Heat-killed Limosilactobacillus reuteri ATCC PTA 6475 prevents bone loss in ovariectomized mice: A preliminary study.

Osteoporosis is an important health problem that occurs due to an imbalance between bone formation and resorption. Hormonal deficiency post-menopause is a significant risk factor. The probiotic Limosilactobacillus reuteri has been reported to prevent ovariectomy (Ovx)-induced bone loss in mice and reduce bone loss in postmenopausal women. Despite the numerous health benefits of probiotics, as they are live bacteria, the administration is not risk-free for certain groups (e.g., neonates and immunosuppressed patients). We evaluated the effects of L. reuteri (ATCC PTA 6475) and its heat-killed (postbiotic) form on Ovx-induced bone loss. Adult female mice (BALB/c) were randomly divided into four groups: group C-control (sham); group OVX-C-Ovx; group OVX-POS-Ovx + heat-killed probiotic; group OVX-PRO-Ovx + probiotic. L. reuteri or the postbiotic was administered to the groups (1.3x109 CFU/day) by gavage. Bacterial morphology after heat treatment was accessed by scanning electron microscopy (SEM). The treatment started one week after Ovx and lasted 28 days (4 weeks). The animals were euthanized at the end of the treatment period. Bone microarchitecture and ileum Occludin and pro-inflammatory cytokines gene expression were evaluated by computed microtomography and qPCR techniques, respectively. The Ovx groups had lower percentage of bone volume (BV/TV) and number of bone trabeculae as well as greater total porosity compared to the control group. Treatment with live and heat-killed L. reuteri resulted in higher BV/TV and trabecular thickness than the Ovx group. The heat treatment caused some cell surface disruptions, but its structure resembled that of the live probiotic in SEM analysis. There were no statistical differences in Occludin, Il-6 and Tnf-α gene expression. Both viable and heat-killed L. reuteri prevented bone loss on ovariectomized mice, independently of gut Occludin and intestinal Il-6 and Tnf-α gene expression.

Gellan-Based Hydrogel as a Drug Delivery System for Caffeic Acid Phenethyl Ester in the Treatment of Oral Candida albicans Infections.

Candida albicans can cause various types of oral infections, mainly associated with denture stomatitis. Conventional therapy has been linked to high recurrence, toxicity, and fungal resistance, necessitating the search for new drugs and delivery systems. In this study, caffeic acid phenethyl ester (CAPE) and gellan gum (GG) were studied as an antifungal agent and carrier system, respectively. First, we observed that different GG formulations (0.6 to 1.0% wt/vol) were able to incorporate and release CAPE, reaching a controlled and prolonged release over 180 min at 1.0% of GG. CAPE-GG formulations exhibited antifungal activity at CAPE concentrations ranging from 128 to >512 µg/mL. Furthermore, CAPE-GG formulations significantly decreased the fungal viability of C. albicans biofilms at short times (12 h), mainly at 1.0% of GG (p < 0.001). C. albicans protease activity was also reduced after 12 h of treatment with CAPE-GG formulations (p < 0.001). Importantly, CAPE was not cytotoxic to human keratinocytes, and CAPE-GG formulations at 1.0% decreased the fungal burden (p = 0.0087) and suppressed inflammation in a rat model of denture stomatitis. Altogether, these results indicate that GG is a promising delivery system for CAPE, showing effective activity against C. albicans and potential to be used in the treatment of denture stomatitis.

Effects of antimicrobial photodynamic therapy with photodithazine® on methicillin-resistant Staphylococcus aureus (MRSA): Studies in biofilms and experimental model with Galleria mellonella.

