Leighann Sherry

Fungal biofilm resistance.

Fungal biofilm infections have become increasingly recognised as a significant clinical problem. One of the major reasons behind this is the impact that these have upon treatment, as antifungal therapy often fails and surgical intervention is required. This places a large financial burden on health care providers. This paper aims to illustrate the importance of fungal biofilms, particularly Candida albicans, and discusses some of the key fungal biofilm resistance mechanisms that include, extracellular matrix (ECM), efflux pump activity, persisters, cell density, overexpression of drug targets, stress responses, and the general physiology of the cell. The paper demonstrates the multifaceted nature of fungal biofilm resistance, which encompasses some of the newest data and ideas in the field.

Biofilm formation is a risk factor for mortality in patients with Candida albicans bloodstream infection—Scotland, 2012–2013

Bloodstream infections caused by Candida species remain a significant cause of morbidity and mortality in hospitalized patients. Biofilm formation by Candida species is an important virulence factor for disease pathogenesis. A prospective analysis of patients with Candida bloodstream infection (n = 217) in Scotland (2012–2013) was performed to assess the risk factors associated with patient mortality, in particular the impact of biofilm formation. Candida bloodstream isolates (n = 280) and clinical records for 157 patients were collected through 11 different health boards across Scotland. Biofilm formation by clinical isolates was assessed in vitro with standard biomass assays. The role of biofilm phenotype on treatment efficacy was also evaluated in vitro by treating preformed biofilms with fixed concentrations of different classes of antifungal. Available mortality data for 134 patients showed that the 30-day candidaemia case mortality rate was 41%, with predisposing factors including patient age and catheter removal. Multivariate Cox regression survival analysis for 42 patients showed a significantly higher mortality rate for Candida albicans infection than for Candida glabrata infection. Biofilm-forming ability was significantly associated with C. albicans mortality (34 patients). Finally, in vitro antifungal sensitivity testing showed that low biofilm formers and high biofilm formers were differentially affected by azoles and echinocandins, but not by polyenes. This study provides further evidence that the biofilm phenotype represents a significant clinical entity, and that isolates with this phenotype differentially respond to antifungal therapy in vitro. Collectively, these findings show that greater clinical understanding is required with respect to Candida biofilm infections, and the implications of isolate heterogeneity.

Biofilm-Forming Capability of Highly Virulent, Multidrug-Resistant Candida auris

The emerging multidrug-resistant yeast pathogen Candida auris has attracted considerable attention as a source of healthcare–associated infections. We report that this highly virulent yeast has the capacity to form antifungal resistant biofilms sensitive to the disinfectant chlorhexidine in vitro.

Antifungal, cytotoxic, and immunomodulatory properties of tea tree oil and its derivative components: potential role in management of oral candidosis in cancer patients

Candida albicans forms oral biofilms that cause disease and are difficult to treat with conventional antifungal agents. Tea tree oil (TTO) is a natural compound with reported antimicrobial and immunomodulatory activities. The aims of the study were to evaluate the antifungal efficacy of TTO and key derivatives against C. albicans biofilms, to assess the toxicological effects of TTO on a clinically relevant oral cell line, and to investigate its impact on inflammation. TTO and its derivatives were examined against 100 clinical strains of C. albicans. Planktonic minimum inhibitory concentrations (MICs) were determined using the CLSI M-27A broth microdilution method. Sessile MICs were determined using an XTT reduction assay. Inhibition, time-kill, and mode of action studies were performed. OKF6-TERT2 epithelial cells were used for cytotoxicity and cytokine expression assays. Planktonic C. albicans isolates were susceptible to TTO, terpinen-4-ol (T-4-ol), and α-terpineol, with an MIC50 of 0.5, 0.25, and 0.25%, respectively. These three compounds also displayed potent activity against the 69 biofilm-forming strains, of which T-4-ol and α-terpineol displayed rapid kill kinetics. For all three compounds, 1 × MIC50 effectively inhibited biofilm growth when C. albicans were treated at 0, 1, and 2 h post adhesion. By scanning electron microscopy analysis and PI uptake, TTO and derivative components were shown to be cell membrane active. TTO and T-4-ol were cytotoxic at 1 × MIC50, whereas at 0.5 × MIC50 T-4-ol displayed no significant toxicity. Transcript and protein analysis showed a reduction of IL-8 when treated with TTO and T-4-ol. These data provide further in vitro evidence that TTO and its derivative components, specifically T-4-ol, exhibit strong antimicrobial properties against fungal biofilms. T-4-ol has safety advantages over the complete essential oil and may be suitable for prophylaxis and treatment of established oropharyngeal candidosis. A clinical trial of T-4-ol is worthy of consideration.

