Gene expression of Paracoccidioides virulence factors after interaction with macrophages and fibroblasts

BACKGROUND Paracoccidioidomycosis (PCM) is a systemic mycosis with high prevalence in Latin America that is caused by thermodimorphic fungal species of the Paracoccidioides genus. OBJECTIVES In this study, we used quantitative polymerase chain reaction (qPCR) to investigate the expression of genes related to the virulence of Paracoccidioides brasiliensis (Pb18) and P. lutzii (Pb01) strains in their mycelial (M) and yeast (Y) forms after contact with alveolar macrophages (AMJ2-C11 cell line) and fibroblasts (MRC-5 cell line). METHODS The selected genes were those coding for 43 kDa glycoprotein (gp43), enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 14-3-3 protein (30 kDa), phospholipase, and aspartyl protease. FINDINGS In the Pb18 M form, the aspartyl protease gene showed the highest expression among all genes tested, both before and after infection of host cells. In the Pb18 Y form after macrophage infection, the 14-3-3 gene showed the highest expression among all genes tested, followed by the phospholipase and gp43 genes, and their expression was 50-fold, 10-fold, and 6-fold higher, respectively, than that in the M form. After fibroblast infection with the Pb18 Y form, the 14-3-3 gene showed the highest expression, followed by the phospholipase and aspartyl protease genes, and their expression was 25-fold, 10-fold, and 10-fold higher, respectively, than that in the M form. Enolase and aspartyl protease genes were expressed upon infection of both cell lines. After macrophage infection with the Pb01 Y form, the 14-3-3 gene showed the highest expression, followed by the phospholipase and aspartyl protease genes, and their expression was 18-fold, 12.5-fold, and 6-fold higher, respectively, than that in the M form. MAIN CONCLUSIONS In conclusion, the data show that the expression of the genes analysed may be upregulated upon fungus-host interaction. Therefore, these genes may be involved in the pathogenesis of paracoccidioidomycosis.

Antibiofilm efficacy of tea tree oil and of its main component terpinen-4-ol against Candida albicans

Abstract Candida infection is an important cause of morbidity and mortality in immunocompromised patients. The increase in its incidence has been associated with resistance to antimicrobial therapy and biofilm formation. The aim of this study was to evaluate the efficacy of tea tree oil (TTO) and its main component - terpinen-4-ol - against resistant Candida albicans strains (genotypes A and B) identified by molecular typing and against C. albicans ATCC 90028 and SC 5314 reference strains in planktonic and biofilm cultures. The minimum inhibitory concentration, minimum fungicidal concentration, and rate of biofilm development were used to evaluate antifungal activity. Results were obtained from analysis of the biofilm using the cell proliferation assay 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and confocal laser scanning microscopy (CLSM). Terpinen-4-ol and TTO inhibited C. albicans growth. CLSM confirmed that 17.92 mg/mL of TTO and 8.86 mg/mL of terpinen-4-ol applied for 60 s (rinse simulation) interfered with biofilm formation. Hence, this in vitro study revealed that natural substances such as TTO and terpinen-4-ol present promising results for the treatment of oral candidiasis.

Effect of Cinnamomum verum leaf essential oil on virulence factors of Candida species and determination of the in-vivo toxicity with Galleria mellonella model

BACKGROUND Essential oils (EO) extracted from Cinnamomum verum has been used as an antimicrobial agents for centuries. The effects of C. verum leaf oil against virulence of microorganisms is not well studied yet. OBJECTIVES This study evaluates the effect of C. verum leaf oil against three virulence factors of Candida albicans, C. tropicalis and C. dubliniensis and its in-vivo toxicity. METHODS Chemical composition of EO was determined using gas chromatography-mass spectrometry (GC-MS). Minimum inhibitory concentration (MIC) was determined using clinical and laboratory standards institute (CLSI) M27-A3 broth microdilution. Effect of EO on initial adhesion was quantified using XTT assay after allowing Candida cells to adhere to the polystyrene surface for 2 h. Biofilm formation of Candida in the presence of EO was quantified using XTT viability assay. Efficacy on reduction of germ tube formation was evaluated using standard protocol. Visualisation of biofilm formation and progression under the EO treatment were done using scanning electron microscope (SEM) and Time lapses microscope respectively. In-vivo toxicity of EO was determined using Galleria mellonella larvae. Chlorhexidine digluconate: positive control. RESULTS Eugenol was the main compound of EO. MIC was 1.0 mg/mL. 50% reduction in initial adhesion was achieved by C. albicans, C. tropicalis and C. dubliniensis with 1.0, > 2.0 and 0.34 mg/mL respectively. 0.5 and 1.0 mg/mL significantly inhibit the germ tube formation. MBIC50 for forming biofilms were ≤ 0.35 mg/mL. 1.0 mg/mL prevent biofilm progression of Candida. SEM images exhibited cell wall damages, cellular shrinkages and decreased hyphal formation. No lethal effect was noted with in-vivo experiment model at any concentration tested. CONCLUSION C. verum leaf oil acts against virulence factors of Candida and does not show any toxicity.

