Efeito de extratos vegetais de Hamamelis virginiana, Persea americana, Cynara scolymus L e Stryphnodendron barbatiman Msobre a expressão fenotípica dos fatores de virulência em biofilmes de Candida albicans / Effect of Hamamelis virginiana, Persea americana, Cynara scolymus L and Stryphnodendron barbatiman M plant extracts on the phenotypic expression of virulence factors in biofilms of the Candida albicans

Objetivo: Analisar a expressão fenotípica de fatores de virulência em biofilmes de Candida albicans frente a extratos glicólicos de plantas. Material e Métodos: Os biofilmes de Candida albicans (ATCC 18804) obtidos a partir de incubação de 48 horas foram expostos por 5 minutos e 24 horas a diferentes concentrações de extratos glicólicos de Hamamelis virginiana e Persea americana, Cynara scolymus L e Stryphnodendron barbatiman M, a fim de verificar a ação antifúngica da proteinase, fosfolipase e hemolisina. Resultados: Todos os extratos foram eficazes na redução do biofilme. Em contato por 5 minutos. os extratos reduziram 50% do biofilme. Após 24 horas. o extrato de Persea americana apresentou o biofilme em 90%, seguido de Cynara scolymus, que o interrompeu em 85%. Houve mudança na intensidade da proteinase após 5 minutos e 24 horas, com uma atividade enzimática média de 0,69 em comparação com o controle de 0,49. Cynara scolymus foi o extrato com maior concentração média de 100 mg/ml; a intensidade da fosfolipase foi alterada com Stryphnodendron barbatiman sendo mais efetivo em 24 horas em relação ao controle (p< 0,0001). A secreção de hemolisina foi modificada por Hamamelis virginiana (12,5 mg/ml) após 5 minutos de exposição e em 24 horas. todos os extratos foram capazes de causar alterações na secreção. Conclusão: Os extratos testados apresentam potencial antifúngico em biofilmes de Candida albicans, implicando em redução significativa dos fatores de virulência. Assim, estes podem ser indicados como uma ferramenta terapêutica alternativa para reduzir a morbidade dessas infecções, já que em ambos os tempos de exposição investigados, eles foram capazes de reduzir a secreção enzimática do fungo (AU)

Objective: Analyze the phenotypic expression of virulence factors in Candida albicans biofilms against plant glycolicextracts. Material and Methods: The biofilms of Candida albicans (ATCC 18804) obtained from incubation for 48 hours were exposed for 5 minutes and 24 hours to different concentrations of glycolic extracts of Hamamelis virginiana and Persea americana, Cynara scolymus L and Stryphnodendron barbatiman M, in order to verify the antifungal activity of the proteinase, phospholipase and hemolysin. Results: All extracts were effective in reducing biofilm. In contact for 5 minutes. the extracts reduced 50% of the biofilm. After 24 hours, the Persea americanaextract showed the biofilm at 90%, followed by Cynara scolymus, which interrupted it at 85%, There was a change in proteinase intensity after 5 minutes and 24 hours. with an average enzymatic activity of 0.69 compared to the control of 0.49. Cynara scolymus was the extract with the highest mean concentration of 100 mg/ml; the phospholipase intensity was changed with Stryphnodendron barbatiman being more effective in 24 hours compared to the control (p< 0.0001). The hemolysin secretion was modified by Hamamelis virginiana (12.5 mg/ml) after 5 minutes of exposure, and in 24 hours. all extracts were capable to cause changes in secretion. Conclusion: The tested extracts have antifungal potential in Candida albicans biofilms, implying a significant reduction in virulence factors. Thus, these can be indicated as an alternative therapeutic tool to reduce the morbidity of these infections, as in both investigated exposure times. they were able to reduce theenzymatic secretion of the fungus (AU)

Effect of ozonized olive oil on oral levels of Candida spp. in patients with denture stomatitis / Efeito do óleo de oliva ozonizado sobre os níveis orais de Candida spp. em pacientes com estomatite protética

Objective: The aim of this study was to evaluate the effect of ozonized olive oil (OZ) on the oral levels of Candida spp. in patients with denture stomatitis. Material and Methods: In vitro tests were performed to validate antifungal activity and to standardize OZ conditions. Antifungal activity was screened against C. albicans and five non-albicans species (C. tropicalis, C. dubliniensis, C. krusei, C. guilliermondii, and C. parapsilosis). Also, the effects on C. albicans planktonic and biofilm were evaluated. After validation, OZ was included in a therapeutic protocol of denture stomatitis in vivo. Thirty patients used OZ and 20 used sodium bicarbonate (SB) for 14 days. After 7 and 14 days, clinical evaluation, isolation and identification of yeasts were performed. Isolates were identified by phenotypic and genotypic tests. Ozonated oil showed in vitro antifungal activity against all species of Candida. Ozonated oil reduced the number of viable cells in C. albicans biofilms. Oral candidal levels were lower in relation to baseline both after after 14 days of treatment with SB and OZ. Results: A total of 493 Candida spp. isolates was obtained and 80% were identified as C. albicans. Remission of denture stomatitis was observed in all patients after 7 days of treatment in both groups. Conclusion: Within the limits of the study we can conclude that ozonized olive oil can be a new alternative for the control of biofilm in patients with denture stomatitis. (AU)

