The influence of substrate surface conditioning and biofilm age on the composition of Enterococcus faecalis biofilms.

AIM: To investigate the null hypothesis that neither the surface conditioning (collagen, serum, saliva) of hydroxyapatite (HA) discs, nor the biofilm age (3 days vs. 21 days) has a significant effect on the cellular and matrix composition of biofilms, using Enterococcus faecalis as the model organism. METHODOLOGY: Sterile HA discs were conditioned with collagen, saliva or serum, and inoculated with E. faecalis to form 3-day and 21-day-old biofilms. Unconditioned discs served as controls. The biofilms were analysed using culture-dependent and independent (confocal microscopy and biochemical analysis) methods, to determine the colony-forming units and the biofilm matrix composition (polysaccharides and proteins), respectively. Statistical analyses were performed using appropriate parametric and nonparametric tests (P = 0.05). RESULTS: Collagen conditioning significantly increased the number of CFUs in the 21-day biofilms, compared to the 3-day biofilms (P < 0.05). Although the biochemical analysis revealed that surface conditioning had no significant effect on the total carbohydrate content in the 21-day biofilms, confocal microscopic analysis revealed that collagen and saliva conditioning selectively increased the polysaccharide content of 21-day biofilms, compared to the 3-day biofilms (P < 0.05). CONCLUSIONS: The results of this study raise an important methodological concern that the substrate conditioning substances and biofilm age differentially influence the cellular and extracellular matrix components of E. faecalis biofilms.

Enteric Gram-negative bacilli suppress Candida biofilms on Foley urinary catheters.

Mixed Candida-bacterial biofilms in urinary catheters are common in hospitalized patients. (i) The aims of this study were to evaluate, quantitatively and qualitatively, the in vitro development of mono- and dual-species biofilms (MSBs and DSBs) of Candida albicans and two enteric gram-negative bacilli (EGNB; Pseudomonas aeruginosa or Escherichia coli) on Foley catheter (FC) discs, (ii) to determine the biofilm growth in tryptic soy broth or glucose supplemented artificial urine (AU) and (iii) to assess the inhibitory effects of EGNB and their lipopolysaccharides (LPS) on Candida biofilm growth. The growth of MSBs and DSBs on FC discs was monitored by cell counts and SEM. The metabolic activity of LPS-treated Candida biofilms was determined by the XTT reduction assay. Candida albicans and EGNB demonstrated significant inter- and intra-species differences in biofilm growth on FC discs (p < 0.01). Pseudomonas aeruginosa suppressed Candida albicans significantly (p < 0.001) in DSBs. Compared with MSBs, DSB of EGNB in glucose supplemented AU demonstrated robust growth. Escherichia coli and its LPS, significantly suppressed Candida biofilm growth, compared with Pseudomonas aeruginosa and its LPS (p < 0.001). Candida albicans and EGNB colonization in FC is significantly increased in AU with glucose, and variably modified by Escherichia coli, Pseudomonas aeruginosa and their corresponding LPS.

Fluconazole resistance in Candida glabrata is associated with increased bud formation and metallothionein production.

Virulence associated with fluconazole (FL) resistance in Candida glabrata is a global problem and has not been well characterized at the proteome level. In this study, a stable FL-resistant (MIC >256 µg ml(-1)) strain of C. glabrata was generated on agar containing FL. Eight phenotypic mutants were characterized by contour-clamped homogeneous electrophoretic field analysis and two-dimensional PAGE. The secondary derivatives of C. glabrata yielded four distinct genotypes with varying chromosomal profiles. Proteomic analysis performed by tandem mass spectrometry for two of the mutants, CG(L2) and CG(S3), demonstrated a total of 25 differentially regulated proteins of which 13 were upregulated and 12 were downregulated or were similar compared with the parental isolate. The mRNA transcript levels of significantly (P<0.001) upregulated genes were determined by quantitative RT-PCR analysis, and their physiological relevance in terms of phenotypic expression of virulence attributes was verified by conventional laboratory methodologies. The data showed that the FL resistance (MIC >256 µg ml(-1)) of CG(S3) was associated with significantly upregulated (P<0.001) mRNA transcript levels of several genes - ERG11, CDR1, CDR2, MFS, MTI, TPR, VPS and EFT2 - in addition to a number of other potentially virulent genes expressed differentially at a lower level. The results demonstrated accentuated phenotypic expression of bud formation of yeast and metallothionein production associated with FL resistance in C. glabrata, which may help the fungus to colonize the host.

Pseudomonas aeruginosa inhibits in-vitro Candida biofilm development.

