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Candida albicans , a major opportunistic fungal pathogen, is frequently found together with Streptococcus mutans in dental biofilms associated with severe childhood caries (tooth decay), a prevalent pediatric oral disease. However, the impact of this cross-kingdom relationship on C. albicans remains largely uncharacterized. Here, we employed a novel quantitative proteomics approach in conjunction with transcriptomic profiling to unravel molecular pathways of C. albicans when cocultured with S. mutans in mixed biofilms. RNA sequencing and iTRAQ (isobaric tags for relative and absolute quantitation)-based quantitative proteomics revealed that C. albicans genes and proteins associated with carbohydrate metabolism were significantly enhanced, including sugar transport, aerobic respiration, pyruvate breakdown, and the glyoxylate cycle. Other C. albicans genes and proteins directly and indirectly related to cell morphogenesis and cell wall components such as mannan and glucan were also upregulated, indicating enhanced fungal activity in mixed-species biofilm. Further analyses revealed that S. mutans -derived exoenzyme glucosyltransferase B (GtfB), which binds to the fungal cell surface to promote coadhesion, can break down sucrose into glucose and fructose that can be readily metabolized by C. albicans , enhancing growth and acid production. Altogether, we identified key pathways used by C. albicans in the mixed biofilm, indicating an active fungal role in the sugar metabolism and environmental acidification (key virulence traits associated with caries onset) when interacting with S. mutans , and a new cross-feeding mechanism mediated by GtfB that enhances C. albicans carbohydrate utilization. In addition, we demonstrate that comprehensive transcriptomics and quantitative proteomics can be powerful tools to study microbial contributions which remain underexplored in cross-kingdom biofilms., (Copyright © 2019 Ellepola et al.)

Pathobiology of dental caries is complex. Data from recent molecular microbiologic studies have further redefined the role of the oral microbiome in the etiology of dental caries. This new information challenges the conventional view on the hegemony of classic cariogenic prokaryotes such as Streptococcus mutans in caries etiology, and raises the intriguing possibility of the participation of the eukaryotic oral fungal pathogen Candida in the caries process. The virulence attributes of Candida species such as their acidogenicity and aciduric nature, the ability to develop profuse biofilms, ferment and assimilate dietary sugars, and produce collagenolytic proteinases are all indicative of their latent cariogenic potential. Based on the above, oral candidal counts have been used by some as a caries risk indicator. On the contrary, other studies suggest that Candida is merely a passenger extant in an acidic cariogenic milieu, and not a true pathogen. In this review, we critically examine the varying roles of Candida, and traditionally accepted cariogens such as the mutans group of streptococci in the pathobiology of dental caries. The weight of available data tends to imply that Candida may play a pivotal role as a secondary agent perpetuating the carious process, especially in dentinal caries., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Streptococcus mutans is a biofilm-forming oral pathogen commonly associated with dental caries. Clinical studies have shown that S. mutans is often detected with Candida albicans in early childhood caries. Although the C. albicans presence has been shown to enhance bacterial accumulation in biofilms, the influence of S. mutans on fungal biology in this mixed-species relationship remains largely uncharacterized. Therefore, we aimed to investigate how the presence of S. mutans influences C. albicans biofilm development and coexistence. Using a newly established haploid biofilm model of C. albicans, we found that S. mutans augmented haploid C. albicans accumulation in mixed-species biofilms. Similarly, diploid C. albicans also showed enhanced biofilm formation in the presence of S. mutans. Surprisingly, the presence of S. mutans restored the biofilm-forming ability of C. albicans bcr1Δ mutant and bcr1Δ/Δ mutant, which is known to be severely defective in biofilm formation when grown as single species. Moreover, C. albicans hyphal growth factor HWP1 as well as ALS1 and ALS3, which are also involved in fungal biofilm formation, were upregulated in the presence of S. mutans. Subsequently, we found that S. mutans-derived glucosyltransferase B (GtfB) itself can promote C. albicans biofilm development. Interestingly, GtfB was able to increase the expression of HWP1, ALS1, and ALS3 genes in the C. albicans diploid wild-type SC5314 and bcr1Δ/Δ, leading to enhanced fungal biofilms. Hence, the present study demonstrates that a bacterial exoenzyme (GtfB) augments the C. albicans counterpart in mixed-species biofilms through a BCR1-independent mechanism. This novel finding may explain the mutualistic role of S. mutans and C. albicans in cariogenic biofilms.

