Antimicrobial efficacy of S-PRG containing toothpastes on S. mutans biofilm development / Eficácia antimicrobiana de dentifrícios contendo S-PRG sobre o desenvolvimento de biofilme de S. mutans

Objective: to investigate the antimicrobial effects of toothpastes containing bioactive surface pre-reacted glass particles (S-PRG) on S. mutans biofilms adherence, initial colonization and maturation. Material and Methods: a reference UA 159 and a clinical S. mutans (SM6) strain were used. Bovine enamel specimens were randomly allocated into the groups (n=5): toothpastes containing 0%; 1%; 5%; 20%; 30% S-PRG; positive control dentifrice (NaF+triclosan); and negative control (distilled water). For biofilm development, samples were placed in a 24-well plate containing artificial saliva (4h), followed by adding 1mL of artificial saliva, BHI broth and 225µL of S. mutans suspension. Treatments with toothpastes were applied previously or after 4h and 24h of biofilm formation. Samples were incubated for 48h at 37°C in 5%CO2 and biofilm was detached and seeded in Petri dishes for determining the number of viable cells. Data were analyzed by ANOVA and Tukey test (5%). Results: significantly lower microorganisms' adherence (p<0.05) was obtained for all S-PRG toothpastes, with similar results to NaF+triclosan for SM6 and 20 and 30%S-PRG groups exhibiting higher inhibition effect than the NaF+Triclosan for UA159. Antibacterial effect on the early-stage biofilm was also observed for the S-PRG groups, but was not superior to the NaF+Triclosan toothpaste. For the mature biofilm, the effective antimicrobial potential of S-PRG toothpastes was observed only for the SM6 clinical strain, but was not higher than the positive control. Conclusion: experimental S-PRG toothpastes were effective to inhibit S. mutans biofilm growth by exhibiting antimicrobial activity, being promising agents to prevent cariogenic biofilm development (AU)

Objetivo: investigar o efeito de dentifrícios contendo S-PRG sobre a colonização inicial e maturação de biofilmes de S. mutans. Material e Métodos: uma cepa de referência (UA 159) e uma cepa clínica de S. mutans (SM6) foram utilizadas. Espécimes de esmalte bovino foram alocados nos grupos (n=5): dentifrícios contendo 0%; 1%; 5%; 20% e 30%S-PRG; controle positivo (NaF+triclosan); e controle negativo (água destilada). Os espécimes foram inseridos em uma placa de 24 poços contendo saliva artificial (4h), seguido por adição de 1mL de saliva artificial, BHI, 225µL de suspensão de S. mutans e foram tratados com suspensões de dentifrícios antes ou depois de 4 e 24h da formação do biofilme. Os espécimes foram incubados por 48h e o biofilme foi removido dos espécimes e semeado em placas de Petri para contagem de UFC/mL. Os dados foram analisados por ANOVA e teste de Tukey (5%). Resultados: houve diminuição na adesão de microrganismos para os grupos tratados com S-PRG (p<0.05). Para SM6, os dentifrícios contendo S-PRG apresentaram resultados semelhantes ao NaF+triclosan e para a cepa UA159 o dentifrício com 30%S-PRG apresentou efeito superior. Efeito antimicrobiano no biofilme recém-formado (4h) foi observado para os grupos contendo S-PRG, mas não foi observado efeito superior ao NaF+Triclosan. Para o biofilme maduro, efeito antimicrobiano do S-PRG foi observado apenas para a cepa clínica, mas não superior ao efeito do NaF+Triclosan. Conclusão: dentifrícios contendo S-PRG foram eficazes na inibição do desenvolvimento de biofilmes de S. mutans, sendo promissores agentes para prevenir o desenvolvimento de biofilme cariogênico. (AU)

Photodynamic inactivation of planktonic cultures of Streptococcus mutans using erythrosine irradiated by LED / Inativação fotodinâmica de culturas planctônicas de Streptococcus mutans usando eritrosina irradiada por LED

Objective: The aim of this in vitro study was to evaluate the efficacy of photodynamic inactivation (PDI) with erythrosine (E), using a light-emitting diode (LED) on planktonic cultures of Streptococcus mutans. Material and Methods: A Streptococcus mutans strain (UA 159) was used to prepare the suspensions containing 107 cells/mL, which was tested under different experimental conditions: a) LED irradiation in the presence of erythrosine as a photosensitizer (E+L+); b) LED irradiation only (P-L+); c) treatment with erythrosine only (E+L-); and d) no LED irradiation or photosensitizer (P) treatment, which served as a control group (P-L-). After treatment, strains were seeded onto MSBS agar for determination of the number of colony-forming units (CFU/mL). Results: The results were submitted to analysis of variance and the Tukey test (p < 0.05). No reduction in the number of CFU/mL was observed in the treatment group with erythrosine (E+L+) when compared to the control (P-L-). Conclusion: PDI using erythrosine did not reduce the number of CFUs per millimeter within the parameters in this study. (AU)

Objetivo: o objetivo deste estudo in vitro foi avaliar a eficácia da inativação fotodinâmica (PDI) com a eritrosina (E), usando diodo de emissão de luz azul (LED) em culturas planctônicas de Streptococcus mutans. Material e métodos: a cepa de Streptococcus mutans (UA 159) foi usada para o preparo das suspensões padrões contendo 107 células/mL, as quais foram testadas em diferentes condições experimentais a) irradiação com LED em presença da eritrosina como fotossensibilizador (E+L+); b) irradiação com LED apenas (F-L+); c) tratamento com eritrosina apenas (E+L-); e d) tratamento sem irradiação com LED ou fotossensibilizador (F), que serviu como grupo controle (F-L-). Após o tratamento, as cepas foram semeadas em ágar MSBS para determinação do número de unidades formadoras de colônias (UFC/mL). Resultados: os resultados foram submetidos à análise de variância e teste de Tukey (p < 0.05). Não foi observada redução no número de UFC/mL no grupo de tratamento com eritrosina (E+L+) quando comparado ao grupo controle (F-L-). Conclusão: a PDI usando etritrosina e LED não reduziu o número de UFCs por milímetro com os parâmetros utilizados neste estudo.(AU)