BACKGROUND: Elucidation of the communal behavior of microbes in mixed species biofilms may have a major impact on understanding infectious diseases and for the therapeutics. Although, the structure and the properties of monospecies biofilms and their role in disease have been extensively studied during the last decade, the interactions within mixed biofilms consisting of bacteria and fungi such as Candida spp. have not been illustrated in depth. Hence, the aim of this study was to evaluate the interspecies interactions of Pseudomonas aeruginosa and six different species of Candida comprising C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis, and C. dubliniensis in dual species biofilm development. RESULTS: A significant reduction in colony forming units (CFU) of C. parapsilosis (90 min), C. albicans and C. tropicalis (90 min, 24 h and 48 h), C. dubliniensis and C. glabrata, (24 h and 48 h) was noted when co-cultured with P. aeruginosa in comparison to their monospecies counterparts (P < 0.05). A simultaneous significant reduction in P. aeruginosa numbers grown with C. albicans (90 min and 48 h), C. krusei (90 min, 24 h and 48 h),C. glabrata, (24 h and 48 h), and an elevation of P. aeruginosa numbers co-cultured with C. tropicalis (48 h) was noted (P < 0.05). When data from all Candida spp. and P. aeruginosa were pooled, highly significant mutual inhibition of biofilm formation was noted (Candida P < 0.001, P. aeruginosa P < 0.01). Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM) analyses confirmed scanty architecture in dual species biofilm in spite of dense colonization in monospecies counterparts. CONCLUSIONS: P. aeruginosa and Candida in a dual species environment mutually suppress biofilm development, both quantitatively and qualitatively. These findings provide a foundation to clarify the molecular basis of bacterial-fungal interactions, and to understand the pathobiology of mixed bacterial-fungal infections.

The post-antifungal effect (PAFE) of amphotericin B, nystatin, ketoconazole and 5-fluorocytosine and its impact on the colonization traits of Candida glabrata.

The post-antifungal effect (PAFE) has been shown to affect Candida pathogenicity, but there is little information on either PAFE or its association with the colonization traits of Candida glabrata. The objective of this study was to determine, in vitro, the PAFE on 14 C. glabrata isolates following exposure to amphotericin B (AMB), nystatin (NYS), ketoconazole (KETO) and 5-fluorocytosine (5FC). In addition, we evaluated the impact of PAFE on yeast adherence to buccal epithelial cells (BEC), cell-surface-hydrophobicity (CSH) and biofilm growth (BG) on denture acrylic surfaces. PAFE was induced following a 1-h exposure of yeasts to (x1-x4MIC) of AMB, NYS, KETO and 5FC in RPMI medium and, measured using automated turbidometry. The BEC adhesion, CSH and BG assays were performed by the methods of Kimura & Pearsall, Sweet et al., and Jin et al., respectively. Significant differences in PAFE (P < 0.001) were observed after exposure to AMB and NYS, but not KETO and 5FC. Following exposure to AMB, NYS, KETO and 5FC, significant inter-strain differences (P < 0.001) were observed in percentage terms in adhesion (39.0%, 43.48%, 38.28%, 35.07%) and biofilm growth (42.86%, 39.86%, 42.81%, 36.38%), respectively. Short exposure of C. glabrata to sub-cidal concentrations of antifungals modulates yeast growth and also affects some of their colonization traits.

Escherichia coli and its lipopolysaccharide modulate in vitro Candida biofilm formation.

Demystification of microbial behaviour in mixed biofilms could have a major impact on our understanding of infectious diseases. The objectives of this study were to evaluate in vitro the interactions of six different Candida species and a Gram-negative coliform, Escherichia coli, in dual-species biofilms, and to assess the effect of E. coli LPS on Candida biofilm formation. A single isolate of E. coli ATCC 25922 and six different species of Candida, Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida krusei ATCC 6258, Candida tropicalis ATCC 13803, Candida parapsilosis ATCC 22019 and Candida dubliniensis MYA-646, were studied using a standard biofilm assay. Each Candida species was co-cultured with E. coli on a polystyrene surface and biofilm formation was quantified by a c.f.u. assay. The biofilm was then analysed by Live/Dead staining and fluorescence microscopy (confocal laser-scanning microscopy, CLSM), whilst scanning electron microscopy (SEM) was employed to visualize the biofilm architecture. The effect of E. coli LPS on Candida biofilm cell activity at defined time intervals was assessed with an XTT reduction assay. A significant quantitative reduction in c.f.u. counts of C. tropicalis (after 90 min), C. parapsilosis (after 90 min and 24 h), C. krusei (after 24 h) and C. dubliniensis (after 24 and 48 h) was noted on incubation with E. coli in comparison with their monospecies biofilm counterparts (P <0.05). On the other hand, a simultaneous and significant reduction in E. coli cell numbers occurred on co-culture with C. albicans (after 90 min), and an elevation of E. coli cell numbers followed co-culture with C. tropicalis (after 24 h) and C. dubliniensis (after 24 h and 48 h) (P <0.05). All quantitative findings were confirmed by SEM and CLSM analyses. By SEM observation, dual-species biofilms demonstrated scanty architecture with reduced visible cell counts at all stages of biofilm development, despite profuse growth and dense colonization in their single-species counterparts. Significantly elevated metabolic activity, as assessed by XTT readings, was observed in E. coli LPS-treated C. tropicalis and C. parapsilosis biofilms (after 48 h), whilst this had the opposite effect for C. dubliniensis (after 24 h) (P <0.05). These data indicate that E. coli and Candida species in a mixed-species environment mutually modulate biofilm development, both quantitatively and qualitatively, and that E. coli LPS appears to be a key component in mediating these outcomes.