Objective: Enterococcus faecalis is a bacterial pathogen that is often associated with endodontic infections. Biofilm formation is a key virulence attribute in the pathogenicity of E. faecalis. In the present study, we comprehensively examined the effect of various culture media and nutrients on the development of E. faecalis biofilms., Design: A reference strain and a clinical isolate of E. faecalis were used in all experiments for comparison. Commonly used liquid culture media with different nutrient compositions were used to support the development of E. faecalis biofilms in a time-dependent assay. E. faecalis biofilms were quantified by colony forming unit (CFU) and crystal violet (CV) assays. Biofilm architecture and cellular viability were evaluated by scanning electron microscopy and confocal laser scanning microscopy., Results: Growth kinetics evaluated by CFU and CV assays and by microscopy showed that E. faecalis biofilms reached maturity at 72h. "Pg broth" (Tryptic Soy Broth with yeast extract, hemen and vitamin K) promoted E. faecalis biofilm formation more than Brain Heart Infusion broth or Tryptic Soy Broth. Addition of 2% glucose enhanced biofilm formation. Thus, it seems that nutrients such as hemen, vitamin K and glucose are important for E. faecalis for the formation of biofilms., Conclusion: The present study demonstrated that nutrient-rich media containing glucose enhances the formation of E. faecalis biofilms, which exhibit maturation at 72h., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Most clinical guidelines for the use of biocides have been developed for planktonic micro-organisms, but in nature, most micro-organisms live as surface-adherent communities or biofilms., Aim: To evaluate the effectiveness of commonly used biocides against Escherichia coli and Candida spp. in three distinct growth phases: planktonic, adhesion and biofilm., Methods: Ultrastructural, architectural and cellular viability changes following a 5 min exposure to biocide were monitored by scanning electron microscopy and confocal laser scanning microscopy using fluorescent dyes. Comparative transcript expression of the antioxidants SOD1 and CAT1 in the planktonic and biofilm phases was evaluated using quantitative real-time polymerase chain reaction., Findings: E. coli and Candida spp. in the planktonic phase were susceptible to all the tested biocides at the recommended concentrations. However, early adhesion and late biofilm phases of both were less susceptible to the biocides, and exceeded the recommended concentrations on several occasions. A short period of biocide exposure failed to fully eradicate the adherent microbial cells, and they recovered from the biocide challenge, forming biofilm on the biocide-treated surfaces. The biofilm phase showed higher expression of SOD1 and CAT1., Conclusion: The recommended concentrations of biocides for clinical disinfection in the hospital setting may not fully eradicate the adhesion or biofilm phases of E. coli and Candida spp. Higher antioxidative capacities in microbial biofilms may be responsible for the resistance of biofilms against clinical biocides., (Copyright © 2011 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

Biofilms are surface-attached, matrix-encased, structured microbial communities which display phenotypic features that are dramatically different from those of their free-floating, or planktonic, counterparts. Biofilms seem to be the preferred mode of growth of microorganisms in nature, and at least 65% of all human infections are associated with biofilms. The most notable and clinically relevant property of biofilms is their greater resistance to antimicrobials compared with their planktonic counterparts. Although both bacterial and fungal biofilms display this phenotypic feature, the exact mechanisms underlying their increased drug resistance are yet to be determined. Advances in proteomics techniques during the past decade have facilitated in-depth analysis of the possible mechanisms underpinning increased drug resistance in biofilms. These studies have demonstrated the ability of proteomics techniques to unravel new targets for combating microbial biofilms. In this review, we discuss the putative drug resistance mechanisms of microbial biofilms that have been uncovered by proteomics and critically evaluate the possible contribution of the new knowledge to future development in the field. We also summarize strategic uses of novel proteomics technologies in studies related to drug resistance mechanisms of microbial biofilms., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: The aim of this study was to evaluate the antimicrobial efficacy of 2 commercially available mouth rinses on a monospecies-biofilm model on orthodontic brackets in vitro., Methods: The antimicrobial effects of the 2 mouth rinses, Listerine (tartar control; IDS Manufacturing, Bangkok, Thailand) and Corsodyl (SmithKline Beecham, Maidenhead, United Kingdom), on the planktonic Streptococcus mutans were tested by maximum inhibitory dilution assay. The cell viability of S mutans biofilm on Damon3 MX brackets (Ormco, Glendora, Calif) after exposure to the 2 mouth rinses was quantified by 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay. Visualization of the biofilm samples was performed by fluorescence microscopy and confocal laser scanning microscopy., Results: The maximum inhibitory dilution assays of S mutans were 1:5 for Listerine and 1:320 for Corsodyl. The optical density values, which were measured by XTT reduction assay from S mutans biofilms after 1 minute of exposure to the different test agents, demonstrated that the cell viability of S mutans biofilms exposed to Listerine was less than that for Corsodyl, which was less than that for brain-heart infusion (P <0.001). Listerine caused more dead cells on the surface of the brackets than did Corsodyl when examined with the 2 microscope systems., Conclusions: Both mouth rinses showed marked antimicrobial effects on the monospecies biofilm in vitro. Listerine showed a stronger bactericidal effect but had less bacterial inhibitory effect than did Corsodyl., (Copyright © 2011 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.)

