Antibiofilm Activity and Biocompatibility of Temporin-SHa: A Promising Antimicrobial Peptide for Control of Fluconazole-Resistant Candida albicans.

The aim of the study was to investigate the effect of antimicrobial peptides (AMPs) Hylin-a1, KR-12-a5, and Temporin-SHa in Candida albicans as well as the biocompatibility of keratinocytes spontaneously immortalized (NOK-si) and human gingival fibroblasts (FGH) cells. Initially, the susceptible (CaS-ATCC 90028) and fluconazole-resistant (CaR-ATCC 96901) C. albicans strains were grown to evaluate the effect of each AMP in planktonic culture, biofilm, and biocompatibility on oral cells. Among the AMPs evaluated, temporin-SHa showed the most promising results. After 24 h of Temporin-SHa exposure, the survival curve results showed that CaS and CaR suspensions reduced 72% and 70% of cell viability compared to the control group. The minimum inhibitory/fungicide concentrations (MIC and MFC) showed that Temporin-SHa was able to reduce ≥50% at ≥256 µg/mL for both strains. The inhibition of biofilm formation, efficacy against biofilm formation, and total biomass assays were performed until 48 h of biofilm maturation, and Temporin-SHa was able to reduce ≥50% of CaS and CaR growth. Furthermore, Temporin-SHa (512 µg/mL) was classified as non-cytotoxic and slightly cytotoxic for NOK-si and FGH, respectively. Temporin-SHa demonstrated an anti-biofilm effect against CaS and CaR and was biocompatible with NOK-si and FGH oral cells in monolayer.

The Effect of Sub-Lethal Successive Applications of Photodynamic Therapy on Candida albicans Biofilm Depends on the Photosensitizer.

This study aimed to evaluate the potential of successive applications of sub-lethal doses of the antimicrobial photodynamic therapy (aPDT) mediated by Photodithazine® (PDZ) and curcumin (CUR) associated with LED in the viability, reactive oxygen species (ROS) production, and gene expression of Candida albicans. The microbial assays were performed using planktonic cultures and biofilms. Ten successive applications (Apl#) were performed: aPDT (P+L+; C+L+), photosensitizer (P+L-; C+L-), and LED (P-L+; C-L+). Control groups were used (P-L-; C-L-). The viability of C. albicans was determined by cultivating treated cultures on agar plates with or without fluconazole (FLU). In addition, the ROS detection and expression of SOD1, CAP1, and ERG11 genes were determined. For planktonic cultures, no viable colonies were observed after Apl#3 (without FLU) and Apl#2 (with FLU) for either photosensitizer. Biofilm treated with P+L+ resulted in the absence of cell viability after Apl#7, while C+L+ showed ~1.40 log10 increase in cell viability after Apl#2, regardless of FLU. For both photosensitizers, after the last application with viable colonies, the production of ROS was higher in the biofilms than in the planktonic cultures, and SOD1 expression was the highest in P+L+. A reduction of CAP1 and ERG11 expression occurred after P+L+, regardless of FLU. C+L+ had a higher level of ROS, and the treatments were non-significant for gene expression. Sub-lethal doses of aPDT mediated by CUR could induce C. albicans resistance in biofilms, while C. albicans cells in biofilms were susceptible to aPDT mediated by PDZ.

Efficacy of Antimicrobial Photodynamic Therapy Mediated by Photosensitizers Conjugated with Inorganic Nanoparticles: Systematic Review and Meta-Analysis.

Antimicrobial photodynamic therapy (aPDT) is a method that does not seem to promote antimicrobial resistance. Photosensitizers (PS) conjugated with inorganic nanoparticles for the drug-delivery system have the purpose of enhancing the efficacy of aPDT. The present study was to perform a systematic review and meta-analysis of the efficacy of aPDT mediated by PS conjugated with inorganic nanoparticles. The PubMed, Scopus, Web of Science, Science Direct, Cochrane Library, SciELO, and Lilacs databases were searched. OHAT Rob toll was used to assess the risk of bias. A random effect model with an odds ratio (OR) and effect measure was used. Fourteen articles were able to be included in the present review. The most frequent microorganisms evaluated were Staphylococcus aureus and Escherichia coli, and metallic and silica nanoparticles were the most common drug-delivery systems associated with PS. Articles showed biases related to blinding. Significant results were found in aPDT mediated by PS conjugated with inorganic nanoparticles for overall reduction of microorganism cultured in suspension (OR = 0.19 [0.07; 0.67]/p-value = 0.0019), E. coli (OR = 0.08 [0.01; 0.52]/p-value = 0.0081), and for Gram-negative bacteria (OR = 0.12 [0.02; 0.56/p-value = 0.0071). This association approach significantly improved the efficacy in the reduction of microbial cells. However, additional blinding studies evaluating the efficacy of this therapy over microorganisms cultured in biofilm are required.

