Antimicrobial and antibiofilm activity of the benzoquinone oncocalyxone A.

Resistance to antimicrobials is a challenging issue that complicates the treatment of infections caused by bacteria and fungi, thus requiring new therapeutic options. Oncocalyxone A, a benzoquinone obtained from Auxemma oncocalyx (Allem) Taub has several biological effects; however, there is no data on its antimicrobial action. In this study, its antimicrobial and antibiofilm activities were evaluated against bacteria and fungi of clinical interest. Strains of Gram-positive and Gram-negative bacteria, and filamentous fungi and yeasts were selected to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of oncocalyxone A. The antibacterial effect of oncocalyxone A was studied using survival curves, atomic force microscopy (AFM), and the involvement of oxidative stress. We examined the inhibitory action of the molecule on biofilm formation and its hemolytic activity against human erythrocytes. Our results showed that among the strains tested, Staphylococcus epidermidis was highly sensitive to the action of oncocalyxone A, with an MIC of 9.43 µg/mL. In most bacterial strains analyzed, a bacteriostatic effect was observed, though the molecule showed no antifungal activity. Antibiofilm activity was observed against the methicillin-resistant S. aureus bacteria. Additionally, results from atomic force microscopy imaging showed that oncocalyxone A significantly altered bacterial morphology. Further, oncocalyxone A showed no hemolytic activity at concentrations ≥151 µg/mL. Together, our results demonstrate the antibacterial and antibiofilm potential of oncocalyxone A, indicating its therapeutic potential against bacterial resistance.

Structural characterization, antifungal and cytotoxic profiles of quaternized heteropolysaccharide from Anadenanthera colubrina.

In the present work, we investigated the minimal inhibitory concentration (MIC) against fungal strains (Fonsecaea pedrosoi, Microsporum canis, Candida albicans, Cryptococcus neoformans), and cytotoxicity to normal cell lines for modified red angico gum (AG) with eterifying agent N-chloride (3-chloro-2-hydroxypropyl) trimethylammonium (CHPTAC). Quaternized ammonium groups were linked to AG backbone using N-(3-chloro-2-hydroxypropyl) trimethylammonium chloride. The chemical features of the quaternized gum derivatives (QAG) were analyzed by: FTIR, elemental analysis, Zeta potential and gel permeation chromatography. The angico quaternizated gum presented a degree of substitution (DS) of 0.22 and Zeta potential of +36.43. For the antifungal test, it was observed that unmodified gum did not inhibit fungal growth. While, QAG inhibited the growth of most fungi used in this study. By AFM technique QAG interacted with the fungal surface, altering wall roughness significantly. The probable affinity of fragments of the QAG structure for the fungal enzyme 5I33 (Adenylosuccinate synthetase) has been shown by molecular docking. Low cytotoxicity was observed for polymers (unmodified gum and QAG). The results demonstrate that the quaternized polymer of AG presented in this study is a quite promising biomaterial for biotechnological applications.

Synergic effect of simvastatin in combination with amphotericin B against environmental strains of Cryptococcus neoformans from northeastern Brazil: a prospective experimental study.

BACKGROUND: Statins are used as cholesterol-lowering drugs and may also have direct antimicrobial effects. OBJECTIVE: To evaluate synergic interactions between simvastatin and both amphotericin B and fluconazole, against environmental strains of Cryptococcus neoformans isolated from captive birds' droppings. DESIGNAND SETTING: Experimental study conducted at Federal University of Piauí, Parnaíba, in collaboration with Federal University of Triângulo Mineiro, Uberaba, Brazil. METHODS: Statin susceptibility tests of Cryptococcus neoformans samples were performed as prescribed in standards. Interactions of simvastatin with amphotericin and fluconazole were evaluated using the checkerboard microdilution method. Presence of these interactions was quantitatively detected through determining the fractional inhibitory concentration index (FICI). RESULTS: Isolates of Cryptococcus neoformans were obtained from 30 of the 206 samples of dry bird excreta (14.5%) that were collected from pet shops and houses. Ten isolates were selected for susceptibility tests. All of them were susceptible to amphotericin and fluconazole. All presented minimum inhibitory concentration (MIC) > 128 µg/ml and, thus, were resistant in vitro to simvastatin. An in vitro synergic effect was shown through combined testing of amphotericin B and simvastatin, such that six isolates (60%) presented FICI < 0.500. Two isolates showed considerable reductions in MIC, from 1 µg/ml to 0.250 µg/ml. No synergic effect was observed through combining fluconazole and simvastatin. CONCLUSION: These results demonstrate that simvastatin should be considered to be a therapeutic alternative, capable of potentiating the action of amphotericin B. However, further studies are necessary to clarify the real effect of simvastatin as an antifungal agent.

