In Vitro Activities of New Triazole Antifungal Agents, Posaconazole and Voriconazole, Against Oral Candida Isolates from Patients Suffering from Denture Stomatitis

Denture stomatitis is often treated with antifungal agents but recurrences or new episodes are common, and certain episodes can be resistant. New triazoles, such as posaconazole and voriconazole, may represent useful alternatives for management. In vitro activities of amphotericin B, nystatin, miconazole, fluconazole, itraconazole, posaconazole and voriconazole against 150 oral Candida (101 C. albicans, 18 C. tropicalis, 12 C. glabrata, 11 C. guilliermondii, 4 C. parapsilosis, 2 Saccharomyces cerevisiae, 1 C. dubliniensis and 1 C. krusei) from 100 denture wearers were tested by the CLSI M27-A3 method. Resistant isolates were retested by Sensititre YeastOne and Etest. Most antifungal agents were very active. However, 4 C. glabrata (33.3%), 2 C. tropicalis (11.1%), 6 C. albicans (5.6%) and 1 C. krusei were resistant to itraconazole. Posaconazole was active against 143 yeast isolates (95.3%): 6 C. albicans (5.9%) and 1 C. tropicalis (5.6%) were resistant. Geometric mean MICs were 0.036 μg/ml for C. parapsilosis, 0.062 μg/ml for C. albicans, 0.085 μg/ml for C. tropicalis, 0.387 μg/ml for C. guilliermondii and 0.498 μg/ml for C. glabrata. Voriconazole was active against 148 isolates (98.7%) with geometric mean MICs ranging from 0.030 μg/ml for C. parapsilosis, 0.042 μg/ml for C. albicans, 0.048 μg/ml for C. tropicalis, 0.082 μg/ml for C. guilliermondii, to 0.137 μg/ml for C. glabrata. Only 2 C. albicans (2%) were resistant to voriconazole showing cross-resistance to other azoles. Posaconazole and voriconazole have excellent in vitro activities against all Candida isolates and could represent useful alternatives for recalcitrant or recurrent candidiasis.     

Wild-Type MIC Distributions and Epidemiologic Cutoff Values for Fluconazole and <Emphasis Type="Italic">Candida</Emphasis>: Time for New Clinical Breakpoints?

Antifungal susceptibility testing of Candida against fluconazole has been standardized by both the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Both CLSI and EUCAST have developed clinical breakpoint (CBP) criteria for fluconazole, but these differ in both magnitude and target species. Studies using the EUCAST method have also defined wild-type minimum inhibitory concentration (MIC) distributions and epidemiologic cutoff values (ECVs or ECOFFs) for the common species of Candida. The ECVs serve as a sensitive means of discriminating wild-type strains from those with acquired resistance mechanisms and include MICs of 1 μg/mL for C. albicans, 2 μg/mL for C. tropicalis and C. parapsilosis, 32 μg/mL for C. glabrata, and 128 μg/mL for C. krusei. Because the CLSI CBPs may be too insensitive to detect emerging resistance among strains of C. albicans, C. tropicalis, and C. parapsilosis, and bisect the WT MIC distribution of C. glabrata, we sought to establish the wild-type MIC distribution and ECVs for fluconazole and Candida spp. The establishment of the wild-type MIC distributions and ECVs for fluconazole using CLSI methods will be useful in resistance surveillance and may prove to be an important step in the development of species-specific CBPs for this important antifungal agent.     