Effects of Bacillus subtilis on Candida albicans: biofilm formation, filamentation and gene expression / Efeitos de Bacillus subtilis sobre Candida albicans: formação de biofilme, filamentação e expressão gênica

Objective: The aim of this study was evaluate the effect of Bacillus subtilis on Candida albicans biofilm formation and filamentation by evaluating the gene expression of ALS3, HWP1, BCR1, EFG1 and TEC1. Material and Methods: Mixed (C. albicans / B.subtilis) and monotypic biofilms were cultured in plates at 37°C for 48 h under shaking for counting viable cells (CFU / mL) and analysis of gene expression by real-time PCR. The C. albicans filamentation assay was performed in medium containing 10% fetal bovine serum at 37°C for 6 hours. Data was analysed by t-Student and Mann­ Whitney tests. Results: B. subtilis reduced the biofilm formation of C. albicans in 1 log when cultured in the same environment (p<0.0001). In addition, it significantly reduced the yeast - hypha transition affecting the morphology of C. albicans. Among all of the analyzed genes, the ALS3 and HWP1 genes were the most affected, achieving 111.1- and 333.3- fold decreases in the C. albicans biofilms associated with B. subtilis, respectively. Conclusion: B. subtilis reduced the biofilm formation and filamentation of C. albicans by negatively regulating the ALS3, HWP1, BCR1, EFG1 and TEC1 genes that are essential for the production of biofilm and hyphae. (AU)

Objetivo: O objetivo deste estudo foi avaliar o efeito de Bacillus subtilis sobre a formação de biofilme e filamentação de Candida albicans através da avaliação da expressão dos genes ALS3, HWP1, BCR1, EFG1 and TEC1. Material e métodos: Biofilmes monotípicos e mistos (C. albicans / B.subtilis) foram cultivados em placas a 37°C por 48 h sob agitação, para a contagem de células viáveis (UFC/mL) e para a análise da expressão gênica por PCR em tempo real. O ensaio de filamentação de C. albicans foi realizado em meio contendo 10% de soro fetal bovino a 37°C por 6 h. Os dados obtidos foram analisados por testes t-Student e Mann­Whitney. Resultados: B.subtilis reduziu em 1 log a formação de biofilme por C. albicans quando cultivados no mesmo ambiente (p<0.0001). Além disso, reduziu significantemente a transição de levedura para hifa, afetando assim, a morfologia de C. albicans. Em relação aos genes analisados, os genes ALS3 e HWP1 foram os mais regulados negativamente, com uma diminuição de 111,1 e 333,3 vezes, respectivamente, na sua expressão em biofilmes de C. albicans associados a B. subtilis. Conclusão: B. subtilis reduziu a filamentação e a formação de biofilme de C. albicans através da regulação negativa dos genes ALS3, HWP1, BCR1, EFG1 e TEC1, que são essenciais na produção de hifas e de biofilme. (AU)

Antimicrobial and mechanical acrylic resin properties with silver particles obtained from Fusarium oxysporum / Propriedades antimicrobiana e mecânica de resina acrílica com partículas de prata obtidas a partir de Fusarium oxysporum

Objective: This study evaluated the effects of the incorporation of silver nanoparticles (AgNPs) obtained from Fusarium oxysporum on heat-activated acrylic resin (HAAR) and their influence on resin's surface roughness, hardness, color alteration and antimicrobial capacity against Candida albicans. Material and Methods: For this, 50 discs of HAAR (2x5 mm) were produced and divided into three groups, Control: HAAR; Ag1: HAAR plus 0.539 mg of AgNPs; and Ag2: HAAR plus 1.1 mg of AgNPs. Knopp hardness (HK), surface roughness (Ra and Rz) and color alteration according to the CIE Lab were measured. Specimens were then evaluated in vitro with regard to C. albicans biofilm formation through formed colony count (CFU/mL). Scanning Electron Microscopy (SEM) and Atomic force microscopy (AFM) analyses were performed. Results: The addition of AgNPs of both concentrations changed Ra, Rz and HK significantly. There was statistically significant difference for L (p=0.00); a*(p=0.00) and b*(p=0.00) parameters. There were no differences between Ag1 and Ag2 biofilm formation, but the comparison of both with the control group presented a significant reduction (p=0.0091) on biofilm formation. SEM and AFM images showed no signs of NPs clustering. Conclusion: It can be concluded tha AgNPs incorporation in HAAR was effective in reducing C. albicans activity, with a slight change in color and hardness of the material, being effective therefore, in regions such as the dental prostheses palate, which have lesser aesthetic appeal. (AU)

Objetivo: Este estudo avaliou os efeitos da incorporação de nanopartículas de prata (AgNPs) obtidas a partir de Fusarium oxysporum em resina acrílica ativada termicamente (RAAT) e sua influência na rugosidade, dureza, cor e capacidade antimicrobiana contra Candida albicans. Material e Métodos: Para isso, 50 discos de RAAT (2x5 mm) foram produzidos e divididos em três grupos, Controle: RAAT; Ag1: RAAT com 0,539 mg de AgNPs; e Ag2: RAAT com 1,1 mg de AgNPs. Foram medidas a dureza Knopp (DK), a rugosidade superficial (Ra e Rz) e a alteração da cor de acordo com o sistema CIE Lab. As amostras foram então avaliadas in vitro em relação à formação de biofilme de C. albicans através da contagem de unidades formadoras de colônia (UFC / mL). Foram realizadas análises de Microscopia Eletrônica de Varredura (MEV) e Microscopia de Força Atômica (AFM). Resultados: A adição de AgNPs de ambas as concentrações alterou significativamente Ra. Rz e DK. Houve diferença estatisticamente significativa para os parâmetros L (p = 0,00); a * (p = 0,00) e b * (p = 0,00). Não houve diferenças entre a formação de biofilme Ag1 e Ag2, mas a comparação entre ambos com o grupo controle apresentou redução significativa (p = 0,0091) na formação de biofilmes. As imagens de MEV e AFM não mostraram sinais de agrupamento de NPs. Conclusão: Pode-se concluir que a incorporação de AgNP no RAAT foi eficaz na redução da atividade de C. albicans, com uma discreta alteração na cor e dureza do material, sendo efetiva, portanto, em regiões linguais de próteses dentárias, que possuem menor apelo estético. (AU)