Synergic effect of simvastatin in combination with amphotericin B against environmental strains of Cryptococcus neoformans from northeastern Brazil: a prospective experimental study

BACKGROUND: Statins are used as cholesterol-lowering drugs and may also have direct antimicrobial effects. OBJECTIVE: To evaluate synergic interactions between simvastatin and both amphotericin B and fluconazole, against environmental strains of Cryptococcus neoformans isolated from captive birds' droppings. DESIGNAND SETTING: Experimental study conducted at Federal University of Piauí, Parnaíba, in collaboration with Federal University of Triângulo Mineiro, Uberaba, Brazil. METHODS: Statin susceptibility tests of Cryptococcus neoformans samples were performed as prescribed in standards. Interactions of simvastatin with amphotericin and fluconazole were evaluated using the checkerboard microdilution method. Presence of these interactions was quantitatively detected through determining the fractional inhibitory concentration index (FICI). RESULTS: Isolates of Cryptococcus neoformans were obtained from 30 of the 206 samples of dry bird excreta (14.5%) that were collected from pet shops and houses. Ten isolates were selected for susceptibility tests. All of them were susceptible to amphotericin and fluconazole. All presented minimum inhibitory concentration (MIC) > 128 µg/ml and, thus, were resistant in vitro to simvastatin. An in vitro synergic effect was shown through combined testing of amphotericin B and simvastatin, such that six isolates (60%) presented FICI < 0.500. Two isolates showed considerable reductions in MIC, from 1 µg/ml to 0.250 µg/ml. No synergic effect was observed through combining fluconazole and simvastatin. CONCLUSION: These results demonstrate that simvastatin should be considered to be a therapeutic alternative, capable of potentiating the action of amphotericin B. However, further studies are necessary to clarify the real effect of simvastatin as an antifungal agent.

Antifungal Proteins from Plant Latex.

Latex, a milky fluid found in several plants, is widely used for many purposes, and its proteins have been investigated by researchers. Many studies have shown that latex produced by some plant species is a natural source of biologically active compounds, and many of the hydrolytic enzymes are related to health benefits. Research on the characterization and industrial and pharmaceutical utility of latex has progressed in recent years. Latex proteins are associated with plants' defense mechanisms, against attacks by fungi. In this respect, there are several biotechnological applications of antifungal proteins. Some findings reveal that antifungal proteins inhibit fungi by interrupting the synthesis of fungal cell walls or rupturing the membrane. Moreover, both phytopathogenic and clinical fungal strains are susceptible to latex proteins. The present review describes some important features of proteins isolated from plant latex which presented in vitro antifungal activities: protein classification, function, molecular weight, isoelectric point, as well as the fungal species that are inhibited by them. We also discuss their mechanisms of action.

SIXTEEN YEARS OF DERMATOMYCOSIS CAUSED BY Candida spp. IN THE METROPOLITAN AREA OF PORTO ALEGRE, SOUTHERN BRAZIL.