Biofilm formation by <Emphasis Type="Italic">Candida albicans</Emphasis> isolated from intrauterine devices

Our survey revealed that infected intrauterine devices (IUDs) recovered from patients suffering from reproductive tract infections (RTIs) were tainted with Candida biofilm composed of a single or multiple species. Scanning electron microscopy (SEM) analysis of C. albicans biofilm topography showed that it consists of a dense network of mono- or multilayer of cells embedded within the matrix of extracellular polymeric substances (EPS). Confocal scanning laser microscopy (CSLM) and atomic force microscopy (AFM) images depicted that C. albicans biofilms have a highly heterogeneous architecture composed of cellular and noncellular elements with EPS distributed in the cell-surface periphery or at cell-cell interface. Biochemical analysis showed that EPS produced by C. albicans biofilm contained significantly reduced total carbohydrate (40%), protein (5%) and enhanced amount of hexosamine (4%) in contrast to its planktonic counterparts. The in vitro activity of antifungal agents amphotericin B, nystatin, fluconazole and chlorhexidine against pre-formed C. albicans biofilm, assessed using XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) reduction assay revealed increased resistance of these infectious biofilm (50% reduction in metabolic activity at a concentration of 8, 16, 64, 128 μg/ml respectively) in comparison to its planktonic form.     

Silver colloidal nanoparticles: antifungal effect against adhered cells and biofilms of Candida albicans and Candida glabrata

The aim of this study was to evaluate the effect of silver nanoparticles (SN) against Candida albicans and Candida glabrata adhered cells and biofilms. SN (average diameter 5 nm) were synthesized by silver nitrate reduction with sodium citrate and stabilized with ammonia. Minimal inhibitory concentration (MIC) tests were performed for C. albicans (n = 2) and C. glabrata (n = 2) grown in suspension following the Clinical Laboratory Standards Institute microbroth dilution method. SN were applied to adhered cells (2 h) or biofilms (48 h) and after 24 h of contact their effect was assessed by enumeration of colony forming units (CFUs) and quantification of total biomass (by crystal violet staining). The MIC results showed that SN were fungicidal against all strains tested at very low concentrations (0.4–3.3 μg ml^?1). Furthermore, SN were more effective in reducing biofilm biomass when applied to adhered cells (2 h) than to pre-formed biofilms (48 h), with the exception of C. glabrata ATCC, which in both cases showed a reduction ～90%. Regarding cell viability, SN were highly effective on adhered C. glabrata and respective biofilms. On C. albicans the effect was not so evident but there was also a reduction in the number of viable biofilm cells. In summary, SN may have the potential to be an effective alternative to conventional antifungal agents for future therapies in Candida-associated denture stomatitis.     

Characterization of Switch Phenotypes in <Emphasis Type="Italic">Candida albicans</Emphasis> Biofilms

The aim of this study was to characterize switch phenotypes in Candida albicans biofilms. Cells of Candida albicans 192887g biofilms (24 h) were resuspended and these together with their planktonic counterparts were separately inoculated on Lee’s medium agar supplemented with arginine and zinc, at 25 °C for 9 days, for colony formation. The different switch phenotypes, as reflected by varying colony morphologies, were then examined for their (i) stability under various growth conditions, (ii) carbohydrate assimilation profiles, (iii) susceptibility to the polyene antifungal, nystatin, (iv) adhering and biofilm-forming ability, (v) filamentation, and (vi) growth rate in yeast nitrogen base medium supplemented with 100 mM glucose. Our data showed that the frequency of phenotypic switching in C. albicans biofilms was approximately 1%. Compared with the planktonic yeasts, cells derived from candidal biofilms generated one of the phenotypes less frequently (Chi-square-tests: P = 0.017). The five phenotypes derived from the biofilm growth demonstrated differing profiles for carbohydrate assimilation, adhesion, biofilm formation, filamentation, and growth rate. These findings reported here, for the first time, imply that phenotypic switching in the candidal biofilms differs from that in the planktonic growth, and affects multiple biological attributes.     

