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Objective This study assessed the effect of tyrosol and chlorhexidine gluconate in combination against Candida albicans, Candida glabrata, and Streptococcus mutans in the planktonic state or forming biofilms in vitro. Materials and Methods Checkerboard assays were performed for determination of minimum inhibitory concentration. Biofilms were cultivated during 24 h on specimens of acrylic resin and hydroxyapatite and treated with the drugs alone or in combination twice a day for 1 min, during 3 days. The antibiofilm effect was determined by quantification of the metabolic activity and cultivable cells. The drug combination was also applied on C. albicans to investigate its action on the number of hyphae. Data were statistically examined by two-way ANOVA and Holm-Sidak test ( P < 0.05). Results The effect of drug combination on planktonic cells was classified as antagonistic for C. albicans and indifferent for the other strains. Also, the drugs were ineffective against the tested biofilms. However, the drug combination showed a synergistic effect in reducing the number of hyphae by C. albicans. Conclusion The combination of tyrosol with chlorhexidine gluconate was only effective in reducing the number of hyphae by C. albicans, a relevant virulence factor of this species. [ABSTRACT FROM AUTHOR]

The contribution of different Candida species in oral fungal infections has stimulated the search for more effective therapies. This study assessed the antibiofilm effects of nanocarriers of miconazole (MCZ) or fluconazole (FLZ) on Candida biofilms, and their cytotoxic effects on murine fibroblasts. Three-species biofilms ( Candida albicans/Candida glabrata/Candida tropicalis ) were formed on 96-well plates, and they were treated with nanocarriers (iron oxide nanoparticles coated with chitosan-"IONPs-CS") of MCZ or FLZ at 39/78/156 µg/mL; antifungals alone at 156 µg/mL and artificial saliva were tested as positive and negative controls, respectively. Biofilms were analyzed by colony forming units (CFU), biomass, metabolic activity, and structure/viability. The cytotoxicity (L929 cells) of all treatments was determined via 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) reduction assay. Data were submitted to one- or two-way ANOVA, followed by Tukey's or Fisher LSD's tests ( p < 0.05). IONPs-CS-MCZ at 78 µg/mL promoted similar antibiofilm and cytotoxic effects compared with MCZ at 156 µg/mL. In turn, IONPs-CS-FLZ at 156 µg/mL was overall the most effective FLZ antibiofilm treatment, surpassing the effects of FLZ alone; this nanocarrier was also less cytotoxic compared with FLZ alone. It can be concluded that both nanocarriers are more effective alternatives to fight Candida biofilms compared with their respective positive controls in vitro, being a promising alternative for the treatment of oral fungal infections.

Nanocarriers have been used as alternative tools to overcome the resistance of Candida species to conventional treatments. This study prepared a nanocarrier of cetylpyridinium chloride (CPC) using iron oxide nanoparticles (IONPs) conjugated with chitosan (CS), and assessed its antifungal and cytotoxic effects. CPC was immobilized on CS-coated IONPs, and the nanocarrier was physico-chemically characterized. Antifungal effects were determined on planktonic cells of Candida albicans and Candida glabrata (by minimum inhibitory concentration (MIC) assays) and on single- and dual-species biofilms of these strains (by quantification of cultivable cells, total biomass and metabolic activity). Murine fibroblasts were exposed to different concentrations of the nanocarrier, and the cytotoxic effect was evaluated by MTT reduction assay. Characterization methods confirmed the presence of a nanocarrier smaller than 313 nm. IONPs-CS-CPC and free CPC showed the same MIC values (0.78 µg mL -1 ). CPC-containing nanocarrier at 78 µg mL -1 significantly reduced the number of cultivable cells for all biofilms, surpassing the effect promoted by free CPC. For total biomass, metabolic activity, and cytotoxic effects, the nanocarrier and free CPC produced statistically similar outcomes. In conclusion, the IONPs-CS-CPC nanocarrier was more effective than CPC in reducing the cultivable cells of Candida biofilms without increasing the cytotoxic effects of CPC, and may be a useful tool for the treatment of oral fungal infections.