The yeasts of the genus Candida infect skin, nails, and mucous membranes of the gastrointestinal and the genitourinary tract. The aim of this study was to determine the prevalence of dermatomycoses caused by Candida spp., and their etiological aspects in the metropolitan area of Porto Alegre, Brazil. A retrospective study with data obtained from tertiary hospital patients, from 1996 to 2011, was performed. The analyzed parameters were date, age, gender, ethnicity, anatomical region of lesions, and the direct examination results. For all the statistical analyses, a = 0.05 was considered. Among positive results in the direct mycological examination, 12.5% of the total of 4,815 cases were positive for Candida spp. The angular coefficient (B) was -0.7%/ year, showing a decrease over the years. The genus Candida was more prevalent in women (15.9% of women versus 5.84% of men), and in addition, women were older than men (54 versus 47 years old, respectively). There was no difference between ethnic groups. The nails were more affected than the skin, with 80.37% of the infections in the nails (72.9% in fingernails and 7.47% in toenails). Our study corroborates the literature regarding the preference for gender, age, and place of injury. Moreover, we found a decrease in infection over the studied period.

SIXTEEN YEARS OF PITYRIASIS VERSICOLOR IN METROPOLITAN AREA OF PORTO ALEGRE, SOUTHERN BRAZIL.

Pityriasis versicolor is the most common of the diseases caused by Malassezia yeasts. The aim of this study is to determine the prevalence of pityriasis versicolor and its etiological aspects in the metropolitan area of Porto Alegre, Brazil. A retrospective cross-sectional study with data from patients of a reference hospital from 1996 to 2011 was performed. Collected data included: date, age, gender, ethnicity, anatomical region of lesion and the direct mycological examination results. Among the positive results in the direct mycological examination, 5.8% (2,239) were positive for pityriasis versicolor. The angular coefficient (B) was -0.3%/year, showing a decrease over the years. The disease was more prevalent in men (7.1% of men versus 5.1% of women that underwent the direct mycological examination); younger age (median 31 years old); "pardo" and black people (3.7% more than expected in the sample); trunk (73.44% of the affected anatomic sites). Lesions in rare sites (groin, genitals, legs, feet and hands) were also observed in this study. In conclusion, due to the decrease in the prevalence of pityriasis versicolor, long-term epidemiological studies in the metropolitan area of Porto Alegre, Brazil, are needed to continue the monitoring of this disease.

Dermatophytosis: a 16-year retrospective study in a metropolitan area in southern Brazil.

INTRODUCTION: Dermatophytoses are considered a public health problem. The objectives of this study were to determine the evolution of their prevalence in the metropolitan area of Porto Alegre, Brazil, and to analyze the dermatophyte species distribution according to body site and demographic characteristics of the patients. METHODOLOGY: This work was a retrospective analysis of data from patients attending a tertiary care hospital during 1996-2011. RESULTS: There were 9,048 cases with cultures positive for dermatophytes. Trichophyton rubrum occurred in 59.6% of the cases, followed by Trichophyton interdigitale (34%), Microsporum canis (2.6%), Epidermophyton floccosum (1.5%), Microsporum gypseum (1.3%), and Trichophyton tonsurans (0.9%). The angular coefficients for T. interdigitale, E. floccosum, T. rubrum, and M. canis were +1.119, +0.211, -0.826 and -0.324% per year, respectively. Males presented higher prevalence of infection (79.3% versus 53.9%). Tinea unguium occurred in 48.5% of the cases, followed by tinea pedis (33.1%). T. rubrum was the predominant species in all regions of the body except the scalp, where M. canis was responsible for 75% of the cases. CONCLUSION: Monitoring of the evolution of dermatophytosis tracks changes in prevalence over the years and may assist practical measures for the public health control of this disease.

Sixteen years o Pityriasis Versicolor in metropolitan area of Porto Alegre, Southern Brazil / Dezesseis anos de Pitiríase Versicolor na Região Metropolitana de Porto Alegre, Sul do Brasil

