Glabrata Biofilms Research Articles

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.

New regulators of biofilm development in Candida glabrata

Biofilm formation plays an important role in fungal pathogenesis. In this work, we used a genetic screen in order to identify and characterize genes involved in the formation of biofilms by the opportunistic fungal pathogen Candida glabrata. We identified the Cst6p transcription factor as a negative regulator of the EPA6 gene that encodes an adhesin central to C. glabrata biofilm formation. Analysis of single and double mutant strains showed that Cst6p acts in a pathway independent of the Yak1/Sir4 pathway also known to regulate expression of EPA6 and consequently biofilm formation. In contrast, we showed that the chromatin remodelling Swi/Snf complex positively regulates biofilm formation in C. glabrata. RT-qPCR experiments demonstrated that EPA6 expression, and thus biofilm formation, depends on the integrity of the Sir complex. Finally, we showed that Swi/Snf-dependent regulation of biofilm formation is adhesin-specific.

Could liposomal amphotericin B (L-AMB) lock solutions be useful to inhibit Candida spp. biofilms on silicone biomaterials?

Candida infections associated with catheters remain difficult to manage. Antifungal lock strategies could be a therapeutic option when the device is difficult to remove or in combination with systemic treatment to increase efficacy. This study deals with the antibiofilm potential of liposomal amphotericin B (L-AMB) used as a lock solution to inhibit Candida albicans, Candida glabrata and Candida parapsilosis biofilms in vitro. Biofilms aged 12 h and 5 days were formed on silicone catheters. L-AMB (200 or 1000 mg/L) was added to biofilms and catheters were incubated for 4, 12 or 24 h at 37°C. L-AMB was then removed by washing. The metabolic activity of yeasts was assessed by the XTT method up to 48 h after the end of the locks to evaluate the persistence of the antibiofilm activity. Controls without antifungal were used as references to calculate the inhibition percentages induced by L-AMB lock solutions. L-AMB (200 and 1000 mg/L) inhibited, for up to 48 h, C. albicans and C. glabrata biofilms by >70%, regardless of the lock duration. The activity of L-AMB (200 mg/L) against C. parapsilosis mature biofilms was lower and less sustained, especially for 4 h locks. L-AMB (1000 mg/L) lock solutions strongly inhibited Candida spp. in young and mature biofilms for up to 48 h after the end of the lock. However, overall eradication of the biofilm was not obtained using 1000 mg/L L-AMB as a single lock. These results suggest the usefulness of systemic treatment combined with an L-AMB lock to control Candida spp. biofilms associated with catheters.

Effect of Amphotericin B Alone or in Combination with Rifampicin or Clarithromycin Against Candida Species Biofilms

Effectiveness of amphotericin B alone or in combination with rifampicin or clarithromycin on the killing of Candida species biofilms was investigated in vitro. Amphotericin B was assayed at 0.005 to 10 mg/ml. Rifampin and clarithromycin were assayed at 10 mg/ml. We studied 7 Candida albicans, 3 Candida parapsilosis, 3 Candida glabrata, 3 Candida krusei and 2 Candida tropicalis strains. Biofilms were developed in 96-well, flat-bottomed microtiter plates for 48 hours. A synergistic effect between amphotericin B and clarithromycin was demonstrated against 66.6% of C. parapsilosis, 66.6% of C. glabrata, and 42.8% of C. albicans biofilms. A synergistic effect between amphotericin B and rifampin was demonstrated against 66.6% of C. parapsilosis, 42.8% of C. albicans, and 33.3% of C. glabrata biofilms. No synergistic effect was observed against C. krusei or C. tropicalis biofilms with any of the combinations. Rifampin or clarithromycin alone did not exert any effect on Candida species biofilms. Rifampin or clarithromycin combinations with amphotericin B might be of interest in the treatment of Candida biofilm-related infections.