Anadenanthera Colubrina vell Brenan: anti-Candida and antibiofilm activities, toxicity and therapeutical action

Abstract: We evaluated the antifungal and antibiofilm potential of the hydroalcoholic extract of bark from Anadenanthera colubrina (vell.) Brenan, known as Angico, against Candida spp. Antifungal activity was evaluated using the microdilution technique through the Minimum Inhibitory and Fungicide Concentrations (MIC and MFC). The antibiofilm potential was tested in mature biofilms formed by Candida species and analyzed through the counting of CFU/mL and scanning electron micrograph (SEM). In vivo toxicity and therapeutic action was evaluated in the Galleria mellonella model. The treatment with the extract, in low doses, was able to reduce the growth of planktonic cells of Candida species. MIC values range between 19.5 and 39 µg/mL and MFC values range between 79 and 625 µg/mL. In addition was able to reduce the number of CFU/mL in biofilms and to cause structural alteration and cellular destruction, observed via SEM. A. colubrina showed low toxicity in the in vivo assay, having not affected the viability of the larvae at doses below 100mg/kg and high potential in the treatment of C. albicans infection. Considering its high antifungal potential, its low toxicity and potential to treatment of infections in in vivo model, A. colubrina extract is a strong candidate for development of a new agent for the treatment of oral candidiasis.

Interactions between Terpinen-4-ol and Nystatin on biofilm of Candida albicans and Candida tropicalis

Abstract The aim of this study was to evaluate the antifungal activity of Terpinen-4-ol associated with nystatin, on single and mixed species biofilms formed by Candida albicans and Candida tropicalis, as well as the effect of terpinen-4-ol on adhesion in oral cells and the enzymatic activity. The minimum inhibitory concentrations and minimum fungicide concentrations of terpinen-4-ol and nystatin on Candida albicans and Candida tropicalis were determined using the microdilution broth method, along with their synergistic activity ("checkerboard" method). Single and mixed species biofilms were prepared using the static microtiter plate model and quantified by colony forming units (CFU/mL). The effect of Terpinen-4-ol in adhesion of Candida albicans and Candida tropicalis in coculture with oral keratinocytes (NOK Si) was evaluated, as well as the enzymatic activity by measuring the size of the precipitation zone, after the growth agar to phospholipase, protease and hemolysin. Terpinen-4-ol (4.53 mg mL-1) and nystatin (0.008 mg mL-1) were able to inhibit biofilms growth, and a synergistic antifungal effect was showed with the drug association, reducing the inhibitory concentration of nystatin up to 8 times in single biofilm of Candida albicans, and 2 times in mixed species biofilm. A small decrease in the adhesion of Candida tropicalis in NOK Si cells was showed after treatment with terpinen-4-ol, and nystatin had a greater effect for both species. For enzymatic activity, the drugs showed no action. The effect potentiated by the combination of terpinen-4-ol and nystatin and the reduction of adhesion provide evidence of its potential as an anti-fungal agent.

Resumo O objetivo desse estudo foi avaliar a atividade antifúngica do Terpinen4-ol associado à nistatina em biofilmes simples e misto, formados por Candida albicans e Candida tropicalis, bem como o efeito do terpinen-4-ol na adesão em células orais e atividade enzimática. As concentrações inibitórias mínimas e as concentrações fungicidas mínimas do terpinen-4-ol e da nistatina em Candida albicans e Candida tropicalis foram determinadas pelo método de microdiluição em caldo, juntamente com a atividade sinérgica (método do tabuleiro de "xadrez"). Biofilmes simples e misto foram preparados usando o modelo de placa de microtitulação estática e quantificados por unidades formadoras de colônias (CFU/mL). O efeito do Terpinen-4-ol na adesão de Candida albicans e Candida tropicalis em co-cultura com queratinócitos orais (NOK Si) foi avaliado, bem como a atividade enzimática, medindo o tamanho da zona de precipitação, após o crescimento em ágar fosfolipase, protease e hemolisina. O terpinen-4-ol (4.53 mg mL-1) e a nistatina (0,008 mg mL-1) conseguiram inibir o crescimento de biofilmes e um efeito antifúngico sinérgico foi demonstrado com a associação de fármaco, reduzindo a concentração inibidora de nistatina até 8 vezes em biofilme simpes de Candida albicans e 2 vezes em biofilme misto. Uma pequena diminuição na adesão de Candida tropicalis em células NOK Si foi mostrada após o tratamento com terpinen-4-ol e a nistatina teve um efeito maior para ambas as espécies. Para a atividade enzimática, as drogas não apresentaram ação. O efeito potencializado pela combinação de terpinen-4-ol e nistatina e a redução de adesão evidenciam seu potencial como agente anti-fúngico.

