Metal resistance in Candida biofilms

Yeasts are often successful in metal-polluted environments; therefore, the ability of biofilm and planktonic cell Candida tropicalis to endure metal toxicity was investigated. Fifteen water-soluble metal ions, chosen to represent groups 6A to 6B of the periodic table, were tested against this organism. With in vitro exposures as long as 24 h, biofilms were up to 65 times more tolerant to killing by metals than corresponding planktonic cultures. Of the most toxic heavy metals tested, only very high concentrations of Hg2+, CrO4 (2-) or Cu2+ killed surface-adherent Candida. Metal-chelator precipitates could be formed in biofilms following exposure to the heavy metals Cu2+ and Ni2+. This suggests that Candida biofilms may adsorb metal cations from their surroundings and that sequestration in the extracellular matrix may contribute to resistance. We concluded that biofilm formation may be a strategy for metal resistance and/or tolerance in yeasts.     

Inhibition on <Emphasis Type="Italic">Candida albicans</Emphasis> biofilm formation using divalent cation chelators (EDTA)

Candida albicans can readily form biofilms on both inanimate and biological surfaces. In this study we investigated a means of inhibiting biofilm formation using EDTA (Ethylenediaminetetra-acetic acid), a divalent cation chelating agent, which has been shown to affect C. albicans filamentation. Candida albicans biofilms were formed in 96-well microtitre plates. Cells were allowed to adhere for 1, 2, and 4 h at 37°C, washed in PBS, and then treated with different concentrations of EDTA (0, 2.5, 25, and 250 mM). EDTA was also added to the standardized suspension prior to adding to the microtiter plate and to a preformed 24 h biofilm. All plates were then incubated at 37°C for an additional 24 h to allow for biofilm formation. The extent and characteristics of biofilm formation were then microscopically assessed and with a semi-quantitative colorimetric technique based on the use of an XTT-reduction assay. Northern blot analysis of the hyphal wall protein (HWP1) expression was also monitored in planktonic and biofilm cells treated with EDTA. Microscopic analysis and colorimetric readings revealed that filamentation and biofilm formation were inhibited by EDTA in a concentration dependant manner. However, preformed biofilms were minimally affected by EDTA (maximum of 31% reduction at 250 mM). The HWP1 gene expression was reduced in EDTA-treated planktonic and biofilm samples. These results indicate that EDTA inhibits C. albicans biofilm formation are most likely through its inhibitory effect on filamentation and indicates the potential therapeutic effects of EDTA. This compound may serve a non-toxic means of preventing biofilm formation on infections with a C. albicans biofilm etiology.     

Candida biofilms on implanted biomaterials: a clinically significant problem

In recent years there has been an increasing appreciation that microbial biofilms are ubiquitous, which has resulted in a number of studies on infectious diseases from a biofilm perspective. Biofilms are defined as structured microbial communities that are attached to a surface and encased in a matrix of exopolymeric material. A wide range of biomaterials used in clinical practice have been shown to support colonization and biofilm formation by Candida spp., and the increase in Candida infections in the last decades has almost paralleled the increase and widespread use of a broad range of medical implant devices, mainly in populations with impaired host defenses. Formation of Candida biofilms has important clinical repercussions because of their increased resistance to antifungal therapy and the ability of cells within biofilms to withstand host immune defenses. Further recognition and understanding of the role of Candida biofilms in human infection should help in the clinical management of these recalcitrant infections.     

Metal ions may suppress or enhance cellular differentiation in Candida albicans and Candida tropicalis biofilms

Candida albicans and Candida tropicalis are polymorphic fungi that develop antimicrobial-resistant biofilm communities that are characterized by multiple cell morphotypes. This study investigated cell type interconversion and drug and metal resistance as well as community organization in biofilms of these microorganisms that were exposed to metal ions. To study this, Candida biofilms were grown either in microtiter plates containing gradient arrays of metal ions or in the Calgary Biofilm Device for high-throughput susceptibility testing. Biofilm formation and antifungal resistance were evaluated by viable cell counts, tetrazolium salt reduction, light microscopy, and confocal laser scanning microscopy in conjunction with three-dimensional visualization. We discovered that subinhibitory concentrations of certain metal ions (CrO(4)(2-), Co(2+), Cu(2+), Ag(+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), AsO(2)(-), and SeO(3)(2-)) caused changes in biofilm structure by blocking or eliciting the transition between yeast and hyphal cell types. Four distinct biofilm community structure types were discerned from these data, which were designated "domed," "layer cake," "flat," and "mycelial." This study suggests that Candida biofilm populations may respond to metal ions to form cell-cell and solid-surface-attached assemblages with distinct patterns of cellular differentiation.     