Identification and Differential Gene Expression of Adhesin-Like Wall Proteins in Candida glabrata Biofilms

An important initial step in biofilm development and subsequent establishment of fungal infections by the human pathogen Candida glabrata is adherence to a surface. Adherence is mediated through a large number of differentially regulated cell wall-bound adhesins. The fungus can modify the incorporation of adhesins in the cell wall allowing crucial adaptations to new environments. In this study, expression and cell wall incorporation of C. glabrata adhesins were evaluated in biofilms cultured in two different media: YPD and a semi-defined medium SdmYg. Tandem mass spectrometry of isolated C. glabrata cell walls identified 22 proteins including six adhesins: the novel adhesins Awp5 and Awp6, Epa3 and the previously identified adhesins Epa6, Awp2 and Awp4. Regulation of expression of these and other relevant adhesin genes was investigated using real-time qPCR analysis. For most adhesin genes, significant up-regulation was observed in biofilms in at least one of the culturing media. However, this was not the case for EPA6 and AWP2, which is consistent with their gene products already being abundantly present in planktonic cultures grown in YPD medium. Furthermore, most of the adhesin genes tested also show medium-dependent differential regulation. These results underline the idea that many adhesins in C. glabrata are involved in biofilm formation and that their expression is tightly regulated and dependent on environmental conditions and growth phase. This may contribute to its potential to form resilient biofilms and cause infection in various host tissues.

Silver colloidal nanoparticles: antifungal effect against adhered cells and biofilms of Candida albicans and Candida glabrata

The aim of this study was to evaluate the effect of silver nanoparticles (SN) against Candida albicans and Candida glabrata adhered cells and biofilms. SN (average diameter 5 nm) were synthesized by silver nitrate reduction with sodium citrate and stabilized with ammonia. Minimal inhibitory concentration (MIC) tests were performed for C. albicans (n = 2) and C. glabrata (n = 2) grown in suspension following the Clinical Laboratory Standards Institute microbroth dilution method. SN were applied to adhered cells (2 h) or biofilms (48 h) and after 24 h of contact their effect was assessed by enumeration of colony forming units (CFUs) and quantification of total biomass (by crystal violet staining). The MIC results showed that SN were fungicidal against all strains tested at very low concentrations (0.4–3.3 μg ml−1). Furthermore, SN were more effective in reducing biofilm biomass when applied to adhered cells (2 h) than to pre-formed biofilms (48 h), with the exception of C. glabrata ATCC, which in both cases showed a reduction ∼90%. Regarding cell viability, SN were highly effective on adhered C. glabrata and respective biofilms. On C. albicans the effect was not so evident but there was also a reduction in the number of viable biofilm cells. In summary, SN may have the potential to be an effective alternative to conventional antifungal agents for future therapies in Candida-associated denture stomatitis.

Detailed comparison of Candida albicans and Candida glabrata biofilms under different conditions and their susceptibility to caspofungin and anidulafungin

Candida biofilm development can be influenced by diverse factors such as substrate, culture medium, carbohydrate source and pH. We have analysed biofilm formation of Candida albicans SC5314 and Candida glabrata ATCC 2001 wild-type strains in the presence of different media (RPMI 1640 versus YNB) and using different pH values (pH 5.6 or 7.0). We determined adhesion and biofilm formation on polystyrene, changes in the expression of adhesin genes during these processes and the susceptibility of mature biofilms to echinocandins. Biofilms formed on polystyrene by both Candida species proved to be influenced strongly by the composition of the medium rather than pH. C. albicans and C. glabrata formed thicker biofilms in RPMI 1640 medium, whereas in YNB medium, both species manifested adhesion rather than characteristic multilayer biofilm architecture. The stimulated biofilm formation in RPMI 1640 medium at pH 7.0 corroborated positively with increased expression of adhesin genes, essential to biofilm formation in vitro, including ALS3 and EAP1 in C. albicans and EPA6 in C. glabrata. The thicker biofilms grown in RPMI 1640 medium were more tolerant to caspofungin and anidulafungin than YNB-grown biofilms. We also observed that mature C. glabrata biofilms were less susceptible in RPMI 1640 medium to echinocandins than C. albicans biofilms. Environmental conditions, i.e. medium and pH, can significantly affect not only biofilm architecture, but also the expression profile of several genes involved during the different stages of biofilm development. In addition, growth conditions may also influence the antifungal-susceptibility profile of fungal populations within biofilm structures. Therefore, before designing any experimental biofilm set-up, it is important to consider the potential influence of external environmental factors on Candida biofilm development.

