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Both Nd:YAG and Er:YAG lasers have been suggested as root canal disinfection aids. The aim of this in vitro study is to compare both wavelengths in terms of irradiation dose required for microbial inactivation, to quantify these irradiation doses and to investigate the influence of certain (laser) parameters on the antimicrobial efficacy. Agar plates containing a uniform layer of Enterococcus faecalis, Candida albicans or Propionibacterium acnes were mounted perpendicularly underneath the laser handpieces (5 mm spot). The Er:YAG laser was operated in single-pulse mode. Pulse energies of 40-400 mJ and pulse lengths of 100, 300, 600, and 1,000 μs were tested. After incubation at 37°C for 48 h, growth on the plates was scored. The pulse energy yielding complete absence of growth over the entire spot area was taken as the total inhibition threshold (TIT). TITs were determined for every species and pulse length. The Nd:YAG laser was operated with pulse trains because single pulses were ineffective. Output power was 15 W and frequency was 100 Hz. Spots were irradiated for 5-120 s. After incubation, the diameters of the inhibition zones were measured. For the Er:YAG laser, TITs varied between 100 and 210 mJ, and differed significantly between species and pulse lengths. Using Nd:YAG irradiation, TITs were around 5,300 J/cm(2) for C. albicans and 7,100 J/cm(2) for P. acnes. No inhibition was observed for E. faecalis. Er:YAG irradiation was superior to Nd:YAG in inactivating microorganisms on agar surfaces.

Previous research has shown that 1% to 10% of sessile Candida albicans cells survive treatment with high doses of miconazole (a fungicidal imidazole). In the present study, we investigated the involvement of sphingolipid biosynthetic intermediates in this survival. We observed that the LCB4 gene, coding for the enzyme that catalyzes the phosphorylation of dihydrosphingosine and phytosphingosine, is important in governing the miconazole resistance of sessile Saccharomyces cerevisiae and C. albicans cells. The addition of 10 nM phytosphingosine-1-phosphate (PHS-1-P) drastically reduced the intracellular miconazole concentration and significantly increased the miconazole resistance of a hypersusceptible C. albicans heterozygous LCB4/lcb4 mutant, indicating a protective effect of PHS-1-P against miconazole-induced cell death in sessile cells. At this concentration of PHS-1-P, we did not observe any effect on the fluidity of the cytoplasmic membrane. The protective effect of PHS-1-P was not observed when the efflux pumps were inhibited or when tested in a mutant without functional efflux systems. Also, the addition of PHS-1-P during miconazole treatment increased the expression levels of genes coding for efflux pumps, leading to the hypothesis that PHS-1-P acts as a signaling molecule and enhances the efflux of miconazole in sessile C. albicans cells.

Candida albicans biofilms are a major cause of voice prosthesis deterioration in laryngectomized patients. The aim of this study was to produce a surface capable of inhibiting C. albicans biofilm formation. Dimethylaminoethylmethacrylate (DMAEMA) and polyethylenimine (PEI) moieties were covalently bound to the surface of polydimethylsiloxane (PDMS) or polymethylmethacrylate (PMMA) and subsequently quaternized. Physicochemical characterization of the grafted surfaces was carried out and their effect on C. albicans cell numbers was assessed using a modified Robbins device to grow the biofilms. Covalently bound quaternized polyDMAEMA (polyDMAEMAq) and PEI (PEIq) inhibited biofilm growth, with reductions up to 92%. Our approach may show promise for future application in medical devices such as catheters and prostheses.

Biofilm formation is often associated with persistent Candida albicans infections. Treatment of these infections is difficult, since sessile C. albicans cells show increased resistance towards antifungal agents. The molecular mechanisms behind biofilm resistance in C. albicans are not yet understood. In the present study, we investigated the transcriptional response in young and mature in vitro-grown biofilms after a short and longer exposure time to high doses of fluconazole or amphotericin B. Treatment of biofilms with high doses of antifungal agents resulted in a drug-specific transcriptional response. Exposure of biofilms to fluconazole induced upregulation of genes encoding enzymes involved in ergosterol biosynthesis (ERG1, ERG3, ERG11 and ERG25). Treatment of biofilms with amphotericin B resulted in an overexpression of KRE1 and SKN1, two genes encoding proteins involved in beta-1,6-glucan biosynthesis. Our data indicate that sessile C. albicans cells show controlled regulation of gene expression, as they quickly mount a drug-specific transcriptional response in the presence of high doses of antifungal agents. These transcriptional changes suggest upregulation of ergosterol biosynthesis (fluconazole) and upregulation of beta-1,6-glucan biosynthesis (amphotericin B) in sessile C. albicans cells that might contribute to a resistant biofilm phenotype., ((c) 2010 Elsevier Masson SAS. All rights reserved.)

