Antimicrobial effects of surface pre-reacted glass-ionomer (S-PRG) eluate against oral microcosm biofilm.

This study investigated the antimicrobial activity of surface pre-reacted glass ionomer eluate (S-PRG) against oral microcosm biofilms collected from the oral cavity of patients. Dental biofilm samples were collected from three volunteers to form microcosm biofilms in vitro. Initially, screening tests were carried out to determine the biofilm treatment conditions with S-PRG eluate. The effects of a daily treatment for 5 min using three microcosm biofilms from different patients was then evaluated. For this, biofilms were formed on tooth enamel specimens for 120 h. Biofilms treated with 100% S-PRG for 5 min per day for 5 days showed a reduction in the number of total microorganisms, streptococci and mutans streptococci. SEM images confirmed a reduction in the biofilm after treatment. Furthermore, S-PRG also reduced lactic acid production. It was concluded that S-PRG eluate reduced the microbial load and lactic acid production in oral microcosm biofilms, reinforcing its promising use as a mouthwash agent.

N-acetylcysteine reduces amphotericin B deoxycholate nephrotoxicity and improves the outcome of murine cryptococcosis.

Cryptococcosis is a life-threatening fungal infection, and its current treatment is toxic and subject to resistance. Drug repurposing represents an interesting approach to find drugs to reduce the toxicity of antifungals. In this study, we evaluated the combination of N-acetylcysteine (NAC) with amphotericin B (AMB) for the treatment of cryptococcosis. We examined the effects of NAC on fungal morphophysiology and on the macrophage fungicidal activity 3 and 24 hours post inoculation. The therapeutic effects of NAC combination with AMB were investigated in a murine model with daily treatments regimens. NAC alone reduced the oxidative burst generated by AMB in yeast cells, but did not inhibit fungal growth. The combination NAC + AMB decreased capsule size, zeta potential, superoxide dismutase activity and lipid peroxidation. In macrophage assays, NAC + AMB did not influence the phagocytosis, but induced fungal killing with different levels of oxidative bursts when compared to AMB alone: there was an increased reactive oxygen species (ROS) after 3 hours and reduced levels after 24 hours. By contrast, ROS remained elevated when AMB was tested alone, demonstrating that NAC reduced AMB oxidative effects without influencing its antifungal activity. Uninfected mice treated with NAC + AMB had lower concentrations of serum creatinine and glutamate-pyruvate transaminase in comparison to AMB. The combination of NAC + AMB was far better than AMB alone in increasing survival and reducing morbidity in murine-induced cryptococcosis, leading to reduced fungal burden in lungs and brain and also lower concentrations of pro-inflammatory cytokines in the lungs. In conclusion, NAC + AMB may represent an alternative adjuvant for the treatment of cryptococcosis.

In vitro effects of selective serotonin reuptake inhibitors on Cryptococcus gattii capsule and biofilm.

Infections caused by Cryptococcus gattii mainly affect immunocompetent individuals and the treatment presents important limitations. This study aimed to validate the efficacy of selective serotonin reuptake inhibitors (SSRI), fluoxetine hydrochloride (FLH), and paroxetine hydrochloride (PAH) in vitro against C. gattii. The antifungal activity of SSRI using the microdilution method revealed a minimal inhibitory concentration (MIC) of 31.25 µg/ml. The combination of FLH or PAH with amphotericin B (AmB) was analyzed using the checkerboard assay and the synergistic effect of SSRI in combination with AmB was able to reduce the SSRI or AmB MIC values 4-8-fold. When examining the effect of SSRI on the induced capsules, we observed that FLH and PAH significantly decreased the size of C. gattii capsules. In addition, the effects of FLH and PAH were evaluated in biofilm biomass and viability. The SSRI were able to reduce biofilm biomass and biofilm viability. In conclusion, our results indicate the use of FLH and PAH exhibited in vitro anticryptococcal activity, representing a possible future alternative for the cryptococcosis treatment.

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.

Does sorghum phenolic extract have antifungal effect?

This study aimed to evaluate the antifungal effect of SC319 sorghum phenolic extract (SPE) on the Aspergillus, Fusarium, Penicillium, Stenocarpella, Colletotrichum, and Macrophomina genera. SPE was extracted by 20% ethanol and used in four assays: (1) against Fusarium verticillioides in solid (PDA) and liquid (PD) potato dextrose media; (2) Minimum Inhibitory Concentration (MIC) assay with 16 fungi isolates; (3) Conidial Germination Rate (CGR) with 14 fungi isolates and (4) Growth Curve (GC) with 11 fungi isolates. There was no reduction in the mycelial growth (colony diameter and dry weight) and in the number of Fusarium verticillioides spores in assay 1 (PDA and PD). The colony's dry weight was almost six times higher in the presence than in the absence of SPE. All SPE samples presented MIC (assay 1) above the maximum concentration tested (5000 µg.mL-1) for the 16 isolates. Also, there was no inhibitory effect of SPE on conidia germination rate (CGR). Oppositely, in GC assay, the control had a higher CFU count than the samples with SPE in 24 h. This result suggests that SPE can delay the fungal growth in the first hours of incubation, which is an important finding that may help reduce the severity of fungal diseases in plants. However, further studies are needed to confirm these results, including sorghum genotypes with different profiles of phenolic compounds. Although the SC319 SPE was not effective as an antifungal agent, it may have potential as a growth promoter of beneficial fungi in the food and pharmaceutical industries.

