Plastic pollution as a novel reservoir for the environmental survival of the drug resistant fungal pathogen Candida auris.

The WHO recently classified Candida auris as a fungal pathogen of "critical concern". Evidence suggests that C. auris emerged from the natural environment, yet the ability of this pathogenic yeast to survive in the natural environment is still poorly understood. The aim of this study, therefore, was to quantify the persistence of C. auris in simulated environmental matrices and explore the role of plastic pollution for facilitating survival and potential transfer of C. auris. Multi-drug resistant strains of C. auris persisted for over 30 days in river water or seawater, either planktonically, or in biofilms colonising high-density polyethylene (HDPE) or glass. C. auris could be transferred from plastic beads onto simulated beach sand, particularly when the sand was wet. Importantly, all C. auris cells recovered from plastics retained their pathogenicity; therefore, plastic pollution could play a significant role in the widescale environmental dissemination of this recently emerged pathogen.

Environmental reservoirs of the drug-resistant pathogenic yeast Candida auris.

Candia auris is an emerging human pathogenic yeast; yet, despite phenotypic attributes and genomic evidence suggesting that it probably emerged from a natural reservoir, we know nothing about the environmental phase of its life cycle and the transmission pathways associated with it. The thermotolerant characteristics of C. auris have been hypothesised to be an environmental adaptation to increasing temperatures due to global warming (which may have facilitated its ability to tolerate the mammalian thermal barrier that is considered a protective strategy for humans against colonisation by environmental fungi with pathogenic potential). Thus, C. auris may be the first human pathogenic fungus to have emerged as a result of climate change. In addition, the release of antifungal chemicals, such as azoles, into the environment (from both pharmaceutical and agricultural sources) is likely to be responsible for the environmental enrichment of resistant strains of C. auris; however, the survival and dissemination of C. auris in the natural environment is poorly understood. In this paper, we critically review the possible pathways through which C. auris can be introduced into the environment and evaluate the environmental characteristics that can influence its persistence and transmission in natural environments. Identifying potential environmental niches and reservoirs of C. auris and understanding its emergence against a backdrop of climate change and environmental pollution will be crucial for the development of effective epidemiological and environmental management responses.

An integrated transcriptomic and metabolomic approach to investigate the heterogeneous Candida albicans biofilm phenotype.

Candida albicans is the most prevalent and notorious of the Candida species involved in bloodstream infections, which is characterised by its capacity to form robust biofilms. Biofilm formation is an important clinical entity shown to be highly variable among clinical isolates. There are various environmental and physiological factors, including nutrient availability which influence the phenotype of Candida species. However, mechanisms underpinning adaptive biofilm heterogeneity have not yet been fully explored. Within this study we have profiled previously characterised and phenotypically distinct C. albicans bloodstream isolates. We assessed the dynamic susceptibility of these differing populations to antifungal treatments using population analysis profiling in addition to assessing biofilm formation and morphological changes. High throughput methodologies of RNA-Seq and LC-MS were employed to map and integrate the transcriptional and metabolic reprogramming undertaken by heterogenous C. albicans isolates in response to biofilm and hyphal inducing serum. We found a significant relationship between biofilm heterogeneity and azole resistance (P < 0.05). In addition, we observed that in response to serum our low biofilm forming (LBF) C. albicans exhibited a significant increase in biofilm formation and hyphal elongation. The transcriptional reprogramming of LBF strains compared to high biofilm forming (HBF) was distinct, indicating a high level of plasticity and variation in stress responses by heterogenous strains. The metabolic responses, although variable between LBF and HBF, shared many of the same responses to serum. Notably, a high upregulation of the arachidonic acid cascade, part of the COX pathway, was observed and this pathway was found to induce biofilm formation in LBF 3-fold. C. albicans is a highly heterogenous bloodstream pathogen with clinical isolates varying in antifungal tolerance and biofilm formation. In addition to this, C. albicans is capable of highly complex and variable regulation of transcription and metabolic pathways and heterogeneity across isolates further increases the complexity of these pathways. Here we have shown with a dual and integrated approach, the importance of studying a diverse panel of C. albicans isolates, which has the potential to reveal distinct pathways that can harnessed for drug discovery.

Our current clinical understanding of Candida biofilms: where are we two decades on?

Clinically we have been aware of the concept of Candida biofilms for many decades, though perhaps without the formal designation. Just over 20 years ago the subject emerged on the back of progress made from the bacterial biofilms, and academic progress pace has continued to mirror the bacterial biofilm community, albeit at a decreased volume. It is apparent that Candida species have a considerable capacity to colonize surfaces and interfaces and form tenacious biofilm structures, either alone or in mixed species communities. From the oral cavity, to the respiratory and genitourinary tracts, wounds, or in and around a plethora of biomedical devices, the scope of these infections is vast. These are highly tolerant to antifungal therapies that has a measurable impact on clinical management. This review aims to provide a comprehensive overight of our current clinical understanding of where these biofilms cause infections, and we discuss existing and emerging antifungal therapies and strategies.

