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

Evaluating Candida albicans biofilm formation and novel antifungal treatment

Candida biofilms have become an increasingly important clinical problem. The widespread use of antibiotics, frequent use of indwelling medical devices, and a trend towards increased patient immuno-suppression has resulted in a creation of opportunity for clinically important yeasts to form biofilms. Whilst there is growing evidence of the importance of Candida biofilms in clinical medicine, not all clinical isolates are able to form biofilms. There is therefore a fundamental gap in understanding exactly what drives biofilm formation and its clinical implications. These structures 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. Therefore, the discovery of alternative antifungal agents to be used in the treatment of fungal biofilms is in great demand for the management of these infections. A panel of Candida albicans bloodstream isolates were assessed for their biofilm forming ability by using the crystal violet assay and measuring cellular surface hydrophobicity. Scanning electron microscopy was used to visualise differences in the clinical biofilms. The impact of amphotericin B (AMB) treatment was determined next by broth microdilution method to assess differences in susceptibility profiles of the clinical isolates. The virulence of these clinical isolates was evaluated in vivo using a Galleria mellonella model and transcriptional analysis used to assess the expression of various genes associated with C. albicans biofilm formation within clinical isolates. Extracellular DNA (eDNA) in clinical biofilms was quantified using a microplate fluorescence assay and chitinase activity measured using a biochemical assay. Moreover, the potential of a novel antimicrobial agent Carbohydrate-derived fulvic acid (CHD-FA) was assessed against a panel of fungal and bacterial species. The mechanism of action of CHD-FA was determined using membrane assays include ATP release, and propidium iodide fluorescence, with various inhibitors used to determine whether CHD-FA activity is affected by known resistance mechanisms. Finally, the immunomodulatory properties of CHD-FA were investigated using ELISA and PCR arrays. The results from this study have shown C. albicans biofilm formation is differential within clinical isolates, where those with high biofilm formation (HBF) predominately consisted of hyphal cells, were more virulent in vivo and had decreased susceptibility to AMB, when compared to those with low biofilm formation (LBF). Furthermore, transcriptional analysis identified a number of genes that positively correlated with C. albicans biofilm formation. The novel agent carbohydrate-derived fulvic acid (CHD-FA) was shown to not only be highly active against C. albicans biofilms, but also against a range or orally relevant bacteria through non-specific membrane activity. Furthermore, CHD-FA was shown to down-regulate a number of pro-inflammatory mediators in an oral epithelial cell line. In conclusion, this study has characterised C. albicans clinical isolates based on their biological characteristics, where clear difference in virulence and antifungal treatment have been shown. It may be possible to develop a panel of genetic markers that could be used as a diagnostic tool for detecting biofilm formation in clinical isolates. CHD-FA is a microbiocidal compound that may serve as a potential novel antiseptic agent for the treatment of oral candidiasis and other candidal biofilm infections, whereby the immunomodulatory properties of CHD-FA could be exploited for controlling inflammation in a number of diseases

Tolerance of Pseudomonas aeruginosa in in vitro biofilms to high level peracetic acid disinfection

Biofilm has been suggested as a cause of disinfection failures in flexible endoscopes where no lapses in the decontamination procedure can be identified. To test this theory, the activity of peracetic acid (PAA), one of the commonly used disinfectants in the reprocessing of flexible endoscopes, was evaluated against both planktonic and sessile communities of Pseudomonas aeruginosa. To investigate the ability of P. aeruginosa biofilm to survive high level PAA disinfection. The susceptibility of planktonic cells of P. aeruginosa and biofilms 24, 48, 96 and 192 h old to PAA was evaluated by estimating their viability using resazurin viability and plate count methods. The biomass of the P. aeruginosa biofilms was also quantified using crystal violet assay. Planktonic cells of P. aeruginosa were treated with 5 - 30 ppm concentration of PAA in the presence of 3.0 g/L of Bovine serum albumin (BSA) for 5 min. Biofilms of P. aeruginosa were also treated with various PAA concentrations (100 - 3000 ppm) for 5 min. Planktonic cells of P. aeruginosa were eradicated by 20 ppm of PAA, whereas biofilms showed an age dependent tolerance to PAA, and 96 h old biofilm was only eradicated at PAA concentration of 2500 ppm. 96 h old P. aeruginosa biofilm survives 5 min treatment with 2000 ppm of PAA, which is the working concentration used in some endoscope washer disinfectors. This implies that disinfection failure of flexible endoscopes could occur when biofilms are allowed to build up in the lumens of endoscopes

New strategic insights into managing fungal biofilms

Fungal infections have dramatically increased in the last decades in parallel with an increase of populations with impaired immunity, resulting from medical conditions such as cancer, transplantation or other chronic diseases. Such opportunistic infections result from a complex relationship between fungi and host, and can range from self-limiting to chronic or life-threatening infections. Modern medicine, characterized by a wide use of biomedical devices, offers new niches for fungi to colonize and form biofilm communities. The capability of fungi to form biofilms is well documented and associated with increased drug tolerance and resistance. In addition, biofilm formation facilitates persistence in the host promoting a persistent inflammatory condition. With a limited availability of antifungals within our arsenal, new therapeutic approaches able to address both host and pathogenic factors that promote fungal disease progression, i.e. chronic inflammation and biofilm-formation, could represent an advantage in the clinical setting. In this paper we discuss the antifungal properties of Myriocin, Fulvic Acid and Acetylcholine in light of their already known anti-inflammatory activity and as candidate dual action therapeutics to treat opportunistic fungal infections

<i>Candida albicans</i> fungaemia following traumatic urethral catheterisation in a paraplegic patient with diabetes mellitus and candiduria treated by caspofungin