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.

Antifungal and antibiofilm effect of duloxetine hydrochloride against Cryptococcus neoformans and Cryptococcus gattii.

Cryptococcosis is an invasive mycosis caused mainly by Cryptococcus gattii and C. neoformans and is treated with amphotericin B (AMB), fluconazole and 5-fluorocytosine. However, antifungal resistance, limited and toxic antifungal arsenal stimulate the search for therapeutic strategies such as drug repurposing. Among the repurposed drugs studied, the selective serotonin reuptake inhibitors (SSRIs) have shown activity against Cryptococcus spp. However, little is known about the antifungal effect of duloxetine hydrochloride (DH), a selective serotonin and norepinephrine reuptake inhibitor (SSNRI), against C. neoformans and C. gattii. In this study, DH inhibited the growth of several C. neoformans and C. gattii strains at concentrations ranging from 15.62 to 62.50 µg/mL. In addition, DH exhibited fungicidal activity ranging from 15.62 to 250 µg/mL. In biofilm, DH treatment reduced Cryptococcus spp. biomass at a level comparable to AMB, with a significant reduction (85%) for C. neoformans biofilms. The metabolic activity of C. neoformans and C. gattii biofilms decreased significantly (99%) after treatment with DH. Scanning electron micrographs confirmed the anti-biofilm activity of DH, as isolated cells could be observed after treatment. In conclusion, DH showed promising antifungal activity against planktonic cells and biofilms of C. neoformans and C. gattii, opening perspectives for further studies with DH in vivo.

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.

Staphylococcus aureus triggers a protective inflammatory response against secondary Cryptococcus gattii infection in a murine model.

Prior infections can provide protection or enhance susceptibility to a subsequent infection through microorganism's interaction or host immunomodulation. Staphylococcus aureus (SA) and Cryptococcus gattii (CG) cause lungs infection, but it is unclear how they interact in vivo. This study aimed to study the effects of the primary SA lung infection on secondary cryptococcosis caused by CG in a murine model. The mice's survival, fungal burden, behavior, immune cells, cytokines, and chemokines were quantified to evaluate murine cryptococcosis under the influence of a previous SA infection. Further, fungal-bacterial in vitro interaction was studied in a culture medium and a phagocytosis assay. The primary infection with SA protects animals from the subsequent CG infection by reducing lethality, improving behavior, and impairing the fungal proliferation within the host. This phenotype was associated with the proinflammatory antifungal host response elicited by the bacteria in the early stage of cryptococcosis. There was no direct inhibition of CG by SA, although the phagocytic activity of macrophages was reduced. Identifying mechanisms involved in this protection may lead to new approaches for preventing and treating cryptococcosis.

The Th2 Response and Alternative Activation of Macrophages Triggered by Strongyloides venezuelensis Is Linked to Increased Morbidity and Mortality Due to Cryptococcosis in Mice.

Cryptococcosis is a systemic mycosis that causes pneumonia and meningoencephalitis. Strongyloidiasis is a chronic gastrointestinal infection caused by parasites of the genus Strongyloides. Cryptococcosis and strongyloidiasis affect the lungs and are more prevalent in the same world regions, i.e., Africa and tropical countries such as Brazil. It is undeniable that those coincidences may lead to the occurrence of coinfections. However, there are no studies focused on the interaction between Cryptococcus spp. and Strongyloides spp. In this work, we aimed to investigate the interaction between Strongyloides venezuelensis (Sv) and Cryptococcus gattii (Cg) in a murine coinfection model. Murine macrophage exposure to Sv antigens reduced their ability to engulf Cg and produce reactive oxygen species, increasing the ability of fungal growth intracellularly. We then infected mice with both pathogens. Sv infection skewed the host's response to fungal infection, increasing lethality in a murine coinfection model. In addition to increased NO levels and arginase activity, coinfected mice presented a classic Th2 anti-Sv response: eosinophilia, higher levels of alternate activated macrophages (M2), increased concentrations of CCL24 and IL-4, and lower levels of IL-1ß. This milieu favored fungal growth in the lungs with prominent translocation to the brain, increasing the host's tissue damage. In conclusion, our data shows that primary Sv infection promotes Th2 bias of the pulmonary response to Cg-infection and worsens its pathological outcomes.

Acute oral toxicity, antinociceptive and antimicrobial activities of kava dried extracts and synthetic kavain.

