzed regarding macro and micromorphological features and nutritional patterns. Results: Fifty-two samples presented abundant chlamydoconidia, which were produced in chains. Macroscopically, these strains developed small glabrous colonies that were firmly attached to the surface of the culture medium, with few or no aerial mycelia and intense rusty yellow pigmentation. Seven strains did not grow with stimulus from thiamine. Samples were heterogeneous regarding urease production and none presented in vitro hair perforation. Conclusions: The observation of T. tonsurans strains with distinct phenotypic features indicates the need to revise the taxonomic criteria for routine identification of this dermatophyte.

This study analyzed the RYP1 gene as a target for the molecular diagnosis of histoplasmosis. This assay detected fungal DNA in 13/13 blood samples from HIV/AIDS-patients with histoplasmosis. Therefore, the detection of RYP1 gene in whole blood sample is a quick and sensitive test to diagnose histoplasmosis.

This study aimed to identify yeasts from the gastrointestinal tract of scarlet ibises (Eudocimus ruber) and from plant material collected from the environment where they live. Then, the isolates phenotypically identified as Candida famata were submitted to molecular identification of their closely related species and evaluated for their antifungal susceptibility and possible resistance mechanisms to antifungal drugs. Cloacal swabs from 20 scarlet ibises kept in captivity at Mangal das Garcas Park (Brazil), pooled stool samples (n = 20) and samples of trunks and hollow of trees (n = 20) obtained from their enclosures were collected. The samples were seeded on Sabouraud agar supplemented with chloramphenicol. The 48 recovered isolates were phenotypically identified as 15 Candida famata, 13 Candida catenulata, 2 Candida intermedia, 1 Candida lusitaniae, 2 Candida guilliermondii, 1 Candida kefyr, 1 Candida amapae, 1 Candida krusei, 8 Trichosporon spp., and 4 Rhodotorula spp. The C. famata isolates were further identified as 3 C. famata, 8 Debaryomyces nepalensis, and 4 C. palmioleophila. All C. famata and C. palmioleophila were susceptible to caspofungin and itraconazole, while one D. nepalensis was resistant to fluconazole and voriconazole. This same isolate and another D. nepalensis had lower amphotericin B susceptibility. The azole resistant strain had an increased efflux of rhodamine 6G and an alteration in the membrane sterol content, demonstrating multifactorial resistance mechanism. Finally, this research shows that scarlet ibises and their environment harbor C. famata and closely related species, including antifungal resistant isolates, emphasizing the need of monitoring the antifungal susceptibility of these yeast species.

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.

Tyrosol is a quorum-sensing molecule of Candida albicans able to induce hyphal development in the early and intermediate stages of biofilm growth. In the present study, we evaluated the effect of high concentrations of exogenous tyrosol on planktonic cells and biofilms of C. albicans (n = 10) and C. tropicalis (n = 10), and investigated whether tyrosol could be synergic to antifungals that target cellular ergosterol. Antifungal susceptibility and drug interaction against planktonic cells were investigated by the broth microdilution method. Tyrosol was able to inhibit planktonic cells, with MIC values ranging from 2.5 to 5.0 mM for both species. Synergism was observed between tyrosol/amphotericin B (11/20 strains), tyrosol/itraconazole (18/20 strains) and tyrosol/fluconazole (18/20 strains). Exogenous tyrosol alone or combined with antifungals at both 10 × MIC and 50 × MIC were able to reduce biofilm of both Candida species. Mature biofilms were susceptible to tyrosol alone at 50 × MIC or combined with amphotericin at both 10 × MIC and 50 × MIC. On the other hand, tyrosol plus azoles at both 10 × MIC and 50 × MIC enhanced biofilm growth.

Aims This study aimed to evaluate the in vitro activity of miltefosine and levamisole against strains of Coccidioides posadasii in the filamentous phase and strains of Histoplasma capsulatum in filamentous and yeast phases. Methods and Results Strains of C. posadasii in the filamentous phase (n = 22) and strains of H. capsulatum in filamentous (n = 40) and yeast phases (n = 13) were, respectively, submitted to broth macrodilution and broth microdilution methods, as described by the Clinical and Laboratory Standards Institute, to determine the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) of miltefosine and levamisole. The effect of the drugs on cell membrane permeability under osmotic stress conditions and total ergosterol production were also assessed, along with quantification of extravasated molecules. The results show the inhibitory effect of levamisole and miltefosine against C. posadasii and H. capsulatum and the effect of these drugs on ergosterol synthesis and the permeability of the plasma membrane using subinhibitory concentrations against strains subjected to osmotic stress. Levamisole was also able to cause the release of nucleic acids. Conclusions Miltefosine and levamisole are capable of inhibiting the in vitro growth of C. posadasii and H. capsulatum, probably by altering the permeability of the cellular membrane. Significance and Impact of the Study This work presents alternatives for the treatment of histoplasmosis and coccidioidomycosis, raising the possibility of the use of miltefosine and levamisole as adjuvants in antifungal therapy, providing perspectives for the design of in vivo studies.

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.