Bioactivity and cellular structure of Candida albicans and Candida glabrata biofilms grown in the presence of fluconazole

ObjectiveThe aim of this study was to evaluate whether fluconazole (FLZ) could affect the bioactivity and cellular structure of Candida albicans or Candida glabrata biofilms grown in the presence of FLZ. Materials and methodsTokens were fabricated using poly(methylmethacrylate) resin (PMMA) in a hot water bath. Salivary pellicles were formed on the PMMA surface, and biofilms of a reference strain and two clinical isolates of C. albicans (ATCC 90028, P01 and P34) and C. glabrata (ATCC 2001, P11 and P31) were developed for a period of 48h. Control and experimental groups were formed. FLZ at the bioavailable concentration in saliva (2.56μg/mL) was added to the medium of the experimental group. The culture mediums of the control and experimental groups were changed after 24h. The bioactivities of the biofilms were evaluated using an XTT reduction colorimetric assay. The cellular structure was analysed by confocal scanning laser microscopy and by transmission electron microscopy. The data were analysed by the independent sample Student's t-test, with the significance level set at 5%. ResultsThe presence of FLZ decreased the bioactivity of all C. albicans biofilms (p<0.001), however, it did not change the cellular structure of C. albicans P34. Regarding the C. glabrata biofilm bioactivity and structure, no statistically significant differences were found between the control and experimental groups. ConclusionFLZ, at the bioavailable concentration present in saliva, interferes with the development of C. albicans biofilms, but does not interfere with the development of C. glabrata biofilms.

Possible role of azole and echinocandin lock solutions in the control of Candida biofilms associated with silicone

Until now, management of candidiasis related to implanted devices has remained problematic. The aim of this study was to investigate antifungal lock strategies against Candida albicans and Candida glabrata biofilms in vitro. Three antifungal agents were used against eight C. albicans and six C. glabrata clinical strains isolated from infected catheters. Caspofungin and micafungin, both echinocandins, as well as the azole posaconazole were tested. An in vitro model of Candida biofilm on 100% silicone catheters was used. Efficacy of the antifungal lock was tested against biofilms aged 12h and 5 days following exposure to caspofungin (5mg/L and 25mg/L), micafungin (5mg/L and 15mg/L) and posaconazole (10mg/L) for 12h. Persistence of antibiofilm activity was investigated 1–3 days following drug elimination. Antifungal lock was considered effective in the event of a significant decrease (P<0.001) in the metabolic activity of the biofilm yeast. The results showed that micafungin had significant inhibitory effectiveness against young and mature C. albicans and C. glabrata biofilms. Moreover, this activity appeared to persist for up to 3 days. Caspofungin displayed similar activity against all C. albicans biofilms, but the activity was less persistent for C. glabrata biofilms. Posaconazole was less effective against C. albicans biofilms, but its activity was sustained. Echinocandin lock therapy could significantly enhance the management of candidiasis in patients with indwelling catheters by combating biofilms and enabling device maintenance in situ.

Efficacy of denture cleansers on Candida spp. biofilm formed on polyamide and polymethyl methacrylate resins