Study of Microbial Interaction Formed by "Candida krusei" and "Candida glabrata": "In Vitro" and "In Vivo" Studies

Abstract Recently, the non-albicans Candida species have become recognized as an important source of infection and oral colonization by association of different species in a large number of immunosuppressed patients. The objective of this study was to evaluate the interactions between C. krusei and C. glabrata in biofilms formed in vitro and their ability to colonize the oral cavity of mouse model. Monospecies and mixed biofilms were developed of each strain, on 96-well microtiter plates for 48 h. These biofilms were analyzed by counting colony-forming units (CFU/mL) and by determining cell viability, using the XTT hydroxide colorimetric assay. For the in vivo study, twenty-four mice received topical applications of monospecie or mixed suspensions of each strain. After 48 h, yeasts were recovered from the mice and quantified by CFU/mL count. In the biofilm assays, the results for the CFU/mL count and the XTT assay showed that the two species studied were capable of forming high levels of in vitro monospecie biofilm. In mixed biofilm, the CFU of C. krusei increased (p=0.0001) and C. glabrata decreased (p=0.0001). The metabolic activity observed in XTT assay of mixed biofilm was significantly reduced compared with a single C. glabrata biofilm (p=0.0001). Agreeing with CFU in vitro count, C. glabrata CFU/mL values recovered from oral cavity of mice were statistically higher in the group with single infection (p=0.0001) than the group with mixed infection. We concluded that C. krusei inhibits C. glabrata and takes advantage to colonize the oral cavity and to form biofilms.

Resumo Recentemente, as espécies não albicans tem se tornado uma importante fonte de infecção e de colonização oral pela associação de espécies em um grande número de pacientes imunossuprimidos. O objetivo desse estudo foi avaliar a interação entre C. krusei e C. glabrata em biofilmes formados in vitro e sua capacidade em colonizar a cavidade oral em modelo de camundongo. Biofilmes monoespécies e mistos foram formados em placas de 96 poços por 48 h. Esses biofilmes foram analisados pela contagem de UFC/mL e pela determinação da viabilidade celular, usando ensaio de XTT. Para o estudo in vivo, vinte e quatro camundongos receberam aplicações tópicas de suspensões monoespécies e mistas de cada espécie. Após 48 h, as leveduras foram recuperadas dos camundongos e quantificadas por UFC/mL. Nos ensaios de biofilme, os resultados da contagem de UFC/mL e do ensaio de XTT mostraram que as duas espécies estudadas foram capazes de formar grande quantidade de biofilme monoespécie in vitro. Nos biofilmes mistos, a UFC/mL de C. krusei aumentou (p=0,0001) e de C. glabrata diminuiu (p=0,0001). A atividade metabólica observada no ensaio de XTT nos biofilmes mistos foi significantemente reduzida comparada com o biofilme formado apenas de C. glabrata (p=0,0001). Concordado com as contagens in vitro, os valores de UFC/mL de C. glabrata recuperados da cavidade oral dos camundongos foram estatisticamente maior no grupo com infecção simples (p=0,0001) do que do grupo com infecção mista. Nós concluímos que C. krusei inibe C. glabrata e possui vantagem em colonizar a cavidade oral e formar biofilmes.

In vitro reduction of pathogenic sporothrix schenckii fungus by photodynamic therapy / Terapia fotodinâmica na redução in vitro do fungo patogênico sporothrix schenckii

Sporotrichosis is a disease that affects the lymph vessels, skin and some internal organs. Most cases are presented as asubacute chronic mycosis caused by the Sporothrix schenckii fungus; fairly common in tropical regions. The aim of thisstudy was to evaluate the susceptibility of Sporothrix schenckii yeast cells to the effects of photodynamic inactivation.For this, the viable cells were separated into four groups: irradiated with photosensitizer group (L+F+); irradiated withoutphotosensitizer group (L+F-), without irradiation and with photosensitizer group (L-F+); and without irradiation andwithout photosensitizer group (L-F-). The methylene blue photosensitizer concentration used was 0.1 mg/mL, and theAluminum Gallium Arsenide laser dose was 26.3 J/cm2. Then, counting of colony forming units (CFUs) was performedin each group. The main result was that the irradiated group with photosensitizer (L+F+) was the one that showed nogrowth of CFUs. Thus, it was concluded that Sporothrix schenckii can be inactivated by use of photodynamic therapy

A esporotricose é uma doença que afeta os vasos linfáticos, pele e alguns órgãos internos. A maioria dos casos seapresenta como uma micose subaguda à crônica, provocada por fungos do complexo Sporothrix schenckii, bastantecomuns em regiões tropicais. O objetivo deste estudo foi avaliar a suscetibilidade aos efeitos da inativação fotodinâmicaem células leveduriformes de fungos do complexo Sporothrix schenckii. Para tal, as células viáveis foram separadas emquatro grupos, sendo estes: grupo irradiado com fotossensibilizador (L+F+); grupo irradiado sem fotossensilizador (L+F-),grupo não irradiado com fotossensibilizador (L-F+); e grupo não irradiado sem fotossensibilizador (L-F-). A concentraçãodo fotossensibilizador azul de metileno utilizada foi de 0,1 mg/mL, e a dosagem do laser de Arseneto de Gálio Alumíniofoi de 26,3 J/cm2. Em seguida, foi realizada a contagem das unidades formadoras de colônias (UFCs) em cada grupo.Como principal resultado, verificou-se que o grupo irradiado com fotossensibilizador (L+F+) foi o único que nãoapresentou crescimento de UFCs. Dessa forma, concluiu-se que fungos do complexo Sporothrix schenckii podem serinativados com o uso da terapia fotodinâmica

