Chitosan microparticles loaded with essential oils inhibit duo-biofilms of Candida albicans and Streptococcus mutans.

OBJECTIVE: Oral candidiasis is a common fungal infection that affects the oral mucosa, and happens when Candida albicans interacts with bacteria in the oral microbiota, such as Streptococcus mutans, causing severe early childhood caries. C. albicans and S. mutans mixed biofilms are challenging to treat with conventional antimicrobial therapies, thus, new anti-infective drugs are required. This study aimed to test a drug delivery system based on chitosan microparticles loaded with geranium and lemongrass essential oils to inhibit C. albicans and S. mutans mixed biofilms. METHODOLOGY: Chitosan microparticles loaded with essential oils (CM-EOs) were obtained by spray-drying. Susceptibility of planktonic were performed according CLSI at 4 to 2,048 µg/mL. Mixed biofilms were incubated at 37ºC for 48 h and exposed to CM-EOs at 256 to 4,096 µg/mL. The antimicrobial effect was evaluated using the MTT assay, with biofilm architectural changes analyzed by scanning electron microscopy. RAW 264.7 cell was used to evaluate compound cytotoxicity. RESULTS: CM-EOs had better planktonic activity against C. albicans than S. mutans. All samples reduced the metabolic activity of mixed C. albicans and S. mutans biofilms, with encapsulated oils showing better activity than raw chitosan or oils. The microparticles reduced the biofilm on the slides. The essential oils showed cytotoxic effects against RAW 264.7 cells, but encapsulation into chitosan microparticles decreased their toxicity. CONCLUSION: This study demonstrates that chitosan loaded with essential oils may provide an alternative method for treating diseases caused by C. albicans and S. mutans mixed biofilm, such as dental caries.

Chitosan microparticles loaded with essential oils inhibit duo-biofilms of Candida albicans and Streptococcus mutans

Abstract Oral candidiasis is a common fungal infection that affects the oral mucosa, and happens when Candida albicans interacts with bacteria in the oral microbiota, such as Streptococcus mutans, causing severe early childhood caries. C. albicans and S. mutans mixed biofilms are challenging to treat with conventional antimicrobial therapies, thus, new anti-infective drugs are required. Objective This study aimed to test a drug delivery system based on chitosan microparticles loaded with geranium and lemongrass essential oils to inhibit C. albicans and S. mutans mixed biofilms. Methodology Chitosan microparticles loaded with essential oils (CM-EOs) were obtained by spray-drying. Susceptibility of planktonic were performed according CLSI at 4 to 2,048 µg/mL. Mixed biofilms were incubated at 37ºC for 48 h and exposed to CM-EOs at 256 to 4,096 µg/mL. The antimicrobial effect was evaluated using the MTT assay, with biofilm architectural changes analyzed by scanning electron microscopy. RAW 264.7 cell was used to evaluate compound cytotoxicity. Results CM-EOs had better planktonic activity against C. albicans than S. mutans. All samples reduced the metabolic activity of mixed C. albicans and S. mutans biofilms, with encapsulated oils showing better activity than raw chitosan or oils. The microparticles reduced the biofilm on the slides. The essential oils showed cytotoxic effects against RAW 264.7 cells, but encapsulation into chitosan microparticles decreased their toxicity. Conclusion This study demonstrates that chitosan loaded with essential oils may provide an alternative method for treating diseases caused by C. albicans and S. mutans mixed biofilm, such as dental caries.

The herbicide paraquat alters growth and melanin production on the Cryptococcus neoformans/Cryptococcus gattii species complex.

Paraquat (1,10-dimethyl-4,4-bipyridinium dichloride; PQ) is a free-radical producing herbicide that affects cell membranes and can upset the environmental balance of microorganisms present in soil, such as Cryptococcus spp. This study aimed to evaluate the in vitro activity of PQ against Cryptococcus spp. in planktonic and biofilm forms, as well as the protective effect of antioxidant agents against the antifungal effect of PQ and the kinetics of melanin production in response to PQ. Susceptibility to PQ was evaluated by microdilution. Cryptococcus sp. strains exposed to PQ were grown in media with ascorbic acid (AA) and glutathione (GSH). Melanin production was assessed in the presence of l-3,4-dihydroxyphenylalanine (l-DOPA) + PQ. The minimum inhibitory concentration of PQ against Cryptococcus spp. ranged from 8 to 256 µg/mL. Furthermore, PQ reduced biofilm formation. AA and GSH restored the fungal growth of Cryptococcus spp. exposed to PQ. In addition, l-DOPA + PQ delayed melanin production by 24 and 48 h for C. deuterogattii and C. neoformans sensu lato, respectively, suggesting that PQ induces a fitness trade-off in melanin production. Taken together, our data suggest that the antifungal effect of PQ against Cryptococcus spp. possibly exerts selective pressures interfering with biofilm formation and melanin production by these yeasts.

Essential oils encapsulated in chitosan microparticles against Candida albicans biofilms.