Although new materials have emerged as options to fabricate removable dental prostheses, the development of Candida biofilms on these materials and the effectiveness of methods to control these pathogenic biofilms are poorly understood. The purpose of this study was to evaluate the efficacy of denture cleansers on Candida single- and dual-species biofilms formed on polyamide resin. Polymethyl methacrylate (PMMA) resin (Acron MC) and polyamide resin (Flexite M.P.) specimens (n=116) were prepared, and their surface roughness was standardized (0.34 ±0.02 μm). Surface free energy (SFE) was measured for some specimens (n=20 per resin), while the remainder were randomly divided by lottery into 24 groups (n=8) for biofilm assay. C. albicans and/or C. glabrata biofilm was formed for 72 hours, and then specimens were treated with an enzymatic cleanser solution (Polident 3 Minutes), a cleanser solution (Corega Tabs), or 0.5% sodium hypochlorite (NaOCl) solution. Water served as the negative control. Remaining adherent microorganisms were removed from the treated specimens by ultrasonic waves, and colony-forming units (CFU) of each microorganism were calculated. SFE data were analyzed by 1-way ANOVA, and Candida species data were analyzed by 3-way ANOVA followed by the Tukey-Kramer test (P=.05). All tested biofilms displayed significantly higher growth on polyamide resin (P<.001), which presented the lowest SFE. Denture cleansers significantly decreased Candida levels; however, the 0.5% NaOCl solution was the only effective cleanser. C. glabrata revealed significantly higher CFU counts under all experimental conditions (P<.001). The highest Candida spp. biofilm growth was shown to occur on polyamide resin when compared with PMMA. Denture cleansers were able to remove Candida spp. biofilm formed on both denture base resins.

Long-term efficacy of denture cleansers in preventing Candida spp. biofilm recolonization on liner surface

This study evaluated the long-term efficacy of denture cleansers against Candida spp. biofilm recolonization on liner surface. Specimens were fabricated of a poly(methyl methacrylate)-based denture liner and had their surface roughness evaluated at baseline and after cleansing treatments. C. albicans or C. glabrata biofilms were formed on liner surface for 48 h, and then the specimens were randomly assigned to one of cleaning treatments: two alkaline peroxides (soaking for 3 or 15 min), 0.5% sodium hypochlorite (10 min) or distilled water (control; 15 min). After the treatments, the specimens were sonicated to disrupt the biofilm, and residual cells were counted (cell/mL). Long-term effectiveness of the cleaning processes was determined by submitting a set of cleaned specimens to biofilm growth conditions for 48 h followed by estimation of cell counts. The topography of specimens after cleaning treatments was analyzed by SEM. Data were analyzed by ANOVA and Tukey's test (α; = 0.05). Results of cell count estimation showed significant differences in cleanliness among the treatments (p < 0.001), and it could be observed by SEM. However, no significant difference (p > 0.05) was observed among the Candida species regarding the recolonization condition. Alkaline denture cleansers showed similar cleaning performance and both differed from the control (p < 0.001). Sodium hypochlorite was the only treatment that removed biofilm efficiently, since no viable cells were found after its use. In conclusion, alkaline peroxide denture cleansers were not effective in removing Candida spp. biofilm from denture liner surfaces and preventing biofilm recolonization.

In Vitro Biofilm Activity of Non-Candida albicans Candida Species

Candidosis has been attributed to C. albicans; however, infections caused by non-Candida albicans Candida (NCAC) species are increasingly being recognised. The ability of Candida to grow as a biofilm is an important feature that promotes both infection and persistence in the host. The biofilms' activity is significant since high activity might be associated with enhanced expression of putative virulence factors, whilst in contrast low activity has previously been suggested as a mechanism for resistance of biofilm cells to antimicrobials. The aim of this study was to determine the metabolic activity of in vitro biofilms formed by different clinical isolates of NCAC species. The in situ total metabolic activity of C. parapsilosis, C. tropicalis and C. glabrata biofilms was determined using 2,3-(2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay, and the number of cultivable cells was also established by CFU (colony forming unit) counts. The biofilm structure was assessed by scanning electron microscopy (SEM). Results showed that total biofilm metabolic activity was species and strain dependent. C. glabrata exhibited the lowest biofilm metabolic activity despite having the highest number of biofilm cultivable cells. Similarly, the metabolic activity of resuspended C. glabrata biofilm and planktonic cells was lower than that of the other species. This study demonstrates the existence of intrinsic activity differences amongst NCAC species, which could have important implications in terms of species relative virulence. Furthermore, the absence of an obvious correlation, between cultivable cells number and total biofilm activity, raises the question about which parameter is the most appropriate for the in vitro assessment of biofilms and their potential clinical significance.

