Total Biomass Quantification Research Articles

Combined Application of Antisense Oligomers to Control Transcription Factors of Candida albicans Biofilm Formation.

Antisense oligomers (ASOs) have been little exploited to control determinants ofCandida albicansvirulence. Biofilm formation is an important virulence factor of C. albicans, that is regulated by a complex network of transcription factors (such as EFG1, BRG1 and ROB1). Thus, the main goal of this work was to project ASOs, based on the 2'-OMethyl chemical modification, to targetBRG1andROB1mRNA and to validate its application either alone or in combination with the EFG1 mRNA target, to reduceC. albicans biofilm formation. The ability of ASOs to control gene expression was evaluate by qRT-PCR. The effect on biofilm formation was determined by the total biomass quantification, and simultaneously the carbohydrates and proteins reduction on extracellular matrix. It was verified that all the oligomers were able to reduce the levels of gene expression and the ability of C. albicans to form biofilms. Furthermore, the combined application of the cocktail of ASOs enhances the inhibition of C. albicans biofilm formation, minimizing biofilm thickness by reducing the quantity of matrix content (protein and carbohydrate). So, our work confirms that ASOs are useful tools for research and therapeutic development on the control of Candida species biofilm formation.

Antimicrobial, Antibiofilm Activities and Synergic Effect of Triterpene 3β,6β,16β-trihydroxyilup-20(29)-ene Isolated from Combretum leprosum Leaves Against Staphylococcus Strains

Antimicrobial resistance is a natural phenomenon and is becoming a huge global public health problem, since some microorganisms not respond to the treatment of several classes of antibiotics. The objective of the present study was to evaluate the antibacterial, antibiofilm, and synergistic effect of triterpene 3β,6β,16β-trihydroxyilup-20(29)-ene (CLF1) against Staphylococcus aureus and Staphylococcus epidermidis strains. Bacterial susceptibility to CLF1 was evaluated by minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) assay. In addition, the effect combined with antibiotics (ampicillin and tetracycline) was verified by the checkerboard method. The biofilms susceptibility was assessed by enumeration of colony-forming units (CFUs) and quantification of total biomass by crystal violet staining. The compound showed bacteriostatic and bactericidal activity against all Staphylococcal strains tested. The synergistic effect with ampicillin was observed only for S. epidermidis strains. Moreover, CLF1 significantly inhibited the biofilm formation and disrupted preformed biofilm of the all strains. Scanning electron microscopy (SEM) images showed changes in the cell morphology and structure of S. aureus ATCC 700698 biofilms (a methicillin-resistant S. aureus strain). Molecular docking simulations showed that CLF1 has a more favorable interaction energy than the antibiotic ampicillin on penicillin-binding protein (PBP) 2a of MRSA, coupled in different regions of the protein. Based on the results obtained, CLF1 proved to be a promising antimicrobial compound against Staphylococcus biofilms.

Farnesol modulation of Rhodotorula mucilaginosa in biofilm and planktonic forms.

Biofilms are important to the virulence of human pathogenic fungi, and some molecules have been found to play key roles in the growth and regulation of fungal biofilms. Farnesol, one of these molecules, is well-described for some microorganisms but is still scarcely known for Rhodotorula spp. This study aimed to evaluate the influence of farnesol on the biofilm of R. mucilaginosa. Initially, screening with 0.2 mM to 2.1 mM of farnesol was evaluated against planktonic forms. A concentration of this compound was then chosen and evaluated for its effect on biofilm in formation and on preformed biofilm after 24, 48 and 72 hours. The impact of farnesol was evaluated by colony-forming units (CFU) counts, determination of metabolic activity and quantification of total biomass. In the presence of 0.9 mM, farnesol was able to decrease the CFU number, at 48 hours, when the biofilm was in formation, although it did not affect the preformed biofilms. Thus, our results show that farnesol exerts a modulating activity during biofilm formation for R. mucilaginosa, with this compound reducing the metabolic activity and total biomass of the biofilms.

