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, particularlyCandida 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 by Candida albicans has shown to be highly variable and is directly associated with pathogenicity and poor clinical outcomes in patients at risk. The aim of this study was to test the hypotheses that the extracellular DNA release by C. albicans is strain dependent and is associated with biofilm heterogeneity. Initially, biofilm formed by C. albicans high biofilm formers (HBF) or low biofilm formers (LBF) were treated with DNase to find whether eDNA play a role in their biofilm formation. Digestion of biofilm eDNA significantly reduced the HBF biofilm biomass by five fold compared to untreated controls. In addition, quantification of eDNA over the period of biofilm formation by SYBR green assay demonstrate a significantly higher level of 2 to 6 fold in HBF compared to LBF. Biochemical and transcriptional analyses showed that chitinase activity and mRNA levels of chitinase genes, a marker of autolysis, were upregulated in 24 h biofilm formation by HBF compared to LBF, indicating autolysis pathway possibly involved in causing variation. The biofilm biomass and eDNA release by single (∆cht2, ∆cht3) and double knockout (∆cht2/∆cht3) chitinase mutants were significantly less compared to their parental strain CA14, confirming the role of chitinases in eDNA release and biofilm formation. Correlation analysis found a positive correlation between chitinases and HWP 1, suggesting eDNA may release during the hyphal growth. Finally, we showed a combinational treatment of biofilms with DNase or chitinase inhibitor (acetazolamide) plus amphotericin B significantly improved antifungal susceptibility by 2 to 8 fold. Collectively, these data show that eDNA release by C. albicans clinical isolates is variable and is associated with differential biofilm formation. Digestion of biofilm eDNA by DNase may provide a novel therapeutic strategies to destabilise biofilm growth and improves antifungal sensitivity.

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.

Approximately 20 % of the UK population wear some form of denture prosthesis, resulting in denture stomatitis in half of these individuals. Candida albicans is primarily attributed as the causative agent, due to its biofilm -forming ability. Recently, there has been increasing evidence of C. albicans biofilm heterogeneity and the negative impact it can have clinically; however, this phenomenon has yet to be studied in relation to denture isolates. The aims of this study were to evaluate C. albicans biofilm formation of clinical denture isolates in a denture environment and to assess antimicrobial activity of common denture cleansers against these tenacious communities. C. albicans isolated from dentures of healthy and diseased individuals was quantified using real-time PCR and biofilm biomass assessed using crystal violet. Biofilm development on the denture substratum poly(methyl methacrylate), Molloplast B and Ufi-gel was determined. Biofilm formation was assessed using metabolic and biomass stains, following treatment with denture hygiene products. Although C. albicans was detected in greater quantities in diseased individuals, it was not associated with increased biofilm biomass. Denture substrata were shown to influence biofilm biomass, with poly(methyl methacrylate) providing the most suitable environment for C. albicans to reside. Of all denture hygiene products tested, Milton had the most effective antimicrobial activity, reducing biofilm biomass and viability the greatest. Overall, our results highlight the complex nature of denture- related disease, and disease development cannot always be attributed to a sole cause. It is the distinct combination of various factors that ultimately determines the pathogenic outcome.

Graphical Overview of Candidozyma auris biology. (a) Climate change and associated environmental stressors are hypothesized to have been a driver for this heat and salt tolerant pathogen to emerge. Exactly how it spreads from the environment into the hospital remains unknown. (b) Within the clinical environment, it is able to colonize and persist on the skin of patients and clinical staff. It is readily transferred and shed from patients onto fomites and medical equipment, where it can survive for prolonged periods. (c) On patients and the wider hospital environment, it can persist as adherent wet and dry surface biofilm communities that are highly tolerant to antifungal agents, antiseptics and disinfectants through inhibition by biofilm properties (e.g. extracellular matrix).

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 yieldedCandida albicans. 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 formationin vitroas 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.