SUMMARYPityriasis versicolor is the most common of the diseases caused by Malasseziayeasts. The aim of this study is to determine the prevalence of pityriasis versicolor and its etiological aspects in the metropolitan area of Porto Alegre, Brazil. A retrospective crosssectional study with data from patients of a reference hospital from 1996 to 2011 was performed. Collected data included: date, age, gender, ethnicity, anatomical region of lesion and the direct mycological examination results. Among the positive results in the direct mycological examination, 5.8% (2,239) were positive for pityriasis versicolor. The angular coefficient (B) was -0.3%/year, showing a decrease over the years. The disease was more prevalent in men (7.1% of men versus 5.1% of women that underwent the direct mycological examination); younger age (median 31 years old); "pardo" and black people (3.7% more than expected in the sample); trunk (73.44% of the affected anatomic sites). Lesions in rare sites (groin, genitals, legs, feet and hands) were also observed in this study. In conclusion, due to the decrease in the prevalence of pityriasis versicolor, long-term epidemiological studies in the metropolitan area of Porto Alegre, Brazil, are needed to continue the monitoring of this disease.

RESUMOPitiríase versicolor é a mais comum das doenças causadas pelas leveduras do gênero Malassezia. O objetivo do trabalho foi determinar a prevalência de pitiríase versicolor e seus aspectos etiológicos na região metropolitana de Porto Alegre, Brasil. Foi realizado um estudo retrospectivo transversal com dados de pacientes de hospital de referência entre 1996 a 2011. Os dados coletados foram: data, idade, gênero, etnia, local anatômico da lesão e resultados do exame micológico direto. Dentre os resultados positivos do exame micológico direto, 5,8% (2.239) foram positivos para pitiríase versicolor. O coeficiente angular (B) foi -0,3%/ ano, mostrando uma diminuição ao longo dos anos. A doença foi mais prevalente em homens (7,1% dos homens versus 5,1% das mulheres que se submeteram ao exame micológico direto); jovens (mediana de 31 anos); pardos e pretos (3,7% acima do esperado para a amostra); tronco (73,4% dos locais anatômicos afetados). Lesões em locais raros (virilha, genitálias, pernas, pés e mãos) também foram observadas no estudo. Em conclusão, devido à diminuição da prevalência de pitiríase versicolor, estudos epidemiológicos de longo prazo são necessários para continuar o monitoramento da doença na região metropolitana de Porto Alegre, Brasil.

Application of 6-nitrocoumarin as a substrate for the fluorescent detection of nitroreductase activity in Sporothrix schenckii.

INTRODUCTION: Sporothrix schenckii is a thermal dimorphic pathogenic fungus causing a subcutaneous mycosis, sporotrichosis. Nitrocoumarin represents a fluorogenic substrate class where the microbial nitroreductase activity produces several derivatives, already used in several other enzyme assays. The objective of this study was the analysis of 6-nitrocoumarin (6-NC) as a substrate to study the nitroreductase activity in Sporothrix schenckii. METHODS: Thirty-five samples of S. schenckii were cultivated for seven, 14 and 21 days at 35 °C in a microculture containing 6-nitrocoumarin or 6-aminocoumarin (6-AC) dissolved in dimethyl sulfoxide or dimethyl sulfoxide as a negative control, for posterior examination under an epifluorescence microscope. The organic layer of the seven, 14 and 21-day cultures was analyzed by means of direct illumination with 365 nm UV light and by means of elution on G silica gel plate with hexane:ethyl acetate 1:4 unveiled with UV light. RESULTS: All of the strains showed the presence of 6-AC (yellow fluorescence) and 6-hydroxylaminocoumarin (blue fluorescence) in thin layer chromatography, which explains the green fluorescence observed in the fungus structure. CONCLUSION: The nitroreductase activity is widely distributed in the S. schenckii complex and 6-NC is a fluorogenic substrate of easy access and applicability for the nitroreductase activity detection.