In Vitro Investigation of Antifungal Activity of Allicin Alone and in Combination with Azoles Against <Emphasis Type="Italic">Candida</Emphasis> Species

Candidiasis is a term describing infections by yeasts from the genus Candida, and the type of infection encompassed by candidiasis ranges from superficial to systemic. Treatment of such infections often requires antifungals such as the azoles, but increased use of these drugs has led to selection of yeasts with increased resistance to these drugs. In this study, we used allicin, an allyl sulfur derivative of garlic, to demonstrate both its intrinsic antifungal activity and its synergy with the azoles, in the treatment of these yeasts in vitro. In this study, the MIC₅₀ and MIC₉₀ of allicin alone against six Candida spp. ranged from 0.05 to 25 μg/ml. However, when allicin was used in combination with fluconazole or ketoconazole, the MICs were decreased in some isolates. Our results demonstrated the existing synergistic effect between allicin and azoles in some of the Candida spp. such as C. albicans, C. glabrata and C. tropicalis, but synergy was not demonstrated in the majority of Candida spp. tested. Nonetheless, In vivo testing needs to be performed to support these findings.     

Distribution Coefficients of Dietary Sugars in Artificial 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 cm² 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.     

Relationship between salivary histatin 5 levels and Candida CFU counts in healthy elderly

Objectives: Few epidemiological studies have confirmed the antifungal activity of histatin 5 in saliva against Candida species. The purpose of this study was to examine the relationship between concentrations of histatin 5 and the number of cultivable Candida in saliva samples from elderly. Methods: Whole saliva samples were obtained from 124 elderly people, 65 years or older, living in a rural community. The concentrations of histatin 5 in saliva samples were determined by the enzyme‐linked immunosorbent assay (ELISA) using a monoclonal antibody. Total colony‐forming units (CFUs) were counted on a selective medium for Candida. Multiple linear regression analysis was performed to determine the independent contribution of explanatory variables to Candida CFUs using age, sex, histatin 5 concentration and type of denture prosthesis as independent variables. Results: Saliva samples from 104 subjects (84%) were candidal colony‐positive. The youngest group (65–69 years old) showed significantly smaller Candida CFU counts than those in the older group. The mean Candida CFU count of denture wearers was significantly higher than that of non‐denture wearers. Significantly negative associations were found between Candida CFU counts and histatin 5 level in the oldest group (p < 0.05) and in the full‐denture wearers (p < 0.01). Multiple linear regression analysis revealed that Candida CFU counts were mostly associated with type of dentures, followed by histatin 5 concentration. Conclusion: Possible activity of histatin 5 against Candida in whole saliva of elderly people was epidemiologically confirmed. The area covered by the prostheses was a strong factor associated with the Candida CFU count.     

Experiments on in vivo biofilm formation and in vitro adhesion of Candida species on polysiloxane liners

doi:10.1111/j.1741‐2358.2009.00329.x
Experiments on in vivo biofilm formation and in vitro adhesion of Candida species on polysiloxane liners Objectives: Microorganisms may colonise polysiloxane soft liners leading to bio‐deterioration. The aim of this study was to investigate in vitro adhesion and in vivo biofilm formation of Candida species on polysiloxane surfaces. Methods: The materials used in this study were Molloplast B, GC Reline soft, Mollosil Plus, Silagum Comfort and Palapress Vario. The in vitro retention of clinical isolates of Candida albicans to the relining and denture‐base materials by microscopic (scanning electron microscopy, SEM), conventional culturing methods and antimicrobial properties of these materials were studied. Candida found on materials and mucosa following long‐term use were identified and quantified, and biofilms covering the surfaces were investigated by SEM. Results: There was a significant decrease in the number of cells attached in vitro to saliva‐coated surfaces compared with non‐treated surfaces. An oral Candida carriage of 78% was found. Candida albicans, C. glabrata, C. intermedia and C. tropicalis were identified. In vivo biofilm formation on the liners appeared as massive colonisation by microorganisms. Conclusions: The results of the in vitro experiments suggest that salivary film influences early colonisation of different C. albicans strains. The film layer also minimises the differences among different strains. The Candida carriage of these patients was similar to denture‐wearing patients without soft liners.     