Liposomal Amphotericin B Displays Rapid Dose-Dependent Activity against Candida albicans Biofilms

ABSTRACT Biofilms formed by Candida albicans bloodstream isolates on catheters are an important clinical problem. Devising chemotherapeutic strategies to treat these in situ is an attractive option. We report here that liposomal amphotericin effectively kills C. albicans biofilms rapidly (12 h) and effectively (>90%) in a dose-dependent manner, whereas caspofungin displays an inverse concentration-dependent effect. This study has implications for considering the effective doses of antifungal agents used for catheter lock therapy.

Carbohydrate derived fulvic acid: an in vitro investigation of a novel membrane active antiseptic agent against Candida albicans biofilms

Carbohydrate derived fulvic acid (CHD-FA) is a heat stable low molecular weight, water soluble, cationic, colloidal material with proposed therapeutic properties. The aim of this study was to evaluate the antifungal activity of CHD-FA against Candida albicans, and to characterize its mode of action. A panel of C. albicans isolates (n = 50) derived from a range of clinical specimens were grown planktonically and as biofilms, and the minimum inhibitory concentrations determined. Scanning electron microscopy was performed to examine ultrastructural changes and different cell membrane assays were used to determine its mode of action. In addition, the role of C. albicans biofilm resistance mechanisms were investigated to determine their effects on CHD-FA activity. CHD-FA was active against planktonic and sessile C. albicans at concentrations 0.125 and 0.25% respectively, and was shown to be fungicidal, acting through disruption of the cell membrane activity. Resistance mechanisms, including matrix, efflux, and stress, had a limited role upon CHD-FA activity. Overall, based on the promising in vitro spectrum of activity and minimal biofilm resistance of the natural and cheap antiseptic CHD-FA, further studies are required to determine its applicability for clinical use.

Polymicrobial Candida biofilms: friends and foe in the oral cavity

The role of polymicrobial biofilm infections in medicine is becoming more apparent. Increasing number of microbiome studies and deep sequencing has enabled us to develop a greater understanding of how positive and negative microbial interactions influence disease outcomes. An environment where this is particularly pertinent is within the oral cavity, a rich and diverse ecosystem inhabited by both bacteria and yeasts, which collectively occupy and coexist within various niches as biofilm communities. Studies within this environment have however tended to be subject to extensive independent investigation, in the context of either polymicrobial bacterial communities or yeast biofilms, but rarely both together. It is clear however that they are not mutually exclusive. Therefore, this review aims to explore the influence of candidal populations on the composition of these complex aggregates and biofilm communities, to investigate their mechanistic interactions to understand how these impact clinical outcomes, and determine whether we can translate how this knowledge can be used to improve patient management.

Utilising polyphenols for the clinical management of Candida albicans biofilms

Polyphenols (PPs) are secondary metabolites abundant in plant-derived foods. They are reported to exhibit antimicrobial activity that may offer an alternative to existing antimicrobials. The aim of this study was to evaluate the antifungal potential of PPs against Candida albicans biofilms that are commonly recalcitrant to antifungal therapy. The antifungal activity of 14 PPs was assessed in terms of planktonic and sessile minimum inhibitory concentrations (PMICs and SMICs, respectively) against various C. albicans clinical isolates. The most active PPs were further tested for their effect on C. albicans adhesion and biofilm growth using standard biomass assays, microscopy and quantitative gene expression. Of the 14 PPs tested, 7 were effective inhibitors of planktonic growth, of which pyrogallol (PYG) was the most effective (PMIC₅₀=78 μg/mL), followed by curcumin (CUR) (PMIC₅₀=100 μg/mL) and pyrocatechol (PMIC₅₀=625 μg/mL). Both PYG and CUR displayed activity against C. albicans biofilms (SMIC₅₀=40 μg/mL and 50 μg/mL, respectively), although they did not disrupt the biofilm or directly affect the cellular structure. Overall, CUR displayed superior biofilm activity, significantly inhibiting initial cell adhesion following pre-coating (P<0.01), biofilm growth (P<0.05) and gene expression (P<0.05). This inhibitory effect diminished with prolonged CUR exposure, although it still inhibited by 50% after 4h adhesion. Overall, CUR exhibited positive antibiofilm properties that could be used at the basis for development of similar molecules, although further cellular and in vivo studies are required to explore its precise mechanism of action.