Staphylococcus aureus infections are a severe health problem due to the high mortality rate. Conventional treatment of these infections is via the administration of antibiotics. However, its indiscriminate use can select resistant microorganisms. Thus, it is necessary to develop alternatives for antibiotic therapy. Antimicrobial Photodynamic Therapy (aPDT), a therapeutic method that associates a photosensitizer (PS), a light source with adequate wavelength to the PS, interacts with molecular oxygen generating reactive oxygen species responsible for cell inactivation, is a viable alternative. This work aimed to analyze, in vitro and in vivo, the action of aPDT with PS Photodithazine® (PDZ) on the methicillin-resistant S. aureus (MRSA) strain. In the in vitro method, the S. aureus biofilm was incubated with PDZ at 50 and 75 µg.mL-1 for 15 min, adopting the light dose of 25, 50, and 100 J/cm2. In addition, PS interaction, formation of reactive oxygen species (ROS), bacterial metabolism, adhesion, bacterial viability, and biofilm structure were evaluated by scanning electron microscopy. Subsequently, the strain was inoculated into models of Galleria mellonella, and the survival curve, health scale, blood cell analysis, and CFU/mL of S. aureus in the hemolymph were analyzed after aPDT. In the in vitro results, bacterial reduction was observed in the different PDZ concentrations, highlighting the parameters of 75 µg.mL-1 of PDZ and 100 J/cm2. As for in vivo results, aPDT increased survival and stimulated the immune system of G. mellonella infected by S. aureus. aPDT proved effective in both models, demonstrating its potential as an alternative therapy in treating MRSA bacterial infections.

Effects of natural antimicrobial compounds propolis and copaiba on periodontal ligament fibroblasts, molecular docking, and in vivo study in Galleria mellonella.

Root canal treatment addresses infectious processes that require control. Occasionally, the radicular pulp is vital and inflamed, presenting a superficial infection. To preserve pulpal remnants, conservative procedures have gained favor, employing anti-inflammatory medications. This study investigated the effects of propolis (PRO), and copaiba oil-resin (COR) associated with hydrocortisone (H) and compared their impact to that of Otosporin® concerning cytotoxic and genotoxic activity, cytokine detection, and toxicity in the Galleria mellonella model. Human periodontal ligament fibroblasts (PDLFs) were exposed to drug concentrations and evaluated by the MTT assay. Associations were tested from concentrations that did not compromise cell density. Genotoxicity was evaluated through micronucleus counting, while cytokines IL-6 and TGF-ß1 were detected in the cell supernatant using ELISA. Molecular docking simulations were conducted, considering the major compounds identified in PRO, COR, and H. Increasing concentrations of PRO and COR were assessed for acute toxicity in Galleria mellonella model. Cellular assays were analyzed using one-way ANOVA followed by Tukey tests, while larval survivals were evaluated using the Log-rank (Mantel-Cox) test (α = 0.05). PRO and COR promoted PDLFs proliferation, even in conjunction with H. No changes in cell metabolism were observed concerning cytokine levels. The tested materials induce the release of AT1R, proliferating the PDFLs through interactions. PRO and COR had low toxicity in larvae, suggesting safety at tested levels. These findings endorse the potential of PRO and COR in endodontics and present promising applications across medical domains, such as preventive strategies in inflammation, shedding light on their potential development into commercially available drugs.

Streptococcus mutans supernatant affects the virulence of Candida albicans.

Candida albicans causes a variety of clinical manifestations through multiple virulence factors that act simultaneously to overcome the immune system and invade the host tissues. Owing to the limited number of antifungal agents available, new candidiasis therapeutic strategies are required. Previous studies have demonstrated that the metabolites produced by Streptococcus mutans lead to a decrease in the number of Candida cells. Here, for the first time, we evaluated whether the C. albicans cells that survived the pretreatment with S. mutans supernatant can modify their virulence factors and their capability to infect Galleria mellonella larvae. Streptococcus mutans supernatant (SM-S) was obtained by filtering the culture supernatant of this bacterium. Then, C. albicans cells were pretreated with SM-S for 24 h, and the surviving cells were evaluated using in vitro and in vivo assays. The C. albicans pretreated with SM-S showed a significant inhibition of hyphal growth, an altered adhesion pattern, and an impaired capability to form biofilms; however, its proteolytic activity was not affected. In the in vivo assays, C. albicans cells previously exposed to SM-S exhibited a reduced ability to infect G. mellonella and a higher amount of circulating hemocytes. Thus, SM-S could inhibit important virulence factors of C. albicans, which may contribute to the development of new candidiasis therapeutic strategies.

PepO and CppA modulate Streptococcus sanguinis susceptibility to complement immunity and virulence.