Candida auris has emerged as a multidrug-resistant nosocomial pathogen over the last decade. Outbreaks of the organism in health care facilities have resulted in life-threatening invasive candidiasis in over 40 countries worldwide. Resistance by C. auris to conventional antifungal drugs such as fluconazole and amphotericin B means that alternative therapeutics must be explored. As such, this study served to investigate the efficacy of a naturally derived polysaccharide called chitosan against aggregative (Agg) and nonaggregative (non-Agg) isolates of C. auris in vitro and in vivo. In vitro results indicated that chitosan was effective against planktonic and sessile forms of Agg and non-Agg C. auris In a Galleria mellonella model to assess C. auris virulence, chitosan treatment was shown to ameliorate killing effects of both C. auris phenotypes (NCPF 8973 and NCPF 8978, respectively) in vivo Specifically, chitosan reduced the fungal load and increased survival rates of infected Galleria , while treatment alone was nontoxic to the larvae. Finally, chitosan treatment appeared to induce a stress-like gene expression response in NCPF 8973 in the larvae likely arising from a protective response by the organism to resist antifungal activity of the compound. Taken together, results from this study demonstrate that naturally derived compounds such as chitosan may be useful alternatives to conventional antifungals against C. auris ., (Copyright © 2020 American Society for Microbiology.)

Background: Novel and new therapeutic strategies capable of enhancing the efficacy of existing antimicrobials is an attractive proposition to meet the needs of society., Objective: This study aimed to evaluate the potentiating effect of a miconazole (MCZ) nanocarrier system, incorporated with iron oxide nanoparticles (IONPs) and chitosan (CS) (IONPs-CS-MCZ). This was tested on three representative complex interkingdom oral biofilm models (caries, denture and gingivitis)., Materials and Methods: The planktonic and sessile minimum inhibitory concentrations (MICs) of IONPs-CS-MCZ against different Candida albicans strains were determined, as well as against all represented bacterial species that formed within the three biofilm models. Biofilms were treated for 24 hours with the IONPs-CS nanocarrier system containing MCZ at 64 mg/L, and characterized using a range of bioassays for quantitative and qualitative assessment., Results: MIC results generally showed that IONPs-CS-MCZ was more effective than MCZ alone. IONPs-CS-MCZ also promoted reductions in the number of CFUs, biomass and metabolic activity of the representative biofilms, as well as altering biofilm ultrastructure when compared to untreated biofilms. IONPs-CS-MCZ affected the composition and reduced the CFEs for most of the microorganisms present in the three evaluated biofilms. In particular, the proportion of streptococci in the biofilm composition were reduced in all three models, whilst Fusobacterium spp. percentage reduced in the gingivitis and caries models, respectively., Conclusion: In conclusion, the IONPs-CS-MCZ nanocarrier was efficient against three in vitro models of pathogenic oral biofilms, showing potential to possibly interfere in the synergistic interactions among fungal and bacterial cells within polymicrobial consortia., Competing Interests: The authors declare no conflicts of interest., (© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Overexposure of microorganisms to conventional drugs has led to resistant species that require new treatment strategies. This study prepared and characterized a nanocarrier of miconazole (MCZ) based on iron oxide nanoparticles (IONPs) functionalized with chitosan (CS), and tested its antifungal activity against biofilms of Candida albicans and Candida glabrata. IONPs-CS-MCZ nanocarrier was prepared by loading MCZ on CS-covered IONPs and characterized by physicochemical methods. Minimum inhibitory concentration (MIC) of the nanocarrier was determined by the microdilution method. Biofilms were developed (48 h) in microtiter plates and treated with MCZ-carrying nanocarrier at 31.2 and 78 μg/mL, in both the presence and absence of an external magnetic field (EMF). Biofilms were evaluated by total biomass, metabolic activity, cultivable cells (CFU), extracellular matrix components, scanning electron microscopy and confocal microscopy. Data were analyzed by two-way ANOVA and Holm-Sidak test (p < 0.05). A nanocarrier with diameter lower than 50 nm was obtained, presenting MIC values lower than those found for MCZ, and showing synergism for C. albicans and indifference for C. glabrata (fractional inhibitory concentration indexes of <0.12 and <0.53, respectively). IONPs-CS-MCZ did not affect total biomass and extracellular matrix. IONPs-CS-MCZ containing 78 μg/mL MCZ showed a superior antibiofilm effect to MCZ in reducing CFU and metabolism for single biofilms of C. albicans and dual-species biofilms. The EMF did not improve the nanocarrier effects. Microscopy confirmed the antibiofilm effect of the nanocarrier. In conclusion, IONPs-CS-MCZ was more effective than MCZ mainly against C. albicans planktonic cells and number of CFU and metabolism of the biofilms., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aim: To assemble, characterize and assess the antifungal effects of a new fluconazole (FLZ)-carrier nanosystem. Materials & methods: The nanosystem was prepared by loading FLZ on chitosan (CS)-coated iron oxide nanoparticles (IONPs). Antifungal effects were evaluated on planktonic cells (by minimum inhibitory concentration determination) and on biofilms (by quantification of cultivable cells, total biomass, metabolism and extracellular matrix) of Candida albicans and Candida glabrata . Results: Characterization results ratified the formation of a nanosystem (<320 nm) with FLZ successfully embedded. IONPs-CS-FLZ nanosystem reduced minimum inhibitory concentration values and, in general, showed similar antibiofilm effects compared with FLZ alone. Conclusion: IONPs-CS-FLZ nanosystem was more effective than FLZ mainly in inhibiting Candida planktonic cells. This nanocarrier has potential to fight fungal infections.