Antibiofilm activity in vitro of Rosmarinus officinalis and Syzygium cumini glycolic extracts on Staphylococcus spp. of dentistry interest / Atividade antibiofilme in vitro dos extratos glicólicos de Rosmarinus officinalis e Syzygium cumini em Staphylococcus spp. de interesse odontológico

Objectives: The aim of this study was to identify the slime production and evaluate the effects of Rosmarinus officinalis (rosemary) and Syzygium cumini (jambolan) glycolic extracts, and 0.12% chlorhexidine (CHX) in biofilms formed by strains of coagulase-positive Staphylococcus - CPS and coagulase negative Staphylococcus - CNS isolated from the oral cavity. Material and Methods: Slime production was evaluated by two methods: the color of colony presented in Congo red agar, and through the amount of slime adhered to polystyrene. Biofilms were grown in acrylic resin discs immersed in broth, inoculated with microbial suspension (106 cells/ml) and incubated at 37°C/48 h. After formation, the biofilms were exposed for 5 minutes to glycol extracts, CHX or saline solution. The viability of biofilms was determined by counting the colony-forming units per milliliter (CFU/ml) in agar, and analyzed statistically by Tukey test (p< 0.05). Results: The strains S. aureus, S. schleiferi and S. epidermidis obtained the highest values of slime adhered to polystyrene. R. officinalis promoted reductions ranging from 12.1% to 78.7% in biofilms formed by isolates of CPS, and 9.2% to 73.7% in the biofilms of CNS. S. cumini reduced 12% to 55.7% in biofilms of CPS, and 7.9% to 71.5% in biofilms of CNS. With exception of S. saprophyticus, glycol extracts produced significant reductions in biofilms. For five isolates studied, R. officinalis produced greater reductions than CHX. Conclusion: R. officinalis and S. cumini showed effective antibiofilm activity against isolates that showed slime production.(AU)

Objetivos: O objetivo deste estudo foi identificar a produção de slime e avaliar os efeitos dos extratos glicólicos de Rosmarinus officinalis (alecrim), Syzygium cumini (jambolão) e 0,12% de clorexidina (CLX) em biofilmes formados por cepas de Staphylococcus coagulase positivo (SCP) e Staphylococcus coagulase negativo (SCN) da cavidade oral. Material e Métodos: A produção de slime foi avaliada por dois métodos: a cor da colônia apresentada em ágar vermelho Congo e pela quantidade de slime aderido ao poliestireno. Os biofilmes foram crescidos em discos de resina acrílica imersos em caldo, inoculados com suspensão microbiana (106 células/ml) e incubados a 37°C/48h. Após a formação, os biofilmes foram expostos durante 5 minutos aos extractos glicólicos, CLX ou solução salina. A viabilidade dos biofilmes foi determinada pela contagem das unidades formadoras de colônias por mililitro (UFC/ml) em ágar e analisada estatisticamente pelo teste de Tukey (p< 0,05). Resultados: As cepas S. aureus, S. schleiferi e S. epidermidis obtiveram os maiores valores de aderência ao poliestireno. R. officinalis promoveu reduções variando de 12,1% a 78,7% em biofilmes formados por isolados de SCP e 9,2% a 73,7% nos biofilmes de SCN. S. cumini reduziu de 12% a 55,7% nos biofilmes de SCP, e 7,9% a 71,5% nos biofilmes de SCN. Com exceção de S. saprophyticus, os extratos glicólicos produziram reduções estatísticas nos biofilmes. Para cinco isolados estudados, R. officinalis produziu maiores reduções do que CLX. Conclusão: R. officinalis e S. cumini mostraram atividade antibiofilme efetiva contra isolados que apresentaram produção de slime.(AU)

Mixed biofilms formed by C. albicans and non-albicans species: a study of microbial interactions

Abstract Most Candida infections are related to microbial biofilms often formed by the association of different species. The objective of this study was to evaluate the interactions between Candida albicans and non-albicans species in biofilms formed in vitro. The non-albicans species studied were:Candida tropicalis, Candida glabrata andCandida krusei. Single and mixed biofilms (formed by clinical isolates of C. albicans and non-albicans species) were developed from standardized suspensions of each strain (107 cells/mL), on flat-bottom 96-well microtiter plates for 48 hour. These biofilms were analyzed by counting colony-forming units (CFU/mL) in Candida HiChrome agar and by determining cell viability, using the XTT 2,3-bis (2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide colorimetric assay. The results for both the CFU/mL count and the XTT colorimetric assay showed that all the species studied were capable of forming high levels of in vitro biofilm. The number of CFU/mL and the metabolic activity of C. albicans were reduced in mixed biofilms with non-albicans species, as compared with a singleC. albicans biofilm. Among the species tested, C. krusei exerted the highest inhibitory action against C. albicans. In conclusion, C. albicans established antagonistic interactions with non-albicans Candida species in mixed biofilms.

Efeitos da terapia fotodinâmica antimicrobiana em leveduras do gênero Candida / Effects of antimicrobial photodynamic therapy on Candida yeasts

O objetivo deste estudo foi avaliar os efeitos da terapia fotodinâmica utilizando azul de metileno em cepas de Candida, Foram estudados: 5 C, albicans, 4 C, tropicalis , 4 C, glabrata, 2 C, parapsilosis, 2 C, kefyr, 1 C, stellatoidea, 1 C, krusei e 1 C, lipolytica, Cada cepa foi submetida a quatro condições experimentais: laser e azul de metileno (L+F+), irradiação com laser (L+F-), tratamento com azul de metileno (L-F+) e tratamento com soro fisiológico como grupo de controle (L-F-), Após o tratamento de cada cepa diluições seriadas e plaqueamento em Sabouraud dextrose agar foram realizadas, Os dados de unidades formadoras de colônias por mililitro (CFU/ml) foram analisados pelos testes de ANOVA e Tukey (p <0,05), Os resultados sugerem que os grupos tratados com laser (L+F+ e L+F-) apresentaram médias de UFC/ml (Log) inferiores aos grupos tratados sem laser (L-F- e L-F+), O grupo com a terapia fotodinâmica (L+F+) apresentou uma média de CFU/ml (Log) semelhante ao grupo (L+F-), Concluiu-se que as cepas de Candida analisadas foram sensíveis à irradiação do laser de baixa potência na presença ou ausencia do azul de metileno...