Streptococcus sanguinis is a ubiquitous commensal species of the oral cavity commonly involved as an opportunistic pathogen in cardiovascular infections. In this study, we investigated the functions of endopeptidase O (PepO) and a C3-degrading protease (CppA) in the systemic virulence of S. sanguinis. Isogenic mutants of pepO and cppA obtained in strain SK36 showed increased susceptibility to C3b deposition and to opsonophagocytosis by human polymorphonuclear neutrophils (PMN). These mutants differ, however, in their profiles of binding to serum amyloid P component (SAP) and C1q, whereas both showed reduced interaction with C4b-binding protein (C4BP) and/or factor H (FH) regulators as compared to SK36. The two mutants showed defects in ex vivo persistence in human blood, serum-mediated invasion of HCAEC endothelial cells, and virulence in a Galleria mellonella infection model. The transcriptional activities of pepO and cppA, assessed by RT-qPCR in nine wild-type strains, further indicated strain-specific profiles of pepO/cppA expression. Moreover, non-conserved amino acid substitutions were detected among the strains, mostly in CppA. Phylogenetic comparisons with homologues of streptococcal species of the oral and oropharyngeal sites suggested that S. sanguinis PepO and CppA have independent ancestralities. Thus, this study showed that PepO and CppA are complement evasion proteins expressed by S. sanguinis in a strain-specific manner, which are required for multiple functions associated with cardiovascular virulence.

Efficacy of bioactive materials in preventing Streptococcus mutans-induced caries on enamel and dentine.

The study investigated the ability of bioactive materials used to restore enamel and dentine specimens to prevent caries. Enamel (n = 50) and dentine (n = 50) specimens were obtained from bovine incisors, prepared, and randomly allocated to one of five groups according to the restorative treatment: alkasite without adhesive system; alkasite with adhesive system; high viscosity glass ionomer cement; resin composite; no restoration; negative control group. Specimens were restored, exposed to a thermal cycling aging protocol, sterilized, and exposed to a cariogenic challenge induced by Streptococcus mutans and then submitted to surface and subsurface microhardness tests and polarized light microscopy to verify the caries lesion development in enamel or dentine surrounding the restorative materials. Data were analyzed using one-way ANOVA. In enamel and dentine, glass ionomer cement, alkasite without and with adhesive system presented a lower percentage surface microhardness loss than resin composite and negative control. Enamel subsurface microhardness presented no statistically significant differences between glass ionomer cement, alkasite without and with adhesive system. Glass ionomer cement also did not present statistically significant differences from resin composite and the negative control. In dentine, glass ionomer cement showed the highest subsurface microhardness values. In conclusion, bioactive restorative materials provide greater protection to enamel and dentine against surface caries development than resin composite.

Microbiological evaluation in invisible aligner chemical cleaning methods against Candida albicans and Streptococcus mutans.

INTRODUCTION: This study aimed to assess the efficacy of chemical agents in removing Candida albicans and Streptococcus mutans biofilm from invisible aligners. METHODS: The samples were made of EX30 Invisalign trays, biofilm was cultured by standardized suspensions of C. albicans ATCC strain and S. mutans clinical strain on the sample. The treatments used were 0.5% sodium hypochlorite (NaClO) (20 minutes), 1% NaClO (10 minutes), chlorhexidine (5 minutes), peroxide (15 minutes), and orthophosphoric acid (15 seconds). The control group received phosphate-buffered saline for 10 minutes. The colony-forming units per milliliter of each microorganism were determined by serial dilutions seeded in plates with selective culture mediums for each one. Data were analyzed by the Kruskal-Wallis and Conover-Iman tests at an α of 0.05. RESULTS: For the C. albicans biofilm group, the control group had 9.7 Log10 of microorganism growth, and all treatment groups had statistically significant biofilm reduction, in which chlorhexidine presented the highest inhibition of 3 Log10, followed by alkaline peroxide and orthophosphoric acid both with 2.6 Log10, 1% NaClO (2.5 Log10), and 0.5% NaClO (2 Log10). As for S. mutans, the control group had 8.9 Log10 of growth, and a total microorganism inhibition was reached by chlorhexidine, 1% NaClO, and orthophosphoric acid, whereas alkaline peroxide inhibited growth to 7.9 Log10 and 0.5% NaClO 5.1 Log10. CONCLUSIONS: Within the limitations, chlorhexidine and orthophosphoric acid had greater efficacy in both biofilms. In addition, 1% NaClO and alkaline peroxide also had significant effects; therefore, their incorporation aligners disinfection protocols are valid.

Metal Nanoparticles to Combat Candida albicans Infections: An Update.

