Role of the biofilm matrix in resistance of Candida biofilms to antifungal agents

The aim of this project was to investigate the possible role of the biofilm matrix as a barrier to drug diffusion in Candida biofilms and in mixed species fungal-bacterial biofilms. The penetration of antifungal agents through single- and mixed-species biofilms containing Candida was investigated using a novel filter disk bioassay. Fluconazole permeated all single-species Candida biofilms more rapidly than flucytosine. Drug penetration was more extensive with C. albicans than with the other species and the rates of diffusion of either drug through biofilms of three strains of C. albicans were similar. In all cases, after 3 to 6h the drug concentration at the distal edge of the biofilm was very high (many times the MIC). Nevertheless, drug penetration failed to produce complete killing of biofilm cells. These results indicate that poor antifungal penetration is not a major drug resistance mechanism for Candida biofilms under these conditions. It has been reported that the production of extracellular matrix by Candida biofilms growing under static incubation conditions is relatively minimal, but increases dramatically when developing biofilms are subjected to a liquid flow. In this study, Candida biofilms were grown under flow conditions in a modified Robbins device (MRD). Biofilms of C. albicans grown in the MRD produced more matrix material than those grown statically, and were significantly more resistant (P<0.001) to amphotericin B. Biofilms of C. tropicalis synthesized large amounts of matrix material even when grown statically, and such biofilms were completely resistant to both amphotericin B and fluconazole. Mixed-species biofilms of C. albicans and S. epidermidis RP62A, when grown statically or in the MRD, were also completely resistant to amphotericin B and fluconazole. Mixed-species biofilms of C. albicans and S. epidermidis M7, on the other hand, were completely drug resistant only when grown under flow conditions. Overall, these findings demonstrate that the matrix can make a significant contribution to drug resistance in Candida biofilms, especially under conditions similar to those found in catheter infections in vivo, and that the composition of the matrix material is an important determinant in resistance

Candida tropicalis biofilms matrix - involvement on its resistance to amphotericin B

Candida tropicalis has emerged as one of the most prevalent fungal pathogens, and its ability to form biofilms has been considered one of the most important virulence factors, since they represent high tolerance to antifungal agents. However, the mechanisms of biofilm resistance to antifungal agents remain poorly understood. Thus, the main goal of this study was to infer about the ability of amphotericin B (AMB) to control and combat C. tropicalis biofilms. Additionally, it was also intended to determine the influence of matrix components in bio- film resistance. AMB was unable to totally prevent biofilm formation and to eradicate C. tropicalis preformed biofilms. Moreover, AMB led to a significant increase of the biofilm production due to an augment of the total protein and carbohydrate contents of the matrix. The C. tropicalis biofilm matrix assumes an important role on its resistance to AMB.This work was supported by the Programa Operacional, Fatores de competitividade and by national funds through Fundacao para a Ciencia e a Tecnologia on the scope of the projects FCT PTDC/SAU-MIC/119069/2010, RECI/EBB-EBI/0179/2012, and PEst-OE/EQB/LA0023/2013. The authors also thank the Project "BioHealth - Biotechnology and Bioengineering approaches to improve health quality; Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER

Biofilms of non-Candida albicans Candida species : 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.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/28341/2006, PDTC/BIO/61112/200

Candida bracarensis: Evaluation of virulence factors and its tolerance to Amphotericin B and Fluconazole

Candida bracarensis is an uncommon Candida species found during an epidemiological study of candidiasis performed in Braga, Portugal. Initially, it was identified as C. glabrata, but recently detailed analyses pointed out their differences. So, little information is still available about C. bracarensis virulence factors and antifungal susceptibilities. Therefore, the main goal of this work is to evaluate the ability of C. bracarensis to form biofilms, to produce hydrolytic enzymes (proteases, phospholipases and hemolysins), as well as its susceptibility to amphotericin B and fluconazole. It was shown, for the first time, that all C. bracarensis strains were able to form biofilms and display proteinase and hemolytic activities. Moreover, although planktonic cells presented antifungal susceptibility, amphotericin B and fluconazole were unable to inhibit biofilm formation and eradicate pre-formed biofilms. Due to the propensity of C. bracarensis to display antifungal resistance and virulence attributes, the control of these emerging pathogens is recommended.This work was supported by the projects PTDC/SAU-MIC/119069/2010, PEst-OE/EQB/LA0023/2013, from Fundação para a Ciência e Tecnologia (FCT), Portugal and ‘‘BioHealth—Biotechnology and Bioengineering approaches to improve health quality’’, Ref. NORTE-07-0124FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER. The authors also acknowledge the project ‘‘Consolidating Research Expertise and Resources on Cellular and Molecular Biotechnology at CEB/IBB’’, Ref. FCOMP-01-0124-FEDER027462