Protection against the intraerythrocytic protozoan parasite Babesia bovis depends on both strong innate and adaptive immune response, this latter involving the presentation of parasite antigens to CD4 + T-lymphocytes by professional antigen-presenting cells. Secretion of Th1 cytokines by CD4 + T cell is also very important for isotype switching to IgG 2 , the best opsonising antibody isotype in cattle, to target extracellular parasites and parasite antigens displayed at the erythrocyte surface. In the field of vaccinology, heterologous prime-boost schemes combining protein-adjuvant formulations with a modified vaccinia Ankara vector expressing the same antigen have demonstrated the induction of both humoral and cellular immune responses. It has been previously demonstrated that MVA-infected dendritic cells can present antigens in the context of MHC II and activate CD4 + T cell. These results support the use of the MVA viral vector for a pathogen like Babesia bovis, which only resides within erythrocytes. In this study, 13-15-months-old Holstein-Friesian steers were immunised with a subunit vaccine as a prime and a modified vaccinia Ankara vector as a boost, both expressing a chimeric multi-antigen (rMABbo - rMVA). This antigen includes the immunodominant B and T cell epitopes of three B. bovis proteins: merozoite surface antigen - 2c (MSA - 2c), rhoptry associated protein 1 (RAP - 1) and heat shock protein 20 (HSP20). Responses were compared with the Babesia bovis live attenuated vaccine used in Argentina (R1A). Eleven weeks after the first immunisation, all bovines were challenged by the inoculation of a virulent B. bovis strain. All groups were monitored daily for hyperthermia and reduction of packed cell volume. Both the rMABbo - rMVA and R1A vaccinated animals developed high titters of total IgG antibodies and an antigen-specific Th1 cellular response before and after challenge. However, all rMABbo - rMVA steers showed clinical signs of disease upon challenge. Only the R1A live vaccine group developed an immune response associated with in vitro neutralising antibodies at a level that significantly inhibited the parasite invasion. The lack of protection observed with this recombinant formulation indicates the need to perform further basic and clinical studies in the bovine model in order to achieve the desired effectiveness. This is the first report in which a novel vaccine candidate against Babesia bovis was constructed based on a recombinant and rationally designed viral vector and evaluated in the biological model of the disease., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