The aim of this study was to evaluate the effects of photodynamic therapy using methylene blue on Candida strains. Were studied: 5 C. albicans, 4 C. tropicalis, 4 C. glabrata 2 C. parapsilosis, 2 C. kefyr, 1 C. stellatoidea, 1 C. krusei and 1 C. lipolytica. Each strain was underwent to four experimental conditions: methylene blue laser (L+P+) irradiation with laser (L+P-), treatment with methylene blue (L-P+), and treatment with saline as control group (L-P-). After treatment of each strain and plating serial dilutions on Sabouraud dextrose agar were performed. The data from colony forming units per milliliter (CFU/ml) were analyzed by ANOVA and Tukey test (p <0.05). The results suggest that the groups treated with laser (L+ P+ and L+P-) showed average CFU/ml (Log) lower than without laser treatment groups (L-F- e L-F+). The group with photodynamic therapy (L+P+) showed an average CFU/ml (Log) similar to the group (L+P-). It was concluded that the strains of Candida analyzed were sensitive to laser irradiation of low power in the presence or absence of methylene blue...

Different Extracts of Zingiber officinale Decrease Enterococcus faecalis Infection in Galleria mellonella

Dried, fresh and glycolic extracts of Zingiber officinale were obtained to evaluate the action against G. mellonella survival assay against Enterococcus faecalis infection. Eighty larvae were divided into: 1) E. faecalis suspension (control); 2) E. faecalis + fresh extract of Z. officinale (FEO); 3) E. faecalis + dried extract of Z. officinale (DEO); 4) E. faecalis + glycolic extract of Z. officinale (GEO); 5) Phosphate buffered saline (PBS). For control group, a 5 μL inoculum of standardized suspension (107 cells/mL) of E. faecalis (ATCC 29212) was injected into the last left proleg of each larva. For the treatment groups, after E. faecalis inoculation, the extracts were also injected, but into the last right proleg. The larvae were stored at 37 °C and the number of dead larvae was recorded daily for 168 h (7 days) to analyze the survival curve. The larvae were considered dead when they did not show any movement after touching. E. faecalis infection led to the death of 85% of the larvae after 168 h. Notwithstanding, in treatment groups with association of extracts, there was an increase in the survival rates of 50% (GEO), 61% (FEO) and 66% (DEO) of the larvae. In all treatment groups, the larvae exhibited a survival increase with statistically significant difference in relation to control group (p=0.0029). There were no statistically significant differences among treatment groups with different extracts (p=0.3859). It may be concluded that the tested extracts showed antimicrobial activity against E. faecalis infection by increasing the survival of Galleria mellonella larvae.

Extratos seco, fresco e glicólico de Zingiber officinale foram obtidos para avaliar suas ações por meio de ensaio de sobrevivência em G. mellonella contra infecção por Enterococcus faecalis. Oitenta larvas foram divididas em: 1) Suspensão de E. faecalis (controle); 2) E. faecalis + extrato fresco de Z. officinale (FEO); 3) E. faecalis + extrato seco de Z. officinale (DEO); 4) E. faecalis + extrato glicólico de Z. officinale (GEO); 5) Solução tampão fosfato salina (PBS). Para o grupo de controle, 5 µL de inóculo de suspensão padronizada (107 células/mL) de E. faecalis (ATCC 29212) foi injetado na última proleg esquerda de cada lagarta. Para os grupos com tratamento, após a injeção de E. faecalis, os extratos foram injetados na última proleg direita. Após as injeções, as lagartas foram armazenadas a 37 °C e o número de animais mortos foi registrado diariamente em 168 h (7 dias) para analisar a curva de sobrevivência. As lagartas foram consideradas mortas quando elas não mostraram qualquer movimento após o toque. A infecção por E. faecalis levou à morte de 85% das lagartas após 168 h. Não obstante, nos grupos de tratamento com associação dos extratos, houve um aumento nas taxas de sobrevivência de 50% (GEO), 61% (FEO) e 66% (DEO) das lagartas. Em todos os grupos com tratamento, as lagartas apresentaram um aumento na sobrevivência, com diferença estatisticamente significativa em relação ao grupo controle (p=0,0029). Não houve diferença estatisticamente significativa entre os tratamentos com os diferentes extratos (p=0,3859). Pode concluir-se que os extratos testados mostraram atividade antimicrobiana contra a infecção por E. faecalis, aumentando a sobrevivência das lagartas de G. mellonella.

Novel in vitro methodology for induction of Enterococcus faecalis biofilm on apical resorption areas.

Context: Teeth with periapical lesion usually present external root resorption around the apical foramen. These areas facilitate adhesion and co-aggregation of microorganisms developing biofilms. Up to the present moment, there is no methodology in the literature that enables the in vitro evaluation of endodontic irrigants and intracanal dressings on biofilms located in apical external root resorptions of human teeth. Aims: This study aimed to describe a new in vitro methodology for Enterococcus faecalis biofilm development in external apical reportion areas of human extracted teeth in different periods of time. Settings and Design: In vitro qualitative laboratory study. Subjects and Methods: Thirty roots from human extracted teeth presenting external apical resorption had their root canal diameters standardized by means of instrumentation. Next, the roots were randomly divided into three groups (n = 30) according to E. faecalis strains (ATCC 29212) exposure time as follows: Group T5, with 5‑day exposure; Group T10, with 10‑day exposure, and Group T15, with 15‑day exposure. The roots were attached to 24‑well culture plates so that only their apices could be in contact with bacteria for induction of biofilm formation. At the end of these exposure times, the roots were qualitatively evaluated with scanning electron microscope to observe the presence of biofilm in external resorptions around the apical foramen. Results: It was found that microorganisms were present in all exposure times, although structures suggesting the presence of biofilm with great conglomerate of bacteria showing structures similar to polysaccharide extensions were observed at the 10th day of exposure. Conclusions: By means of this new methodology, it was possible to observe biofilm formation in the areas of external apical resorption after 10 days of exposure.