Unlike various disinfectants, antifungals have not been commonly incorporated so far in medical devices, such as catheters or prostheses, to prevent biofilm formation by Candida spp. In the present study, five antimycotics were added to polydimethyl siloxane (PDMS) disks via admixture (nystatin) or impregnation (trimethylsilyl-nystatin (TMS-nystatin), miconazole, tea tree oil (TTO), zinc pyrithione). Nystatin-medicated PDMS disks exhibited a concentration-dependent inhibitory effect on biofilm formation in a microtiter plate (MTP) but not in a Modified Robbins Device (MRD). This observation, together with HPLC data and agar diffusion tests, indicates that a small fraction of free nystatin is released, which kills Candida albicans cells in the limited volume of a MTP well. In contrast, biofilm inhibition amounted to more than one log unit in the MRD on disks impregnated with miconazole, TTO, and zinc pyrithione. It is hypothesized that the reduction in biofilm formation by these compounds in a flow system occurs through a contact-dependent effect.

In order to prevent biofilm formation by Candida albicans, several cationic peptides were covalently bound to polydimethylsiloxane (PDMS). The salivary peptide histatin 5 and two synthetic variants (Dhvar 4 and Dhvar 5) were used to prepare peptide functionalized PDMS using 4-azido-2,3,5,6-tetrafluoro-benzoic acid (AFB) as an interlinkage molecule. In addition, polylysine-, polyarginine-, and polyhistidine-PDMS surfaces were prepared. Dhvar 4 functionalized PDMS yielded the highest reduction of the number of C. albicans biofilm cells in the Modified Robbins Device. Amino acid analysis demonstrated that the amount of peptide immobilized on the modified disks was in the nanomole range. Poly-d-lysine PDMS, in particular the homopeptides with low molecular weight (2500 and 9600) showed the highest activity against C. albicans biofilms, with reductions of 93% and 91%, respectively. The results indicate that the reductions are peptide dependent.

In the present study six assays for the quantification of biofilms formed in 96-well microtiter plates were optimised and evaluated: the crystal violet (CV) assay, the Syto9 assay, the fluorescein diacetate (FDA) assay, the resazurin assay, the XTT assay and the dimethyl methylene blue (DMMB) assay. Pseudomonas aeruginosa, Burkholderia cenocepacia, Staphylococcus aureus, Propionibacterium acnes and Candida albicans were used as test organisms. In general, these assays showed a broad applicability and a high repeatability for most isolates. In addition, the estimated numbers of CFUs present in the biofilms show limited variations between the different assays. Nevertheless, our data show that some assays are less suitable for the quantification of biofilms of particular isolates (e.g. the CV assay for P. aeruginosa).

Background: Candida albicans biofilms are commonly found on indwelling medical devices. However, the molecular basis of biofilm formation and development is not completely understood. Expression analysis of genes potentially involved in these processes, such as the ALS (Agglutinine Like Sequence) gene family can be performed using quantitative PCR (qPCR). In the present study, we investigated the expression stability of eight housekeeping genes potentially useful as reference genes to study gene expression in Candida albicans (C. albicans) biofilms, using the geNorm Visual Basic Application (VBA) for Microsoft Excel. To validate our normalization strategies we determined differences in ALS1 and ALS3 expression levels between C. albicans biofilm cells and their planktonic counterparts., Results: The eight genes tested in this study are ranked according to their expression stability (from most stable to least stable) as follows: ACT1 (beta-actin)/PMA1 (adenosine triphosphatase), RIP (ubiquinol cytochrome-c reductase complex component), RPP2B (cytosolic ribosomal acidic protein P2B), LSC2 (succinyl-CoA synthetase beta-subunit fragment), IMH3 (inosine-5'-monophosphate dehydrogenase fragment), CPA1 (carbamoyl-phosphate synthethase small subunit) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Our data indicate that five genes are necessary for accurate and reliable normalization of gene expression data in C. albicans biofilms. Using different normalization strategies, we found a significant upregulation of the ALS1 gene and downregulation of the ALS3 gene in C. albicans biofilms grown on silicone disks in a continous flow system, the CDC reactor (Centre for Disease Control), for 24 hours., Conclusion: In conclusion, we recommend the use of the geometric mean of the relative expression values from the five housekeeping genes (ACT1, PMA1, RIP, RPP2B and LSC2) for normalization, when analysing differences in gene expression levels between C. albicans biofilm cells and planktonic cells. Validation of the normalization strategies described above showed that the ALS1 gene is overexpressed and the ALS3 gene is underexpressed in C. albicans biofilms grown on silicone in the CDC reactor for 24 hours.

A double-blind randomized trial was conducted among 36 laryngectomees to assess the influence of a buccal bioadhesive slow-release tablet containing miconazole nitrate on the lifetime of the Provox voice prosthesis. All patients colonized with Candida spp and treated with miconazole showed a significant decrease of colonization at the end of the study. Intratracheal phonatory pressures were remarkably higher after 2 months of follow-up in the placebo group. No local or systemic adverse reactions to miconazole were observed during this study. Patient compliance was acceptable according to regular miconazole determination in saliva samples. The device lifetime was significantly higher in patients treated with miconazole even after 1 year of follow-up. The use of a buccal bioadhesive slow-release tablet containing an antimycotic agent proves to be an adequate method of preventing fungal colonization and deterioration of silicone voice prostheses.