This study synthesized and characterized a chlorhexidine (CHX)-carrier nanosystem based on iron oxide magnetic nanoparticles (IONPs) and chitosan (CS), and evaluated its antimicrobial effect on mono- and dual-species biofilms of Candida albicans and Streptococcus mutans. CHX was directly solubilized in CS-coated IONPs and maintained under magnetic stirring for obtaining the IONPs-CS-CHX nanosystem. Antimicrobial susceptibility testing for planktonic cells was performed by determining the minimum inhibitory concentration (MIC) of the nanosystem and controls. The effects of the IONPs-CS-CHX nanosystem on the formation of mono- and dual-species biofilms, as well as on pre-formed biofilms were assessed by quantification of total biomass, metabolic activity and colony-forming units. Data were analyzed by the Kruskal-Wallis' test or one-way analysis of variance, followed by the Student-Newman-Keuls' or Holm-Sidak's tests (α = 0.05), respectively. Physico-chemical results confirmed the formation of a nanosystem with a size smaller than 40 nm. The IONPs-CS-CHX nanosystem and free CHX showed similar MIC values for both species analyzed. In general, biofilm quantification assays revealed that the CHX nanosystem at 78 μg/mL promoted similar or superior antibiofilm effects compared to its counterpart at 39 μg/mL and free CHX at 78 μg/mL. These findings highlight the potential of CS-coated IONPs as preventive or therapeutic agents carrying CHX to fight biofilm-associated oral diseases., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Medical applications and biotechnological advances, including magnetic resonance imaging, cell separation and detection, tissue repair, magnetic hyperthermia and drug delivery, have strongly benefited from employing iron oxide nanoparticles (IONPs) due to their remarkable properties, such as superparamagnetism, size and possibility of receiving a biocompatible coating. Ongoing research efforts focus on reducing drug concentration, toxicity, and other side effects, while increasing efficacy of IONPs-based treatments. This review highlights the methods of synthesis and presents the most recent reports in the literature regarding advances in drug delivery using IONPs-based systems, as well as their antimicrobial activity against different microorganisms. Furthermore, the toxicity of IONPs alone and constituting nanosystems is also addressed.

The aim of this study was to evaluate the effect of farnesol on the production of acids and hydrolytic enzymes by biofilms of Streptococcus mutans and Candida albicans . The present study also evaluated the time-kill curve and the effect of farnesol on matrix composition and structure of single-species and dual-species biofilms. Farnesol, at subinhibitory concentrations, showed a significant reduction in S. mutans biofilm acid production, but did not alter C. albicans hydrolytic enzyme production. The number of cultivable cells of both microorganisms was significantly reduced after 8 h of contact with farnesol. Extracellular matrix protein content was reduced for biofilms formed in the presence of farnesol. In addition, confocal laser scanning and scanning electron microscopy displayed structural alterations in all biofilms treated with farnesol, which included reduction in viable cells and extracellular matrix. In conclusion, farnesol showed favorable properties controlling some virulence factors of S. mutans and C. albicans biofilms. These findings should stimulate further studies using this quorum-sensing molecule, combined with other drugs, to prevent or treat biofilm-associated oral diseases., Competing Interests: Compliance with Ethical StandardsThe authors declare that they have no conflict of interest.

Aim: This study examined the antifungal activity of the combination of tyrosol and farnesol against Candida albicans and Candida glabrata in the planktonic state or forming biofilms., Methods and Results: The effect of drug association against Candida planktonic cells was assessed by the fractional inhibitory concentration index. Mono- and dual-species biofilms were developed during 24 h and then treated with the compounds for 3 days, with two daily treatments of 1 min each. After, the total biomass, metabolic activity and the number of cultivable cells were quantified. Planktonic cells of the two species showed a similar susceptibility to the drug combination, however, a synergistic effect was only verified for C. glabrata. Regarding biofilm susceptibility, significant reductions in C. glabrata biomass, metabolism of C. albicans and mixed biofilms, and cultivable cells of single biofilms were verified for the drug combination, indicating an additive effect. For all other experiments, the effects were classified as indifferent., Conclusion: The combined use of tyrosol and farnesol was advantageous for some of the analysed parameters against Candida species., Significance and Impact of the Study: These findings may contribute to the development of oral care products containing tyrosol and farnesol to combat oral infections caused by Candida species., (© 2017 The Society for Applied Microbiology.)

Purpose: To assess the effect of tyrosol on the production of hydrolytic enzymes (by Candida biofilm cells) and acid (by Streptococcus mutans biofilms), as well as to quantify single and mixed biofilms of these species formed on acrylic resin (AR) and hydroxyapatite (HA)., Methods: Candida and S. mutans biofilms were formed on AR and HA in the presence of tyrosol during 48 hours. Next, acid proteinase, phospholipase and hemolytic activities of Candida biofilm cells were determined, while acid production by S. mutans biofilms was assessed by pH determination. The effect of tyrosol on mature biofilms (96 hours) was evaluated through quantification of total biomass, metabolic activity, number of colony-forming units and composition of biofilms' extracellular matrix. Data were analyzed by one- and two-way ANOVA, followed by Tukey's and Holm-Sidak's tests (α = 0.05)., Results: Treatments with tyrosol were not able to significantly reduce hydrolytic enzymes and acid production by Candida and S. mutans. Tyrosol only significantly reduced the metabolic activity of single biofilms of Candida species., Clinical Significance: Tyrosol on its own had a limited efficacy against single and mixed-species oral biofilms. Its use as an alternative antimicrobial for topical therapies still demands more investigation., Competing Interests: The authors declared no conflict of interest. This study was supported by São Paulo Research Foundation (FAPESP; grant numbers 2013/10285-2, 2013/03273-8 and 2014/05507-9), Brazil.