Development of an in vitro periodontal biofilm model for assessing antimicrobial and host modulatory effects of bioactive molecules

BackgroundInflammation within the oral cavity occurs due to dysregulation between microbial biofilms and the host response. Understanding how different oral hygiene products influence inflammatory properties is important for the development of new products. Therefore, creation of a robust host-pathogen biofilm platform capable of evaluating novel oral healthcare compounds is an attractive option. We therefore devised a multi-species biofilm co-culture model to evaluate the naturally derived polyphenol resveratrol (RSV) and gold standard chlorhexidine (CHX) with respect to anti-biofilm and anti-inflammatory properties.MethodsAn in vitro multi-species biofilm containing S. mitis, F. nucleatum, P. gingivalis and A. actinomycetemcomitans was created to represent a disease-associated biofilm and the oral epithelial cell in OKF6-TERT2. Cytotoxicity studies were performed using RSV and CHX. Multi-species biofilms were either treated with either molecule, or alternatively epithelial cells were treated with these prior to biofilm co-culture. Biofilm composition was evaluated and inflammatory responses quantified at a transcriptional and protein level.ResultsCHX was toxic to epithelial cells and multi-species biofilms at concentrations ranging from 0.01-0.2%. RSV did not effect multi-species biofilm composition, but was toxic to epithelial cells at concentrations greater than 0.01%. In co-culture, CHX-treated biofilms resulted in down regulation of the inflammatory chemokine IL-8 at both mRNA and protein level. RSV-treated epithelial cells in co-culture were down-regulated in the release of IL-8 protein, but not mRNA.ConclusionsCHX possesses potent bactericidal properties, which may impact downstream inflammatory mediators. RSV does not appear to have bactericidal properties against multi-species biofilms, however it did appear to supress epithelial cells from releasing inflammatory mediators. This study demonstrates the potential to understand the mechanisms by which different oral hygiene products may influence gingival inflammation, thereby validating the use of a biofilm co-culture model.

Investigating the biological properties of carbohydrate derived fulvic acid (CHD-FA) as a potential novel therapy for the management of oral biofilm infections

BackgroundA number of oral diseases, including periodontitis, derive from microbial biofilms and are associated with increased antimicrobial resistance. Despite the widespread use of mouthwashes being used as adjunctive measures to control these biofilms, their prolonged use is not recommended due to various side effects. Therefore, alternative broad-spectrum antimicrobials that minimise these effects are highly sought after. Carbohydrate derived fulvic acid (CHD-FA) is an organic acid which has previously demonstrated to be microbiocidal against Candida albicans biofilms, therefore, the aims of this study were to evaluate the antibacterial activity of CHD-FA against orally derived biofilms and to investigate adjunctive biological effects.MethodsMinimum inhibitory concentrations were evaluated for CHD-FA and chlorhexidine (CHX) against a range of oral bacteria using standardised microdilution testing for planktonic and sessile. Scanning electron microscopy was also employed to visualise changes in oral biofilms after antimicrobial treatment. Cytotoxicity of these compounds was assessed against oral epithelial cells, and the effect of CHD-FA on host inflammatory markers was assessed by measuring mRNA and protein expression.ResultsCHD-FA was highly active against all of the oral bacteria tested, including Porphyromonas gingivalis, with a sessile minimum inhibitory concentration of 0.5%. This concentration was shown to kill multi-species biofilms by approximately 90%, levels comparable to that of chlorhexidine (CHX). In a mammalian cell culture model, pretreatment of epithelial cells with buffered CHD-FA was shown to significantly down-regulate key inflammatory mediators, including interleukin-8 (IL-8), after stimulation with a multi-species biofilm.ConclusionsOverall, CHD-FA was shown to possess broad-spectrum antibacterial activity, with a supplementary function of being able to down-regulate inflammation. These properties offer an attractive spectrum of function from a naturally derived compound, which could be used as an alternative topical treatment strategy for oral biofilm diseases. Further studies in vitro and in vivo are required to determine the precise mechanism by which CHD-FA modulates the host immune response.