Efficacy of curcumin-mediated antibacterial photodynamic therapy for oral antisepsis: A systematic review and network meta-analysis of randomized clinical trials.

BACKGROUND: The presence of oral microorganisms resistant to traditional treatment is increasing, thus a search for new therapies is needed. In this context, antimicrobial photodynamic therapy (aPDT) is an approach for the treatment of antibiotic resistant andnon resistant microorganisms. Therefore, the aim of the present study was to conduct a systematic review and meta-analysis of randomized clinical trials of aPDT for oral antisepsis against oral polymicrobial biofilms. METHODS: PubMed, Science Direct, Scopus, SciELO, Lilacs, Cochrane Library and Embase databases were searched. In total, five articles were included for qualitative analysis and four articles were used for quantitative analyses. Bias assessment of the eligible articles was made using the RoB 2 criteria. Network meta-analysis was performed using the random-effect model. Subgroup's analysis was also conducted. The groups evaluated were aPDT, exposure to light only and no treatment at all (control group). The quality of evidence was assessed by CINeMA approach. RESULTS: aPDT mediated by curcumin had significant results in the reducing bacterial load (0.31-0.49 log10 UFC/ I2=0%) when compared with the control group. The included articles were classified as low risk of bias, despite biases detected by allocation and blinding. Moreover, quantitative analysis between aPDT and control group and between light and control group were classified with low risk of confidence rating, while the results from aPDT versus light were classified as moderate risk of confidence rating. CONCLUSION: aPDT has significant efficacy for oral antisepsis, however more randomized clinical trials will be needed to validate the present results.

Use of Photodynamic Therapy Associated with Antimicrobial Peptides for Bacterial Control: A Systematic Review and Meta-Analysis.

Considering the challenges related to antimicrobial resistance, other strategies for controlling infections have been suggested, such as antimicrobial photodynamic therapy (aPDT) and antimicrobial peptides (AMP). This study aims to perform a systematic review and meta-analysis to obtain evidence on the antimicrobial effectiveness of aPDT associated with AMP and establish in vitro knowledge on this topic for further study designs. The PubMed, Scopus, Web of Science, Science Direct, Scielo, and Cochrane Library databases were searched. Two independent and calibrated researchers (Kappa = 0.88) performed all the systematic steps according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). The odds ratio (OR) was used as the effect measure. The Peto method was used to perform the meta-analysis due to the sparse data. Twenty studies were included in the present review. The result was significant (OR = 0.14/p = 0.0235/I-squared = 0%), showing better outcomes of aPDT associated with peptides than those of aPDT alone for controlling the microbial load. Only 20% of the studies included evaluated this approach in a biofilm culture. Combined treatment with aPDT and AMP highly increased the ability of microbial reduction of Gram-positive and Gram-negative bacteria. However, additional blind studies are required to evaluate the efficacy of this therapy on microbial biofilms.

Lactobacillus casei reduces the extracellular matrix components of fluconazole-susceptible Candida albicans biofilms.

Fluconazole-sensitive (CaS) and -resistant (CaR) C. albicans were grown as single-species and dual-species biofilms with Lactobacillus casei (Lc) and Lactobacillus rhamnosus (Lr). Single-species Lc and Lr were also evaluated. Biofilm analysis included viable plate counts, the extracellular matrix components, biomass, and structural organization. Lc reduced the viability of CaS, water-soluble polysaccharides, and eDNA in CaS + Lc biofilm. Lc biofilm presented more eDNA than CaS. The total biomass of CaS + Lc biofilm was higher than the single-species biofilms. The viability of Lc and Lr was reduced by CaR dual-species biofilms. The total and insoluble biomass in CaS + Lr was higher than in single-species CaS biofilms. Lc hindered the growth of CaS, and their association hampered matrix components linked to the structural integrity of the biofilm. These findings allow understanding of how the implementation of probiotics influences the growth of C. albicans biofilms and thereby helps with the development of novel approaches to control these biofilms.