Candidiasis is an opportunistic mycosis with high annual incidence worldwide. In these infections, Candida albicans is the chief pathogen owing to its multiple virulence factors. C. albicans infections are usually treated with azoles, polyenes and echinocandins. However, these antifungals may have limitations regarding toxicity, relapse of infections, high cost, and emergence of antifungal resistance. Thus, the development of nanocarrier systems, such as metal nanoparticles, has been widely investigated. Metal nanoparticles are particulate dispersions or solid particles 10-100 nm in size, with unique physical and chemical properties that make them useful in biomedical applications. In this review, we focus on the activity of silver, gold, and iron nanoparticles against C. albicans. We discuss the use of metal nanoparticles as delivery vehicles for antifungal drugs or natural compounds to increase their biocompatibility and effectiveness. Promisingly, most of these nanoparticles exhibit potential antifungal activity through multi-target mechanisms in C. albicans cells and biofilms, which can minimize the emergence of antifungal resistance. The cytotoxicity of metal nanoparticles is a concern, and adjustments in synthesis approaches or coating techniques have been addressed to overcome these limitations, with great emphasis on green synthesis.

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)

Effects of α and ß-adrenergic signaling on innate immunity and Porphyromonas gingivalis virulence in an invertebrate model.

To investigate the role of adrenergic signalling (AS) in the host immune response and Porphyromonas gingivalis virulence, we compared norepinephrine (NE) and isoproterenol (ISO) responses in Galleria mellonella. P. gingivalis infection was evaluated by survival; humoral immune responses (i.e. melanization and cecropin and gloverin mRNA expression); cellular immune responses (i.e. haemocyte count, nodulation by histology); and P. gingivalis recovery (CFU/mL). P. gingivalis was cultivated in the presence of ISO (PgISO) or NE and injected into the larvae for survival evaluation. Finally, we co-injected ISO and PgISO to evaluate the concomitant effects on the immune response and bacterial virulence. None of the ligands were toxic to the larvae; ISO increased haemocyte number, even after P. gingivalis infection, by mobilizing sessile haemocytes in a ß-adrenergic-specific manner, while NE showed the opposite effect. ISO treatment reduced larval mortality and the number of recovered bacteria, while NE increased mortality and showed no effect on bacterial recovery. ISO and NE had similar effects on melanization and decreased the expression of cecropin. Although co-cultivation with NE and ISO increased the gene expression of bacterial virulence factors in vitro, only the injection of PgISO increased larval death, which was partially reversed by circulating ISO. Therefore, α- and ß-adrenergic signalling had opposite effects after P. gingivalis infection. Ultimately, the catecholamine influence on the immune response overcame the effect of more virulent strains. The effect of AS directly on the pathogen found in vitro did not translate to the in vivo setting.

Farnesol as a potentiator of antimicrobial photodynamic inactivation on Enterococcus faecalis.

Enterococcus faecalis is related to the recurrence of endodontic infections and approaches to intracanal disinfection are necessary. Farnesol, an alcohol commonly found in propolis, has antimicrobial properties, and can enhance the efficacy of some antibiotic therapies. The objective was to evaluate whether farnesol can increase the efficacy of the antimicrobial photodynamic inactivation (aPDI) on E. faecalis, investigating its action on planktonic growth, biofilms, and cell permeability. Planktonic cells and biofilms of E. faecalis were pre-treated with farnesol (0.25 mM) 2 h before aPDI. Methylene blue (1 mg/mL) and laser (660 nm) were employed in the aPDI. As a result, farnesol was able to increase the antimicrobial activity of aPDI in both planktonic and biofilm stages, reaching cell reductions of 4.6 to 6 log10 CFU and 1.3 to 3 log10 CFU, respectively, when compared to aPDI isolated. The efficacy of farnesol in enhancing the anti-biofilm activity of aPDI was also confirmed by electron microscopy, in which a smaller number of bacterial cells and extracellular matrix were verified in the combined therapy compared to aPDI alone. The potentiating action of farnesol was associated with its effects in increasing the cell permeability and methylene blue uptake by the bacterial cells. Therefore, farnesol can be a promising potentiator of aPDI against E. faecalis.

Cryptococcus spp. and Cryptococcosis: focusing on the infection in Brazil.