Synergistic activity of lysozyme and antifungal agents against Candida albicans biofilms on denture acrylic surfaces.

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.

Interspecies variation in Candida biofilm formation studied using the Calgary biofilm device.

An in vitro assay to study multiple Candida biofilms, in parallel, has been carried out using the Calgary biofilm device (CBD). We here report: i) standardization of the CBD for Candida albicans biofilm formation, ii) kinetics of C. albicans biofilm formation, iii) biofilm formation by five Candida species, and iv) effect of dietary carbohydrates on biofilm formation. The biofilm metabolic activity on all CBD pegs was similar (p=0.6693) and C. albicans biofilm formation revealed slow growth up to 36 h and significantly higher growth up to 48 h (p<0.001). Significant differences in total biofilm metabolic activity were seen for glucose, fructose and lactose grown C. albicans compared with sucrose and maltose grown yeasts. Candida krusei developed the largest biofilm mass (p<0.05) relative to C. albicans, C. glabrata, C. dubliniensis and C. tropicalis. Scanning electron microscopy revealed that C. krusei produced a thick multilayered biofilm of pseudohyphal forms embedded within the polymer matrix, whereas C. albicans, C. dubliniensis and C. tropicalis biofilms consisted of clusters or chains of cells with sparse extracellular matrix material. We conclude that CBD is a useful, simple, low cost miniature device for parallel study of Candida biofilms and factors modulating this phenomenon.

Differential phospholipase gene expression by Candida albicans in artificial media and cultured human oral epithelium.

Phospholipases B1, B2, C and D of Candida albicans play a significant role in the host invasive process. Hence we evaluated the in vitro expression of PLB1, PLB2, PLC1 and PLD1 in phospholipase-positive (PL(+)) and -deficient (PL(-)) C. albicans isolates in egg yolk agar (EYA), yeast peptone dextrose broth (YPD), and in a model of oral candidiasis based on reconstituted human oral epithelium (RHOE). The growth of Candida was then determined in YPD and its cellular invasion was investigated using the RHOE model. The PL(+) group demonstrated PLB1, PLB2, PLC1 and PLD1 expression in both EYA and YPD, in contrast to the PL(-) group, which expressed only PLB2 and PLD1. Although PL(+) isolates grew profusely in the RHOE model, they expressed only PLB2, PLC1 and PLD1, and not PLB1. Gene expression investigations could not be carried out with PL(-) isolates due to their inability to grow in the RHOE model. Significant growth differences in YPD medium were also observed within the PL(+) and PL(-) groups. Taken together, these findings indicate that phospholipase gene expression in C. albicans is differentially affected by their growth milieu, and this in turn may modulate the disease outcomes in vivo.

Water concentration in self-etching primers affects their aggressiveness and bonding efficacy to dentin.

Water is required to ionize acid resin monomers for demineralization of tooth substrates. We tested the null hypothesis that altering the water concentration in two-step self-etching primers has no effect on their aggressiveness and bonding efficacy to dentin. Five experimental self-etching primers were prepared with resin-water-ethanol volume ratios of 9-0-1, 8-1-1, 7-2-1, 5-4-1, and 3-6-1. They were applied to smear-layer-covered dentin, followed by a bonding resin and composite build-ups for microtensile bond testing and TEM examination of tracer penetration. Increasing water concentration from 0-60 vol% improved acidic monomer ionization that was manifested as increasing hybrid layer thickness. However, significantly higher bond strength was observed in the 7-2-1 group, with minimal nanoleakage in the corresponding hybrid layer. When self-etching primers are formulated, a balance must be achieved to provide sufficient water for adequate ionization of the acidic monomers, without lowering the resin concentration too much, to optimize their bonding efficacy to dentin.