Coronal bacterial leakage in root canals filled with single cone technique and different endodontic sealers / Infiltração coronária microbiana em canais radiculares obturados pela técnica do cone único com diferentes cimentos endodônticos

Objective: To evaluate coronal bacterial leakage comparing five endodontic sealers (AH Plus, Apexit Plus, Copaifera sp oil, EndoREZ and Polifil), and comparing root canals filled with EndoREZ sealer/ EndoREZ® Points and EndoREZ sealer/conventional gutta-percha points. Material & Methods: 84 human teeth were prepared and filled with gutta-percha points using the single cone technique. Roots were randomly divided into 6 groups: Apexit Plus, AH Plus, Copaifera sp oil, Polifil, EndoREZ, and EndoREZ/EndoREZ Points. After setting time, the roots were incorporated in a leakage model, which upper chamber contained a suspension of Streptococcus mutans, and lower chamber a broth. Leakage was assessed for turbidity in lower chamber for 60 days. Statistic analysis was performed using the nonparametric Kaplan-Meier method (p<0.05). Results: All experimental groups presented leakage during the study’s period. The medium time of leakage was: Apexit Plus and AH Plus 6.3 days, Polifil 5.1 days, Copaifera 1.2 days, and both EndoREZ groups infiltrated in the first day. Conclusions: There was no statistically significant difference between the sealers Apexit Plus, AH Plus and Polifil, but they prevented leakage better than Copaifera sp oil and both EndoREZ groups. However, none of the tested sealers was capable of resisting coronal bacterial leakage for more than 22 days.

Objetivo: Avaliar a infiltração coronária microbiana de cinco cimentos endodônticos (AH Plus, Apexit Plus, Copaiba, EndoREZ and Polifil), e comparar canais obturados com cimento EndoREZ/ cones EndoREZ e canais com cimento EndoREZ/ cones de guta-percha. Material e Métodos: 84 raízes de dentes humanos uniradiculados tiveram seus canais preparados e obturados pela técnica do cone único. As raízes foram divididas em 6 grupos: Apexit Plus, AH Plus, Copaiba, Polifil, EndoREZ e EndoREZ/ cones EndoREZ. Após endurecimento dos cimentos, as raízes foram adaptadas a um modelo de infiltração, cuja câmara superior continha uma suspensão de Streptococcus mutans, e a inferior um meio de cultura, deixando a porção apical da raiz imersa. A infiltração foi verificada diariamente pelo turvamento na câmara inferior, por um período de 60 dias. Os dados foram avaliados pela análise estatística não paramétrica Kaplan-Meier (p<0,05). Resultados: Todos os grupos experimentais apresentaram infiltração no período do experimento, contudo o tempo máximo foi de 22 dias. O tempo médio de infiltração foi: Apexit Plus 6,3 dias, AH Plus 6,3 dias, Polifil 5,1 dias, Copaiba 1,2 dias, e em ambos os grupos do cimento EndoREZ todos os espécimes infiltraram no primeiro dia. Conclusão: Não houve diferença estatisticamente significante entre os cimentos Apexit Plus, AH Plus e Polifil, mas estes apresentaram melhores resultados que Copaifera e ambos os grupos do EndoREZ. Porém, nenhum cimento foi capaz de impedir a infiltração coronária microbiana por mais de 22 dias.

Correlation of phospholipase and proteinase production of Candida with in vivo pathogenicity in Galleria mellonella

An essential factor to the virulence of the genus Candida is the ability to produce enzymes and this may be crucial in the establishment of fungal infections. AIM:This study investigated in vitro enzymatic activities of Candida species and their virulence in an in vivo Galleria mellonella experimental model. METHODS: Twenty-four clinical strains of Candida spp. isolated from the human oral cavity were evaluated, including the following species: C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. norvegensis, C. lusitaniae and C. guilliermondii. All Candida strains were tested in vitro for production of proteinase and phospholipase. The Candida strains were also injected into Galleria mellonella larvae to induce experimental candidiasis, and after 24 hours, the survival rate was assessed. RESULTS: Phospholipase and proteinase activity were observed in 100% of the C. albicans strains. In the non-albicans species, proteinase and phospholipase activity were observed in 25 and 43% of the studied strains, respectively. The most pathogenic Candida species in G. mellonella were C. albicans, C. dubliniensis and C. lusitaniae, whereas C. glabrata was the least virulent species. Furthermore, a positive significant correlation was found between both enzymatic activities with virulence in G. mellonella. CONCLUSIONS: The virulence of Candida strains in G. mellonella is related to the quantity of proteinases and phospholipases production of each strain.

Efeitos clínicos e radiográficos do laser em baixa intensidade após a extração de terceiros molares inclusos / Clinical and radiographic effects of low intensity laser after extraction of unerupted third molars

Introdução: O laser em baixa intensidade tem sido indicado como tratamento coadjuvante no pós-operatório da cirurgia de extração dentária. Objetivo: O objetivo deste trabalho foi avaliar os efeitos clínicos e radiográficos do laser em baixa intensidade na cirurgia de exodontia de terceiros molares inclusos. Material e método: Oito pacientes foram submetidos à extração dos terceiros molares inferiores inclusos. O dente esquerdo foi tratado com laser durante a cirurgia e por mais dois dias do pós-operatório (Grupo Laser). A cirurgia do dente direito foi realizada após 15 dias da cirurgia do dente esquerdo e não recebeu laserterapia (Grupo Controle). A avaliação clínica do pós-operatório foi baseada na medida do edema e na análise de questionário para avaliação da dor. Após 40 dias de cada cirurgia, foram feitas radiografias periapicais digitais para medida das densidades ópticas da reparação óssea, por meio do programa Image J. Os dados obtidos na medida do edema e na análise de densidade óptica foram submetidos ao teste estatístico t de Student. Resultado: O nível de dor dos pacientes no pós-operatório foi menor no Grupo Laser em relação ao Grupo Controle. Entretanto, na medida do edema e na análise de densidade óptica das radiografias, não houve diferença estaticamente significante do Grupo Laser em relação ao Grupo Controle. Conclusão: De acordo com os parâmetros utilizados neste estudo, concluiu-se que a aplicação do laser em baixa intensidade promoveu analgesia no pós-operatório, porém não teve efeito sobre o edema e a reparação óssea.