Piper methysticum G. Forst, popularly known as kava, is a traditional medicinal plant widely used for the treatment of anxiety and insomnia. The aim of this study was to investigate new therapeutic applications of this plant. Nociceptive response induced by heat (hot-plate) was used as pain model. Susceptibility of different strains to kava ethanolic dried extracts was evaluated by broth microdilution method. Acute oral toxicity was performed according to Organisation for Economic Cooperation and Development (OECD) guideline. Administration of kava dried extracts and kavain inhibited the nociceptive response in the hot-plate model and did not affect the time mice spent in the rota-rod apparatus. The samples showed no significant antibacterial activity, however slight antifungal activity was verified. The extracts may be considered of low oral acute toxicity. Kava extracts exhibited promising antinociceptive activity in model of nociceptive pain, which should be deeper explored as a new therapeutic application of kava.

Randomized, phase 1/2, double-blind pioglitazone repositioning trial combined with antifungals for the treatment of cryptococcal meningitis - PIO study.

BACKGROUND: Cryptococcosis affects more than 220,000 patients/year, with high mortality even when the standard treatment [amphotericin B (AMB), 5-flucytosin (5-FC) and fluconazole] is used. AMB presents high toxicity and 5-FC is not currently available in Brazil. In a pre-clinical study, pioglitazone (PIO - an antidiabetic drug) decreased AMB toxicity and lead to an increased mice survival, reduced morbidity and fungal burden in brain and lungs. The aim of this trial is to evaluate the efficacy and safety of PIO combined with standard antifungal treatment for human cryptococcosis. METHODS: A phase 1/2, randomized, double blind, placebo-controlled trial will be performed with patients from Belo Horizonte, Brazil. They will be divided into three groups (placebo, PIO 15 mg/day or PIO 45 mg/day) and will receive an additional pill during the induction phase of cryptococcosis' treatment. Our hypothesis is that treated patients will have increased survival, so the primary outcome will be the mortality rate. Patients will be monitored for survival, side effects, fungal burden and inflammatory mediators in blood and cerebrospinal fluid. The follow up will occur for up 60 days. CONCLUSIONS: We expect that PIO will be an adequate adjuvant to the standard cryptococcosis' treatment. TRIAL REGISTRATION: ICTRP/WHO (and International Clinical Trial Registry Plataform (ICTRP/WHO) (http://apps.who.int/trialsearch/Trial2.aspx?TrialID=RBR-9fv3f4), RBR-9fv3f4 (http://www.ensaiosclinicos.gov.br/rg/RBR-9fv3f4). UTN Number: U1111-1226-1535. Ethical approvement number: CAAE 17377019.0.0000.5149.

Pioglitazone as an adjuvant of amphotericin B for the treatment of cryptococcosis.

Approximately 180,000 people worldwide die from cryptococcosis each year, probably due to the ineffectiveness and toxicity of drugs currently available to treat the disease. Amphotericin B (AMB) is effective for killing the fungus, but has serious adverse effects linked to excessive production of reactive oxygen species which compromise renal function. Pioglitazone (PIO) is a peroxisome proliferator-activated receptor-γ agonist widely repositioned as an adjuvant of various drugs that have toxic effects due to its antioxidant and anti-inflammatory effects. This study evaluated PIO in combination with AMB for the treatment of cryptococcosis. PIO was found to reduce serum creatinine and glutamic-oxalacetic transaminase levels in mice treated with PIO+AMB. In vitro, PIO was able to control harmful oxidative bursts induced by AMB without compromising the antifungal effect. In vivo, PIO+AMB increased the survival rate compared with AMB alone, and improved the morbidity of the animals. PIO+AMB was more efficient than AMB alone for inhibiting fungal transmigration from the lungs to the brain, and killing yeasts that reached the central nervous system, avoiding the establishment of meningoencephalitis. In a phagocytosis assay, PIO did not influence the engulfment and fungicidal activity of macrophages induced by AMB, but reduced the oxidative bursts after the reduction of fungal burden, pointing to control of the pathogen without leading to excessive stress which can be damaging to the host. In conclusion, PIO+AMB was found to ameliorate cryptococcosis in a murine model, indicating that it is a promising therapeutic adjuvant for combating and controlling this fungal infection.

Antimicrobial and Immunomodulatory Activities of Dried Extracts of Echinacea Purpurea

Abstract The use of Echinacea purpurea (EP), a plant native from North America, is widely diffused throughout the world, presenting many pharmacological applications, mainly for the treatment of infections of respiratory and urinary tracts. Due to the widespread commercialization of EP-based products, an effective evaluation of their pharmacological properties is essential to assure efficacy during clinical use. In this study, in vitro tests were performed to evaluate the antimicrobial activity of dried extracts of EP by the microdilution method. In addition, a phagocytosis model was employed to assess the immunomodulatory potential of the extracts. The increase in reactive oxygen species production, as well as the intracellular proliferation rate of Cryptococcus gatti after phagocytosis by macrophages in the presence of EP dried extracts were also evaluated. The analyzed samples showed no significant antibacterial activity; however, a slight antifungal activity was verified. Positive effects of EP extracts on the modulation of cellular immune response were observed in different experiments, indicating that this mechanism may contribute to the control and treatment of infections.