Proteomics of drug resistance in Candida glabrata biofilms

Candida glabrata is a fungal pathogen that causes a variety of mucosal and systemic infections among compromised patient populations with higher mortality rates. Previous studies have shown that biofilm mode of the growth of the fungus is highly resistant to antifungal agents compared with the free-floating or planktonic mode of growth. Therefore, in the present study, we used 2-D DIGE to evaluate the differential proteomic profiles of C. glabrata under planktonic and biofilm modes of growth. Candida glabrata biofilms were developed on polystyrene surfaces and age-matched planktonic cultures were obtained in parallel. Initially, biofilm architecture, viability, and antifungal susceptibility were evaluated. Differentially expressed proteins more than 1.5-fold in DIGE analysis were subjected to MS/MS. The transcriptomic regulation of these biomarkers was evaluated by quantitative real-time PCR. Candida glabrata biofilms were highly resistant to the antifungals and biocides compared with the planktonic mode of growth. Candida glabrata biofilm proteome when compared with its planktonic proteome showed upregulation of stress response proteins, while glycolysis enzymes were downregulated. Similar trend could be observed at transcriptomic level. In conclusion, C. glabrata biofilms possess higher amount of stress response proteins, which may potentially contribute to the higher antifungal resistance seen in C. glabrata biofilms.

In vitro Evaluation of Antibiotic Lock Technique for the Treatment ofCandida albicans,C. glabrata, andC. tropicalisBiofilms

Candidaemia associated with intravascular catheter-associated infections is of great concern due to the resulting high morbidity and mortality. The antibiotic lock technique (ALT) was previously introduced to treat catheter-associated bacterial infections without removal of catheter. So far, the efficacy of ALT against Candida infections has not been rigorously evaluated. We investigated in vitro activity of ALT against Candida biofilms formed by C. albicans, C. glabrata, and C. tropicalis using five antifungal agents (caspofungin, amphotericin B, itraconazole, fluconazole, and voriconazole). The effectiveness of antifungal treatment was assayed by monitoring viable cell counts after exposure to 1 mg/mL solutions of each antibiotic. Fluconazole, itraconazole, and voriconazole eliminated detectable viability in the biofilms of all Candida species within 7, 10, and 14 days, respectively, while caspofungin and amphotericin B did not completely kill fungi in C. albicans and C. glabrata biofilms within 14 days. For C. tropicalis biofilm, caspofungin lock achieved eradication more rapidly than amphotericin B and three azoles. Our study suggests that azoles may be useful ALT agents in the treatment of catheter-related candidemia.

Architectural analysis, viability assessment and growth kinetics of Candida albicans and Candida glabrata biofilms

The human fungal pathogen Candida is able to form biofilms in almost all the medical devices in current use. Indeed, biofilm formation is a major virulence attribute of microorganisms and account for a majority of human infections. Therefore, understanding processes appertaining to biofilm development is an important prerequisite for devising new strategies to prevent or eradicate biofilm-related infections. In the present study we used an array of both conventional and novel analytical tools to obtain a comprehensive view of Candida biofilm development. Enumeration of colony forming units, colorimetric (XTT) assay, Scanning Electron Microscopy (SEM) and novel Confocal Laser Scanning Microscopy (CLSM) coupled with COMSTAT software analyses were utilised to evaluate growth kinetics; architecture and viability of biofilms of a reference (ATCC) and a clinical strain each of two Candida species, C. albicans and C. glabrata. Biofilm growth kinetics on a polystyrene substrate was evaluated from the initial adhesion step (1.5 h) up to 72 h. These analyses revealed substantial inter- and intra-species differences in temporal organisation of Candida biofilm architecture, spatiality and cellular viability, while reaching maturity within a period of 48 h, on a polystyrene substrate. There were substantial differences in the growth kinetics upon methodology, although general trend seemed to be the same. Detailed architectural analysis provided by COMSTAT software corroborated the SEM and CSLM views. These analyses may provide a strong foundation for down stream molecular work of fungal biofilms.