The aim of this study was to investigate the morphological features and distribution of biofilms on Invisalign orthodontic appliances, in a sample of 'slow' and 'fast' plaque formers using scanning electron microscopy (SEM). Fifty-six Chinese male/female volunteers (aged 19-39 years) were screened for their plaque-forming rate using the plaque percentage index (PPI) coupled with digital photography and computer-based image analysis, after a period of 48 hours of abstinence from oral hygiene procedures. Eleven volunteers (seven males/four females) representing the lowest and highest ends of the plaque formation spectrum were chosen as slow and fast plaque formers, respectively. The subjects wore a full-coverage splint appliance, in which four tiles of Invisalign material were embedded. These tiles were collected at intervals of 1, 3, 6, 12, 24, and 48 hours, as well as 3, 7, and 14 days, immediately fixed in 10 per cent paraformaldehyde in 0.2 M cacodylate buffer solution and prepared for SEM. The surface configuration of the Invisalign appliance was visualized, as well as the chronological pattern of biofilm formation. Significance between fast and slow plaque formers was determined using a Student's t-test. Colonization appeared to centre initially on the raised edges or textured surfaces of the appliance, and initial adhesion was quicker and more abundant in the fast plaque-forming group. In the later stages of biofilm development, both groups showed no discernible differences in biofilm accrual on the surfaces, but the fast group displayed a more complex biofilm structure. More recessed and sheltered areas of the appliance, such as the cusp tips and attachment dimples, harboured more biofilm than the flat surfaces. Hence, it seems that the novel Invisialign orthodontic appliance is a useful tool to investigate the features of biofilm formation in time-course studies.

Biofilm formation is a major virulence attribute of Candida albicans and is directly associated with therapeutic failure. One method by which Candida acquires antifungal resistance is the expression of drug-resistance genes. This study aimed to evaluate the transcriptional regulation of several genes associated with antifungal resistance of C. albicans under planktonic, recently adhered and biofilm growth modes and in C. albicans biofilms in response to antifungal agents. Initially, the antifungal susceptibility of C. albicans cultures in different growth modes was evaluated by standard antifungal susceptibility testing. Next, to assess CDR1, CDR2, MDR1, ERG11, FKS1 and PIL1 expression, RNA was harvested from cells in each growth mode, and from biofilms after drug treatment, and subjected to quantitative real-time RT-PCR (qRT-PCR). Biofilm C. albicans was more resistant to antifungals than recently adhered cells and stationary-phase planktonic cultures. Transcriptional expression of CDR1, CDR2, MDR1, ERG11 and FKS1 was lower in recently adhered C. albicans than in the stationary-phase planktonic cultures. In contrast, PIL1 levels were significantly increased in recently adhered and biofilm modes of growth. The expression of MDR1 in biofilms greatly increased on challenge with amphotericin B but not with the other drugs tested (P<0.01). ERG11 was significantly upregulated by ketoconazole (P<0.01). Caspofungin and amphotericin B significantly upregulated FKS1 expression, whereas they significantly downregulated PIL1 expression (P<0.01). These results indicate that the expression of drug-resistance genes is associated with higher drug resistance of Candida biofilms, and lay a foundation for future large-scale genome-wide expression analysis.

Objective: the aim of this study was to evaluate the antimicrobial efficacy of Traditional Chinese Medicine Fructus mume on a monospecies-biofilm model established on orthodontic brackets in vitro., Materials and Methods: the antimicrobial effect of Fructus mume aqueous extract on the planktonic Streptococcus mutans (S. mutans) was tested by microdilution method (MIC). The cell viability of S. mutans biofilm on Damon3 MX bracket (Ormco, USA) after exposed to Fructus mume extract was quantified by XTT reduction assay. Visualization of the samples was performed by fluorescence microscope and confocal laser scanning microscopy (CLSM)., Results: HPLC analysis revealed that the main compounds of Fructus mume are organic acids. The MIC of Fructus mume extract on the planktonic S. mutans was 50mg/mL. The optical density (OD) values, measured by XTT reduction assay from S. mutans biofilms after 1-min exposure to different test agents, demonstrated that the cell viability of S. mutans biofilms exposed to 250mg/mL Fructus mume extract