Antimicrobial Activity of Two Root Canal Filling Pastes in Deciduous Teeth: an In Vitro Study / Avaliação "in vitro" da Atividade Antimicrobiana de Duas Pastas Obturadoras de Canais Radiculares de Dentes Decíduos

O tratamento endodôntico de dentes decíduos é de suma importância para que se possa preservar a dentição primária e, assim, promoverdesenvolvimento craniofacial adequado, oclusão normal e qualidades estéticas. No entanto, para que isso ocorra, é necessário o uso de pastasobturadoras com propriedades biológicas importantes para a preservação do dente. O objetivo deste estudo foi avaliar in vitro a atividadeantimicrobiana de duas pastas obturadoras utilizadas em dentes decíduos, Vitapex® e Calcipex®, em espécies microbianas comumenteencontradas em infecções endodônticas (Candida albicans, Enterococcus faecalis, Streptococcus mutans, Streptococcus sanguinis, Escherichiacoli e Staphylococcus aureus), utilizando o teste de difusão em ágar. Foram utilizadas placas de BHI (Brain Heart Infusion) e confeccionadosquatro pontos equidistantes, imediatamente preenchidos com o Vitapex® cimentos e Calcipex®. O gluconato de clorexidina a 1% (CHX) e águadestilada foram utilizados como controle positivo e negativo, respectivamente. Após a incubação das placas a 37 °C durante 24 h, o diâmetrodas zonas de inibição do crescimento bacteriano ao redor dos poços foi medido (em milímetros) com um compasso de calibre digital com luzrefletida. Este teste foi realizado em triplicata e os dados foram submetidos à análise de variância e teste de Tukey (? = 0,05). Ambas as pastastiveram atividade antimicrobiana, mas Calcipex® foi mais eficiente para a maioria das cepas. O halo formado nos experimentos contendoas cepas de S. mutans e S. sanguinis foi semelhante para ambas as pastas. No presente estudo, podemos sugerir que Calcipex® apresentou amelhor propriedade antimicrobiana em relação aos microrganismos estudados.

Anti dermatophytic therapy: prospects for the discovery of new drugs from natural products

Millions of people and animals suffer from superficial infections caused by a group of highly specialized filamentous fungi, the dermatophytes, which only infect keratinized structures. With the appearance of AIDS, the incidence of dermatophytosis has increased. Current drug therapy used for these infections is often toxic, long-term, and expensive and has limited effectiveness; therefore, the discovery of new anti dermatophytic compounds is a necessity. Natural products have been the most productive source for new drug development. This paper provides a brief review of the current literature regarding the presence of dermatophytes in immunocompromised patients, drug resistance to conventional treatments and new anti dermatophytic treatments.

Environmental isolation, biochemical identification, and antifungal drug susceptibility of Cryptococcus species

Introduction The incidence of opportunistic fungal infections has increased in recent years and is considered an important public health problem. Among systemic and opportunistic mycoses, cryptococcosis is distinguished by its clinical importance due to the increased risk of infection in individuals infected by human immunodeficiency virus. Methods To determine the occurrence of pathogenic Cryptococcus in pigeon excrement in the City of Araraquara, samples were collected from nine environments, including state and municipal schools, abandoned buildings, parks, and a hospital. The isolates were identified using classical tests, and susceptibility testing for the antifungal drugs (fluconazole, itraconazole, voriconazole, and amphotericin B) independently was also performed. After collection, the excrement samples were plated on Niger agar and incubated at room temperature. Results A total of 87 bird dropping samples were collected, and 66.6% were positive for the genus Cryptococcus. The following species were identified: Cryptococcus neoformans (17.2%), Cryptococcus gattii (5.2%), Cryptococcus ater (3.5%), Cryptococcus laurentti (1.7%), and Cryptococcus luteolus (1.7%). A total of 70.7% of the isolates were not identified to the species level and are referred to as Cryptococcus spp. throughout the manuscript. Conclusions Although none of the isolates demonstrated resistance to antifungal drugs, the identification of infested areas, the proper control of birds, and the disinfection of these environments are essential for the epidemiological control of cryptococcosis. .