Expression ofCgCDR1,CgCDR2, andCgERG11inCandida glabratabiofilms formed by bloodstream isolates

Many studies have shown that Candida biofilms are highly resistant to fluconazole. The mechanisms of acquired fluconazole resistance of Candida glabrata in non-biofilms have been elucidated, but the relevance of them relative to C. glabrata in biofilms are unknown. We examined by real-time PCR the expression of CgCDR1, CgCDR2, and CgERG11 of four bloodstream isolates of C. glabrata during the early (6 h), intermediate (15 h), and mature (48 h) phases of biofilm development. We found high levels of biofilm formation in vitro. At 6 and 15 h, the biofilms exhibited, in comparison to planktonic cells, approximately 1.5- and 3.3-fold upregulation of CgCDR1 and 0.5- and 3.1-fold upregulation of CgCDR2, respectively. However, at 48 h, neither gene was upregulated. In comparison, the expression of CgERG11 did not significantly increase during any of the three phases. Our results indicate a temporary increase in the expression of both CgCDR1 and CgCDR2 during the intermediate phase of C. glabrata biofilm development. In addition, the data collected at 48 h suggest that CgCDR1, CgCDR2, and CgERG11 may not play a role in the azole resistance of C. glabrata in mature biofilms.

Biofilms of non-Candida albicans Candidaspecies: quantification, structure and matrix composition

Most cases of candidiasis have been attributed to C. albicans, but recently, non- Candida albicans Candida (NCAC) species have been identified as common pathogens. The ability of Candida species to form biofilms has important clinical repercussions due to their increased resistance to antifungal therapy and the ability of yeast cells within the biofilms to withstand host immune defenses. Given this clinical importance of the biofilm growth form, the aim of this study was to characterize biofilms produced by three NCAC species, namely C. parapsilosis, C. tropicalis and C. glabrata. The biofilm forming ability of clinical isolates of C. parapsilosis, C. tropicalis and C. glabrata recovered from different sources, was evaluated by crystal violet staining. The structure and morphological characteristics of the biofilms were also assessed by scanning electron microscopy and the biofilm matrix composition analyzed for protein and carbohydrate content. All NCAC species were able to form biofilms although these were less extensive for C. glabrata compared with C. parapsilosis and C. tropicalis. It was evident that C. parapsilosis biofilm production was highly strain dependent, a feature not evident with C. glabrata and C. tropicalis. Scanning electron microscopy revealed structural differences for biofilms with respect to cell morphology and spatial arrangement. Candida parapsilosis biofilm matrices had large amounts of carbohydrate with less protein. Conversely, matrices extracted from C. tropicalis biofilms had low amounts of carbohydrate and protein. Interestingly, C. glabrata biofilm matrix was high in both protein and carbohydrate content. The present work demonstrates that biofilm forming ability, structure and matrix composition are highly species dependent with additional strain variability occurring with C. parapsilosis.