Twenty traditional Chinese medicines (TCM) were evaluated for their antimicrobial activity against four common oral bacteria. TCMs were tested for sensitivity against Streptococcus mitis, Streptococcus sanguis, Streptococcus mutans and Porphyromonas gingivalis. Aliquots of suspension of each bacterial species were inoculated onto a horse blood agar plate with TCMs soaked separately on 6mm paper disks. The plates were incubated for 48h anaerobically and the mean diameters of growth inhibition of three different areas obtained. 0.2% (w/v) chlorhexidine was used as a positive control. Broth microdilution assay was used to determine minimum inhibitory concentration and minimum bactericidal concentration. Fructus armeniaca mume was effective against all four bacteria. Thirteen TCMs demonstrated antimicrobial activity against Porphyromonas gingivalis, including Cortex magnoliae officinalis, Cortex phellodendri, Flos caryophylli, Flos lonicerae japonicae, Fructus armeniaca mume, Fructus forsythiae suspensae, Herba cum radice violae yedoensitis, Herba menthae haplocalycis, Pericarpium granati, Radix et rhizoma rhei, Radix gentianae, Ramulus cinnamomi cassia and Rhizoma cimicifugae. Cortex phellodendri showed antimicrobial activity against Streptococcus mutans, while Radix et rhizoma rhei was effective against Streptococcus mitis and Streptococcus sanguis. Fructus armeniaca mume had inhibitory effects against Streptococcus mitis, Streptococcus sanguis, Streptococcus mutans and Porphyromonas gingivalis in vitro.

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.

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.

Candida is the most common human fungal pathogen that causes a variety of afflictions from superficial mucosal infections to deep mycoses. Biofilm formation is a major virulence factor of Candida, and more than 300 articles have been published on Candida biofilms over the past two decades. However, most of these data are on monospecies biofilms of Candida, and information on mixed-species Candida biofilms or bacteria-Candida combinations is still scarce. Yet, in nature, the yeast exist in a mixed milieu either in the oral cavity or in other habitats with a multitude of bacteria colonising mucosal surfaces within a shared community. This mini review describes the current knowledge on candidal-candidal or bacterial-candidal interactions in mixed-species biofilms. The underlying mechanisms of these interactions appear to depend on several factors relating to biofilm development, such as species and strains of organisms, nutritional factors, aerobiosis and related environmental factors. Although the fundamental nature of these interactions appears to be commensalism and antagonism, the emerging evidence based on novel molecular, proteomic and imaging tools indicates these biological mechanisms to be far more complex than hitherto recognised. Demystifying the mechanisms underlying the growth and development of mixed-species communities involving Candida will undoubtedly yield useful data for the effective management of microbial infections in general.

Biofilm formation involving profuse hyphal growth is a major characteristic of Candida spp. and confers higher antifungal resistance than its planktonic mode of growth. We investigated the antifungal susceptibility of Candida albicans and its hyphal mutants (Delta efg1/efg1, Delta cph1/cph1 and DeltaDelta cph1/cph1 efg1/efg1) to commonly used antifungals during planktonic, adhesion and biofilm modes of growth. The minimum inhibitory concentration (MIC) of each antifungal agent was determined for a lower inoculum (1x10(3) cells/mL) and higher inoculum (1x10(7) cells/mL) of planktonic Candida. Furthermore, MICs of C. albicans biofilms and adhesion modes of growth were determined with a standard XTT assay. Candida albicans in adhesion and biofilm modes of growth, but not in planktonic mode, were resistant to all five antifungal agents tested. Although Delta efg1/efg1 and DeltaDelta cph1/cph1 efg1/efg1 mutants formed less biofilm than wild-type C. albicans SC5314, they were similarly resistant to caspofungin. However, these mutants were more sensitive to amphotericin B and nystatin than the wild-type. Adhesion per se confers increased resistance to antifungal agents, which is further pronounced in the biofilm mode of Candida. Filamentation does not appear to be a major determinant of the antifungal resistance in Candida biofilms.