Silver colloidal nanoparticles : effect on matrix composition and structure of Candida albicans and Candida glabrata biofilms

Aim : The aim of this study was to assess the effect of different silver nanoparticles (SN) concentrations on the matrix composition and structure of Candida albicans and Candida glabrata biofilms. Methods and Results : Candida biofilms were developed in 6-well microtiter plates during 48 h. After, these biofilms were exposed to 13·5 or 54 μg SN ml−1 for 24 h. Then, extracellular matrices were extracted from biofilms and analysed chemically in terms of proteins, carbohydrates and DNA. To investigate the biofilm structure, scanning electron microscopy (SEM) and epifluorescence microscopy were used. SN interfered with the matrix composition of Candida biofilms tested in terms of protein, carbohydrate and DNA, except for the protein content of C. albicans biofilm. By SEM, Candida biofilms treated with SN revealed structural differences, when compared with the control groups. Further, SN showed a trend of agglomeration within the biofilms. Epifluorescence microscopy images suggest that SN induced damage on cell walls of the Candida isolates tested. Conclusions : In general, irrespective of concentration, SN affected the matrix composition and structure of Candida biofilms and these findings may be related to the mechanisms of biocide action of SN. Significance and Impact of the Study : This study reveals new insights about the behaviour of SN when in contact with Candida biofilms. SN may contribute to the development of therapies to prevent or control Candida infections.We thank David Williams (Cardiff University, Cardiff, UK) for providing strain 324LA/94. The authors also thank CAPES (grant BEX 1221/10-8) and FAPESP (2009/15146-5), Brazil, for supporting Douglas Roberto Monteiro work. The colloidal suspension of silver nanoparticles used herein was prepared and characterized by LIEC-CMDMC and INCTMN/FAPESP-CNPq, Sao Carlos, Brazil

The role of secreted aspartyl proteinases in Candida tropicalis invasion and damage of oral mucosa

Candida virulence attributes include the ability to colonize and invade host tissues, and the secretion of hydrolytic enzymes. Although Candida albicans is regarded as the principal fungi causing infections in humans, other species, particularly Candida tropicalis, are increasingly being recognized as human pathogens. Relatively little is known, however, about the virulence attributes associated with C. tropicalis. The present study aimed to investigate epithelial infection by C. tropicalis using a reconstituted human oral epithelium (RHOE) together with confocal laser scanning microscopy and real-time PCR. A comparison of clinical strains was made in terms of tissue colonization, invasion and C. tropicalis secreted aspartyl proteinase (SAPT) gene expression. All C. tropicalis strains were able to colonize RHOE in a strain-dependent manner. After 12 h of infection, C. tropicalis was found to be highly invasive, with extensive tissue damage occurring after 24 h. Real-time PCR of C. tropicalis SAPT1-4 genes showed that expression was strain-dependent, with SAPT2-4 transcripts being frequently detected and SAPT1 rarely detected. Tissue invasion and damage was not inhibited by the presence of pepstatin A. Accordingly, and given that an increase in infection time was not accompanied with an increase in SAPT gene expression, it can be suggested that the proteinases are not involved in invasion and damage of RHOE by C. tropicalis. In summary, C. tropicalis can be considered as highly invasive with the ability to induce significant tissue damage. These features, however, do not appear to be related to specific SAPT gene expression.We would like to thank Mrs Kath Allsopp for processing and sectioning the tissue samples. This work was supported by grant SFRH/BD/28341/2006 from 'Fundacao para a Ciencia e Tecnologia' (FCT), Portugal

Effect of farnesol on structure and composition of staphylococcus epidermidis biofilm matrix