The aim of the study was to produce and characterize chitosan microparticles loaded with essential oils (CMEOs), evaluate the essential oil (EO) release profile and the CMEOs' anti-Candida activity. The chitosan microparticles (CMs) loaded with lemongrass essential oil (LEO) and geranium essential oil (GEO) were produced by the spray-drying method and characterized regarding CMEO morphological and physicochemical parameters and EO encapsulation efficiency (EE) and release profile. The planktonic activity was quantified by broth microdilution, and the activity against biofilm was quantified by biomass formation measurement. The LEO and GEO compositions were analyzed by gas chromatography combined with mass spectrometry (GC/MS), finding the main components citral (83.17%) and citronellol (24.53%). The CMs and CMEOs showed regular distribution and spherical shape (1 to 15 µm), without any morphological and physical modifications after EO incorporation. EE% ranged from 12 to 39%. In vitro release tests demonstrated the EO release rates, after 144 h, were 33% and 55% in PBS and HCl media, respectively. The minimum inhibitory concentration (MIC) values for CMEOs were lower than for CMs and pure EOs (P < 0.05). The higher CMEO biofilm inhibition percentage demonstrates the efficiency of microparticles against Candida biofilm. These results indicate that CMEOs are promising compounds that have antibiofilm activity against C. albicans.

In vitro inhibitory effect of statins on planktonic cells and biofilms of the Sporothrix schenckii species complex.

Introduction. Sporotrichosis, caused by species of the Sporothrix schenckii complex, is the most prevalent subcutaneous mycosis in many areas of Latin America. Statins are a class of drugs widely used for lowering high sterol levels through their action on 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme in the synthesis of sterol.Aim. In this study, the antifungal activity of statins (simvastatin, atorvastatin, pravastatin) against planktonic cells and biofilms of S. schenckii complex species was evaluated, as well as the interaction of pravastatin with classical antifungals (amphotericin B, itraconazole, terbinafine).Methodology. Eighteen strains of Sporothrix species were used. The antifungal susceptibility assay was performed using the broth microdilution method. Mature biofilms were exposed to statins and metabolic activity was measured by the XTT reduction assay.Results. MICs of statins ranged from 8 to 512 µg ml-1 and from 8 to 256 µg ml-1 for filamentous and yeast forms, respectively. Regarding mature biofilms, MICs of 50 % inhibition (SMIC50) were 128 µg ml-1 for simvastatin and atorvastatin and >2048 µg ml-1 for pravastatin. MICs of 90 % inhibition (SMIC90) were 512 µg ml-1 for simvastatin and >2048 µg ml-1 for atorvastatin and pravastatin.Conclusion. These results highlight the antifungal and antibiofilm potential of statins against S. schenckii complex species.

Antifungal activity of different molecular weight chitosans against planktonic cells and biofilm of Sporothrix brasiliensis.

Sporotrichosis, caused by Sporothrix schenckii complex species, is the most prevalent subcutaneous mycosis in many areas of Latin America. Chitosan has been used as an antifungal agent; however the effects of the molecular weight (MW) of chitosan (i.e. high (HMW), medium (MMW) and low (LMW) molecular weight chitosan) on S. brasiliensis has not been well described, particularly on biofilms. Effects on the planktonic form activity of S. brasiliensis were quantified by broth microdilution, while anti-biofilm activity was quantified by measuring metabolic activity via XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide and biomass formation (crystal violet). The molecular weight of chitosan modulated its effect on the planktonic form of S. brasiliensis, presenting lower MIC values for LMW chitosan. With regards both the adhesive and mature phases of biofilm, the LMW chitosan reduced biomass and metabolic activity most effectively. This study confirms the effects of the molecular weight and deacetylation degree of chitosan on its antifungal properties for potentially pathogenic fungi.

Potassium iodide and miltefosine inhibit biofilms of Sporothrix schenckii species complex in yeast and filamentous forms.

This study aimed to evaluate the yeast biofilm growth kinetics and ultrastructure of Sporothrix schenckii complex and assess their mature biofilm susceptibility in filamentous and yeast forms to potassium iodide (KI) and miltefosine (MIL). Yeast biofilms were evaluated by crystal violet staining, XTT reduction assay and microscopic techniques. Susceptibility of planktonic and sessile cells was analyzed by broth microdilution. S. schenckii complex in yeast form produced biofilms, with an optimum maturation at 96 h, showing multilayered blastoconidia embedded in extracellular matrix. KI and MIL minimum inhibitory concentration (MIC) ranges against planktonic cells were 62,500-250,000 µg/ml and 0.125-4 µg/ml, respectively. KI and MIL reduced biofilm metabolic activity by 75.4% and 67.7% for filamentous form and 55.1% and 51.6% for yeast form, respectively. This study demonstrated that S. schenckii complex forms biofilms in vitro, and potassium iodide and miltefosine inhibit Sporothrix spp. biofilms in both filamentous and yeast forms.

Effect of the molecular weight of chitosan on its antifungal activity against Candida spp. in planktonic cells and biofilm.

Difficulties in the treatment of Candida spp. invasive infections are usually related to the formation of biofilms. The aim of this study was to determine the effects of molecular weight (MW) of chitosan (using high (HMW), medium (MMW) and low (LMW) molecular weight chitosan) on Candida albicans, Candida tropicalis and Candida parapsilosis sensu stricto. The deacetylation degree (DD) and molecular weight M were measured by potentiometric titration and viscosimetry, respectively. The planktonic shape activity was quantified by broth microdilution, and the activity against biofilm was quantified by metabolic activity through XTT 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]- 2H-tetrazolium hydroxide and biomass formation (crystal violet). The influence of chitosan MW on the planktonic form of Candida spp. was strain dependent. Fungal growth decreased with increasing chitosan MW for C. tropicalis and C. parapsilosis, while chitosan MW did not modulate the effect for C. albicans. With regard to the formation of biofilms, in both the adhesion and mature phases, the biomass and metabolic activities of Candida spp. were reduced by about 70% and 80%, respectively for each phase.