Aspects related to biofilm production and antifungal susceptibility of clinically relevant yeasts of the genus Trichosporon.

Trichosporonosis corresponds to a systemic fungal disease that leads to high mortality rates and is frequently associated with medical devices. It affects immunosuppressed patients in particular and is strongly linked to acquired human immunodeficiency, organ and tissue transplants, and malignant hematologic diseases such as leukemia and lymphomas. Trichosporon infections have been increasingly reported worldwide; however, little information is available either about their characteristics or the causative microorganism. Thus, the aims of the present study were: to investigate 59 yeasts of the genus Trichosporon by verifying the biofilm formation capacity of isolates; to analyze the susceptibility patterns of planktonic cells against the antifungals fluconazole, itraconazole, amphotericin-B, voriconazole, and caspofungin by comparing European Committee for Antimicrobial Susceptibility Testing (EUCAST) broth microdilution technique with the commercial method Etest; and to assess the susceptibility patterns of biofilm cells (sessile) against the same antifungals through broth microdilution. The ability to form biofilm on the surface of polystyrene plates was noted for all isolates, and 54.3% of samples were considered strong producers. Comparison between the antifungal susceptibility techniques evidenced that Etest showed higher and discordant minimum inhibitory concentrations (MICs) from those obtained by the microdilution method, especially for fluconazole, itraconazole, and caspofungin. Considering the susceptibility of biofilms, most species had high MIC50 and MIC90 against the tested antifungals, showing 4-to-66-fold higher concentrations for amphotericin B and 2-to-33-fold greater concentrations for caspofungin. These results highlight the importance of further studies with Trichosporon spp. for comparison between laboratory findings and in vivo response, considering both the susceptibility tests and the behavior of biofilm cells against drugs.

This study investigated 59 isolates of the medically important yeast Trichosporon in relation to their ability to form biofilms and the susceptibility of biofilms to antifungal agents. All isolates were able to produce biofilms and biofilms showed lower antifungal susceptibility.

Comparative analysis of biofilm formation by Candida albicans and Candida krusei in different types of contact lenses / Análise comparativa da formação de biofilmes, por Candida albicans e Candida krusei, em diferentes tipos de lentes de contato

ABSTRACT Objective: To evaluate the Candida krusei and Candida albicans biofilm formation abilities on 5 different types of contact lenses and compare their metabolic activities and biomass. Methods: After biofilm formation by both the test species, their metabolic activity was assessed by the 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide reduction assay with menadione, while the biomass was determined by staining with 0.4% crystal violet dye for further statistical analysis. Results: Both the Candida species could form biofilms on different types of contact lenses, with greater metabolic activities and lower biomass formation in rigid gas permeable lenses. Conclusion: Biofilm formation with greater metabolic activity and greater biomass were expected on soft contact lenses considering their surface hydrophobicity. However, the results demonstrated a greater metabolic activity on rigid contact lenses. This result has a great significance with regards to the increasing risk of microbial keratitis, although further studies are warranted to better elucidate the formation of biofilms on different types of contact lens materials in the future.

RESUMO Objetivo: Avaliar a capacidade de formação de bio­filmes de Candida krusei e Candida albicans em cinco tipos de lentes de contato, comparando atividade metabólica e biomassa dos mesmos. Métodos: Após a formação de biofilme de ambas as espécies, a atividade metabólica foi avaliada por ensaio de redução 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-te­tra­zolium-5-carboxanilide com Menadiona, e a biomassa foi avaliada por coloração com Cristal Violeta 0,4% para posterior análise estatística. Resultados: Ambas as espécies de Candida foram capazes de formar biofilmes nos diferentes tipos de lentes de contato, havendo em lentes rígidas gás permeável maior atividade metabólica e menor biomassa formada. Conclusão: Esperava-se a obtenção de biofilmes de maior atividade metabólica e maior biomassa em lentes de contato gelatinosas com base no fundamento da Hidrofobicidade Superficial. Porém, o resultado apontou para maior atividade metabólica em lentes de contato rígidas. Apesar de observados resultados significativos, trata-se de um assunto de grande importância frente ao aumento do número de ceratites microbianas, mostrando-se necessários outros estudos para melhor elucidar a formação de biofilmes em diferentes tipos de materiais de lentes de contato.

Comparative analysis of biofilm formation by Candida albicans and Candida krusei in different types of contact lenses.