Silver nanoparticles: influence of stabilizing agent and diameter on antifungal activity against Candida albicans and Candida glabrata biofilms

Aim: The purpose of this work was to evaluate the size‐dependent antifungal activity of different silver nanoparticles (SN) colloidal suspensions against Candida albicans and Candida glabrata mature biofilms. Methods and Results: The research presented herein used SN of three different average sizes (5, 10 and 60 nm), which were synthesized by the reduction of silver nitrate through sodium citrate and which were stabilized with ammonia or polyvinylpyrrolidone. Minimal inhibitory concentration (MIC) assays were performed using the microdilution methodology. The antibiofilm activity of SN was determined by total biomass quantification (by crystal violet staining) and colony forming units enumeration. MIC results showed that all SN colloidal suspensions were fungicidal against the tested strains at very low concentrations (0·4–3·3 μg ml^?1). With regard to biomass quantification, SN colloidal suspensions were very effective only against C. glabrata biofilms, achieving biomass reductions around 90% at a silver concentration of 108 μg ml^?1. In general, all SN suspensions promoted significant log₁₀ reduction of the mean number of cultivable biofilm cells after exposure to silver concentrations at or higher than 108 μg ml^?1. Moreover, the results showed that the particle size and the type of stabilizing agent used did not interfere in the antifungal activity of SN against Candida biofilms. Conclusions: This study suggests that SN have antifungal therapeutic potential, but further studies are still required namely regarding formulation and delivery means. Significance and Impact of the Study: SN may contribute to the development of new strategies for the improvement of oral health and quality of life particularly of the complete denture wearers.     

Anticandidal Efficacy of Cinnamon Oil Against Planktonic and Biofilm Cultures of <Emphasis Type="Italic">Candida parapsilosis</Emphasis> and <Emphasis Type="Italic">Candida orthopsilosis</Emphasis>

Candida parapsilosis is yeast capable of forming biofilms on medical devices. Novel approaches for the prevention and eradication of the biofilms are desired. This study investigated the anticandidal activity of sixteen essential oils on planktonic and biofilm cultures of C. parapsilosis complex. We used molecular tools, enumeration of colony-forming units, the colourimetric MTT assay, scanning electron microscopy (SEM) and a chequerboard assay coupled with software analyses to evaluate the growth kinetics, architecture, inhibition and reduction in biofilms formed from environmental isolates of the Candida parapsilosis complex; further, we also evaluated whether essential oils would interact synergistically with amphotericin B to increase their anticandidal activities. Of the environmental C. parapsilosis isolates examined, C. parapsilosis and C. orthopsilosis were identified. Biofilm growth on polystyrene substrates peaked within 48 h, after which growth remained relatively stable up to 72 h, when it began to decline. Details of the architectural analysis assessed by SEM showed that C. parapsilosis complex formed less complex biofilms compared with C. albicans biofilms. The most active essential oil was cinnamon oil (CO), which showed anticandidal activity against C. orthopsilosis and C. parapsilosis in both suspension (minimum inhibitory concentration—MIC—250 and 500 μg/ml) and biofilm (minimum biofilm reduction concentration—MBRC—1,000 and 2,000 μg/ml) cultures. CO also inhibited biofilm formation (MBIC) at concentrations above 250 μg/ml for both species tested. However, synergism with amphotericin B was not observed. Thus, CO is a natural anticandidal agent that can be effectively utilised for the control of the yeasts tested.     

Species and material considerations in the formation and development of microalgal biofilms