A 58-year-old paraplegic male, with long-term indwelling urethral catheter, developed catheter block. The catheter was changed, but blood-stained urine was drained intermittently. A long segment of the catheter was seen lying outside his penis, which indicated that the balloon of Foley catheter had been inflated in urethra. The misplaced catheter was removed and a new catheter was inserted correctly. Gentamicin 160 mg was given intravenously; meropenem 1 gram every eight hours was prescribed; antifungals were not given. Twenty hours later, this patient developed distension of abdomen, tachycardia, and hypotension; he was not arousable. Computed tomography of abdomen revealed inflamed uroepithelium of right renal pelvis and ureter, 4 mm lower ureteric calculus with gas in right ureter proximally, and vesical calculus containing gas in its matrix. Urine and blood culture yielded &lt;i&gt;Candida albicans&lt;/i&gt;. Identical sensitivity pattern of both isolates suggested that the source of the bloodstream infection was most likely urine. Both isolates formed consistently high levels of biofilm formation in vitro as assessed using a biofilm biomass stain, and high levels of resistance to voriconazole were observed. Both amphotericin B and caspofungin showed good activity against the biofilms. HbA1c was 111 mmol/mol. This patient was prescribed human soluble insulin and caspofungin 70 mg followed by 50 mg daily intravenously. He recovered fully from candidemia

The application of phenotypic microarray analysis to anti-fungal drug development

Candida albicans metabolic activity in the presence and absence of acetylcholine was measured using phenotypic microarray analysis. Acetylcholine inhibited C. albicans biofilm formation by slowing metabolism independent of biofilm forming capabilities. Phenotypic microarray analysis can therefore be used for screening compound libraries for novel anti-fungal drugs and measuring antifungal resistance

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

Gaining insights from Candida biofilm heterogeneity: one size does not fit all

Despite their clinical significance and substantial human health burden, fungal infections remain relatively under-appreciated. The widespread overuse of antibiotics and the increasing requirement for indwelling medical devices provides an opportunistic potential for the overgrowth and colonization of pathogenic Candida species on both biological and inert substrates. Indeed, it is now widely recognized that biofilms are a highly important part of their virulence repertoire. Candida albicans is regarded as the primary fungal biofilm forming species, yet there is also increasing interest and growing body of evidence for non-Candida albicans species (NCAS) biofilms, and interkingdom biofilm interactions. C. albicans biofilms are heterogeneous structures by definition, existing as three-dimensional populations of yeast, pseudo-hyphae, and hyphae, embedded within a self-produced extracellular matrix. Classical molecular approaches, driven by extensive studies of laboratory strains and mutants, have enhanced our knowledge and understanding of how these complex communities develop, thrive, and cause host-mediated damage. Yet our clinical observations tell a different story, with differential patient responses potentially due to inherent biological heterogeneity from specific clinical isolates associated with their infections. This review explores some of the recent advances made in an attempt to explore the importance of working with clinical isolates, and what this has taught us

A Prospective Surveillance Study of Candidaemia : Epidemiology, Risk Factors, Antifungal Treatment and Outcome in Hospitalized Patients

Funding This work was supported by the Wellcome Trust Strategic Award for Medical Mycology and Fungal Immunology 097377/Z/11/Z. Data collection was supported by a grant from Pfizer. GR was also supported by a research fellowship grant from Gilead Sciences. The collection of the isolates was funded by a Gilead Fellowship to GR. Acknowledgments We are grateful to microbiology colleagues throughout Scotland for submitting isolates. Antimicrobial sensitivity testing was performed by the Mycology Reference Laboratory, Public Health England, Bristol.Peer reviewedPublisher PD

Biofilms formed by Candida albicans bloodstream isolates display phenotypic and transcriptional heterogeneity that are associated with resistance and pathogenicity

Background: Candida albicans infections have become increasingly recognised as being biofilm related. Recent studies have shown that there is a relationship between biofilm formation and poor clinical outcomes in patients infected with biofilm proficient strains. Here we have investigated a panel of clinical isolates in an attempt to evaluate their phenotypic and transcriptional properties in an attempt to differentiate and define levels of biofilm formation.&lt;p&gt;&lt;/p&gt; Results: Biofilm formation was shown to be heterogeneous; with isolates being defined as either high or low biofilm formers (LBF and HBF) based on different biomass quantification. These categories could also be differentiated using a cell surface hydrophobicity assay with 24 h biofilms. HBF isolates were more resistance to amphotericin B (AMB) treatment than LBF, but not voriconazole (VRZ). In a Galleria mellonella model of infection HBF mortality was significantly increased in comparison to LBF. Histological analysis of the HBF showed hyphal elements intertwined indicative of the biofilm phenotype. Transcriptional analysis of 23 genes implicated in biofilm formation showed no significant differential expression profiles between LBF and HBF, except for Cdr1 at 4 and 24 h. Cluster analysis showed similar patterns of expression for different functional classes of genes, though correlation analysis of the 4 h biofilms with overall biomass at 24 h showed that 7 genes were correlated with high levels of biofilm, including Als3, Eap1, Cph1, Sap5, Plb1, Cdr1 and Zap1.&lt;p&gt;&lt;/p&gt; Conclusions: Our findings show that biofilm formation is variable amongst C. albicans isolates, and categorising isolates depending on this can be used to predict how pathogenic the isolate will behave clinically. We have shown that looking at individual genes in less informative than looking at multiple genes when trying to categorise isolates at LBF or HBF. These findings are important when developing biofilm-specific diagnostics as these could be used to predict how best to treat patients infected with C. albicans. Further studies are required to evaluate this clinically.&lt;p&gt;&lt;/p&gt