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.     

Diclofenac exhibits synergism with azoles against planktonic cells and biofilms of Candida tropicalis

Abstract

This study aimed to evaluate the effect of diclofenac on minimum inhibitory concentrations of antifungals against planktonic cells and biofilms of Candida tropicalis. Susceptibility testing of planktonic cells was evaluated using the broth microdilution assay and checkerboard method. Biofilm formation by C. tropicalis in the presence of diclofenac, alone or in combination with antifungals, was also evaluated, and scanning electron microscope (SEM) and confocal microscope (CLSM) analyses were performed. Diclofenac showed an MIC of 1024?μg?ml^?1 against planktonic cells. The MICs of fluconazole and voriconazole against azole-resistant isolates were reduced 8- to 32-fold and 16- to 256-fold, respectively, when in combination with diclofenac. When in combination with fluconazole or voriconazole, diclofenac reduced the antifungal concentration necessary to inhibit C. tropicalis biofilm formation. In conclusion, diclofenac presents synergism with fluconazole and voriconazole against resistant C. tropicalis strains and improves the activity of these azole drugs against biofilm formation.     

In vitro activity of amphotericin B and anidulafungin against Candida spp. biofilms

Invasive infections caused by Candida spp. are increasing worldwide and are becoming an important cause of morbidity and mortality in immunocompromised patients. A large number of manifestations of candidiasis are associated with the formation of biofilms on inert or biological surfaces. Candida spp. biofilms are recalcitrant to treatment with conventional antifungal therapies. The aim of this study was dual 1) to determine the prevalence of biofilm producers among clinical isolates from catheter (16 C. albicans ) and blood culture (2 C. albicans and 30 C. tropicalis), and 2) to determine the activity of amphotericin B and anidulafungin against C. albicans and C. tropicalis biofilms of 24 and 48 hours of maturation. Biofilms were developed using a 96-well microtitre plate model and production and activity of antifungal agents against biofilms were determined by the tetrazolium (XTT) reduction assay. Of catheter and blood isolates, 62.5 and 56.25%, respectively, produced biofilms. By species, 68.42% of C. albicans and 53.33% of C. tropicalis were biofilm producers. C. albicans biofilms showed more resistance to amphotericin B and anidulafungin than their planktonic counterparts. Complete killing of biofilms was never achieved, even at the highest concentrations of the drugs tested. Anidulafungin displayed more activity than amphotericin B against C. albicans biofilms of 24 hours of maturation (GM MIC 0.354 vs. 0.686 microg/ml), but against C. tropicalis biofilms amphotericin B was more active (GM MIC 11.285 vs. 0.476 microg/ml). In contrast, against biofilms with 48 hours maturation, amphotericin B was more active against both species.     

Activity of anidulafungin against Candida biofilms

Currently, no standardized method to study the in vitro activity of antifungal agents on biofilms is available, thus, the comparison among different authors is difficult. The studies discussed in this review use the XTT reduction to measure the activity of antifungals on biofilms of 24 h of maturation. To date, biofilm anidulafungin MICs of 47 isolates of Candida spp. (25 Candida albicans, 16 Candida tropicalis, 5 Candida dubliniensis and 1 Candida parapsilosis) have been published. The geometric mean MIC of anidulafungin on biofilms of Candida spp. is of 1.18 microg/ml. Against isolates of species with great capacity of biofilm formation, the geometric mean MIC is 0.325 (C. albicans), 2 (C. parapsilosis) and 0.5 microg/ml (C. dubliniensis). No echinocandin has activity on C. tropicalis biofilms. In addition, anidulafungin can be used for lock therapy of catheters since it is the echinocandin with the least in vitro paradoxical effect.     