APPLICATION OF 6-NITROCOUMARIN AS A SUBSTRATE FOR THE FLUORESCENT DETECTION OF NITROREDUCTASE ACTIVITY IN Sporothrix schenckii / Aplicação de 6-nitrocumarina como substrato fluorescente para detecção de atividade nitroredutásica em Sporothrix schenckii

Introduction Sporothrix schenckii is a thermal dimorphic pathogenic fungus causing a subcutaneous mycosis, sporotrichosis. Nitrocoumarin represents a fluorogenic substrate class where the microbial nitroreductase activity produces several derivatives, already used in several other enzyme assays. The objective of this study was the analysis of 6-nitrocoumarin (6-NC) as a substrate to study the nitroreductase activity in Sporothrix schenckii. Methods Thirty-five samples of S. schenckii were cultivated for seven, 14 and 21 days at 35 °C in a microculture containing 6-nitrocoumarin or 6-aminocoumarin (6-AC) dissolved in dimethyl sulfoxide or dimethyl sulfoxide as a negative control, for posterior examination under an epifluorescence microscope. The organic layer of the seven, 14 and 21-day cultures was analyzed by means of direct illumination with 365 nm UV light and by means of elution on G silica gel plate with hexane:ethyl acetate 1:4 unveiled with UV light. Results All of the strains showed the presence of 6-AC (yellow fluorescence) and 6-hydroxylaminocoumarin (blue fluorescence) in thin layer chromatography, which explains the green fluorescence observed in the fungus structure. Conclusion The nitroreductase activity is widely distributed in the S. schenckii complex and 6-NC is a fluorogenic substrate of easy access and applicability for the nitroreductase activity detection. .

Introdução Sporothrix schenckii é um fungo dimórfico térmico, agente etiológico de micose subcutânea, a esporotricose. Nitrocumarina representa classe de substratos fluorogênicos em que a atividade nitroredutásica microbiana produz vários derivados, já utilizados em vários outros ensaios enzimáticos. O objetivo deste estudo foi analisar 6-nitrocumarina (6-NC) como substrato para estudo da atividade nitroredutásica em Sporothrix schenckii. Métodos Trinta e cinco isolados de S. schenckii foram cultivados por sete, 14 e 21 dias a 35 °C em um microcultivo contendo 6-nitrocumarina ou 6-aminocumarina (6-AC) solubilizada em dimetilsulfóxido ou dimetilsulfóxido como controle negativo, para posterior análise em microscópio de epifluorescência. A fase orgânica da cultura de sete, 14 e 21 dias foi analisada por meio de iluminação direta com luz UV de 365 nm e por eluição em placas de sílica gel G com hexano:acetato de etila 1:4 e revelada com luz UV. Resultados Todos os isolados mostraram a presença de 6-AC (fluorescência amarela) e 6-hidroxilaminocumarina (fluorescência azul) em cromatografia em camada delgada, que explica a fluorescência verde observada na estrutura dos fungos. Conclusão A atividade nitroredutásica é amplamente distribuída no complexo S. schenckii e 6-NC é um substrato fluorogênico de fácil obtenção e aplicabilidade para detecção da atividade nitroredutásica. .

A case of relapsed chromoblastomycosis due to Fonsecaea monophora: antifungal susceptibility and phylogenetic analysis.

Chromoblastomycosis is a chronic cutaneous and subcutaneous mycosis. The management of this infection continues to be challenging because there is no consensus on the therapeutic regimen. We report here a case of a 69-year-old male patient with cauliflower-like lesions on his left leg and foot. He had already been treated with itraconazole at a dose of 200 mg/day for 5 months, with mycological cure for all the affected areas. However, the lesions relapsed at both sites, and treatment with itraconazole was resumed at the dose previously used. Initially, direct mycological examination, cultural, and microculture slide observation were performed. Afterward, sequencing of the ITS1-5.8S rDNA-ITS2 region of the fungal DNA and evaluation of its susceptibility to antifungal agents alone and in combination were performed. In direct mycological examination, the presence of sclerotic cells was verified, and the fungus was identified as Fonsecaea based on cultural and microscopic examinations. Identification as Fonsecaea monophora was confirmed after sequencing of the ITS region and phylogenetic analysis. The isolate was susceptible to itraconazole and terbinafine. The combinations of amphotericin B and terbinafine and terbinafine and voriconazole were synergistic. The use of drugs for which the causative agent is susceptible to singly or in combination may be an alternative for the treatment of mycosis. Furthermore, the identification of the agent by molecular techniques is important for epidemiological purposes. To the best of our knowledge, this is the first case of relapsed chromoblastomycosis caused by F. monophora in Brazil.