Consecutive treatments with photodynamic therapy and nystatin altered the expression of virulence and ergosterol biosynthesis genes of a fluconazole-resistant Candida albicans in vivo.

This investigation assessed the effect of five consecutive daily topical treatments of antimicrobial photodynamic therapy (aPDT), nystatin (NYS), and an association of treatments on a fluconazole-resistant strain of Candida albicans colonizing the tongues of mice. After the last treatments application, colonies of C. albicans were recovered from the tongues and used to determine their fluconazole susceptibility. After 24 hours of the last treatment, the mice tongues were processed to evaluate the expression of C. albicans genes related to the virulence and ergosterol production. The fluconazole susceptibility test yielded a resistance profile similar for all treatment groups and the control group (no treatment). The treatments aPDT, NYS, NYS+aPDT, and aPDT+NYS promoted a reduction in ALS1, EFG1, CAP1, SOD1, SAP1, and LIP3 expression. The expression of HWP1 was higher in the three groups containing nystatin. In contrast, the treatments produced a significative increase in CAT1 gene expression, mainly in the groups in which aPDT was performed. The expression of genes related to ergosterol production was significantly reduced by the treatments evaluated (aPDT, NYS, NYS+aPDT, and aPDT+NYS). Thus, the consecutive topical treatments performed on mice tongues promoted a reduction in the expression of virulence and ergosterol biosynthesis genes of a fluconazole-resistant C. albicans.

Successive applications of Antimicrobial Photodynamic Therapy effects the susceptibility of Candida albicans grown in medium with or without fluconazole.

Antimicrobial Photodynamic Therapy (aPDT) was introduced as a therapy due to resistance that microorganisms have developed to conventional drugs. The study aimed to evaluate the potential of successive applications of aPDT in effecting Candida albicans susceptibility and also whether the presence of fluconazole effected the recovery of the fungi in the culture medium. Planktonic cultures and biofilm were subjected to successive applications of Photodithazine-mediated (25 mg/L) LED-associated aPDT (660 nm, 34 mW/cm2). Plating was performed on Sabouraud Dextrose Agar supplemented or not with fluconazole to recover colony-forming units per milliliter (CFU/mL). Surviving cells were recovered, recultivated, and again exposed to the treatment. The treatments were performed until not enough colonies were available for recultivation and continuation of the protocol. The complete inactivation of the fungus was obtained after three and five applications for planktonic culture and biofilm, respectively. A reduction of 6.3 log10 was observed after third applications in the planktonic cultures grown on medium without fluconazole, while there was a 7 log10 reduction of these cultures grown on fluconazole medium. However, a reduction of 6.1 log10 occurred for biofilms after fifth applications for cultures grown on medium without fluconazole, while a reduction of 6.7 log10 was observed for cultures grown on medium with the antifungal. Thus, aPDT was potentiated by fluconazole. C. albicans in planktonic and biofilm cultures are susceptible to successive applications of PDZ-mediated aPDT, and tolerance to aPDT is higher in the biofilm.

Antimicrobial photodynamic therapy effectiveness against susceptible and methicillin-resistant Staphylococcus aureus biofilms.