Phytochemical composition of Avicennia marina leaf extract, its antioxidant, antimicrobial potentials and inhibitory properties on Pseudomonas fluorescens biofilm

Mangroves are widely used for the extraction of natural compounds for the purpose to be used in traditional medicine and the pharmaceutical sector. In this study, Avicennia marina leaves (gathered in Safaga, Red Sea, Egypt) were dried and extracted by ethanol, acetone and ethyl acetate. The antimicrobial activity of the three organic extracts was examined against different fish and human pathogens. The mangrove ethyl acetate extract (MEE) which gave the highest antimicrobial activity was further evaluated, as it exhibited a promising antioxidant activity determined by the DPPH test with IC50 of 50.3 µg/ml. Its total phenolic and flavonoid contents were determined to be 109 and 23 mg/g, respectively. In addition, MEE antibiofilm activity was assessed by total biomass quantification using microplate assay and observed under light microscope. MEE showed a highly promising antibiofilm activity, where it succeeded not only in preventing initial cell attachment and biofilm formation by the fish pathogen Pseudomonas fluorescens, but also in disrupting the preformed biofilm with IC50 of 42.0 and 45.8 mg/ml, respectively. Furthermore, its chemical composition was determined by GC–MS analysis demonstrating that its major constituents are alcohol, fatty acids and their derivatives. Overall, the current study confirms the promising antimicrobial, antioxidant activities of MEE and reports biofilm inhibition and eradication activities of A. marina ethyl acetate extract against Pseudomonas fluorescens.

Cashew nut shell liquids: Antimicrobial compounds in prevention and control of the oral biofilms

ObjectiveThe aim was to evaluate the antibacterial and antibiofilm activity of natural (n-CNSL) and technical (t-CNSL) cashew nut shell liquid against streptococci and enterococci related to dental caries and chronic apical periodontitis, respectively. Material and methodsMinimum inhibitory concentrations (MIC) and minimal bactericidal concentration (MBC) were determined to assess the antimicrobial effect of both CNSLs (n-CSNL and t-CNSL) against S. oralis ATCC 10557, S. sobrinus ATCC 6715, S. parasanguinis ATCC 903, S. mutans UA 159 and E. faecalis ATCC 19433. The antibiofilm activity was evaluated by total biomass quantification, colony forming unit (CFU) counting and scanning electron microscopy (SEM). Furthermore, cytotoxic effect of the substances was evaluated on L929 and HaCat cell lines by MTS assay. ResultsThe n-CNSL and t-CNSL showed inhibitory and bactericidal effect against all strains tested in this study, with MIC and MBC values ranging from 1.5 to 25 μg/mL. Overall, both CNSLs showed significant reduction in biomass quantification and enumeration of biofilm-entrapped cells for the strains analyzed, in biofilm formation and preformed biofilms (p < 0.05). In biofilm inhibition assay, the t-CNSL and n-CNSL showed reduction in biomass and CFU number for all bacteria, except in cell viability of S. parasanguinis treated with t-CNSL (p > 0.05). Indeed, SEM images showed a reduction in the amount of biomass, bacterial cells and changes in cellular morphology of S. mutans. ConclusionIn conclusion, both substances showed effective antibacterial and antibiofilm activity against the strains used in the study, except in viability of S. parasanguinis cells treated with t-CNSL.

Anticaries activity of GERM CLEAN in Streptococcus mutans and Candida albicans dual-species biofilm.

To evaluate the antimicrobial effects of a peptide containing novel oral spray GERM CLEAN on dual-species biofilm formed by Streptococcus mutans and Candida albicans and to investigate whether GERM CLEAN inhibits the demineralization procedure of bovine enamel in vitro. The antimicrobial effects of GERM CLEAN on dual-species biofilm were analyzed by initial adherence rate calculation, water-insoluble exopolysaccharides quantification, total biomass quantification, and colony-forming units (CFUs) counting. Scanning electron microscopy and confocal laser scanning microscopy were applied to evaluate the impacts of GERM CLEAN on the biofilm structure. Further, the effects of GERM CLEAN on acidogenicity of dual-species were appraised via glycolytic pH drop analysis and hydroxyapatite dissolution measurement. The percentage of Surface Microhardness Reduction (%SMHR) evaluation, Atomic Force Micrograph (AFM) examination, and Transverse Microradiography (TMR) analysis after pH cycling were used to determine whether GERM CLEAN inhibited the demineralization of bovine enamel. GERM CLEAN decreased the adherence rate, water-insoluble EPS production, biofilm formation, and acidogenicity of the dual-species. Moreover, GERM CLEAN significantly inhibited the demineralization status of bovine enamels. This peptide containing novel oral spray GERM CLEAN has antimicrobial potential toward the dual-species. GERM CLEAN can also impede the demineralization procedure of enamel.