Aim: This study aimed to evaluate the effect of tyrosol on the formation of single and mixed biofilms of Candida albicans ATCC 10231, Candida glabrata ATCC 90030 and Streptococcus mutans ATCC 25175 formed on acrylic resin (AR) and hydroxyapatite (HA) surfaces., Methods and Results: Single and mixed biofilms were formed on AR and HA in the presence of tyrosol at 50, 100 and 200 mmol l(-1), during 48 h. Next, antimicrobial activity was assessed through metabolic activity (XTT reduction assay) and the number of colony-forming units (CFUs). Scanning electron microscopy observations were performed in order to analyse biofilm structure. Tyrosol, mainly at 200 mmol l(-1), significantly decreased the metabolic activity and number of CFUs for all single and mixed-species biofilms formed on both surfaces. SEM images suggested cell damage caused by tyrosol., Conclusion: Tyrosol showed inhibitory effects against biofilms formed by important oral pathogens., Significance and Impact of the Study: This is the first study showing the antibiofilm effect of tyrosol on Candida species and Strep. mutans in single and mixed cultures. These results may be useful in the development of topical therapies focused on preventing biofilm-associated oral diseases, such as denture stomatitis and dental caries., (© 2016 The Society for Applied Microbiology.)

The aim of this study was to evaluate the effect of the QS molecule farnesol on single and mixed species biofilms formed by Candida albicans and Streptococcus mutans. The anti-biofilm effect of farnesol was assessed through total biomass quantification, counting of colony forming units (CFUs) and evaluation of metabolic activity. Biofilms were also analyzed by scanning electron microscopy (SEM). It was observed that farnesol reduced the formation of single and mixed biofilms, with significant reductions of 37% to 90% and 64% to 96%, respectively, for total biomass and metabolic activity. Regarding cell viability, farnesol treatment promoted significant log reductions in the number of CFUs, ie 1.3-4.2 log10 and 0.67-5.32 log10, respectively, for single and mixed species biofilms. SEM images confirmed these results, showing decreases in the number of cells in all biofilms. In conclusion, these findings highlight the role of farnesol as an alternative agent with the potential to reduce the formation of pathogenic biofilms.

The prevention of adhesion of Candida cells to acrylic surfaces can be regarded as an alternative to prevent denture stomatitis. The use of quorum sensing molecules, such as tyrosol, could potentially interfere with the adhesion process. Therefore, the aim of this study was to assess the effect of tyrosol on adhesion of single and mixed cultures of Candida albicans and Candida glabrata to acrylic resin surfaces. Tyrosol was diluted in each yeast inoculum (10(7) cells/ml in artificial saliva) at 25, 50, 100, and 200 mM. Then, each dilution was added to wells of 24-well plates containing the acrylic specimens, and the plates were incubated at 37°C for 2 h. After, the effect of tyrosol was determined by total biomass quantification, metabolic activity of the cells and colony-forming unit counting. Chlorhexidine gluconate (CHG) was used as a positive control. Data were analyzed using analysis of variance (ANOVA) and the Holm-Sidak post hoc test (α = 0.05). The results of total biomass quantification and metabolic activity revealed that the tyrosol promoted significant reductions (ranging from 22.32 to 86.16%) on single C. albicans and mixed cultures. Moreover, tyrosol at 200 mM and CHG significantly reduced (p < 0.05) the number of adhered cells to the acrylic surface for single and mixed cultures of both species, with reductions ranging from 1.74 to 3.64-log10. In conclusion, tyrosol has an inhibitory effect on Candida adhesion to acrylic resin, and further investigations are warranted to clarify its potential against Candida infections., (© The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)