肉桂醛对根管内白色念珠菌生物膜作用的体外研究

目的研究肉桂醛对体外白色念珠菌生物膜的影响。方法采用琼脂扩散法进行肉桂醛和洗必泰对白色念珠菌敏感性的比较;MTT法评价肉桂醛对白色念珠菌生物膜及细胞黏附的影响。结果 2 048μg/mL肉桂醛与2%洗必泰抑菌环直径比较差异无统计学意义(P>0.05);4 096μg/mL肉桂醛对白色念珠菌生物膜的抑菌率达93.02%;不同浓度肉桂醛对60、90和120 min的白色念珠菌细胞粘附都具有抑制作用。结论肉桂醛对体外白色念珠菌生物膜有较明显的抑制作用。     

In vitro activity of eugenol against <Emphasis Type="Italic">Candida albicans</Emphasis> biofilms

Most manifestations of candidiasis are associated with biofilm formation occurring on the surfaces of host tissues and medical devices. Candida albicans is the most frequently isolated causative pathogen of candidiasis, and the biofilms display significantly increased levels of resistance to the conventional antifungal agents. Eugenol, the major phenolic component of clove essential oil, possesses potent antifungal activity. The aim of this study was to investigate the effects of eugenol on preformed biofilms, adherent cells, subsequent biofilm formation and cell morphogenesis of C. albicans. Eugenol displayed in vitro activity against C. albicans cells within biofilms, when MIC₅₀ for sessile cells was 500 mg/L. C. albicans adherent cell populations (after 0, 1, 2 and 4 h of adherence) were treated with various concentrations of eugenol (0, 20, 200 and 2,000 mg/L). The extent of subsequent biofilm formation were then assessed with the tetrazolium salt reduction assay. Effect of eugenol on morphogenesis of C. albicans cells was observed by scanning electron microscopy (SEM). The results indicated that the effect of eugenol on adherent cells and subsequent biofilm formation was dependent on the initial adherence time and the concentration of this compound, and that eugenol can inhibit filamentous growth of C. albicans cells. In addition, using human erythrocytes, eugenol showed low hemolytic activity. These results indicated that eugenol displayed potent activity against C. albicans biofilms in vitro with low cytotoxicity and therefore has potential therapeutic implication for biofilm-associated candidal infections.     

Metal Ions May Suppress or Enhance Cellular Differentiation in Candida albicans and Candida tropicalis Biofilms

Candida albicans and Candida tropicalis are polymorphic fungi that develop antimicrobial-resistant biofilm communities that are characterized by multiple cell morphotypes. This study investigated cell type interconversion and drug and metal resistance as well as community organization in biofilms of these microorganisms that were exposed to metal ions. To study this, Candida biofilms were grown either in microtiter plates containing gradient arrays of metal ions or in the Calgary Biofilm Device for high-throughput susceptibility testing. Biofilm formation and antifungal resistance were evaluated by viable cell counts, tetrazolium salt reduction, light microscopy, and confocal laser scanning microscopy in conjunction with three-dimensional visualization. We discovered that subinhibitory concentrations of certain metal ions (CrO₄²⁻, Co²⁺, Cu²⁺, Ag⁺, Zn²⁺, Cd²⁺, Hg²⁺, Pb²⁺, AsO₂⁻, and SeO₃²⁻) caused changes in biofilm structure by blocking or eliciting the transition between yeast and hyphal cell types. Four distinct biofilm community structure types were discerned from these data, which were designated “domed,” “layer cake,” “flat,” and “mycelial.” This study suggests that Candida biofilm populations may respond to metal ions to form cell-cell and solid-surface-attached assemblages with distinct patterns of cellular differentiation.     

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.     