Cryptococcosis is a global fungal infection caused by the Cryptococcus neoformans/Cryptococcus gattii yeast complex. This infection is acquired by inhalation of propagules such as basidiospores or dry yeast, initially causing lung infections with the possibility of progressing to the meninges. This infection mainly affects immunocompromised HIV and transplant patients; however, immunocompetent patients can also be affected. This review proposes to evaluate cryptococcosis focusing on studies of this mycosis in Brazilian territory; moreover, recent advances in the understanding of its virulence mechanism, animal models in research are also assessed. For this, literature review as realized in PubMed, Scielo, and Brazilian legislation. In Brazil, cryptococcosis has been identified as one of the most lethal fungal infections among HIV patients and C. neoformans VNI and C. gattii VGII are the most prevalent genotypes. Moreover, different clinical settings published in Brazil were described. As in other countries, cryptococcosis is difficult to treat due to a limited therapeutic arsenal, which is highly toxic and costly. The presence of a polysaccharide capsule, thermo-tolerance, production of melanin, biofilm formation, mechanisms for iron use, and morphological alterations is an important virulence mechanism of these yeasts. The introduction of cryptococcosis as a compulsory notification disease could improve data regarding incidence and help in the management of these infections.

Systemic Infection by Non-albicans Candida Species Affects the Development of a Murine Model of Multiple Sclerosis.

Candidiasis may affect the central nervous system (CNS), and although Candida albicans is predominant, non-albicans Candida species can also be associated with CNS infections. Some studies have suggested that Candida infections could increase the odds of multiple sclerosis (MS) development. In this context, we investigated whether systemic infection by non-albicans Candida species would affect, clinically or immunologically, the severity of experimental autoimmune encephalomyelitis (EAE), which is an animal model used to study MS. For this, a strain of C. glabrata, C. krusei, and C. parapsilosis was selected and characterized using different in vitro and in vivo models. In these analysis, all the strains exhibited the ability to form biofilms, produce proteolytic enzymes, and cause systemic infections in Galleria mellonella, with C. glabrata being the most virulent species. Next, C57BL/6 mice were infected with strains of C. glabrata, C. krusei, or C. parapsilosis, and 3 days later were immunized with myelin oligodendrocyte glycoprotein to develop EAE. Mice from EAE groups previously infected with C. glabrata and C. krusei developed more severe and more prevalent paralysis, while mice from the EAE group infected with C. parapsilosis developed a disease comparable to non-infected EAE mice. Disease aggravation by C. glabrata and C. krusei strains was concomitant to increased IL-17 and IFN-γ production by splenic cells stimulated with fungi-derived antigens and with increased percentage of T lymphocytes and myeloid cells in the CNS. Analysis of interaction with BV-2 microglial cell line also revealed differences among these strains, in which C. krusei was the strongest activator of microglia concerning the expression of MHC II and CD40 and pro-inflammatory cytokine production. Altogether, these results indicated that the three non-albicans Candida strains were similarly able to reach the CNS but distinct in terms of their effect over EAE development. Whereas C. glabrata and C. Krusei aggravated the development of EAE, C. parapsilosis did not affect its severity. Disease worsening was partially associated to virulence factors in C. glabrata and to a strong activation of microglia in C. krusei infection. In conclusion, systemic infections by non-albicans Candida strains exerted influence on the experimental autoimmune encephalomyelitis in both immunological and clinical aspects, emphasizing their possible relevance in MS development.

Novas estratégias terapêuticas para o controle de biofilmes orais: terapia fotodinâmica e sistema carreador de antimicrobianos / New therapeutic strategics for the control of oral biofilms: photodynamic therapy and drug delivery system