Avaliação in vitro da atividade de fosfolipase e proteinase de cepas de candida albicans isolada da cavidade bucal de pacientes diabéticos / In vitro evaluation of phospholipase and proteinase of candida albicans isolated from oral cavity of diabetic patients

Leveduras do gênero Candida são comuns na cavidade bucal e podem causar candidose na presença de fatores predisponentes, especialmente em paciente diabético, o qual é caracterizado por um aumento anormal da glicose no sangue. A manifestação da doença está relacionada a este conjunto de fatores locais, tais como a presença de próteses dentárias, pH salivar, fluxo salivar e tabaco. A redução da saliva é um dos principais fatores de risco para o aparecimento de infecção e o controle glicêmico inadequado causado por diabetes, em associação com todos estes fatores, pode aumentar a incidência de infecções. Os objetivos deste estudo foram: 1) isolar e identificar cepas de Candida albicans da mucosa bucal de pacientes diabéticos 2) avaliar os fatores de virulência: proteinase e fosfolipase. Amostras microbiológicas foram coletadas a partir de locais da mucosa bucal e semeadas em CHROMagar para posterior identificação de C. albicans por PCR. Foram realizados testes da atividade de proteinase e fosfolipase para todos os isolados de C. albicans. Neste estudo, 22 isolados foram identificados como C. albicans. Em relação às atividades de proteinases, todas as cepas de C. albicans foram capazes de produzir proteinase, enquanto que para fosfolipase, apenas 4,5% dos isolados não produziram esta exoenzima. Portanto, C. albicans presente na cavidade bucal de pacientes diabéticos tem potencial patogênico e pode participar de processos infecciosos e inflamatórios, causando lesões e invadindo os tecidos orais.

Candida yeasts are common in the oral cavity and can cause candidosis in the presence of predisposing factors, especially diabetes, which is characterized by an abnormal increasing in blood glucose concentration. The manifestation of the disease is related to a set of local factors such as the presence of dental prostheses, salivary pH, salivary flow and tobacco. The reduction in saliva is a major risk factor for the onset of infection and poor glycemic control caused by diabetes in association with all these factors further increases the incidence of candidosis. The objectives of this study were: 1) to isolate and identify Candida albicans strains from oral mucosa sites of diabetic patients 2) to evaluate the virulence factors: proteinase and phospholipase. Thus, microbial samples were collected from oral mucosa sites and seeded in CHROMagar for subsequent identification of C. albicans by PCR. For the phenotypic tests, all strains of C. albicans were evaluated for their proteinase and phospholipase productions. In this study, 22 isolates were identified as C. albicans. In regard to the proteinase activities, all strains of C. albicans were able to produce proteinase, while only 4.5% from those isolates were not able to produce phospholipase activity. In conclusion, C. albicans present in the oral cavity of diabetic patients is potentially pathogenic and can participate in infectious and inflammatory processes, causing injury and invading oral tissues.

Conventional and alternative antifungal therapies to oral candidiasis

Candida-associated denture stomatitis is the most common form of oral candidal infection, with Candida albicans being the principal etiological agent. Candida adheres directly or via an intermediary layer of plaque-forming bacteria to denture acrylic. Despite antifungal therapy to treat denture stomatitis, infection is reestablished soon after the treatment ceases. In addition, many predisposing factors have been identified as important in the development of oral candidiasis, including malnourishment, common endocrine disorders, such as diabetis mellitus, antibacterial drug therapy, corticosteroids, radiotherapy and other immunocompromised conditions, such as acquired immunodeficiency syndrome (AIDS). These often results in increased tolerance to the most commonly used antifungals. So this review suggests new therapies to oral candidiasis.