Antifungal and antibiofilm activities of the essential oil of leaves from Lippia gracilis Schauer against phytopathogenic fungi.

This study aimed to evaluate the antifungal and antibiofilm effects of essential oil (EO) from leaves of Lippia gracilis and its major constituents, thymol and carvacrol, against phytopathogenic fungi. The leaves of L. gracilis were hydrodistilled to obtain the EO and the chemical composition was determined by GC/MS analysis. The antifungal activity of EO of L. gracilis was evaluated on the vegetative and mycelial growth of Colletotrichum gloeosporioides, Colletotrichum lindemuthianum, Fusarium oxysporum and Fusarium solani. In addition, the ability of the oil to inhibit fungal biofilm formation was assessed by total biomass quantification using crystal violet staining, analysis of metabolic activity, and scanning electron microscopy (SEM). Moreover the antifungal and antibiofilm activities of the monoterpenes, thymol and carvacrol, present in EO of L. gracilis were evaluated against F. oxysporum. The analysis of the chemical composition of EO extracted from L. gracilis, revealed the presence of monoterpenes (94·13%), which included carvacrol (48·57%) and thymol (7·78%), and 4 sesquiterpenes (3·74%). In general, EO showed significant antifungal activity and inhibited the formation of fungal biofilms. Furthermore, thymol and carvacrol showed significant antifungal and antibiofilm activities against F. oxysporum. SEM images showed structural changes in fungal morphology upon treatment with EO of L. gracilis. The results presented in this study showed promising antifungal and antibiofilm effects of EO of L. gracilis and its major components, carvacrol and thymol. These findings indicate that the EO extracted from L. gracilis, and the monoterpenes, carvacrol and thymol have a great potential as antifungal and antibiofilm agents. Furthermore, this is the first report of the antibiofilm activity of the EO of L. gracilis and its major components against phytopathogenic fungi.

Effect of phenotypic switching on biofilm traits in Candida tropicalis

Candida tropicalis can undergo multiple forms of phenotypic switching. We have reported a switching system in C. tropicalis that is associated with changes in virulence attributes. We aimed to assess biofilm formation by distinct switch states of C. tropicalis and evaluate whether their sessile cells exhibit altered virulence traits. C. tropicalis strains included the parental phenotype (a clinical isolate) and four switch phenotypes (crepe, rough, revertant of crepe and revertant of rough). Biofilm formation and adhesion capability of sessile cells on polystyrene were assessed through quantification of total biomass. Filamentous forms were characterized by direct counting of sessile cells. A virulence assay was conducted using the Galleria mellonella infection model. Switch variants (crepe and rough) and their revertant counterparts produced higher biofilm biomass (P < 0.05) than the parental strain. Additionally, filamentous forms were enriched among sessile cells of switched strains compared to those observed for sessile cells of the parental strain, with the exception of the revertant of rough. Sessile cells of switched strains showed higher adhesion to polystyrene compared to the parental strain. Sessile cells of the crepe variant and its revertant strain (RC) exhibited higher virulence against G. mellonella larvae than sessile cells of the parental strain. Our findings indicate that switching events in C. tropicalis affect biofilm development and that sessile cells of distinct switch states may exhibit increased adhesion ability and enhanced virulence towards G. mellonella larvae.