Effects of chelating agent and environmental stresses on microbial biofilms: relevance to clinical microbiology

Aims: To determine the effect of pH, temperature, desiccation, ethylenediaminetetraacetic acid (EDTA) and desferrioxamine B (DFO) on Panton‐Valentine leukocidin‐positive community acquired methicillin‐susceptible Staphylococcus aureus (PVL +ve CA‐MSSA) biofilm formation. Methods and Results: Biofilms from PVL +ve CA‐MSSA (clinical isolate) were subjected to pH, temperature, desiccation, EDTA and DFO. PVL +ve CA‐MSSA were more resistant to pH and heat than their planktonic equivalents. Desiccation studies demonstrated that PVL +ve CA‐MSSA biofilms were more refractory to the treatment than planktonic cells. Significant inhibition of PVL +ve CA‐MSSA biofilm formation was observed in the presence of 1 mmol l^?1 EDTA. Low concentrations (2·5 μmol l^?1) of DFO enhanced the growth of PVL +ve CA‐MSSA biofilms. At higher concentrations (1 mmol l^?1), DFO did inhibit the growth but not as much as EDTA. A combination of EDTA and DFO inhibited PVL +ve CA‐MSSA biofilm formation at lower concentrations than either alone. Conclusions: This study demonstrates that PVL +ve CA‐MSSA biofilms are resistant to environmental stress but their growth can inhibited effectively by a mixture of EDTA and DFO. Significance and Impact of the Study: The inhibition of biofilm formation by PVL +ve CA‐MSSA using chelating agents has not been previously reported and provides a practical approach to achieve the disruption of these potentially important biofilms formed by an emerging pathogen.     

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

Aim: 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. Methods and Results: 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 log₁₀ 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. Conclusions: This study suggests that SN have antifungal therapeutic potential, but further studies are still required namely regarding formulation and delivery means. Significance and Impact of the Study: 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.     

Candida albicans biofilm formation is associated with increased anti-oxidative capacities

Candida albicans is a common, opportunistic, human fungal pathogen that causes a variety of mucosal and systemic afflictions. It exists in nature both in the biofilm or the sessile phase, as well as in the free-floating or the planktonic phase. Candida biofilms, in particular, display unique characteristics that confer survival advantages over their planktonic counterparts, such as their recalcitrance to common antifungals. The mechanisms underlying Candida biofilm formation and their attributes are poorly understood. In this study, we used a 2-DE-based approach to characterize the protein markers that are differentially expressed in Candida biofilms in comparison to their planktonic counterparts. Using tandem mass spectrometric analysis, we have identified a significant number of proteins including alkyl hydroperoxide reductase, thioredoxin peroxidase, and thioredoxin involved in oxidative stress defenses that are upregulated in the biofilm phase. These proteomic findings were further confirmed by real-time PCR and lucigenin-based chemiluminescence assays. In addition, we demonstrate that a drug target for the new antifungal agent echinocandin, is abundantly expressed and significantly upregulated in Candida biofilms. Taken together, these data imply that the biofilm mode, Candida, compared with their planktonic counterparts, exhibits traits that can sustain oxidative stress (anti-oxidants), and thereby exert resistance to commonly used antifungals.     

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.     

Ambroxol influences voriconazole resistance of Candida parapsilosis biofilm

The ability to form biofilm on different surfaces is typical of most Candida species. Microscopic structure and genetic aspects of fungal biofilms have been the object of many studies because of very high resistance to antimycotic agents because of the scarce permeability of the external matrix and to the alterations in cell metabolism. In our study, 31 isolates of Candida parapsilosis, isolated from bloodstream infections, were tested for their ability to produce biofilm and were found to be good producers. The susceptibility to voriconazole, assayed by colorimetrical XTT assay, revealed a very elevated minimum inhibitory concentrations for sessile cells in comparison with planktonic ones. The addition of ambroxol, a mucolytic agent, increased the susceptibility of biofilm forming cells to voriconazole. Expression of the efflux pump genes CDR and MDR was analyzed in biofilms alone or treated with ambroxol, evidencing a role of ambroxol in the expression of genes involved in azole resistance mechanisms of C.?parapsilosis biofilms. In conclusion, our data seem to encourage the use of different substances in combination with classical antimycotics, with the aim of finding a solution to the increasing problem of the resistance of biofilms formed on medical devices by nonalbicans Candida species.