OBJECTIVE: To evaluate the Candida krusei and Candida albicans biofilm formation abilities on 5 different types of contact lenses and compare their metabolic activities and biomass. METHODS: After biofilm formation by both the test species, their metabolic activity was assessed by the 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide reduction assay with menadione, while the biomass was determined by staining with 0.4% crystal violet dye for further statistical analysis. RESULTS: Both the Candida species could form biofilms on different types of contact lenses, with greater metabolic activities and lower biomass formation in rigid gas permeable lenses. CONCLUSION: Biofilm formation with greater metabolic activity and greater biomass were expected on soft contact lenses considering their surface hydrophobicity. However, the results demonstrated a greater metabolic activity on rigid contact lenses. This result has a great significance with regards to the increasing risk of microbial keratitis, although further studies are warranted to better elucidate the formation of biofilms on different types of contact lens materials in the future.

Effectiveness of disinfecting substances of gutta-percha cones / Efetividade de agentes desinfectantes de cones de guta percha

Aim:To demonstrate the effectiveness of disinfecting substances with 2% and 5% Sodium Hypochlorite and 2% Chlorhexidine Gluconate at each of the pre-established times of 0:15 and 0:30 seconds, and 1, 2, 5, and 10 minutes. Methods: This study selected 96 gutta-percha cones that were contaminated with Enterococcus Faecalis, dried and treated with the aforementioned substances and applied at pre-established times. Subsequently, these were transferred to sterile Brain Heart Infusion broth and placed in a bacteriological incubator at 37°C for 24 hours to evaluate microbial growth, as well as in a nutrient agar medium in Petri dishes. Half of the cone was transferred to individual filter paper packages and exposed to the environment in a dental clinic at Universidade José do Rosário Velano, for 7 days, with subsequent evaluation for microbial growth. The bacterial phenotype test was performed using Gram stain and growth in 6.5% saline solution. The results were submitted to statistical analysis using the Kruskal Wallis H test, with a significance level of 5%. Results:The substances were effective at all times tested and individual storage supported disinfection. In the statistics test, the p-value was greater than 0.05, as there was no variability in the data configurations. Conclusion: The disinfection of gutta-percha cones and individual storage was an effective protocol to be adopted with 2% and 5% Sodium Hypochlorite and 2% Chlorhexidine.

Objetivo: Demonstrar a eficácia de substâncias desinfetantes, Hipoclorito de Sódio a 2% e 5% e Gluconato de Clorexidina 2% em cada um dos tempos pré-estabelecidos de 15 e 30 segundos, e 1,2, 5 e 10 minutos.Métodos: Este estudo selecionou 96 cones de guta-percha,contaminados com Enterococcus Faecalis, secos e tratados com as substâncias citadas e aplicadas em tempos pré-estabelecidos. Posteriormente, estes foram transferidos para tubos contendo caldo Infusão Cérebro Coração estéril e colocados estufa bacteriológica a 37°C por 24 horas para avaliar o crescimento microbiano, também verificado em meio ágar nutriente em Placas de Petri. Metade dos cones foram transferidos para embalagens individuais de papel de filtro, e expostas ao ambiente da clínica odontológica da Universidade José do Rosário Velano por 7 dias, com posterior avaliação do crescimento microbiano. O teste do fenótipo bacteriano foi realizado pela coloração de Gram e crescimento em solução salina a 6,5%. Os resultados foram submetidos à análise estatística por meio do Teste H de Kruskal Wallis, com nível de significância de 5%.Resultados: As substâncias foram eficazes em todos os tempos testados e o armazenamento individual favoreceu a desinfecção. No teste estatístico, o valor de p foi maior que 0,05, pois não houve variabilidade nas configurações dos dados.Conclusão: A desinfecção dos cones com Hipoclorito de Sódio 2% e 5% e Clorexidina 2% a partir de 15 segundos, e o armazenamento individual foram protocolos eficazes para serem adotados.

Glucosyl-1,2,3-triazoles derived from eugenol and analogues: Synthesis, anti-Candida activity, and molecular modeling studies in CYP-51.