The development of microalgal biofilms has received very limited study despite its relevance in the design of photobioreactors where film growth may be advantageous for biomass separation or disadvantageous in fouling surfaces. Here, the effects of species selection, species control, and substrate properties on biofilms of Scenedesmus obliquus and Chlorella vulgaris were investigated. Experiments were conducted in batch culture and in continuous culture modes in a flow cell. Cell growth was monitored using confocal laser scanning microscopy and gravimetrically. Species selection and species control had significant effects on biofilm development. On non-sterile wastewater, C. vulgaris shifted from primarily planktonic (23.7% attachment) to primarily sessile (79.8% attachment) growth. The biofilms that developed in non-sterile conditions were thicker (52 ± 19 μm) than those grown in sterile conditions (7 ± 6 μm). By contrast, S. obliquus attained similar thicknesses (54 ± 31 and 53 ± 38 μm) in both sterile and non-sterile conditions. Neither species was able to dominate a non-sterile biofilm. The effect of substrate surface properties was minimal. Both species grew films of similar thickness (∼30 μm for S. obliquus, <10 μm for C. vulgaris) on materials ranging from hydrophilic (glass) to hydrophobic (polytetrafluoroethylene). Surface roughness created by micropatterning the surface with 10 μm grooves did not translate into long-term increases in biofilm thickness. The results indicate that species selection and control are more important than surface properties in the development of microalgal biofilms.     

Green synthesis of selenium nanoparticles and evaluate their effect on the expression of ERG3, ERG11 and FKS1 antifungal resistance genes in Candida albicans and Candida glabrata

Drug resistance in Candida species has been considerably increased in the last decades. Given the opposition to antifungal agents, toxicity and interactions of the antimicrobial drugs, identifying new antifungal agents seems essential. This study assessed the antifungal effects of nanoparticles (NPs) on the standard strains of Candida albicans and Candida glabrata and determined the expression genes, including ERG3, ERG11 and FKS1. Selenium nanoparticles (Se-NPs) were biosynthesized with a standard strain of C. albicans and approved by several methods including, ultraviolet-visible spectrophotometer, X-ray diffraction technique, Fourier-transform infrared analysis, field-emission scanning electron microscopy and EDX diagram. The antifungal susceptibility testing performed the minimum inhibitory concentrations (MICs) using the CLSI M27-A3 and M27-S4 broth microdilution method. The expression of the desired genes was examined by the real-time PCR assay between untreated and treated by antifungal drugs and Se-NPs. The MICs of itraconazole, amphotericin B and anidulafungin against C. albicans and C. glabrata were 64, 16 and 4 µg ml⁻¹. In comparison, reduced the MIC values for samples treated with Se-NPs to 1 and 0·5 µg ml⁻¹. The results obtained from real-time PCR and analysis of the ∆∆C_q values showed that the expression of ERG3, ERG11 and FKS1 genes was significantly down-regulated in Se-NPs concentrations (P < 0·05). This study's evidence implies biosafety Se-NPs have favourable effects on the reducing expression of ERG3, ERG11 and FKS1 antifungal resistance genes in C. albicans and C. glabrata.     

Environmental pH modulates biofilm formation and matrix composition in Candida albicans and Candida glabrata

Abstract

Candida species are fungal opportunistic pathogens capable of colonizing and infecting various human anatomical sites, where they have to adapt to distinct niche-specific pH conditions. The aim of this study was to analyse the features of Candida albicans and Candida glabrata biofilms developed under neutral and vaginal acidic (pH 4) conditions. C. albicans produced thicker and more filamentous biofilms under neutral than under acidic conditions. On the other hand, the formation of biofilms by C. glabrata was potentiated by the acidic conditions suggesting the high adaptability of this species to the vaginal environment. In general, both species developed biofilms containing higher amounts of matrix components (protein and carbohydrate) under neutral than acidic conditions, although the opposite result was found for one C. glabrata strain. Overall, this study contributes to a better understanding of the modulation of C. albicans and C. glabrata virulence by specific pH conditions.     

Synergistic effects of low doses of histatin 5 and its analogues on amphotericin B anti-mycotic activity