In vitro method to study antifungal perfusion in Candida biofilms.

Antimycotic perfusion through Candida biofilms was demonstrated by a modification of a simple in vitro diffusion cell bioassay system. Using this model, the perfusion of three commonly used antifungal agents, amphotericin B, fluconazole, and flucytosine, was investigated in biofilms of three different Candida species (i.e., Candida albicans, Candida parapsilosis, and Candida krusei) that were developed on microporous filters. Scanning electron microscopy revealed that C. albicans formed a contiguous biofilm with tightly packed blastospores and occasional hyphae compared with C. parapsilosis and C. krusei, which developed confluent biofilms displaying structural heterogeneity and a lesser cell density, after 48 h of incubation on nutrient agar. Minor structural changes were also perceptible on the superficial layers of the biofilm after antifungal perfusion. The transport of antifungals to the distal biofilm-substratum interface was most impeded by C. albicans biofilms in comparison to C. parapsilosis and C. krusei. Fluconazole and flucytosine demonstrated similar levels of perfusion, while amphotericin B was the least penetrant through all three biofilms, although the latter appeared to cause the most structural damage to the superficial cells of the biofilm compared with the other antifungals. These results suggest that the antifungal perfusion through biofilm mode of growth in Candida is dependent both on the antimycotic and the Candida species in question, and in clinical terms, these phenomena could contribute to the failure of Candida biofilm-associated infections. Finally, the in vitro model we have described should serve as a useful system to investigate the complex interactions that appear to operate in vivo within the biofilm-antifungal interphase.

Adhesion and cell-surface-hydrophobicity of sequentially isolated genetic isotypes of Candida albicans in an HIV-infected Southern Chinese cohort.

Objectives of the study were to investigate the variability in yeast adhesion and cell-surface-hydrophobicity (CSH) during human immunodeficiency virus (HIV) disease progression, using a total of 60 sequential Candida albicans isolated from oral rinse samples of seven HIV-infected individuals with (4) and without (3) clinical symptoms of oropharyngeal candidosis. Significant differences in the adhesion to buccal epithelial cells (BECs) during sequential visits were observed for all genetic isotypes in five of the seven individuals and three isotypes belonging to the sixth individual. A single isotype of patient HK1 and another of HK4 (genotype I) demonstrated significant variations in their CSH during sequential visits whereas no such differences were noted for the remaining genotypes. On Spearman correlation analysis an isotype from HK1 demonstrated a significant increased adherence to BECs and CSH during HIV disease progression whereas no such correlation was noted for the remaining isotypes studied. No significant differences in adherence to BECs or CSH values were observed between the symptomatic oral candidosis and the asymptomatic carrier group. Further, on regression analysis only the single isotype of HK1 demonstrated a significant positive correlation between adherence to BECs and CSH whereas no such correlation was observed when all tested Candida isolates were pooled and evaluated as a single, large group.

'Genotypic shuffling' of sequential clones of Candida albicans in HIV-infected individuals with and without symptomatic oral candidiasis.

Although HIV-infected individuals harbour multiple strains of oral Candida albicans, little is known of their micro-evolution over time. Therefore, a prospective study was conducted with 16 HIV-infected ethnic Chinese individuals with and without symptoms of oropharyngeal candidiasis to evaluate the genotype distribution of oral C. albicans isolates during HIV disease progression. Oral-rinse samples were obtained from all individuals and up to five C. albicans colonies were selected for each visit, over a 12 month period of multiple visits. After identification of isolates using standard mycological criteria, the genetic similarities of yeast isolates within and between sequential clones of C. albicans were assessed by DNA fingerprinting through random amplification of polymorphic DNA (RAPD). The results of RAPD gel profiles and the lineage of each isolate were further analysed using commercially available software. RAPD studies revealed the prevalence of up to 14 different genotypes per individual during the study period, with multiple genotypes isolated simultaneously from a single oral rinse. Computer analysis of RAPD profiles revealed that yeasts isolated over sequential visits from symptomatic individuals demonstrated a striking level of relatedness compared with isolates from asymptomatic individuals. Genetically identical C. albicans strains also formed 'loosely' connected subclusters that overlapped multiple visits, implying genetic 'shuffling' in these isolates during disease progression. These data point to varying evolutionary genetic trends in C. albicans associated with symptomatic oral candidiasis and asymptomatic carriage in HIV disease.