ABSTRACTS OF POSTER PRESENTATIONS

s of poster presentations 44 P010 Antifungal agents induces alteration in the matrix composition of candida glabrata biofilms Rodrigues, Celia (Minho University, Departement of Biological Engineering Department, Braga ); Fonseca, Elza (Minho University, Departement of Biological Engineering Department, Braga , AUT); Goncalves, Bruna (Minho University, Departement of Biological Engineering Department, Braga ); Bogas, Diana (Minho University, Departement of Biological Engineering Department, Braga ); Silva, Sonia (Minho University, Departement of Biological Engineering Department, Braga ); Azeredo, Joana (Minho University, Departement of Biological Engineering Department, Braga ); Henriques, Mariana (Minho University, Departement of Biological Engineering Department, Braga ) Candida glabrata has emerged as the second most prevalent pathogen, after Candida albicans, in mucosal and invasive fungal infection. Its ability to form biofilms has been considered one of the most important virulence factors, since they present a high resistance to antifungal agents used in fungal infections treatment. Moreover, there is a lack of information about the physiological response of C. glabrata biofilms to antifungal agents. Thus, the aim of this study was to evaluate the effect of different antifungal agents on C. glabrata biofilm composition and the influence in related resistance genes expression. For that C. glabrata biofilms were formed in the presence of fluconazole (Flu), voriconazole (Vrz) and amphotericin B (AmB). Biofilm matrix composition was evaluated in terms of polysaccharides, proteins and ergosterol and ERG genes expression was also assessed. As expected C. glabrata biofilms are more resistant to Flu, Vrz and AmB than planktonic cells. . Although in a strain dependent manner, polysaccharides were increased in the presence of the antifungals, in opposition to proteins, which decreased in the presence of AmB and Vrz. Due to the interaction of these agents with ergosterol, even in different ways, we evaluated, for the first time, the presence of this compound in the extracelluar matrix. It was noticed that ergosterol was, in fact, present in all the matrices and in general it increased with the presence of the drugs. Therefore, there is an obvious answer of biofilm cells to the stress induced by the different agents, that caused and alteration of matrix composition. In order to determine if the increased concentration of ergosterol in the biofilm matrix was caused by an up-regulation of proteins responsible for ergosterol synthesis, ERG expression was evaluated.. Although ERG gene expression was very strain dependent is was possible to verify that some genes, as ERG11 and ERG6, were upregulated in biofilm cells. Interestingly one strain was unable to express ERG genes when grown in the presence of Vrz. It was then possible to conclude that biofilm cells upon exposure to antifungal agents overexpress ERG genes, which seems to contribute to an increase in ergosterol concentration in the biofilm matrix.

Mixed Candida albicans and Candida glabrata populations associated with the pathogenesis of denture stomatitis

Oral yeasts are an important component of the resident microbial ecology of the oral cavity, but they are also associated with various forms of oral candidosis, such as denture stomatitis. Although Candida albicans is the predominant oral fungal pathogen, other species may also play an integral role in pathogenesis. The aim of this study was to examine the mycological ecology in patients with denture stomatitis, using an improved sampling technique, to determine whether species diversity and species quantity were related to oral pathology. Thirty-seven patients attending the Glasgow Dental Hospital were enrolled in this study following informed consent. A full clinical history was obtained, including details of their oral hygiene practices and the levels of erythema based on Newton's classification scale. Oral rinse, denture sonicate, and swab samples were taken, which were processed for quantitative and qualitative analysis of oral yeasts. The proportion of patients with no inflammation or Newton's Types I, II, and III were 31, 33, 25, and 14%, respectively. Denture sonication was a superior sampling procedure, with statistically greater quantities of yeasts isolated using this methodology (P < 0.01). The predominant oral yeasts isolated were C. albicans (75%) and Candida glabrata (30%), which were isolated in higher proportions in patients with the highest grades of inflammation (100 and 80%), and in combination from 80% of these patients. This study has demonstrated that mixed C. albicans and C. glabrata biofilms may play an important role in the pathogenesis associated with severe inflammation in denture wearers.

In vitro activity of terpenes against Candida biofilms

The antibiofilm activity of 10 terpenes was tested in vitro against three Candida species by 24-h treatment of biofilms aged 1–5 days. Treatment of 24-h-old Candida albicans biofilms with carvacrol, geraniol or thymol (0.06%) resulted in >80% inhibition. Carvacrol (0.03%) inhibition was ≥75% independent of the age of the C. albicans biofilm. Carvacrol (0.125%) inhibition was >75% against Candida glabrata and Candida parapsilosis biofilms. Geraniol (≥0.125%) and thymol (0.06% or 0.125%) inhibition was >75% against C. parapsilosis biofilms whatever their age. This study demonstrates the antibiofilm activity of terpenes and points out the exceptional efficiency of carvacrol, geraniol and thymol, which could represent candidates in the treatment of candidiasis associated with medical devices.