Introduction: The low intensity laser therapy (LLLT) has been indicated as coadjuvant treatment of postoperative dental extraction surgery. Objective: The aim of this study was to evaluate the clinical and radiological findings of LLLT in the surgery for extraction of unerupted third molars. Material and method: Eight patients were submitted to extraction of mandibular third molar. The left tooth was treated with laser during surgery and for another 2 days after surgery (Laser Group). The right tooth surgery was performed after 15 days and did not receive laser therapy (control group). Clinical evaluation of the postoperative period was based on the measuring of edema and analysis of a questionnaire to assess pain. After 40 days of each surgery, digital periapical radiographs were made and measured the optical density of bone repair were analyzed using the Image J. The data obtained in the measurement of edema and analysis of optical density were tested using Student t test. Result: The level of pain in postoperative patients was lower in the laser group compared to the control group. However, in the measurement of edema and analysis of optical density of radiographs there was no statistically significant difference in the laser group compared to control. Conclusion: According to the parameters used in this study, we can concluded that the application of LLLT promoted analgesia postoperatively, but did not show effects on edema and bone repair.

Influence of artificial saliva in biofilm formation of Candida albicans in vitro

Due to the increase in life expectancy, new treatments have emerged which, although palliative, provide individuals with a better quality of life. Artificial saliva is a solution that contains substances that moisten a dry mouth, thus mimicking the role of saliva in lubricating the oral cavity and controlling the existing normal oral microbiota. This study aimed to assess the influence of commercially available artificial saliva on biofilm formation by Candida albicans. Artificial saliva I consists of carboxymethylcellulose, while artificial saliva II is composed of glucose oxidase, lactoferrin, lysozyme and lactoperoxidase. A control group used sterile distilled water. Microorganisms from the oral cavity were transferred to Sabouraud Dextrose Agar and incubated at 37°C for 24 hours. Colonies of Candida albicans were suspended in a sterile solution of NaCl 0.9%, and standardisation of the suspension to 106 cells/mL was achieved. The acrylic discs, immersed in artificial saliva and sterile distilled water, were placed in a 24-well plate containing 2 mL of Sabouraud Dextrose Broth plus 5% sucrose and 0.1 mL aliquot of the Candida albicans suspension. The plates were incubated at 37°C for 5 days, the discs were washed in 2 mL of 0.9% NaCl and placed into a tube containing 10 mL of 0.9% NaCl. After decimal dilutions, aliquots of 0.1 mL were seeded on Sabouraud Dextrose Agar and incubated at 37°C for 48 hours. Counts were reported as CFU/mL (Log10). A statistically significant reduction of 29.89% (1.45 CFU/mL) of Candida albicans was observed in saliva I when compared to saliva II (p = 0.002, considering p≤0.05).

Interação entre Escherichia coli e Candida albicans em biofilmes formados in vitro: análise da viabilidade celular por método colorimétrico / Interaction between Escherichia coli and Candida albicans in biofilms formed in vitro: analysis of cell viability by the colorimetric assay

As interações entre fungos e bactérias estão presentes na natureza e têm grande relevância médica e ambiental. O objetivo deste estudo foi avaliar os efeitos de Escherichia coli sobre os biofilmes de Candida albicans formados in vitro. Foram desenvolvidos biofilmes heterotípicos com a associação de C. albicans e E. coli e biofilmes monotípicos deC. albicans (grupo controle). Foi utilizada uma suspensão padronizada de C. albicans contendo 107 células.mL?1 para formação dos biofilmes. Para analisar os efeitos de E. coli no biofilme de C. albicans, foram testadas diferentes densidades celulares da suspensão de E. coli (107, 106 e 105 células.mL?1). Após 90 minutos e 24 horas da formação do biofilme, a viabilidade celular de C. albicans foi quantificada utilizando-se o ensaio colorimétrico XTT (2 metoxi 4 nitro 5 sulfofenil 5 fenilalanina carbonil 2H tetrazolium hidróxido). Os dados foram submetidos à Análise deVariância ANOVA e ao teste de Tukey, com significância de 5% (p < 0,05). Após 90 minutos da formação do biofilme, observou-se que a viabilidade celular de C. albicans dos biofilmes heterotípicos foi semelhante ao monotípico. A análise de 24 horas demonstrou que a viabilidade celular de C. albicans foi maior no biofilme monotípico emrelação aos biofilmes heterotípicos; entretanto, essa diferença não foi estatisticamente significante. Além disso, não foram observadas diferenças estatísticas entre as densidades celulares de E. coli testadas nos biofilmes heterotípicos. Conclui-se que, dentro dos parâmetros utilizados, E. coli não inibiu a formação de biofilme por C. albicans.

The interactions between fungi and bacteria are present in nature and has medical and environmental importance. The aim of this study was to evaluate the effects of interaction between ATCC strains Candida albicans and Escherichia coli through the study of biofilms in vitro. For this study were developed biofilms formed by the association between C.albicans and E.coli and monotypic biofilms of C. albicans (control group). To evaluate the effects of E. coli in biofilms of C. albicans, we tested different cell densities of E.coli suspension (107, 106 and 105 cells.mL?1). After 90 minutes and 24 hours of biofilm formation, cell viability of C. albicans was quantified using the XTT (2 methoxy 4 nitro 5 sulfophenyl 5 phenylamino carbonyl 2H tetrazolium hydroxide) colorimetric assay. Data were submitted to ANOVA and Tukey?s test, with 5% of significance. After 90 minutes of Candidal biofilm formation, we observedthat cell viability of C. albicans heterotypic biofilms was similar to monotypic biofilm (control group). For the analysis of 24 hours, it was found that cell viability of C. albicans was higher in the monotypic biofilm if compared to heterotypic biofilm, however this difference was not statistically significant. In addition, there were no statisticaldifferences between E. coli different cell densities tested in heterotypic biofilms. According to the methods used, we can conclude that E. coli did not inhibit the biofilm formation by C. albicans.