Candida species are the most important fungal pathogens in humans and cause a variety of superficial and systemic diseases. Biofilm formation is a major virulence attribute contributing to Candida pathogenicity. Although the concentration and distribution of nutrients as well as antifungals across the biofilm thickness play a pivotal role in the development and persistence of Candida biofilms, only limited information is available on the latter aspects of Candida biofilms. Therefore, we attempted to characterize the diffusion coefficient (De) of common dietary sugars such as glucose, galactose, and sucrose in Candida albicans biofilms using horizontal attenuated total reflection-Fourier transform infrared spectroscopy (HATR-FTIR). Artificial Candida biofilms were formed using agarose polymers. De of three sugars tested, glucose, galactose, and sucrose in this artificial Candida biofilm model was found to be 4.08E-06 +/- 3.63E-08, 4.08E-06 +/- 3.70E-08, and 5.38E-06 +/- 4.52E-08 cm(2) s(-1), respectively. We demonstrate here the utility of HATR-FTIR for the determination of diffusion of solutes such as dietary sugars across Candida biofilms.

Unlabelled: Denture related oral candidiasis is a recalcitrant fungal infection not easily resolved by topical antifungals. The antimycotic protein lysozyme, in saliva is an important host defense mechanism although its activity against Candida biofilms on denture acrylic has not been evaluated., Objectives: (i) To establish a clinically relevant denture acrylic assay model to develop standardized Candida albicans biofilms, and (ii) assess the inhibitory effects of lysozyme alone and, the latter combined with antifungals (nystatin, amphotericin B, ketoconazole and 5-fluorocytosine) on sessile Candida cells and, finally (iii) to visualize the accompanying ultrastructural changes., Design: The rotating-disc biofilm reactor was used to develop standardized 48 h Candida biofilms on acrylic discs in YNB/100 mM glucose medium and the biofilm metabolic activity was monitored using a tetrazolium reduction assay., Results: The biofilm metabolic activity was similar in 18 identical denture acrylic discs (p<0.05) thus validating the rotating-disc biofilm model. Very low concentrations of lysozyme (6.25 microg/ml) significantly (p<0.01) inhibited Candida biofilm formation indicating that lysozyme may likely regulate intra-oral Candida biofilm development. Although 100 microg/ml lysozyme killed 45% of sessile Candida cells, further increasing its concentration (up to 240 microg/ml) had no such effect. Nystatin, amphotericin B, and ketoconazole in association with 100 microg/ml lysozyme exhibited effective synergistic killing of biofilm Candida in comparison to drug-free controls. Scanning electron and confocal scanning laser microscopy analysis confirmed the latter trends., Conclusion: Our results indicate that agents found in biological fluids such as lysozyme could be a safe adjunct to antifungals in future treatment strategies for recalcitrant candidal infections.

Candida is the major fungal pathogen of humans causing a variety of afflictions ranging from superficial mucosal diseases to deep seated mycoses. Biofilm formation is a major virulence factor in the pathogenicity of Candida, and Candida biofilms are difficult to eradicate especially because of their very high antifungal resistance. Consequently, research into the pathogenicity of Candida has focused on the prevention and management of biofilm development, their architecture, and antifungal resistance. Although studies have shed some light, molecular mechanisms that govern biofilm formation and pathogenicity still await full clarification. This review outlines the key features of what is currently known of Candida biofilm development, regulation and antifungal resistance and, their proteomics.

Biofilm formation is a major virulence attribute of Candida pathogenicity which contributes to higher antifungal resistance. We investigated the roles of cell density and cellular aging on the relative antifungal susceptibility of planktonic, biofilm, and biofilm-derived planktonic modes of Candida. A reference and a wild-type strain of Candida albicans were used to evaluate the MICs of caspofungin (CAS), amphotericin B (AMB), nystatin (NYT), ketoconazole (KTC), and flucytosine (5FC). Standard, NCCLS, and European Committee on Antibiotic Susceptibility Testing methods were used for planktonic MIC determination. Candida biofilms were then developed on polystyrene wells, and MICs were determined with a standard 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide assay. Subsequently, antifungal susceptibility testing was performed for greater inoculum concentrations and 24- and 48-h-old cultures of planktonic Candida. Furthermore, Candida biofilm-derived planktonic cells (BDPC) were also subjected to antifungal susceptibility testing. The MICs for both C. albicans strains in the planktonic mode were low, although on increasing the inoculum concentration (up to 1 x 10(8) cells/ml), a variable MIC was noted. On the contrary, for Candida biofilms, the MICs of antifungals were 15- to >1,000-fold higher. Interestingly, the MICs for BDPC were lower and were similar to those for planktonic-mode cells, particularly those of CAS and AMB. Our data indicate that higher antifungal resistance of Candida biofilms is an intrinsic feature possibly related to the biofilm architecture rather than cellular density or cellular aging.

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.