Standardization and characterization of antigens for the diagnosis of aspergillosis.

The aim of this study was to develop and characterize antigens for the diagnosis of aspergillosis. Nine strains of Aspergillus species Aspergillus fumigatus , Aspergillus flavus , and Aspergillus niger were grown in Sabouraud and Smith broth to produce exoantigens. The antigens were tested by immunodiffusion against sera from patients with aspergillosis and other systemic mycoses. The protein fraction of the antigens was detected by SDS-PAGE; Western blot and representative bands were assessed by mass spectrometry coupled to a nano Acquity UltraPerformance LC and analyzed by the Mascot search engine. Concurrently, all sera were tested with Platelia Aspergillus EIA. The most reactive antigens to sera from patients infected by A. fumigatus were produced by A. fumigatus MG2 Sabouraud and pooled A. fumigatus Sabouraud samples, both with a sensitivity of 93% and specificity of 100% and 97%, respectively. Aspergillus niger and A. flavus antigens were reactive against A. niger and A. flavus sera, each one with a sensitivity and specificity of 100%. Two proteins, probably responsible for antigenic activity, ß-glucosidase in A. fumigatus and α-amylase in A. niger were attained. The commercial kit had a specificity of 22%, sensitivity of 100%, positive predictive value of 48%, and negative predictive value of 100%. The antigens produced showed high sensitivity and specificity and can be exploited for diagnostics of aspergilloma.

A case of Exophiala spinifera infection in Southern Brazil: Molecular identification and antifungal susceptibility.

We report a case of an 80-year-old Brazilian man, farmer, with lesions on the dorsum of the hand. A direct mycological examination, cultivation and microculture slide observation was performed. The sequencing of ITS1-5.8S rDNA-ITS2 region was carried out and the etiological agent confirmed as Exophiala spinifera. The in vitro susceptibility of this isolate to antifungal agents alone and in combination was evaluated. This is the third case of phaeohyphomycosis caused by Exophiala spinifera in Brazil.

Antifungal activity of Pterocaulon alopecuroides (Asteraceae) against chromoblastomycosis agents.

Plants of the genus Pterocaulon (Asteraceae) are popularly used in the treatment of skin diseases caused by fungi and bacteria. The aim of this work was to investigate the in vitro activity of the crude methanolic extract obtained from the aerial parts of Pterocaulon alopecuroides (Lam.) against some agents of chromoblastomycosis, a chronic fungal infection of the skin and of the subcutaneous tissue caused by traumatic inoculation of the aetiological agent. The extract was active against all the strains tested showing a minimum inhibitory concentration between 625 and 2500 microg ml(-1). The assessment of fungistatic/fungicidal activity demonstrated that the extract was fungistatic against Fonsecaea spp. and fungicidal against all the other fungi. Our results indicate that the identification of bioactive components present in the crude methanolic extract of P. alopecuroides against chromoblastomycosis agents can be an important strategy to manage this mycosis in the future.

In vitro antifungal activity of 2-(2'-hydroxy-5'-aminophenyl)benzoxazole in Candida spp. strains.

The development of azole antifungals has allowed for the treatment of several fungal infections. However, the use of these compounds is restricted because of their hepatotoxicity or because they need to be administered together with other drugs in order to prevent resistance to monotherapy. Benzoxazole derivatives are among the most thriving molecular prototypes for the development of antifungal agents. 2-(2'-hydroxyphenyl) benzoxazoles are versatile molecules that emit fluorescence and have antibacterial, antiviral and antifungal properties. 2-(2'-hydroxy-5'-aminophenyl) benzoxazole (HAMBO) was tested against Candida yeast. The inhibition provided by HAMBO was lower than that of fluconazole, showing low antifungal activity against Candida spp., but equivalent to that of benzoxazoles tested in similar studies. HAMBO showed fungistatic activity against all analysed strains. This class of novel benzoxazole compounds may be used as template to produce better antifungal drugs.