Promethazine inhibits efflux, enhances antifungal susceptibility and disrupts biofilm structure and functioning in Trichosporon.

Trichosporon spp. are emerging opportunistic fungi associated with invasive infections, especially in patients with haematological malignancies. The present study investigated the in vitro inhibition of efflux pumps by promethazine (PMZ) as a strategy to control T. asahii and T. inkin. Planktonic cells were evaluated for antifungal susceptibility to PMZ, as well as inhibition of efflux. The effect of PMZ was also studied in Trichosporon biofilms. PMZ inhibited T. asahii and T. inkin planktonic cells at concentrations ranging from 32 to 256 µg ml-1. Subinhibitory concentrations of PMZ inhibited efflux activity in Trichosporon. Biofilms were completely eradicated by PMZ. PMZ potentiated the action of antifungals, affected the morphology, changed the amount of carbohydrates and proteins and reduced the amount of persister cells inside biofilms. The results showed indirect evidences of the occurrence of efflux pumps in Trichosporon and opens a perspective for the use of this target in the control of trichosporonosis.

The Potential of Phenothiazines against Endodontic Pathogens: A Focus on Enterococcus-Candida Dual-Species Biofilm.

Persistent apical periodontitis occurs when the endodontic treatment fails to eradicate the intraradicular infection, and is mainly caused by Gram-positive bacteria and yeasts, such as Enterococcus faecalis and Candida albicans, respectively. Phenothiazines have been described as potential antimicrobials against bacteria and fungi. This study aimed to investigate the antimicrobial potential of promethazine (PMZ) and chlorpromazine (CPZ) against E. faecalis and C. albicans dual-species biofilms. The susceptibility of planktonic cells to phenothiazines, chlorhexidine (CHX) and sodium hypochlorite (NaOCl) was initially analyzed by broth microdilution. Interaction between phenothiazines and CHX was examined by chequerboard assay. The effect of NaOCl, PMZ, CPZ, CHX, PMZ + CHX, and CPZ + CHX on biofilms was investigated by susceptibility assays, biochemical and morphological analyses. Results were evaluated through one-way ANOVA and Tukey's multiple comparison post-test. PMZ, alone or in combination with irrigants, was the most efficient phenothiazine, capable of reducing cell counts, biomass, biovolume, carbohydrate and protein contents of dual-species biofilms. Neither PMZ nor CPZ increased the antimicrobial activity of CHX. Further investigations of the properties of phenothiazines should be performed to encourage their use in endodontic clinical practice.

Enterococcus faecalis and Candida albicans dual-species biofilm: establishment of an in vitro protocol and characterization.

Enterococcus faecalis is the most important agent of persistent apical periodontitis, and recently, Candida albicans has also been implicated in periapical infections. This study aimed to optimize an in vitro E. faecalis and C. albicans dual-species biofilm protocol for endodontic research. Different physicochemical conditions for biofilm formation were tested. Susceptibility assays to antimicrobials, biochemical composition and an ultra-morphological structure analyses were performed. Reproducible dual-species biofilms were established in BHI medium at 35 °C, for 48 h and in a microaerophilic atmosphere. An increase in biomass and chitin content was detected after vancomycin treatment. Structural analysis revealed that the dual-species biofilm was formed by both microorganisms adhered to the substrate. The proposed protocol could be useful for the study of interkingdom relationships and help to find new strategies against periapical infections.

Antimicrobial effectiveness of grape seed extract against Enterococcus faecalis biofilm: A Confocal Laser Scanning Microscopy analysis.

This study evaluated the antimicrobial effectiveness of 6.5% Vitis vinifera grape seed extract (GSE) against Enterococcus faecalis biofilm using confocal laser scanning microscopy (CLSM). Saline solution (SS), 5.25% sodium hypochlorite (NaOCl) and 2% chlorhexidine (CHX) were used for comparison. Dentin discs were inoculated with E. faecalis strain establishing a 3-week-old biofilm. Discs (n = 10) were exposed to 5.25% NaOCl, 2% CHX, 6.5% GSE and SS (negative control) for 10 min. Discs were stained with the fluorescent LIVE/DEAD-BacLight™ dye and analysed using CLSM. The proportion of dead cells in biofilm was analysed using one-way anova and Tukey tests (P < 0.05). A higher proportion of dead cells was found in GSE group compared with CHX and SS (P < 0.05). NaOCl group was associated with the highest proportion of dead cells (P < 0.05). GSE presented antimicrobial activity against E. faecalis; however, NaOCl was the most effective irrigant solution. GSE was more effective than CHX and SS.