Chemical composition, antioxidant, antimicrobial and antibiofilm activities of Vitex gardneriana schauer leaves's essential oil

This study aimed to determinate the chemical composition and evaluate the antimicrobial and antioxidant activity of the essential oil obtained from leaves of V. gardneriana. The Vitex gardneriana leaves's were hydrodistilled to obtain the essential oil and the chemical composition determined by GC/MS analysis. The antimicrobial activities were determined by microdilution method. The activity of essential oil on biofilm was evaluated by quantification of total biomass and enumeration of biofilm-entrapped viable cells. The antioxidant activity was assessed by DPPH free radical assay, ferrous ion chelating assay, ferric-reducing antioxidant power and β-carotene bleaching assay. Furthermore, the essential oil was tested on viability of health human, animal cells and the microcrustacean Artemia sp. The essential oil showed high content of sesquiterpenes and very low content of monoterpenes. Regarding activity on planktonic cells, the essential oil reduced the growth of the all species tested but showed MIC values only to S. aureus (0.31%). In general, the essential oil reduced significantly the biofilm biomass and the number of viable cells of bacteria and yeasts, mainly on biofilm formation. The essential oil showed a potential antioxidant activity, mainly on β-carotene oxidation. Moreover, the essential oil reduced the cell viability of murine fibroblasts but not show viability reduction of human keratinocytes. Furthermore, the oil not show toxicity against the microcrustacean. Thus, the essential oil from V. gardneriana leaves may be considered as an important alternative against biofilms formed by bacteria and yeasts related to infections, as well as a natural antioxidant and non-toxic substance on human cells.

Antibiofilm effect of chlorhexidine-carrier nanosystem based on iron oxide magnetic nanoparticles and chitosan.

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.

Antibacterial activity of a novel antimicrobial peptide [W7]KR12-KAEK derived from KR-12 against Streptococcus mutans planktonic cells and biofilms

The aims of this study were to describe the synthesis of a novel synthetic peptide based on the primary structure of the KR-12 peptide and to evaluate its antimicrobial and anti-biofilm activities against Streptococcus mutans. The antimicrobial effect of KR-12 and [W7]KR12-KAEK was assessed by determining the minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations. The evaluation of anti-biofilm activity was assessed through total biomass quantification, colony forming unit counting and scanning electron microscopy. [W7]KR12-KAEK showed MIC and MBC values ranging from 31.25 to 7.8 and 62.5 to 15.6 μg ml−1, respectively. Furthermore, [W7]KR12-KAEK significantly reduced biofilm biomass (50–100%). Regarding cell viability, [W7]KR12-KAEK showed reductions in the number of CFUs at concentrations ranging from 62.5 to 7.8 μg ml−1 and 500 to 62.5 μg ml−1 with respect to biofilm formation and preformed biofilms, respectively. SEM micrographs of S. mutans treated with [W7]KR12-KAEK suggested damage to the bacterial surface. [W7]KR12-KAEK is demonstrated to be an antimicrobial agent to control microbial biofilms.

Antibacterial effect on mature biofilms of oral streptococci and antioxidant activity of 3β,6β,16β-trihydroxylup-20(29)-ene from Combretum leprosum

This study aimed to evaluate the antioxidant effect and antibacterial activity of the triterpene 3β,6β,16β-trihydroxylup-20(29)-ene (CLF1) isolated from Combretum leprosum leaves on mature biofilms from Streptococcus mutans and S. parasanguinis, both involved in dental caries. In order to determine the bacterial death time curve promoted by CLF1, minimum inhibitory concentration and minimum bactericidal concentration were also assessed. The susceptibility of mature biofilms from both species to CLF1 was investigated through total biomass quantification and enumeration of biofilm-entrapped colony forming units. Moreover, the effect of CLF1 on S. mutans biofilm architecture was investigated by scanning electron microscopy. Additionally, the antioxidant properties of CLF1 were assessed by scavenging of 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) free radical, ferrous ion chelating assay, ferric-reducing antioxidant power, and β-carotene bleaching assay. The minimum inhibitory concentration and minimum bactericidal concentration of CLF1 for both species were 3.9 and 15.6 μg/mL, respectively, and no bacterial growth was detected at 24 h treatment. Moreover, CLF1 reduced the biomass and viability of biofilms from both species. Electron micrographs of S. mutans biofilms confirmed such results and showed damages on bacterial surface. Regarding antioxidant activity, CLF1 showed efficient antioxidant ability in scavenging DPPH radical and inhibiting β-carotene oxidation. In summary, CLF1 should be considered as an effective molecule against infections caused by S. mutans and S. parasanguinis biofilms, as well as a natural antioxidant.