Os biofilmes orais possuem grande relevância clínica por estarem associados com o desenvolvimento de cárie dentária e candidose bucal, que são doenças infecciosas frequentemente encontradas na população. O presente trabalho foi dividido em dois estudos: Estudo 1 que teve como objetivo analisar os efeitos da terapia fotodinâmica antimicrobiana (TFDa), mediada por Fotoenticine (FTC) e Azul de Metileno (AM), sobre biofilmes microcosmos de cárie dentária; e Estudo 2 cujo objetivo foi avaliar o gellan gum como biomaterial para carreador do antifúngico Ester fenetil do ácido caféico (CAPE) contra Candida albicans. No estudo 1, amostras de dentina cariada foram coletadas de diferentes pacientes para formar biofilmes microcosmos in vitro. Os biofilmes foram tratados com FTC ou AM associado à irradiação LED a 660 nm (28,5 J/cm²). Os dados foram analisados pela contagem de Unidades Formadoras de Colônias (UFC/mL). Além disso, a biomassa, estrutura do biofilme e produção de ácidos pelos microrganismos foram determinadas por análises microscópicas ou espectrofotométricas. Os biofilmes de diferentes pacientes apresentaram variações na composição microbiana, sendo formados por estreptococos, lactobacilos e leveduras. No geral, a TFDa diminuiu 3,7 Log10 do total de microrganismos, 2,8 Log10 de estreptococos, 3,2 Log10 de lactobacilos e 3,2 Log10 de leveduras, e atingiu a erradicação de estreptococos do grupo mutans. A TFDa também foi capaz de reduzir a biomassa, desagregar os biofilmes e diminuir a concentração de ácidos em 1,1 a 1,9 mmol de lactato/L. Em relação ao estudo 2, inicialmente, foram preparadas formulações do CAPE em diferentes concentrações de gellan gum (0,6 a 1%). As formulações foram avaliadas em relação ao sistema de liberação e ação antifúngica contra C. albicans. Verificou-se que concentrações mais altas de gellan (0,9 e 1%) levaram a uma liberação mais prolongada do CAPE em relação as concentrações mais baixas. Os valores de concentração inibitória mínima do CAPE sobre C. albicans foram aumentados quando esse composto foi incorporado no gellan. As formulações de CAPE em gellan apresentaram atividade antifúngica tanto em culturas planctônicas como em biofilmes de C. albicans, sendo esses efeitos dependentes do tempo de tratamento. O CAPE e suas formulações em gellan também levaram a uma diminuição da atividade proteolítica de C. albicans. Concluiu-se que a TFDa mediada por Fotoenticine e o sistema carreador de gellan gum podem ser estratégias terapêuticas promissoras para o controle dos biofilmes na cavidade bucal, podendo ser usadas respectivamente no tratamento da cárie e candidose. (AU)

Dental caries and oral candidiasis are infectious diseases frequently found in the population. The present work is divided into two studies, study 1 time as objective: To analyze the effects of antimicrobial photodynamic therapy (aPDT), mediated by Fotoenticine (FTC) and Methylene Blue (MB), on dental caries microcosm biofilms. In study 2, the objective was to evaluate gellan gum as a biomaterial to carry the antifungal caffeic acid phenethyl ester (CAPE) on Candida albicans. To conduct study 1, carious dentin samples were collected from different patients to form in vitro microcosm biofilms. The biofilms were treated with FTC or MB associated with 660 nm red LED irradiation, with energy dose of 28.5 J/cm² and power dose of 40 mW/cm². The data were analyzed by the count of Colony Forming Units (CFU/mL). In addition, the biomass, biofilm structure and acid production of the microorganisms were determined by microscopic or spectrophotometric analysis. The biofilms from different patients showed variations in microbial composition, being formed by streptococci, lactobacilli, and yeasts. Overall, aPDT decreased 3.7 Log10 of total microorganisms, 2.8 Log10 of streptococci, 3.2 Log10 of lactobacilli and 3.2 Log10 of yeasts, and achieved eradication of mutans group streptococci. PDTa was also able to reduce biomass, disaggregate biofilms, and decrease acid concentration by 1.1 to 1.9 mmol lactate/L. For study 2 of this, first the standards of CAPE were determined, such as minimum inhibitory concentration, and absorption peak, then CAPE was incorporated into gellan gum, and then the standard curve test and analysis of CAPE release was performed, finally the formulations were tested on planktonic culture and biofilm of different strains of C. albicans, it was also analyzed the action of this drug on the production of Sap. The MIC found varied from 32 to 64 µg/mL, the release tests showed a gradual release in the higher formulations, finally in the CFU/mL count both in planktonic culture and biofilm the formulations were able to inhibit the fungus. With this it is concluded that both aPDT for oral microcosm and gellan gum as caregiver of CAPE for Candida albicans inhibition are promising. (AU)

Susceptibility of Dental Caries Microcosm Biofilms to Photodynamic Therapy Mediated by Fotoenticine.