This work describes the synthesis, anti-Candida, and molecular modeling studies of eighteen new glucosyl-1,2,3-triazoles derived from eugenol and correlated phenols. The new compounds were characterized by combined Fourier Transform Infrared, 1 H and 13 C nuclear magnetic resonance and spectroscopy of high-resolution mass spectrometry. The synthesized compounds did not show significant cytotoxicity against healthy fibroblast human cells (MCR-5) providing interesting selectivity indexes (SI) to active compounds. Considering the antifungal activity, nine compounds showed anti-Candida potential and the peracetylated triazoles 17 and 18 were the most promising ones. Eugenol derivative 17 was active against three species of Candida at 26.1-52.1 µM. This compound was four times more potent than fluconazole against Candida krusei and less toxic (SI > 6.6) against the MCR-5 cells than fluconazole (SI > 3.3) considering this strain. Dihydroeugenol derivative 18 showed similar activity to 17 and was four times more potent and less toxic than fluconazole against C. krusei. The deacetylated glucosides and non-glucosylated corresponding derivatives did not show considerable antifungal action, suggesting that the acetyl groups are essential for their anti-Candida activity. Molecular docking coupled with molecular dynamics showed that 14α-lanosterol demethylase is a feasible molecular target, since 17 and 18 could bind to this enzyme once deacetylated in vivo, thereby acting as prodrugs. Also, these studies demonstrated the importance of hydrophobic substituents at the phenyl ring.

Leishmanicidal and antimicrobial activity of primin and primin-containing extracts from Miconia willdenowii.

As a part of an ongoing bioprospective project, searching for potential medicinal plants from the Brazilian Atlantic Forest, Miconia willdenowii was selected for its potential leishmanicidal and antimicrobial activities. The crude ethanolic extract of M. willdenowii showed an inhibition of 99.7% of the promastigote forms of Leishmania amazonensis at the concentration of 80 µg/mL. Further investigation of its antimicrobial activity against pathogenic fungi and Gram positive and negative bacteria, revealed a significant antimicrobial activity. A bioguided study with its liquid-liquid partition fractions revealed the hexane fraction (Hex) as the most active against Leishmania, inhibiting 99.2% and 46.9% of the protozoan at concentrations of 40 and 20 µg/mL, respectively. Hex also showed significant antimicrobial activity against Staphylococcus aureus and Candida krusei with IC50 of 15.6 and 62.5 µg/mL, respectively. Purification of Hex led to the isolation of 2-methoxy-6-pentyl-benzoquinone (1, also known as primin) as the active metabolite, probably responsible for the observed antimicrobial and anti-leishmania effects. Primin (1) disclosed leishmanicidal activity (IC50 = 1.25 µM), showing higher potency than the standard drug amphotericin B (IC50 = 5.08 µM), with additional antifungal effects against all tested fungi species. Compound 1 also showed significant activity against S. aureus (IC50 = 8.94 µM), showing a comparable potency with the reference drug chloramphenicol (IC50 = 6.19 µM), but with a potential cytotoxicity towards peripheral human blood mononuclear cells (CC50 = 255.15 µM). Here in, the antimicrobial and anti-L. amazonensis effects of M. willdenowii are reported for the first time, as well as Primin (1) as its probable bioactive metabolite.

Candida spp. and phagocytosis: multiple evasion mechanisms.

Invasive fungal infections are a global health problem, mainly in hospitals, where year by year hundreds of patients die because of these infections. Commensal yeasts may become pathogenic to human beings, affecting mainly immunocompromised patients. During infectious processes, the immune system uses phagocytes to eliminate invader microorganisms. In order to prevent or neutralize phagocyte attacks, pathogenic yeasts can use virulence factors to survive, as well as to colonize and infect the host. In this review, we describe how Candida spp., mainly Candida albicans, interact with phagocytes and use several factors that contribute to immune evasion. Polymorphism, biofilm formation, gene expression and enzyme production mediate distinct functions such as adhesion, invasion, oxidative stress response, proteolysis and escape from phagocytes. Fungal and human cells have similar structures and mechanisms that decrease the number of potential targets for antifungal drugs. Therefore, research on host-pathogen interaction may aid in the discovery of new targets and in the development of new drugs or treatments for these diseases and thus to save lives.