Candida tropicalis biofilm and human epithelium invasion is highly influenced by environmental pH.

The main goal of this study was to investigate the role of pH on Candida tropicalis virulence determinants, namely the ability to form biofilms and to colonize/invade reconstituted human vaginal epithelia. Biofilm formation was evaluated by enumeration of cultivable cells, total biomass quantification and structural analysis by scanning electron microscopy and confocal laser scanning microscopy. Candida tropicalis human vaginal epithelium colonization and invasiveness were examined qualitatively by epifluorescence microscopy and quantitatively by a novel quantitative real-time PCR protocol for Candida quantification in tissues. The results revealed that environmental pH influences C. tropicalis biofilm formation as well as the colonization and potential to invade human epithelium with intensification at neutral and alkaline conditions compared to acidic conditions. For the first time, we have demonstrated that C. tropicalis biofilm formation and invasion is highly influenced by environmental pH.

Effect of Voriconazole on Candida tropicalis Biofilms: Relation with ERG Genes Expression.

Candida tropicalis has emerged as the third most prevalent fungal pathogens and its ability to form biofilms has been considered one of the most important virulence factors, since biofilms represent high tolerance to antifungal agents. However, the mechanisms of C. tropicalis biofilm resistance to antifungals remain poorly understood. Thus, the main aim of this work was to infer about the effect of voriconazole on the formation and control of C. tropicalis biofilms and disclose its relationship with ERG genes' expression. Planktonic cells tolerance of several C. tropicalis clinical isolates to voriconazole was determined through of antifungal susceptibility test, and the effect of this azole against C. tropicalis biofilm formation and pre-formed biofilms was evaluated by cultivable cells determination and total biomass quantification. ERG genes expression was analyzed by quantitative real-time polymerase chain reaction. This work showed that C. tropicalis resistance to voriconazole is strain dependent and that voriconazole was able to partially control biofilm formation, but was unable to eradicate C. tropicalis pre-formed biofilms. Moreover, C. tropicalis biofilms resistance to voriconazole seems to be associated with alterations of sterol content in the cell membrane, resulting in ERG genes overexpression. Voriconazole is unable to control C. tropicalis biofilms, and the overexpression of ERG genes is a possible mechanism of biofilm resistance.

Is biofilm removal properly assessed? Comparison of different quantification methods in a 96-well plate system.

Various methods have been reported to quantify total biofilm or different components of biofilm; however, these methods are often confusedly used, leading to discrepancies and misleading results. In this study, different methods for quantification of biofilm, including those for total biomass, total amount of bacterial cells, viable cell number, and amount of extracellular polymeric substances, were systematically compared in microtiter plates. To evaluate which method is suitable for assessment of biofilm removal and for bacterial killing, biofilm samples were treated with various cleaners possessing removing and/or killing capacities. It was found that most of the methods tested in this study in general exhibited high reproducibility and repeatability. Crystal Violet staining was a simple but reliable method for total biomass quantification. Total bacteria cell numbers could be reliably quantified by the fluorescent DNA-binding dye Acridine Orange. Viable cells could be quantified by either an ATP-based assay or a proliferation assay. Both of these viability methods showed a broad detection range and led to precise measurement. For quantification of proteins in the biofilm, staining with fluorescein isothiocyanate was most suitable. Furthermore, it was revealed that a combination of different methods is required to determine if a cleaner kills or removes biofilm.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7396-9) contains supplementary material, which is available to authorized users.

Biofilm formation by Candida albicans and Streptococcus mutans in the presence of farnesol: a quantitative evaluation

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.