Photodynamic therapy (PDT) mediated by Fotoenticine® (FTC), a new photosensitizer derived from chlorin e-6, has shown in vitro inhibitory activity against the cariogenic bacterium Streptococcus mutans. However, its antimicrobial effects must be investigated on biofilm models that represent the microbial complexity of caries. Thus, we evaluated the efficacy of FTC-mediated PDT on microcosm biofilms of dental caries. Decayed dentin samples were collected from different patients to form in vitro biofilms. Biofilms were treated with FTC associated with LED irradiation and analyzed by counting the colony forming units (log10 CFU) in selective and non-selective culture media. Furthermore, the biofilm structure and acid production by microorganisms were analyzed using microscopic and spectrophotometric analysis, respectively. The biofilms from different patients showed variations in microbial composition, being formed by streptococci, lactobacilli and yeasts. Altogether, PDT decreased up to 3.7 log10 CFU of total microorganisms, 2.8 log10 CFU of streptococci, 3.2 log10 CFU of lactobacilli and 3.2 log10 CFU of yeasts, and reached eradication of mutans streptococci. PDT was also capable of disaggregating the biofilms and reducing acid concentration in 1.1 to 1.9 mmol lactate/L. It was concluded that FTC was effective in PDT against the heterogeneous biofilms of dental caries.

The Anti-Biofilm Efficacy of Caffeic Acid Phenethyl Ester (CAPE) In Vitro and a Murine Model of Oral Candidiasis.

Candida albicans is the main fungal species associated with the development of oral candidiasis. Currently, therapeutic options for these infections are limited by the adverse effects of antifungal drugs and by the emergence of drug resistant strains. Thus, the development of new antifungal agents is needed for the prevention and treatment of oral Candida infections. Caffeic acid phenethyl ester (CAPE) is a natural compound from propolis polyphenolic groups that exhibits many pharmacological properties. In this study, we investigated whether CAPE can have antifungal and immunomodulatory effects on oral candidiasis. Preliminary tests to assess the antifungal activity of CAPE were performed using the Minimum Inhibitory Concentration (MIC) assay that demonstrated inhibition in a range from 16 to 32 µg/mL, confirming its antifungal activity on several C. albicans strains isolated from the oral cavity. Subsequently, we analyzed Candida spp biofilms formed in vitro, in which CAPE treatment at 5 x MIC caused a reduction of 68.5% in the total biomass and ~2.60 Log in the viable cell count (CFU/mL) in relation to the untreated biofilm (p<0.0001). Next, RNA was extracted from untreated and CAPE-treated biofilms and analyzed by real-time qPCR. A series of genes analyzed (ALS1, ECE1, EPA1, HWP1, YWP1, BCR1, BGR1, CPH1, EFG1, NDT80, ROB1, TEC1, UME6, SAP2, SAP5, PBL2, and LIP9) were downregulated by CAPE compared to the untreated control group (p<0.0001). In in vivo studies using Galleria mellonella, the treatment with CAPE prolonged survival of larvae infected by C. albicans by 44.5% (p < 0.05) and accompanied by a 2.07-fold increase in the number of hemocytes. Flow cytometry revealed the most prominent increases were in types P2 and P3 hemocytes, granular cells, which phagocytize pathogens. In addition, CAPE treatment decreased the fungal load in the hemolymph and stimulated the expression of antifungal peptide genes such as galiomicin and gallerimycin. The antifungal and immunomodulatory activities observed in G. mellonella were extended to a murine model of oral candidiasis, in which CAPE decreased the levels of C. albicans colonization (~2 log CFU/mL) in relation to the untreated control group. In addition, CAPE treatment significantly reduced pseudomembranous lesions, invasion of hyphae on epithelium surfaces, tissue damage and inflammatory infiltrate (p < 0.05). CAPE was also able to increase the expression of ß-defensin 3 compared to the infected and untreated group by 3.91-fold (p < 0.0001). Taken together, these results show that CAPE has both antifungal and immunomodulatory effects, making it a promising natural antifungal agent for the treatment and prevention of candidiasis and shows impact to oral candidiasis.

Probiotic Effects of Lactobacillus paracasei 28.4 to Inhibit Streptococcus mutans in a Gellan-Based Formulation.