BACKGROUND: Staphylococcus aureus have a great ability to become rapidly resistant to conventional antimicrobial therapies. This study evaluated the efficacy of antimicrobial photodynamic therapy (aPDT) mediated by Curcumin (Cur) and light-emitting diode (LED) in the inactivation of biofilms of methicillin susceptible and resistant S. aureus (MSSA and MRSA, respectively). METHODS: Biofilms were treated with Cur (20, 40 or 80 µM) and illuminated with LED source (455 ± 3 nm; 5.28 J/cm2) (aPDT groups), or treated either with Cur or LED only. Other samples were not exposed to Cur or LED (negative control). The biofilms viability after all experimental conditions were evaluated by counting the number of colonies (CFU/mL) and XTT assay. Additional samples were also evaluated by LIVE/DEAD® staining using confocal laser scanning microscopy (CLSM). Data were analyzed by ANOVAs followed by the Games-Howell post hoc test (α = 0.05). RESULTS: For both strains, all aPDT groups significantly reduced both CFU/mL and metabolic activity of biofilms compared to the negative control (p < 0.001). The results were enhanced when 80 µM of Cur was used. CLSM images showed that both bacteria biofilms submitted to aPDT had a large number of red-stained colonies, especially at aPDT80. In general, MRSA biofilms tended to be less susceptible to aPDT than MSSA biofilms. CONCLUSIONS: It can be concluded that aPDT mediated by Cur and LED was an efficient method to inactivate 48 -h biofilms of both S. aureus strains.

Can surface protection prevent the loss of hardness on dentin and composite resin surfaces exposed to erosive challenges? / ¿Puede la protección de la superficie prevenir la pérdida de dureza en las superficies de dentina y resina compuesta expuestas a erosión?

Objective: This study investigated the effect of endogenous erosion on the microhardness of dentine and a nanofilled composite resin. Procedures for preventing erosion were also studied. Materials and Methods: 90 bovine dentine specimens were divided into three groups in accordance with the method for preventing: negative control, topical application of fluoride and resin-modified glass ionomer varnish. 120 composite resin specimens were distributed into four groups, which also included a resin sealant, among the preventive procedures. Specimens were then randomly divided into three sub-groups according to the exposure to simulate gastric acid solution and subsequent remineralization: negative control, 9 and 18 cycles. Surface analysis was carried out by measuring the Knoop hardness. The data obtained were statistically analyzed using 2-way ANOVA and Tukey test. Result: The mean hardness of dentine and of the composite specimens resin exhibited lower hardness after 18 cycles. However, the resin-modified glass ionomer varnish resulted in greater values compared to the other preventive procedures. Conclusion: A resin-modified glass ionomer varnish seems to be a promising method for minimizing the damage caused by endogenous acid, but its protection can be reduced depending on the intensity of the erosive challenge.

Objetivo: Este estudio investigó el efecto de la erosión endógena sobre la microdureza de la dentina y una resina compuesta de nanorrelleno. También se estudiaron los procedimientos para prevenir la erosión. Materiales and Métodos:90 muestras de dentina bovina se dividieron en tres grupos de acuerdo con el método para prevenir: control negativo, aplicación tópica de fluoruro y barniz de ionómero de vidrio modificado con resina. Se distribuyeron 120 muestras de resina compuesta en cuatro grupos, que también incluían un sellador de resina, entre los procedimientos preventivos. Las muestras se dividieron al azar en tres subgrupos de acuerdo con la exposición para simular la solución de ácido gástrico y la remineralización posterior: control negativo, 9 y 18 ciclos. El análisis de la superficie se realizó midiendo la dureza Knoop. Los datos obtenidos se analizaron estadísticamente mediante ANOVA de 2 vías y prueba de Tukey. Resultados: La dureza media de la dentina y de la resina de muestras compuestas exhibió una dureza más baja después de 18 ciclos. Sin embargo, el barniz de ionómero de vidrio modificado con resina resultó en valores mayores en comparación con los otros procedimientos preventivos. Conclusión: Un barniz de ionómero de vidrio modificado con resina parece ser un método prometedor para minimizar el daño causado por el ácido endógeno, pero su protección puede reducirse dependiendo de la intensidad del desafío erosivo.