The increase in the use of antifungal agents for prophylaxis and therapy has led to the development of antifungal drug resistance. Drug combinations may prevent or delay resistance development. The aim of the present study was to investigate whether naturally and designed cationic antifungal peptides act synergistically with commonly used antimycotics. No enhanced activity was found upon addition of dhvar4, a designed analogue of the human salivary peptide histatin 5, or PGLa to fluconazole or 5–flucytosine, respectively. In contrast, strong synergism of amphotericin B with the peptides was found against several Aspergillus, Candida, and Cryptococcus strains, and against an amphotericin B-resistant C. albicans laboratory mutant in the standardised broth microdilution assays according to the NCCLS standard method M27–T. Amphotericin B showed synergism with dhvar5, another designed analogue of histatin 5, and with magainin 2 against all seven tested strains. Combinations of amphotericin B with histatin 5, dhvar4, and PGLa showed synergism against four of the seven strains. The growth inhibitory activity of amphotericin B was enhanced by sub-MIC concentrations of peptide, but its haemolytic activity remained unaffected, suggesting that its cytotoxicity to host cells was not increased and that peptides may be suitable candidates for combination therapy.     

Characterization of a multifunctional feather-degrading <Emphasis Type="Italic">Bacillus subtilis</Emphasis> isolated from forest soil

In this study, we isolated and characterized a novel feather-degrading bacterium that shows keratinolytic, antifungal and plant growth-promoting activities. A bacterium S8 was isolated from forest soil and confirmed to belong to Bacillus subtilis by BIOLOG system and 16S rRNA gene analysis. The improved culture conditions for the production of keratinolytic protease were 0.1% (w/v) sorbitol, 0.3% (w/v) KNO₃, 0.1% (w/v) K₂HPO₄, 0.06% (w/v) KH₂PO₄ and 0.04% (w/v) MgCl₂·6H₂O (pH 8.0 and 30°C), respectively. In the improved medium containing 0.1% (w/v) feather, keratinolytic protease production was around 53.3 ± 0.3 U/ml at 4 day; this value was 10-fold higher than the yield in the basal feather medium (5.3 ± 0.1 U/ml). After cultivation for 5 days in the improved medium, intact feather was completely degraded. Feather degradation resulted in free –SH group, soluble protein and amino acids production. The concentration of free –SH group in the culture medium was 15.5 ± 0.2 μM at 4 days. Nineteen amino acids including all essential amino acids were produced in the culture medium; the concentration of total amino acid produced was 3360.4 μM. Proline (2809.9 μM), histidine (371.3 μM) and phenylalanine (172.0 μM) were the major amino acids released in the culture medium. B. subtilis S8 showed the properties related to plant growth promotion: hydrolytic enzymes, ammonification, indoleacetic acid (IAA), phosphate solubilization, and broad-spectrum antimicrobial activity. Interestingly, the strain S8 grown in the improved medium produced IAA and antifungal activity, indicating simultaneous production of keratinolytic and antifungal activities and IAA by B. subtilis S8. These results suggest that B. subtilis S8 could be not only used to improve the nutritional value of feather wastes but also is useful in situ biodegradation of feather wastes. Furthermore, it could also be a potential biofertilizer or biocontrol agent applicable to crop plant soil.     

Definitions and Epidemiology of Candida Species not Susceptible to Echinocandins

Antifungal susceptibility testing of Candida against the echinocandin antifungal agents (anidulafungin [ANF], caspofungin [CSF], micafungin [MCF]) has been standardized by the Clinical and Laboratory Standards Institute (CLSI) Subcommittee on Antifungal Testing. The CLSI proposed a single set of clinical breakpoints (CBPs) for all three echinocandins and all species of Candida: susceptible, minimum inhibitory concentration (MIC) ≤ 2 μg/mL; nonsusceptible, MIC > 2 μg/mL. Subsequently, these CBPs have been shown to lack sensitivity in detecting strains of Candida with acquired resistance mechanisms associated with treatment failure. Studies using the CLSI method have defined wild-type (WT) MIC distributions and epidemiologic cutoff values (ECVs) for each echinocandin and the common species of Candida. The ECVs serve as a sensitive means of discriminating WT strains from those with acquired resistance mechanisms. WT MIC distributions revealed ECV ranges of 0.03 to 0.25 μg/mL for all major species except C. parapsilosis (1–4 μg/mL) and C. guilliermondii (4–16 μg/mL). These ECVs reliably differentiate WT strains of each species from non-WT strains containing fks mutations. These data, coupled with additional biochemical, clinical, pharmacokinetic, and pharmacodynamic considerations, have resulted in new CBPs of ≤0.25 μg/mL (susceptible), 0.5 μg/mL (intermediate), and ≥1 μg/mL (resistant) for ANF, CSF, and MCF for C. albicans, C. tropicalis, and C. krusei. For these agents and C. parapsilosis, the new CBPs are ≤2 μg/mL (susceptible), 4 μg/mL (intermediate), and ≥8 μg/mL (resistant). For C. glabrata, the CBPs for ANF and CSF are ≤0.12 μg/mL (susceptible), 0.25 μg/mL (intermediate), and ≥0.5 μg/mL (resistant), whereas those for MCF are ≤0.06 μg/mL, 0.12 μg/mL, and ≥0.25 μg/mL, respectively. Application of both ECVs and the lower species-specific CBPs for the echinocandins has proven useful in both resistance surveillance and clinical care and will serve as an important step in international harmonization of in vitro susceptibility testing of this important antifungal class.     

