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.

Human serum potentiates the expression of genes associated with antifungal drug resistance in C. albicans biofilms on central venous catheters.

Candida albicans is a major agent of fungaemias and frequently causes systemic disease through seeded, blood stream dissemination. These infections, particularly common in hospitalized patients with central venous catheters (CVCs), appear to persevere due to biofilm reservoirs of the yeast that tend to develop on the device. Although it is known that candidal biofilms are intrinsically resistant to antifungals compared with their planktonic counterparts, there is a paucity of data on the expression of antifungal drug resistance genes (DRGs) in candidal biofilms in CVC reservoirs. Furthermore, notwithstanding the fact that CVCs are constantly bathed in human serum, there are no studies on the effect of the latter on the DRG expression in candidal biofilms. Hence, we developed in vitro biofilms of three different C. albicans strains on silicone CVC discs immersed in human serum and evaluated the temporal expression of nine antifungal DRGs. In an attempt to evaluate the effect of hyphal elements on DRG expression, we incorporated a hyphal mutant (HM) and its wild-type (WT) counterpart, as well as a fresh clinical isolate in the studies. Human serum significantly up-regulated DRG transcripts in Candida biofilms on CVCs, at different stages of biofilm growth, while the WT strain over-expressed more DRGs than the HM strain. Here, we report, for the first time, that both human serum and the hyphal elements of the yeast have a profound modulatory effect on DRG expression in C. albicans biofilms on CVCs.

Quantitative analysis of salivary and biofilm bacteria associated with cavitated and non-cavitated carious lesions in pre-school children.

OBJECTIVE: To quantify and compare Streptococcus mutans (S. mutans) and Lactobacillus fermentum (L. fermentum) in saliva and biofilm of caries-free children to those with cavitated and non-cavitated lesions. DESIGN: One hundred and thirty-five 3-4 years old children were grouped (n = 45 in each group) according to their caries status: Clinical examination was done by a calibrated examiner. Biofilm and saliva were collected to quantify the microorganisms using qRT-PCR. The decayed-missing-filled surfaces (dmfs) was calculated by adding the number of decayed (ICDAS-II score 3-6), filled (ICDAS-II score 7 and 8) and missing (ICDAS-II score 9) surfaces due to caries. The correlation between the bacterial amounts and the number of carious surfaces was evaluated using Spearman's correlation coefficient. The levels and proportions of the microorganisms were compared using the Kruskal-Wallis test at an α-level of 0.05. RESULTS: The quantity of S. mutans and L. fermentum was significantly higher in saliva and biofilm of children with cavitated lesions, followed by those with non-cavitated lesions and the lowest in caries-free children. Also, salivary and biofilm S. mutans, along with biofilm L. fermentum levels, significantly correlated with the number of non-cavitated surfaces; while salivary and biofilm S. mutans and L. fermentum levels significantly correlated with the number of cavitated surfaces. Additionally, dmfs scores significantly correlated with the salivary and biofilm S. mutans and L. fermentum levels. CONCLUSIONS: S. mutans and L. fermentum in saliva and biofilm samples are associated with caries lesion severity.

Fluconazole resistance in Candida albicans is induced by Pseudomonas aeruginosa quorum sensing.

Microorganisms employ quorum sensing (QS) mechanisms to communicate with each other within microbial ecosystems. Emerging evidence suggests that intraspecies and interspecies QS plays an important role in antimicrobial resistance in microbial communities. However, the relationship between interkingdom QS and antimicrobial resistance is largely unknown. Here, we demonstrate that interkingdom QS interactions between a bacterium, Pseudomonas aeruginosa and a yeast, Candida albicans, induce the resistance of the latter to a widely used antifungal fluconazole. Phenotypic, transcriptomic, and proteomic analyses reveal that P. aeruginosa's main QS molecule, N-(3-Oxododecanoyl)-L-homoserine lactone, induces candidal resistance to fluconazole by reversing the antifungal's effect on the ergosterol biosynthesis pathway. Accessory resistance mechanisms including upregulation of C. albicans drug-efflux, regulation of oxidative stress response, and maintenance of cell membrane integrity, further confirm this phenomenon. These findings demonstrate that P. aeruginosa QS molecules may confer protection to neighboring yeasts against azoles, in turn strengthening their co-existence in hostile polymicrobial infection sites.

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.

Reverse transcriptase polymerase chain reaction (RT-PCR) detection of HLP gene expression in Candida glabrata and its possible role in in vitro haemolysin production.