Antimicrobial activity of Calendula officinalis, Camellia sinensis and chlorhexidine against the adherence of microorganisms to sutures after extraction of unerupted third molars

OBJECTIVE: The objective of this study was to compare the antimicrobial effect of mouthwashes containing Calendula officinalis L., Camellia sinensis (L.) Kuntze and 0.12 percent chlorhexidine digluconate on the adherence of microorganisms to suture materials after extraction of unerupted third molars. MATERIAL AND METHODS: Eighteen patients with unerupted maxillary third molars indicated for extraction were selected (n=6 per mouthwash). First, the patients were subjected to extraction of the left tooth and instructed not to use any type of antiseptic solution at the site of surgery (control group). After 15 days, the right tooth was extracted and the patients were instructed to use the Calendula officinalis, Camellia sinensis or chlorhexidine mouthwash during 1 week (experimental group). For each surgery, the sutures were removed on postoperative day 7 and placed in sterile phosphate-buffered saline. Next, serial dilutions were prepared and seeded onto different culture media for the growth of the following microorganisms: blood agar for total microorganism growth; Mitis Salivarius bacitracin sucrose agar for mutans group streptococci; mannitol agar for Staphylococcus spp.; MacConkey agar for enterobacteria and Pseudomonas spp., and Sabouraud dextrose agar containing chloramphenicol for Candida spp. The plates were incubated during 24-48 h at 37ºC for microorganism count (CFU/mL). RESULTS: The three mouthwashes tested reduced the number of microorganisms adhered to the sutures compared to the control group. However, significant differences between the control and experimental groups were only observed for the mouthwash containing 0.12 percent chlorhexidine digluconate. CONCLUSIONS: Calendula officinalis L. and Camellia sinensis (L.) Kuntze presented antimicrobial activity against the adherence of microorganisms to sutures but were not as efficient as chlorhexidine digluconate.

Effect of photodynamic therapy on clinical isolates of Staphylococcus spp

Staphylococcus spp. are opportunistic microorganisms known for their capacity to develop resistance against antimicrobial agents. The objective of this study was to evaluate the effect of photodynamic therapy (PDT) on 20 Staphylococcus strains isolated from the human oral cavity, including S. aureus, S. schleiferi, S. epidermidis, S. capitis, S. haemolyticus, and S. lentus. A suspension of each Staphylococcus strain (10(6) cells/mL) was submitted to PDT using methylene blue and a low power laser. The isolated effects of methylene blue, laser treatment and ciprofloxacin were also evaluated. After the experimental treatments, 0.1 mL aliquots of the suspensions were seeded onto BHI agar for determination of the number of colony-forming units (CFU/mL). The results were analyzed by analysis of variance and Tukey's test (p < 0.05). The mean reduction in bacterial counts of the strains submitted to PDT ranged from 4.89 to 6.83 CFU (log10)/mL, with the observation of a decreasing susceptibility to treatment of S. schleiferi, S. haemolyticus, S. epidermidis, S. capitis, S. aureus, and S. lentus. The results showed that PDT was effective in reducing the number of viable cells of all clinical Staphylococcus isolates studied.

Presence of Candida spp. in the oral cavity of heart transplantation patients

Candida spp. can lead to infections or even fungal sepsis particularly among immunocompromized individuals. OBJECTIVE: The aim of the present study was to analyze the presence of Candida spp. among patients subjected to orthotopic heart transplantation. MATERIAL AND METHODS: Oral rinses from 50 patients subjected to orthotopic heart transplantation, aged 13 to 70 years, 40 males and 10 females, were examined. Sex-age-oral conditions matched-control included 50 individuals who were not subjected to any kind of transplantation and were not immunocompromized for any other reason. Counts of yeasts were expressed as median values of logarithm of cfu/mL and were statistically compared by Mann-Whitney's test. The heart transplant and control groups were compared for the presence of Candida spp. by chi-square test (p<0.05). RESULTS: The results showed statistically significant difference (p=0.001) in the prevalence of Candida spp. between the transplantation and control groups. Counts of yeasts (cfu/mL) in the transplanted group were significantly higher than in the control group (p=0.005). Candida albicans was the most prevalent species isolated from both groups. CONCLUSIONS: It was concluded that Candida yeast counts were higher in the heart transplant recipients than in the controls. There was higher variation of Candida species among the heart transplant patients and the most frequently isolated samples were: Candida albicans, Candida glabrata and Candida tropicalis. Isolates of Candida dubliniensis was not found in either of the groups.

Susceptibility of planktonic cultures of Streptococcus mutans to photodynamic therapy with a light-emitting diode

The objective of this study was to evaluate the effect of photodynamic therapy with erythrosine and rose bengal using a light-emitting diode (LED) on planktonic cultures of S. mutans. Ten S. mutans strains, including nine clinical strains and one reference strain (ATCC 35688), were used. Suspensions containing 10(6) cells/mL were prepared for each strain and were tested under different experimental conditions: a) LED irradiation in the presence of rose bengal as a photosensitizer (RB+L+); b) LED irradiation in the presence of erythrosine as a photosensitizer (E+L+); c) LED irradiation only (P-L+); d) treatment with rose bengal only (RB+L-); e) treatment with erythrosine only (E+L-); and f) no LED irradiation or photosensitizer treatment, which served as a control group (P-L-). After treatment, the strains were seeded onto BHI agar for determination of the number of colony-forming units (CFU/mL). The results were submitted to analysis of variance and the Tukey test (p < 0.05). The number of CFU/mL was significantly lower in the groups submitted to photodynamic therapy (RB+L+ and E+L+) compared to control (P-L-), with a reduction of 6.86 log10 in the RB+L+ group and of 5.16 log10 in the E+L+ group. Photodynamic therapy with rose bengal and erythrosine exerted an antimicrobial effect on all S. mutans strains studied.