Objetivo: O objetivo deste estudo foi avaliar o efeito do óleo ozonizado (OZ) sobre os níveis orais de Candida spp. em pacientes com estomatite protética. Material e Métodos: Testes in vitro foram realizados para validar a atividade antifúngica e padronizar as condições do OZ. A atividade antifúngica foi avaliada contra C. albicans e cinco espécies não-albicans (C. tropicalis, C. dubliniensis, C. krusei, C. guilliermondii e C. parapsilosis). Além disso, os efeitos sobre C. albicans planctônico e biofilme foram avaliados. Após validação, o OZ foi incluído em um protocolo terapêutico de estomatite protética in vivo. Trinta pacientes usaram OZ e 20 usaram bicarbonato de sódio (SB) por 14 dias. Após 7 e 14 dias, foram realizadas avaliações clínicas, isolamento e identificação de leveduras. Os isolados foram identificados por testes fenotípicos e genotípicos. O óleo ozonizado mostrou atividade antifúngica in vitro contra todas as espécies de Candida. O óleo ozonizado reduziu o número de células viáveis em biofilmes de C. albicans. Os níveis orais de candidíase foram menores em relação aos valores basais após 14 dias de tratamento com SB e OZ. Resultados: Um total de 493 Candida spp. isolados foram obtidos e 80% foram identificados como C. albicans. A remissão da estomatite protética foi observada em todos os pacientes após 7 dias de tratamento em ambos os grupos. Conclusão: Dentro dos limites do estudo podemos concluir que o óleo de oliva ozonizado pode ser uma nova alternativa para o controle do biofilme em pacientes com estomatite protética.(AU)

Molecular identification of Pichia guilliermondii, Debaryomyces hansenii and Candida palmioleophila

Traditional phenotypic methods and commercial kits based on carbohydrate assimilation patterns are unable to consistently distinguish among isolates of Pichia guilliermondii, Debaryomyces hansenii and Candida palmioleophila. As result, these species are often misidentified. In this work, we established a reliable method for the identification/differentiation of these species. Our assay was validated by DNA sequencing of the polymorphic region used in a real-time PCR assay driven by species-specific probes targeted to the fungal ITS 1 region. This assay provides a new tool for pathogen identification and for epidemiological, drug resistance and virulence studies of these organisms.

Opportunistic microorganisms in patients undergoing antibiotic therapy for pulmonary tuberculosis

Antimicrobial therapy may cause changes in the resident oral microbiota, with the increase of opportunistic pathogens. The aim of this study was to compare the prevalence of Candida, Staphylococcus, Pseudomonas and Enterobacteriaceae in the oral cavity of fifty patients undergoing antibiotic therapy for pulmonary tuberculosis and systemically healthy controls. Oral rinsing and subgingival samples were obtained, plated in Sabouraud dextrose agar with chloramphenicol, mannitol agar and MacConkey agar, and incubated for 48 h at 37ºC. Candida spp. and coagulase-positive staphylococci were identified by phenotypic tests, C. dubliniensis, by multiplex PCR, and coagulase-negative staphylococci, Enterobacteriaceae and Pseudomonas spp., by the API systems. The number of Candida spp. was significantly higher in tuberculosis patients, and C. albicans was the most prevalent specie. No significant differences in the prevalence of other microorganisms were observed. In conclusion, the antimicrobial therapy for pulmonary tuberculosis induced significant increase only in the amounts of Candida spp.

HAART therapy does not reduce the proteinase and phospholipase secretion by oral Candida albicans isolated from HIV-positive patients

Production of exoenzymes, specifically the proteinase and phospholipase, is considered one of the most important of pathogenicity mechanisms of C. albicans, which is crucial for tissue invasion. This study aimed at evaluating the production of these exoenzymes in 50 oral C. albicans isolates from HIV-positive (HIV+) patients treated with highly active anti-retroviral therapy (HAART), and from 50 control individuals. For testing the production of phospholipase and proteinase, the culture media containing egg yolk and bovine albumin were used, respectively. The results were obtained by measuring the diameter of the colony and divided by the diameter of colony plus the precipitation zone, defined as Pz. Data were statistically analyzed by Student’s t test (5). Statistically significant difference (p = 0.001) was observed between the mean values of Pz for proteinase in isolates from HIV+ patients (Pz = 0.358±0.295) and from control group (Pz = 0.660±0.370). The same results were observed for phospholipase production (Pz = 0.399±0.227 for HIV+ group; Pz =0.635±0.292 control group). Both enzymes were highly produced by C. albicans isolated from HIV+ patients when compared with those secreted by C. albicans obtained from control group, suggesting that HAART did not reduce the secretion of these enzymes by this pathogenic fungus infecting HIV+ patients.