The effect of a selective octopamine antagonist, epinastine, on pharyngeal pumping in Caenorhabditis elegans

This paper investigates the effect of epinastine, a selective octopamine antagonist in invertebrates, in Caenorhabditis elegans. Specifically, its ability to block the inhibitory action of octopamine on C. elegans-isolated pharynx was assayed. Isolated pharynxes were stimulated to pump by the addition of 500 nM 5-hydroxytryptamine (5-HT) (113 ± 2 per 30 s, n = 15). Octopamine inhibited the 5-HT-induced pumping in a concentration-dependent manner (threshold 1–5 μM) with a 61 ± 11% inhibition with 50 μM (n = 5). Epinastine (0.1 μM) antagonized the inhibitory response to octopamine (P < 0.001; n = 15). Tyramine also inhibited pharyngeal pumping induced by 5-HT but was less potent than octopamine. Tyramine, 50 μM to 1 mM, gave a transient inhibition e.g. of 40 ± 5% at 50 μM (n = 5). A higher (10 μM) concentration of epinastine was required to block the tryamine response compared with octopamine. It is concluded that epinastine selectively antagonizes the effect of octopamine on C. elegans pharynx. Further studies are required to test its selectivity for octopamine in other tissues and other nematodes.     

Synthetic antimicrobial β‐peptide in dual‐treatment with fluconazole or ketoconazole enhances the in vitro inhibition of planktonic and biofilm Candida albicans

Fungal infections are a pressing concern for human health worldwide, particularly for immunocompromised individuals. Current challenges such as the elevated toxicity of common antifungal drugs and the emerging resistance towards these could be overcome by multidrug therapy. Natural antimicrobial peptides, AMPs, in combination with other antifungal agents are a promising avenue to address the prevailing challenges. However, they possess limited biostability and susceptibility to proteases, which has significantly hampered their development as antifungal therapies. β‐peptides are synthetic materials designed to mimic AMPs while allowing high tunability and increased biostability. In this work, we report for the first time the inhibition achieved in Candida albicans when treated with a mixture of a β‐peptide model and fluconazole or ketoconazole. This combination treatment enhanced the biological activity of these azoles in planktonic and biofilm Candida, and also in a fluconazole‐resistant strain. Furthermore, the in vitro cytotoxicity of the dual treatment was evaluated towards the human hepatoma cell line, HepG2, a widely used model derived from liver tissue, which is primarily affected by azoles. Analyses based on the LA‐based method and the mass‐action law principle, using a microtiter checkerboard approach, revealed synergism of the combination treatment in the inhibition of planktonic C. albicans. The dual treatment proved to be fungicidal at 48 and 72 h. Interestingly, it was also found that the viability of HepG2 was not significantly affected by the dual treatments. Finally, a remarkable enhancement in the inhibition of the highly azole‐resistant biofilms and fluconazole resistant C. albicans strain was obtained. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.