Streptococcus mutans is considered to be a major bacterium involved in dental caries, and the control of virulence mechanisms is fundamental to prevent disease. Probiotics present a promising preventive method; however, the use of probiotics requires its incorporation into delivery materials to facilitate oral colonization. Thus, we performed a comprehensive study examining preventive effects of Lactobacillus paracasei 28.4-enriched gellan hydrogel materials to inhibit S. mutans in planktonic and biofilm states, addressing its influence in the production of extracellular polysaccharides (EPS) and altered gene expression of several cariogenic virulence factors. L. paracasei 28.4, a strain isolated from the oral cavity of a caries-free individual, was incorporated in three gellan hydrogels (0.5%, 0.75%, and 1% w/v). The pretreatment with probiotic-gellan formulations provided a release of L. paracasei cells over 24 h that was sufficient to inhibit the planktonic growth of S. mutans, independent of the gellan concentrations and pH variations. This pretreatment also had inhibitory activity against S. mutans biofilms, exhibiting a reduction of 0.57 to 1.54 log10 in CFU/mL (p < 0.0001) and a decrease of 68.8 to 71.3% in total biomass (p < 0.0001) compared with the control group. These inhibitory effects were associated with the decreased production of EPS by 80% (p < 0.0001) and the downregulation of luxS, brpA, gbpB, and gtfB genes. The gellan formulation containing L. paracasei 28.4 exhibited probiotic effects for preventing S. mutans growth, biofilm formation, and production of cariogenic factors to suggest possible use in tooth decay prevention.

Chitosan enhances the antimicrobial photodynamic inactivation mediated by Photoditazine® against Streptococcus mutans.

Chitosan (CS), a biopolymer with intrinsic antimicrobial activity, can increase antimicrobial photodynamic inactivation (aPDI). The aim of this study was to evaluate the capacity of CS to potentiate the efficacy of Photoditazine® (PDZ)-mediated aPDI of the cariogenic bacterium Streptococcus mutans. CS effectively augmented the effects of aPDI, with reductions of approximately 4.5 logs in both planktonic and biofilm states. The combined treatment was also capable of reducing the number of S. mutans cells and amount of extracellular matrix in biofilms formed on enamel surfaces, which were characterized using scanning electron microscopy analysis. Furthermore, CS increased the absorption of PDZ by S. mutans cells. The combination of CS with PDZ-mediated aPDI is hence a promising antimicrobial approach against S. mutans and may be useful to control dental caries.

Exploring the Galleria mellonella model to study antifungal photodynamic therapy.

BACKGROUND: Antimicrobial photodynamic therapy (aPDT) shows antimicrobial activity on yeast of the genus Candida. In aPDT, the depth at which the light penetrates the tissue is extremely important for the elaboration of the treatment. The aim of this study was to evaluate the action of aPDT on experimental candidiasis and the laser impact in the tissue using Galleria mellonella as the infection model. METHODS: G. mellonella larvae were infected with different Candida albicans strains. After 30 min, they were treated with methylene blue-mediated aPDT and a low intensity laser (660 nm). The larvae were incubated at 37 °C for seven days and monitored daily to determine the survival curve, using the Log-rank test (Mantel Cox). To evaluate the distribution of the laser as well as its depth of action in the larva body, the Interactive 3D surface PLOT of Image J was used. The effects of aPDT on the immune system were also evaluated by the quantification of hemocytes in the hemolymph of G. mellonella after 6 h of Candida infection (ANOVA and Tukey's test). RESULTS: In both the ATCC 18,804 strain and the C. albicans clinical strain 17, aPDT prolonged the survival of the infected G. mellonella larvae by a lethal fungal dose. There was a statistically significant difference between the aPDT and the control groups in the ATCC strain (P = 0.0056). The depth of laser action in the insect body without the photosensitizer was 2.5 mm and 2.4 mm from the cuticle of the larva with the photosensitizer. In the larvae, a uniform distribution of light occurred along 32% of the body length for the group without the photosensitizer and in 39.5% for the group with the photosensitizer. In the immunological analysis, the infection by C. albicans ATCC 18,804 in G. mellonella led to a reduction in the number of hemocytes in the hemolymph. The aPDT and laser treatment induced a slight increase in the number of hemocytes. CONCLUSION: Both aPDT and laser treatment positively influenced the treatment of experimental candidiasis. G. mellonella larvae were a useful model for the study of light tissue penetration in antimicrobial photodynamic therapy.