Geraniol Inhibits Both Planktonic Cells and Biofilms of the Candida parapsilosis Species Complex: Highlight for the Improved Efficacy of Amphotericin B, Caspofungin and Fluconazole plus Geraniol.

The Candida parapsilosis species complex poses a recognized threat to the nosocomial environment. In the scenario of the global rise of resistant strains to antifungals, geraniol, a terpene isolated from different essential oils, has shown promising antimicrobial activity. We evaluated: 1- the effects of geraniol against the Candida parapsilosis species complex, in planktonic and biofilm forms; 2- the strains' susceptibility to clinical antifungals and 3- the geraniol interaction with antifungals. Eighteen isolates were subjected to in vitro susceptibility testing by the broth microdilution protocol, using geraniol, amphotericin B, caspofungin, itraconazole and fluconazole to determine the minimum inhibitory concentration (MIC) and subsequently we measured the fungicidal activity. Geraniol was tested against biofilms by the measurement of the metabolic activity and biomass. Pharmacological interactions were performed by the checkerboard method. Geraniol's MIC range was between 256 and 512 µg/ml. MIC range for clinical antifungals was ≤ 0.031-4 µg/ml. Geraniol also showed antibiofilm activity with average reductions of metabolic activity (38.33%) and biomass (30.69%), at MIC concentration. Furthermore, geraniol showed synergistic/additive effects with antifungals. Briefly, geraniol inhibits both planktonic cells and biofilms of the Candida parapsilosis species complex and besides it improves the efficacy of amphotericin B, caspofungin and fluconazole.

Geraniol inhibits Candida parapsilosis species complex both in planktonic and biofilm growth. In addition, it shows synergistic/additive effects with the antifungals amphotericin B and caspofungin, besides additive activity with fluconazole.

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.

Vancomycin enhances growth and virulence of Trichosporon spp. planktonic cells and biofilms.

Invasive fungal infections (IFIs) are important worldwide health problem, affecting the growing population of immunocompromised patients. Although the majority of IFIs are caused by Candida spp., other fungal species have been increasingly recognized as relevant opportunistic pathogens. Trichosporon spp. are members of skin and gut human microbiota. Since 1980's, invasive trichosporonosis has been considered a significant cause of fungemia in patients with hematological malignancies. As prolonged antibiotic therapy is an important risk factor for IFIs, the present study investigated if vancomycin enhances growth and virulence of Trichosporon. Vancomycin was tested against T. inkin (n = 6) and T. asahii (n = 6) clinical strains. Planktonic cells were evaluated for their metabolic activity and virulence against Caenorhabditis elegans. Biofilms were evaluated for metabolic activity, biomass production, amphotericin B tolerance, induction of persister cells, and ultrastructure. Vancomycin stimulated planktonic growth of Trichosporon spp., increased tolerance to AMB, and potentiates virulence against C. elegans. Vancomycin stimulated growth (metabolic activity and biomass) of Trichosporon spp. biofilms during all stages of development. The antibiotic increased the number of persister cells inside Trichosporon biofilms. These cells showed higher tolerance to AMB than persister cells from VAN-free biofilms. Microscopic analysis showed that VAN increased production of extracellular matrix and cells in T. inkin and T. asahii biofilms. These results suggest that antibiotic exposure may have a direct impact on the pathophysiology of opportunistic trichosporonosis in patients at risk. LAY ABSTRACT: This study showed that the vancomycin stimulated Trichosporon growth, induced morphological and physiological changes on their biofilms, and also enhanced their in vivo virulence. Although speculative, the stimulatory effect of vancomycin on fungal cells should be considered in a clinical scenario.

Anthraquinones from Aloe spp. inhibit Cryptococcus neoformans sensu stricto: effects against growing and mature biofilms.

This study aimed to evaluate the in vitro effect of aloe emodin, barbaloin and chrysophanol on growing and mature biofilms of Cryptococcus neoformans sensu stricto. The compounds were added at the moment of inducing biofilm growth or after growth for 72 h to evaluate their effects on growing and mature biofilms, respectively. Then, biofilm biomass was evaluated by crystal violet staining and metabolic activity by the XTT reduction assay. Morphological alterations were also evaluated by laser scanning confocal microscopy. Aloe emodin and barbaloin affected growing biofilms and disrupted mature biofilms, reducing metabolic activity by > 60% and biomass by > 70%. Chrysophanol only inhibited mature biofilms, but to a lesser extent. In conclusion, anthraquinones, especially aloe emodin and barbaloin, show a relevant effect against growing and mature biofilms of C. neoformans sensu stricto.

Antifungal effects of the flavonoids kaempferol and quercetin: a possible alternative for the control of fungal biofilms.

This study aimed to determine the minimum inhibitory concentration (MIC) of kaempferol and quercetin against planktonic and biofilm forms of the Candida parapsilosis complex. Initially, nine C. parapsilosis sensu stricto, nine C. orthopsilosis and nine C. metapsilosis strains were used. Planktonic susceptibility to kaempferol and quercetin was assessed. Growing and mature biofilms were then exposed to the flavonoids at MIC or 10xMIC, respectively, and theywere also analyzed by confocal laser scanning microscopy. The MIC ranges were 32-128 µg ml-1 for kaempferol and 0.5-16 µg ml-1 for quercetin. Kaempferol and quercetin decreased (P < 0.05) the metabolic activity and biomass of growing biofilms of the C. parapsilosis complex. As for mature biofilms, the metabolic effects of the flavonoids varied, according to the cryptic species, but kaempferol caused an overall reduction in biofilm biomass. Microscopic analyses showed restructuring of biofilms after flavonoid exposure. These results highlight the potential use of these compounds as sustainable resources for the control of fungal biofilms.

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.