Staphylococcus epidermidis is the most frequent cause of nosocomial sepsis and catheter-related infections in which biofilm formation is considered to be one of the main virulence mechanisms. Moreover, their increased resistance to conventional antibiotic therapy enhances the need to develop new therapeutical agents. Farnesol, a natural sesquiterpenoid present in many essential oils, has been described as impairing bacterial growth. The aim of this study was to evaluate the effect of farnesol on the structure and composition of biofilm matrix of S. epidermidis. Biofilms formed in the presence of farnesol (300 μM) contained less biomass, and displayed notable changes in the composition of the biofilm matrix. Changes in the spacial structure were also verified by confocal scanning laser microscopy (CSLM). The results obtained by the quantification of extracellular polymers and by wheat germ agglutinin (WGA) fluorescent detection of glycoproteins containing β(1→4)-N-acetyl-d-glucosamine support the hypothesis that farnesol causes disruption of the cytoplasmic membrane and consequently release of cellular content.Fernanda Gomes and Pilar Teixeira fully acknowledge the financial support of Fundacao para a Ciencia e Tecnologia (FCT) through the grants SFRH/BD/32126/2006 and SFRH/BPD/26803/2006, respectively

Presence of extracellular DNA in the Candida albicans biofilm matrix and its contribution to biofilms

DNA has been described as a structural component of the extracellular matrix (ECM) in bacterial biofilms. In Candida albicans, there is a scarce knowledge concerning the contribution of extracellular DNA (eDNA) to biofilm matrix and overall structure. This work examined the presence and quantified the amount of eDNA in C. albicans biofilm ECM and the effect of DNase treatment and the addition of exogenous DNA on C. albicans biofilm development as indicators of a role for eDNA in biofilm development. We were able to detect the accumulation of eDNA in biofilm ECM extracted from C. albicans biofilms formed under conditions of flow, although the quantity of eDNA detected differed according to growth conditions, in particular with regards to the medium used to grow the biofilms. Experiments with C. albicans biofilms formed statically using a microtiter plate model indicated that the addition of exogenous DNA (>160 ng/ml) increases biofilm biomass and, conversely, DNase treatment (>0.03 mg/ml) decreases biofilm biomass at later time points of biofilm development. We present evidence for the role of eDNA in C. albicans biofilm structure and formation, consistent with eDNA being a key element of the ECM in mature C. albicans biofilms and playing a predominant role in biofilm structural integrity and maintenance.National Institute of Dental & Craniofacial ResearchFundação para a Ciência e Tecnologia (FCT) - SFRH/BD/28222/2006National Institute of Allergy and Infectious Disease

The role of extracellular polymers on Staphylococcus epidermidis biofilm biomass and metabolic activity

Staphylococcus epidermidis is now well established as being a major nosocomial pathogen, associated with indwelling medical devices. Its major virulence factor is related with the ability to adhere to indwelling medical devices, with consequent biofilm formation. The present study aimed to evaluate the role of polysaccharides and proteins on biofilm biomass and metabolic activity of five S. epidermidis clinical isolates. For this purpose, S. epidermis biofilms, formed on acrylic coupons, were characterized in terms of total biofilm biomass, determined through crystal violet assay, cell concentration, established by colony forming units (CFU) enumeration, and biofilm matrix composition, which was assessed for polysaccharides and proteins content. Biofilm metabolic activity was evaluated by two distinct methods: glucose uptake and XTT reduction assays. According to the results, S. epidermidis strains revealed different abilities for biofilm formation. In fact, some strains were able to form thicker biofilms than others and this is important because biofilm formation is considered one of the major virulence factors of S. epidermidis species. S. epidermidis 1457 was the strain that produced the larger amount of biofilm and strain LE7 was the lowest biofilm producer, and these were also the highest and the lowest polysaccharides producers, respectively. This suggests a certain degree of correlation between exopolysaccharides production and total amount of biomass formed. Besides, comparing the results obtained, in terms of exopolysaccharides production and biofilm cellular activity, it seems clear that a strong production of exopolysaccharides can lead to a decrease in the metabolic activity of cells, which was the case of S. epidermidis 1457. The protein concentration also varied among strains, with the biofilm matrix of S. epidermidis 9142 presenting a higher concentration of proteins comparing to the remaining strains. This fact indicates the different levels of importance that matrix proteins can have on biofilm composition among strains albeit overall, it is suggested that extracellular protein production it is not a determinant factor for biofilm total biomass, despite its qualitative value. In conclusion, this work provided a reliable approach for a better understanding of S. epidermidis biofilms composition and metabolic activity

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.TheauthorsthanktheFCTfortheStrategic Project of the UID/BIO/04469/2013 unit, FCT and European Union funds (FEDER/COMPETE) for the project RECI/BBBEBI/0179/2012 (FCOMP-01-0124-FEDER-027462). We also would like to acknowledge Pfizer , S.A. for the kindly donation of voriconazole