Atividade antimicrobiana do extrato de semente de uva Vitis vinifera sobre biofilme de Enterococcus faecalis: análise com microscopia confocal / Antimicrobial effectiveness of grape seed extract against Enterococcus faecalis biofilm: confocal microscopy analysis

As limitações dos irrigantes químicos utilizados em endodontia têm incentivado novas pesquisas acerca de substâncias alternativas para serem utilizadas no tratamento endodôntico. Estudos anteriores sugerem que o extrato de semente de uva (Vitis vinifera), tem atividade antimicrobiana assim como é eficaz no combate a cepas de Enterococcus faecalis. No entanto, os efeitos deste novo irrigante em biofilmes microbianos ainda não foram elucidados. Dessa forma, o objetivo deste estudo foi avaliar e quantificar o grau de desinfecção da dentina contaminada por biofilme de E. faecalis após irrigação com extrato de semente de uva Vitis vinífera 6,5% por meio de análise microscopia confocal a laser. A clorexidina (CHX) 2% e o hipoclorito de sódio (NaOCl) 5,25% foram utilizados como irrigantes para comparação. Foram confeccionadas quarenta amostras de discos de dentina que foram contaminadas com E. faecalis e incubadas por um período de 21 dias para formação do biofilme. As amostras foram divididas em diferentes grupos de acordo com o irrigante testado após transcorrido o período de contaminação: grupo NaOCl, grupo CHX, grupo Vitis vinifera e grupo solução salina (controle). Após a irrigação, os discos de dentina foram corados com o corante fluorescente LIVE/DEAD BacLight e analisados por microscopia confocal à laser para determinar a proporção de células mortas no biofilme. A aquisição das imagens e quantificação do biofilme foi realizada utilizando-se o software LAS X. Uma distribuição normal dos dados foi confirmada pelo teste Shapiro- Wilk (p>0,05). Por esse motivo, a análise estatística foi realizada utilizando-se One-way análise de variância. Comparações Post hoc pair-wise foram realizadas utilizando-se o teste Tukey para múltiplas comparações (P<0.05). O grupo no qual o NaOCl foi utilizado como irrigante apresentou maior quantidade de células bacterianas mortas do que os dos outros irrigantes (P<0,05). Já o grupo da Vitis vinifera demonstrou maior morte bacteriana quando comparada a CHX e ao grupo controle (P<0,05). Não foram observadas diferenças entre a CHX e o grupo controle (P>0,05). A irrigação com o extrato de semente de uva, vitis vinífera, apresentou atividade antimicrobiana sobre o biofilme de E. faecalis sendo esta inferior a apresenta pelo NaOCl e superior a apresentada pela CHX.

The limitations of chemical irrigants used in endodontics have encouraged further research on alternative substances to be used in endodontic treatment. Previous studies have suggested that grape seed extract (Vitis vinifera), an irrigant of natural origin, is effective against strains of Enterococcus faecalis. The aim of this study was to evaluate and quantify the degree of disinfection of dentin contaminated by E. faecalis biofilm after irrigation with Vitis vinifera grape seed extract 6,5% and to compare it with chlorhexidine (CHX) 2% and Sodium hypochlorite (NaOCl) 5,25% by confocal microscopy laser. Forty samples of dentin disks that will be contaminated with E. faecalis and incubated for a period of 21 days for biofilm formation were prepared. The samples were divided into different groups according to the irrigant tested after the period of contamination: NaOCl group, CHX group, Vitis vinifera group and control group . After irrigation, the dentin disks were stained with the fluorescent dye LIVE / DEAD BacLight and analyzed by laser confocal microscopy to determine the proportion of dead cells in the biofilm. The acquisition of the images and quantification of the biofilm was performed using the software LAS X. Normal distribution of the data was confirmed by Shapiro- Wilk test (p>0.05). Therefore, statistical analysis was performed using One-way analysis of variance. Post hoc pair-wise comparisons were performed using Tukey's test for multiple comparisons (P<0.05). NaOCl presented a higher amount of bacterial cells killed than the other irrigators (P <0.05). Irrigation with grape seed extract, Vitis vinifera, showed antimicrobial activity on the E. faecalis biofilm, being lower than that presented by NaOCl and higher than that presented by CHX.