Phenotypic diversity of oral C. albicans isolated on single and sequential visits in an HIV-infected Chinese cohort.

HIV-infected individuals maintain multiple oral C. albicans strains over time that are thought to undergo microevolution in terms of both phenotypic and genotypic features. To study this phenomenon, a 12-month prospective study was conducted in a cohort of 16 HIV-infected ethnic Chinese individuals with (A) and without (B) symptoms of oropharyngeal candidiasis to evaluate the phenotype distribution among oral C. albicans isolates during disease progression. Oral rinse samples were obtained and up to five C. albicans colony-forming units were selected per each visit, during the one year period of multiple visits. The isolates were phenotyped using two commercially available biotyping kits, the API 20C system, API ZYM system, and a plate test for resistance to boric acid. A total of 261 C. albicans strains in group A were differentiated into 67 biotypes, while 42 biotypes were seen amongst the 182 isolates from group B. The major biotypes in the two groups were similar and were in decreasing order of prevalence J1R, J1S, J6S, J6R, J2S, K1S, J10R, K1R, and K6R; 48 different biotypes were seen in group A and 24 in group B, with some uniquely represented in each group, leading to a significant association between the prevalence of the biotypes J1S and J2S and symptomatic candidiasis (p<0.05). Taken together this study illustrates the wide phenotypic spectrum of oral C. albicans associated with HIV-infection.

Oral colonisation by aerobic and facultatively anaerobic Gram-negative rods and yeast in Tibetans living in Lhasa.

Sample groups of children (n=50) and adults (n=38) were selected from pools of 207 children, (11-13-year olds from two primary schools) and 94 adults (25-44-year olds from four governmental agencies) who were the subjects of an oral health survey among Tibetans living in Lhasa, Tibet Autonomous Region. Mean ages of the study groups of children (38% females) and adults (61% females) were 11.6+/-0.9 and 37.1+/-6.1 years, respectively. All had lived in Tibet since birth. Oral rinse samples were selective cultured to isolate, quantify and speciate aerobic and facultatively anaerobic Gram-negative rods (using the API 20E kit) and yeasts (using API 20C AUX and API ZYM kits). For children, the isolation rates for oral coliform bacteria and yeasts were 84 and 14%, respectively, for adults, the respective rates were 26 and 40%. The corresponding quantities of coliforms/yeasts for children and adults were 0.4+/-1.6 x 10(3)c.f.u./15.8+/-72.3 and 0.2+/-0.6 x 10(3)c.f.u./57.2+/-137.5c.f.u. per millilitre oral rinse, respectively. Aerobic and facultatively anaerobic Gram-negative rods and Stenotrophomonas maltophilia, a free-living saprophytic and ubiquitous bacterial species of wide geographic distribution, were significantly more frequently recovered from the children's oral rinses. The isolation rates of facultatively anaerobic Gram-negative rods in adults and yeasts in both groups were similar to those found in similar cohorts from southern China in earlier studies. Randomly amplified polymeric DNA analysis showed that the S. maltophilia spp. isolated from children were of several different clonal types and were school specific. This study shows that the colonisation rate of facultatively anaerobic Gram-negative rods in adults and yeasts in both groups are similar to those in populations living at lower altitudes, the native young, urban Tibetans appear to exhibit a high oral carriage rate of S. maltophilia spp.

Subgingival microbiota of renal transplant recipients.

Renal transplant patients undergoing immunosuppressive therapy may experience periodontal side-effects such as gingival overgrowth. This study evaluated the subgingival microbiota of renal transplant recipients with or without periodontal tissue destruction who may have concurrent gingival enlargement. Subgingival paper point samples taken from the deepest probing sites of 38 subjects (one per patient) were examined using direct microscopy and culture techniques. A complex microflora comprising gram-positive and gram-negative cocci, rods and filaments, fusiforms, curved rods and spirochetes was observed using microscopy. Yeasts were occasionally detected. Significantly higher proportions of gram-positive morphotypes, including gram-positive cocci, were observed in samples from periodontally healthy patients. The predominant cultivable microflora from anaerobic culture comprised several species of facultative and obligate anaerobes. Colonization of the subgingival sites by 'foreign' microbes that are normally dermal, intestinal or vaginal flora was detected in up to 50% of the samples. High mean proportions of lost or unidentified species were also occasionally noted. The results showed that the subgingival biofilm of renal transplant recipients with chronic periodontitis comprised mainly gram-negative rods and spirochetes. Besides the usual predominant cultivable subgingival microbiota associated with periodontitis, the high prevalence of unidentified and 'foreign' microbes indicates the possibility of subgingival microbial alteration in renal transplant patients.