Effect of tyrosol on adhesion of Candida albicans and Candida glabrata to acrylic surfaces.

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.

Effects of fluconazole on Candida glabrata biofilms and its relationship with ABC transporter gene expression

Candida glabrata has emerged as the second most prevalent fungal pathogen and its ability to form biofilms has been considered one of the most important virulence factors, since biofilms present a high tolerance to antifungal agents used in fungal infection treatment. The mechanisms of biofilm tolerance to antifungal agents remain poorly understood. Thus, the aim of this study was to evaluate the effects of fluconazole (FLU) on the formation and control of C. glabrata biofilms and its relation with the expression of genes encoding for ABC transporters, CDR1, SNQ2, and PDR1. For that, minimal inhibitory concentration values for seven C. glabrata strains were determined and the effect of FLU against C. glabrata biofilms was evaluated by total biomass quantification and viable cell enumeration. Matrices from biofilms were analyzed in terms of protein, carbohydrate and DNA content. ABC transporter gene expression was analyzed for quantitative real-time PCR. In addition to the high amounts of proteins and carbohydrates detected in the extracellular matrices in the presence of FLU, this work showed that the overexpression of efflux pumps is a possible mechanism of biofilm tolerance to FLU and this phenomenon alters the structure of C. glabrata biofilms by creating cell clusters.

Antifungal activity of silver nanoparticles in combination with nystatin and chlorhexidine digluconate against Candida albicans and Candida glabrata biofilms

Although silver nanoparticles (SN) have been investigated as an alternative to conventional antifungal drugs in the control of Candida-associated denture stomatitis, the antifungal activity of SN in combination with antifungal drugs against Candida biofilms remains unknown. Therefore, the aim of this study was to evaluate the antifungal efficacy of SN in combination with nystatin (NYT) or chlorhexidine digluconate (CHG) against Candida albicans and Candida glabrata biofilms. The drugs alone or combined with SN were applied on mature Candida biofilms (48 h), and after 24 h of treatment their antibiofilm activities were assessed by total biomass quantification (by crystal violet staining) and colony forming units enumeration. The structure of Candida biofilms was analysed by scanning electron microscopy (SEM) images. The data indicated that SN combined with either NYT or CHG demonstrated synergistic antibiofilm activity, and this activity was dependent on the species and on the drug concentrations used. SEM images showed that some drug combinations were able to disrupt Candida biofilms. The results of this study suggest that the combination of SN with NYT or CHG may have clinical implications in the treatment of denture stomatitis. However, further studies are needed before recommending the use of these drugs safely in clinical situations.

Silver nanoparticles: influence of stabilizing agent and diameter on antifungal activity against Candida albicans and Candida glabrata biofilms

The purpose of this work was to evaluate the size-dependent antifungal activity of different silver nanoparticles (SN) colloidal suspensions against Candida albicans and Candida glabrata mature biofilms. The research presented herein used SN of three different average sizes (5, 10 and 60 nm), which were synthesized by the reduction of silver nitrate through sodium citrate and which were stabilized with ammonia or polyvinylpyrrolidone. Minimal inhibitory concentration (MIC) assays were performed using the microdilution methodology. The antibiofilm activity of SN was determined by total biomass quantification (by crystal violet staining) and colony forming units enumeration. MIC results showed that all SN colloidal suspensions were fungicidal against the tested strains at very low concentrations (0·4-3·3 μg ml(-1) ). With regard to biomass quantification, SN colloidal suspensions were very effective only against C. glabrata biofilms, achieving biomass reductions around 90% at a silver concentration of 108 μg ml(-1) . In general, all SN suspensions promoted significant log(10) reduction of the mean number of cultivable biofilm cells after exposure to silver concentrations at or higher than 108 μg ml(-1) . Moreover, the results showed that the particle size and the type of stabilizing agent used did not interfere in the antifungal activity of SN against Candida biofilms. This study suggests that SN have antifungal therapeutic potential, but further studies are still required namely regarding formulation and delivery means. SN may contribute to the development of new strategies for the improvement of oral health and quality of life particularly of the complete denture wearers.