Although haemolysins are known to be putative virulence factors contributing to pathogenicity in Candida species, the haemolytic activity of Candida glabrata and its genetic expression is ill understood at present. Thus, we studied a total of 34 Candida glabrata isolates for their in vitro haemolytic activity using a previously described plate assay system. The mRNA expression of HLP, a putative haemolysin gene, of these isolates was also evaluated using a semi-quantitative, non-competitive RT-PCR assay. All 34 C. glabrata isolates exhibited both partial (alpha) and complete (beta) haemolytic activity to varying degrees. In parallel with the haemolytic activity, all isolates were also positive for HLP mRNA expression. The expression levels of HLP mRNA (as relative units) ranged from 1.01 to 1.82, with a mean value of 1.32. On regression analysis of latter values and the haemolytic activity (in terms of the dimension of the haemolytic zone in the plate assay) of the C. glabrata isolates a highly significant positive correlation was noted (r=0.759, p<0.0001). Taken together, our data illustrate not only the phenotypic characteristics of haemolysin(s) and HLP expression of a battery of C. glabrata clinical isolates, but also, for the first time, evidence for a role of HLP in haemolysis.

'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.

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.

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.

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.

Sjögren's syndrome sufferers have increased oral yeast levels despite regular dental care.

AIM: To investigate the prevalence and quantity of oral yeasts and their association with oral candidiasis in Sjögren's syndrome (SS) patients receiving regular dental care. MATERIALS AND METHODS: Yeasts in oral rinse and full-mouth supra-gingival plaque samples from 25 primary SS, 27 secondary SS and 29 control subjects were selectively cultured. All yeasts except single-species isolates were genotyped using pulsed field gel electrophoresis (PFGE). RESULTS: Ten (19%) SS sufferers had symptomless candidiasis. SS subjects had a higher prevalence (73%vs 7%) and quantity of yeasts than controls in both oral rinse and plaque samples (P < 0.05). The prevalence of yeasts in plaque was associated with candidiasis regardless of denture wearing (P < or = 0.04). Candida albicans was the predominant yeast isolated. PFGE showed 20 (66% of total) C. albicans isolate pairs, i.e. C. albicans species isolated from plaque and oral rinse samples of the same individual, were of closely related genetic clonal types (P < 0.01). CONCLUSIONS: Despite effective oral hygiene, more SS subjects than controls had detectable levels of oral yeasts and their presence in supra-gingival plaque was associated with candidiasis. Candida albicans colonized supra-gingival biofilm even in well-maintained SS individuals, posing a challenge to the control of oral candidiasis.

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.

Development of novel oligonucleotide probes for seven Actinomyces species and their utility in supragingival plaque analysis.

OBJECTIVE: The traditional, biochemical and enzymatic methods of identifying Actinomyces species are frequently confounded by the similar phenotypic characteristics shared by the different members of this genus. Therefore, we developed novel species-specific oligonucleotide probes to accurately speciate seven pathogenic Actinomyces species, namely, Actinomyces bovis, A. gerencseriae, A. israelii, A. meyeri, A. naeslundii, A. odontolyticus and A. viscosus. METHODS: A pair of universal primers and seven 15- to 19-base oligonucleotide probes with a tail of 20 thymidines on the 5' end were developed. The variable regions of 16S ribosomal DNA of 36 strains of Actinomyces belonging to the above species were amplified and labeled with digoxigenin, and an oligonucleotide-DNA hybridization assay was performed to examine the specificity and sensitivity of these probes. RESULTS: All seven, newly developed probes were specific and sensitive, and accurately detected 36 reference and wild type strains belonging to Actinomyces species, without cross-reactions. The probe for A. naeslundii detected all strains belonging to the genospecies 1 (12 strains) and catalase-negative genospecies 2 (four strains); it failed to detect catalase-positive A. naeslundii genospecies 2 (previous A. viscosus serotype II) (two strains). However, the latter strains of catalase-positive A. naeslundii genospecies 2 were correctly detected by the probe developed for A. viscosus. The new probes were then field tested using supragingival plaque samples from 28 healthy preschool children. Whilst A. odontolyticus was detected in almost all samples (96.4%), A. gerencseriae, A. meyeri, catalase-negative A. naeslundii and catalase-positive A. naeslundii genospecies 2 were detected in < 50% samples. CONCLUSION: We conclude that the developed oligonucleotide probes, complementary to the variable regions of 16S rDNA, would be of potential value for differentiating Actinomyces spp. in clinical samples from the oral cavity and other ecosystems where such species may abound.