Effect of Dental Erosion and Methods for its Control on the Marginal and Internal Adaptation of Restorations with Different Adhesive Systems / Efeito da Erosão Dental e de Métodos para seu Controle na Adaptação Marginal e Interna de Restaurações Confeccionadas com Diferentes Sistemas Adesivos

The dentin exposed to erosive challenges is often superficially protected to prevent progression of the lesion. This study investigated the marginal and internal adaptation of composite resin restorations made on surfaces that had first been treated with different methods for controlling erosion. Cavities with margins in dentin were prepared in bovine incisors (n=360) and were divided into three groups according to the method for controlling the erosive challenge: negative control, topical application of fluoride and resin-modified glass ionomer varnish. The specimens were then randomly divided into three sub-groups according to the exposure to simulated gastric acid solution (DES) (5% HCl, pH=2.2) and subsequent remineralization (RE): negative control, 9 and 18 cycles of DES-RE. Finally, teeth were divided into four groups, depending on the bonding agent used for composite resin restoration (n=10): conventional etch-and-rinse adhesive system (2 and 3 steps) and self-etching (1 and 2 steps). Front and internal images of the interface tooth/restoration were recorded in stereoscopic microscope (15x) to quantify the percentage of adhesive failures. Despite the promising results of the resin-modified glass ionomer varnish after 9 cycles; no protective material prevented increased internal defects after 18 erosive cycles. More continuous internal margins were noted with etch-andrinse acid systems after more intense erosion. The maintenance of internal margins in eroded substrates was positively influenced by the resinmodified glass ionomer varnish and, under the most aggressive challenge, by the use of etch-and-rinse adhesives systems. (AU)

A dentina exposta a desafios erosivos é muitas vezes superficialmente protegida para prevenir a progressão da lesão. Este estudo investigou a adaptação marginal e interna de restaurações de resina composta realizadas em superfícies que foram previamente tratadas com diferentes métodos para controlar a erosão. Cavidades com margens em dentina foram preparadas em dentes incisivos bovinos (n=360) e divididas em três grupos de acordo com o método de controle do desafio erosivo: controle negativo, aplicação tópica de flúor e verniz de ionômero de vidro modificado por resina. Os espécimes foram então divididos aleatoriamente em três subgrupos de acordo com a exposição à solução de ácido gástrico simulada (DES) (HCl a 5%, pH = 2,2) e posterior remineralização (RE): controle negativo, 9 e 18 ciclos de DES-RE. Por fim, os dentes foram divididos em quatro grupos, de acordo com o agente de união utilizado para a restauração da resina composta (n=10): sistema adesivo convencional (2 e 3 passos) e autocondicionante (1 e 2 passos). Imagens frontais e internas da interface dente/restauração foram registradas em microscópio estereoscópico (15x) para quantificar a porcentagem de falhas adesivas. Apesar dos resultados promissores do verniz de ionômero de vidro modificado por resina após 9 ciclos; nenhum material de proteção impediu o aumento de defeitos internos após 18 ciclos erosivos. Margens internas mais contínuas foram observadas com sistemas adesivos convencionais após erosão mais intensa. A manutenção das margens internas em substratos erodidos foi influenciada positivamente pelo verniz de ionômero de vidro modificado por resina e, sob o desafio mais agressivo, pelo uso de sistemas de adesivos convencionais. (AU)

Can surface protection prevent damage in margins of composite resin restorations after simulated endogenous erosion?

Aim: The study investigated the effect of using surface protection agents in the adaptation of external and internal margins of restorations subjected to simulated erosion. Methods: Cavities with margins in dentin were prepared in bovine incisors (n=120). Adhesive restorations were placed using a three-step etch&rinse adhesive system and nanofilled composite resin. The specimens were divided into four groups, according to the surface protection: negative control, topical application of fluoride (TAF), resin sealant and resin-modified glass ionomer varnish (RMGI varnish). Afterwards, they were divided into three sub-groups, according to the exposure to a simulated solution of gastric acid (DES) (5% HCl, pH=2,2) and subsequent remineralization (RE): negative control, 9 and 18 cycles of DES-RE. The evaluation of the tooth-restoration interface was performed on the internal and frontal images with the aid of a stereoscopic microscope (15x), and the percentage of continuous margins without adhesive failures was quantified. Results: In the external margins, only those groups with surface protection using sealants (resin and glass-ionomer) did not exhibit a significant decrease in the percentage of continuous margins after the erosive challenges. After 18 cycles of DES-RE, the use of resin-modified glass ionomer varnish resulted in the highest percentage of continuous margins. Conclusion: It was concluded that physically covering the surfaces with a sealing agent preserved the marginal adaptation of composite resin restorations exposed to endogenous erosive challenges