Evaluation of alternative methods for the disinfection of toothbrushes

The aim of this study was to evaluate alternative methods for the disinfection of toothbrushes considering that most of the previously proposed methods are expensive and cannot be easily implemented. Two-hundred toothbrushes with standardized dimensions and bristles were included in the study. The toothbrushes were divided into 20 experimental groups (n = 10), according to microorganism considered and chemical agent used. The toothbrushes were contaminated in vitro by standardized suspensions of Streptococcus mutans, Streptococcus pyogenes, Staphylococcus aureus or Candida albicans. The following disinfectants were tested: 0.12 percent chlorhexidine digluconate, 50 percent white vinegar, a triclosan-containing dentifrice solution, and a perborate-based tablet solution. The disinfection method was immersion in the disinfectant for 10 min. After the disinfection procedure, the number of remaining microbial cells was evaluated. The values of cfu/toothbrush of each group of microorganism after disinfection were compared by Kruskal-Wallis ANOVA and Dunn's test for multiple comparisons (5 percent). The chlorhexidine digluconate solution was the most effective disinfectant. The triclosan-based dentifrice solution promoted a significant reduction of all microorganisms' counts in relation to the control group. As to the disinfection with 50 percent vinegar, a significant reduction was observed for all the microorganisms, except for C. albicans. The sodium perborate solution was the less effective against the tested microorganisms. Solutions based on triclosan-containing dentifrice may be considered effective, nontoxic, cost-effective, and an easily applicable alternative for the disinfection of toothbrushes. The vinegar solution reduced the presence of S. aureus, S. mutans and S. pyogenes on toothbrushes.

Avaliação da microbiota bucal de pacientes com anorexia nervosa e bulimia nervosa / Evaluation of oral microbiota of patients with Anorexia Nervosa and Bulimia Nervosa

Os Transtornos alimentares (TA) como Anorexia Nervosa (AN) e Bulimia Nervosa (BN) são acompanhados de inúmeras alterações sistêmicas e bucais relacionadas ao comprometimento do estado nutricional e às práticas compensatórias inadequadas para o controle do peso. O objetivo deste estudo foi avaliar diversidade microbiológica existente na cavidade bucal de pacientes com estes transtornos, por meio de técnicas de cultivo e utilizando métodos moleculares independentes de cultivo. Foram incluídos no estudo 32 pacientes anoréxicos e 27 bulímicos, pareados com 59 indivíduos controle. Amostras de enxágüe bucal foram semeadas para a avaliação da prevalência de leveduras do gênero Candida, estafilococos, enterococos, estreptococcos do grupo mutans (EGM), lactobacilos, enterobactérias/pseudomonas. Espécies de Candida, estafilococos, enterococos, enterobactérias/pseudomonas foram identificadas pelo sistema API. Amostras de biofilme supragengival foram coletadas e utilizadas somente nos procedimentos moleculares. As contagens de microrganismos nos grupos foram comparadas por ANOVA/Mann-Whitney (5%). Houve diferença estatisticamente significantes (p<0,05) para as contagem de leveduras do gênero Candida, estafilococos, enterococos, EGM e lactobacilos entre o grupo TA e controle, mas não houve diferenças significativas para a prevalência de enterobactérias/pseudomonas (p=0,312). Pequena diferença entre os grupos foi observada na diversidade de espécies dos microrganismos estudados pelo método de cultivo. Avaliação molecular foi realizada pela ribotipagem por seqüenciamento do 16S rRNA bacteriano e regiões D1/D2 do 28S rRNA. Foram avaliados cerca de 3000 clones do grupo TA e 1500 clones do controle. Sessenta e duas espécies ou filotipos de bactérias foram detectados, sendo que 22 identificações foram encontrados somente no grupo de estudo, 6 apenas no grupo controle e 34 em ambos os grupos.

Eating disorders such as nervous Anorexia and Bulimia Nervosa have several clinical and oral alterations related to the nutritional state involvement and the inadequate compensatory practices for weight control. The aim of this study was to evaluate the microbial diversity in the oral cavity of patients with Anorexia Nervosa and Bulimia nervosa by cultivation techniques and cultivation independent molecular methods. The study included 32 patients and 27 bulimic anorexics, matched with 59 control subjects. Oral rinse samples were cultured to assess the prevalence of Candida species, staphylococci, enterococci, streptococci mutans (EGM), lactobacilli, Enterobacteriaceae / Pseudomonas. Candida species, staphylococci, enterococci, Enterobacteriaceae / Pseudomonaswere identified by API systems. Supragingival biofilm samples were collected and used only in molecular procedures. Counts of microorganisms in the groups were compared by ANOVA / Mann-Whitney (5%). There was a statistically significant (p <0.05) for the counting of yeasts, staphylococci, enterococci, and lactobacilli EGM between TA and control groups, but there were no significant differences inthe prevalence of Enterobacteriaceae / Pseudomonas (p = 0.312). Few differences between the groups were observed in the species diversity of organisms studied by the method of cultivation. Molecular analysis was performed by ribotyping by sequencing the 16S rRNA bacterial and D1/D2 regions of 28S rRNA. About 3000 clones of the TA group and 1500 clones of control were evaluated. Sixty-two species or filotypes of bacteria were detected, with 22 identifications were found only in the study group, only 6 in the control group and 34 in both groups. Microorganisms related to caries and periodontal diseases were found in both groups.