Evidências científicas da colonização de Candida spp. em bolsas periodontais / Scientific evidence of Candida spp. colonization in periodontal pockets

Objetivo: o objetivo do presente trabalho é apresentar uma breve revisão de literatura a respeito da colonização de Candida spp. em bolsas periodontais, seus principais fatores de virulência e possível influência sobreas doenças periodontais. Revisão de Literatura: Apesar de a mucosa bucal ser considerada o principal reservatóriode Candida spp, este micro-organismo pode estar coagregado a bactérias do biofilme dental, sendo considerado um fator importante para o processo decolonização de bolsas periodontais. Além disso, possui vários fatores de virulência relevantes na patogênese dadoença periodontal, tais como a capacidade de aderir ao epitélio e invadir o tecido conjuntivo gengival. Também pode inibir a função de neutrófilos polimorfonucleares, bem como produzir enzimas como colagenases e proteinases, que são capazes de degradar imunoglobulinas.Considerações finais: Os fatores de virulência de Candida spp. associada à suscetibilidade do hospedeiro poderiam desempenhar um papel importante nasalterações inflamatórias associadas com as doenças periodontais destrutivas

The role of immune system in the development of periodontal disease: a brief review / O papel do sistema imune no desenvolvimento da doença periodontal: uma breve revisão

Periodontitis is a highly complex and multi-factorial disease. This review summarizes some immunological factors involved in the development and control of this oral disease, such as: the participation of inflammatory cells in local inflammation, the synthesis of chemotaxis proteins with activation of the complement system and a range of antimicrobial peptides, such as defensins, cathelicidin and saposins. The integration of pathogen-associated molecular patterns (PAMPs) from microorganisms with their surface receptors in the immune cells, induces the production of several cytokines and chemokines that presents either a pro- and/or anti-inflammatory role by stimulating the secretion of a great variety of antibody subtypes and the activation of mechanisms of controlling the disease, such as the regulatory T cells. Although several studies have tried to clarify some of the immune mechanisms involved in periodontal disease, more studies must be conducted to understand its development and progression and consequently to discover new alternatives for the prevention and treatment of this severe inflammatory disease.

A periodontite é uma doença altamente complexa e multifatorial. Esta breve revisão reúne alguns fatores imunológicos envolvidos no desenvolvimento e controle desta doença oral, tais como: a participação de células inflamatórias no local da inflamação, a síntese de proteínas quimiotáticas através da ativação do sistema complemento e a presença de alguns dos peptídeos antimicrobianos, como defensinas, catelicidinas e saposinas. A interação de padrões moleculares associados à patógenos (PAMPs) de microrganismos com seus receptores de superfície, em células imunológicas, induz a produção de várias citocinas e quimiocinas que apresentam função pró- e/ou anti-inflamatória estimulando a secreção de uma grande variedade de subtipos de anticorpos e a ativação de mecanismos de controle da doença, como as células T reguladoras. Embora vários trabalhos tentem esclarecer alguns dos mecanismos imunológicos envolvidos na doença periodontal, estudos adicionais são necessários para ampliar conhecimentos sobre o desenvolvimento, a progressão e, consequentemente, para se descobrir novas alternativas de prevenção e tratamento desta grave doença inflamatória.

Alternative low doses and routes of administering a prostaglandin F2alpha analogue to induce luteolysis in Nelore cows

The present study was conducted in order to verify the efficacy of lower doses and alternative routes of a prostaglandin F2 alpha analogue, luprostiol (PGF), for the induction of luteolysis and the precipitation of estrus in nonlactating Nelore cows (Bos taurus indicus). A conventional dose (15 mg) of PGF was compared to doses lower than the conventional dose, which ranges from 10 to 50%, that were administered intramuscularly (IM), intravulvosubmucosally (IVSM), or in the Bai-hui acupuncture site located within the lumbosacral area. The cows were administered PGF 8 day after estrus in the presence of a corpus luteum, and randomly assigned to the following groups: G1 (positive control), 15 mg, IM (n = 23); G2, 7.5 mg, IM (n = 23); G3, 3.75 mg, IM (n = 24); G4, 7.5 mg, IVSM (n = 25); G5, 3.75 mg, Bai-hui acupoint (n = 24); and G6, 1.5 mg, Bai-hui acupoint (n = 25). The results indicated that 50% of a conventional dose of PGF (7.5 mg) resulted in a complete luteal regression (plasma progesterone <1 ng/ml) at Hour 48, and hastened estrus, regardless of whether or not PGF was administered IM or IVSM. Comparatively, 10 or 25% of the conventional dose, even when administered to the Bai-hui acupoint, resulted in an initial reduction in the concentration of progesterone at Hour 24, followed by an increase observed at Hour 48. In conclusion, 25% of a conventional PGF dose administered via the Bai-hui acupoint proved inadequate to induce a complete luteal regression, whereas 50% of a conventional dose administered IM or IVSM was found to be the minimal dose required to induce effectively a complete luteal regression, and to precipitate the onset of estrus in nonlactating Nelore cows.