Candida glabrata among Candida spp. from environmental health practitioners of a Brazilian Hospital

Abstract The incidence of the species Candida albicans and non-albicans Candida was evaluated in a Brazilian Tertiary Hospital from the environment and health practitioners. In a 12-month period we had a total positivity of 19.65% of Candida spp. The most recurring non-albicans Candida species was C. glabrata (37.62%), generally considered a species of low virulence, but with a higher mortality rate than C. albicans. Subsequently, C. parapsilosis (25.74%) and C. tropicalis (16.86%) were the second and third most commonly isolated species. Considering the total samples collected from the emergency room and from the inpatient and the pediatric sector, 19.10% were positive for Candida spp., with the predominance of non-albicans Candida species (89.42%). The high percentage of positivity occurred in the hands (24.32%) and the lab coats (21.88%) of the health care assistants. No sample of C. albicans presented a profile of resistance to the drugs. All the non-albicans Candida species presented a decreased susceptibility to miconazole and itraconazole, but they were susceptible to nystatin. Most of the isolates were susceptible to fluconazole and amphotericin B. As expected, a high resistance rate was observed in C. glabrata and C. krusei, which are intrinsically less susceptible to this antifungal agent. The contamination of environmental surfaces by Candida spp. through hand touching may facilitate the occurrence of Candida infections predominantly in immunocompromised patients. In addition to that, the antifungal agents used should be carefully evaluated considering local epidemiologic trends in Candida spp. infections, so that therapeutic choices may be better guided.

Candida glabrata among Candida spp. from environmental health practitioners of a Brazilian Hospital.

The incidence of the species Candida albicans and non-albicans Candida was evaluated in a Brazilian Tertiary Hospital from the environment and health practitioners. In a 12-month period we had a total positivity of 19.65% of Candida spp. The most recurring non-albicans Candida species was C. glabrata (37.62%), generally considered a species of low virulence, but with a higher mortality rate than C. albicans. Subsequently, C. parapsilosis (25.74%) and C. tropicalis (16.86%) were the second and third most commonly isolated species. Considering the total samples collected from the emergency room and from the inpatient and the pediatric sector, 19.10% were positive for Candida spp., with the predominance of non-albicans Candida species (89.42%). The high percentage of positivity occurred in the hands (24.32%) and the lab coats (21.88%) of the health care assistants. No sample of C. albicans presented a profile of resistance to the drugs. All the non-albicans Candida species presented a decreased susceptibility to miconazole and itraconazole, but they were susceptible to nystatin. Most of the isolates were susceptible to fluconazole and amphotericin B. As expected, a high resistance rate was observed in C. glabrata and C. krusei, which are intrinsically less susceptible to this antifungal agent. The contamination of environmental surfaces by Candida spp. through hand touching may facilitate the occurrence of Candida infections predominantly in immunocompromised patients. In addition to that, the antifungal agents used should be carefully evaluated considering local epidemiologic trends in Candida spp. infections, so that therapeutic choices may be better guided.

New Eugenol Glucoside-based Derivative Shows Fungistatic and Fungicidal Activity against Opportunistic Candida glabrata.

A new series of glucosides modified in their saccharide units were synthesized, evaluated against Candida sp., and compared to prototype 1, an eugenol tetracetyl glucoside previously synthesized and shown to be active against Candida glabrata. Among the new glucosides, benzyl derivative 5 was the most promising, showing fungistatic activity at IC50 18.1 µm against Candida glabrata (threefold higher than fluconazole) and fungicidal activity with a low IC90 value of 36.2 µm. Moreover, the cytotoxic activity of compound 5 (CC50 : 580.9 µm), tested in peripheral blood mononuclear cells, suggests its potential as an agent to treat Candida glabrata infections, with a selectivity index of 32. The new eugenol glucoside 5 may be considered as a novel structural pattern in the development of new anti-Candida drugs.

Synthesis and antimicrobial activity of 6-triazolo-6-deoxy eugenol glucosides.

A new series of 1,2,3-triazole eugenol glucosides were synthesized. The new compound structures were confirmed by MS, (1)H NMR and (13)C NMR. All of the synthesized compounds were screened for antimicrobial and cytotoxic activity. Five compounds exerted significant activity against the Gram-negative bacteria Salmonella typhimurium with low IC50 values (49.73-68.53 µΜ), and seven compounds were active against the Gram-positive bacteria Micrococcus luteus (42.89-210.94 µM). In vitro cytotoxicity on mouse spleen cells was also evaluated. One compound bearing a phenyl substituent at the triazole ring showed good activity against Salmonella typhimurium (49.73 µM) and low toxicity to normal cells (CC50=157.83 µM). Thus, the compounds herein can be considered for further modification for improving their antibacterial activity or obtaining novel antibacterial drug candidates.