In vitro bacterial vaginosis biofilm community manipulation using endolysin therapy.

Bacterial vaginosis (BV) affects approximately 26% of women of childbearing age globally, presenting with 3-5 times increased risk of miscarriage and two-fold risk of pre-term birth. Antibiotics (metronidazole and clindamycin) are typically employed to treat BV; however the success rate is low due to the formation of recalcitrant polymicrobial biofilms. As a novel therapeutic, promising results have been obtained in vitro using Gardnerella endolysins, although to date their efficacy has only been demonstrated against simple biofilm models. In this study, a four-species biofilm was developed consisting of Gardnerella vaginalis, Fannyhessea vaginae, Prevotella bivia and Mobiluncus curtisii. Biofilms were grown in NYC III broth and treated using antibiotics and an anti-Gardnerella endolysin (CCB7.1) for 24 h. Biofilm composition, viability and structure were assessed using colony counts, live/dead qPCR and scanning electron microscopy. All species colonised biofilms to varying degrees, with G. vaginalis being the most abundant. Biofilm composition remained largely unchanged when challenged with escalated concentrations of conventional antibiotics. A Gardnerella-targeted endolysin candidate (CCB7.1) showed efficacy against several Gardnerella species planktonically, and significantly reduced viable G. vaginalis within polymicrobial biofilms at 1 to 4X pMIC (p < 0.05 vs. vehicle control). Collectively, this study highlights the resilience of biofilm-embedded pathogens against the currently used antibiotics and provides a polymicrobial model that allows for more effective pre-clinical screening of BV therapies. The Gardnerella-specific endolysin CCB7.1 demonstrated significant activity against G. vaginalis within polymicrobial biofilms, altering the overall community dynamic and composition.

Investigating Dual-Species Candida auris and Staphylococcal Biofilm Antiseptic Challenge.

Candida auris can persistently colonize human skin, alongside a diverse bacterial microbiome. In this study we aimed to investigate the efficacy of antiseptic activities on dual-species interkingdom biofilms containing staphylococci to determine if antiseptic tolerance was negatively impacted by dual-species biofilms. Chlorhexidine, povidone iodine, and hydrogen peroxide (H2O2), were able to significantly reduce biofilm viable cell counts following exposure at 2%, 10%, and 3%, respectively. Notably, H2O2-treated biofilms were able to significantly recover and considerably repopulate following treatment. Fortunately, inter-kingdom interactions in dual-species biofilms of C. auris and staphylococci did not increase the tolerance of C. auris against antiseptics in vitro. These data indicate mixed infections are manageable with chlorhexidine and povidone iodine, but caution should be exercised in the consideration of H2O2.

Screening the Tocriscreen™ bioactive compound library in search for inhibitors of Candida biofilm formation.

Biofilms formed by Candida species present a significant clinical problem due to the ineffectiveness of many conventional antifungal agents, in particular the azole class. We urgently require new and clinically approved antifungal agents quickly for treatment of critically ill patients. To improve efficiency in antifungal drug development, we utilized a library of 1280 biologically active molecules within the Tocriscreen 2.0 Micro library. Candida auris NCPF 8973 and Candida albicans SC5314 were initially screened for biofilm inhibitory activity using metabolic and biomass quantitative assessment methods, followed up by targeted evaluation of five selected hits. The initial screening (80% metabolic inhibition rate) revealed that there was 90 and 87 hits (approx. 7%) for C. albicans and C. auris, respectively. Additionally, all five compounds selected from the initial hits exhibited a biofilm inhibition effect against several key Candida species tested, including C. glabrata and C. krusei. Toyocamycin displayed the most potent activity at concentrations as low as 0.5 µg/mL, though was limited to inhibition. Darapladib demonstrated an efficacy for biofilm inhibition and treatment at a concentration range from 8 to 32 µg/mL and from 16 to 256 µg/mL, respectively. Combinational testing with conventional antifungals against C. albicans strains demonstrated a range of synergies for planktonic cells, and notably an anti-biofilm synergy for darapladib and caspofungin. Together, these data provide new insights into antifungal management possibilities for Candida biofilms.

Cell Viability Assays for Candida auris.

Cell viability assays are useful for assessing the efficacy of antifungal therapeutics and disinfection strategies in vitro. In recent years these assays have been fundamental for the testing of conventional and novel therapies against the nosocomial fungal pathogen Candida auris. Here we provide detailed descriptions of methods for assessing cellular viability of Candida auris in vitro, such as metabolic assays (XTT and resazurin), colony-forming unit counting, live/dead quantitative PCR, and fluorescent staining for microscopic analyses.

Candida albicans and Enterococcus faecalis biofilm frenemies: When the relationship sours.

The opportunistic yeast Candida albicans and lactic acid bacteria Enterococcus faecalis are frequently co-isolated from various infection sites on the human body, suggesting a common interkingdom interaction. While some reports suggest an antagonism, the reason for their co-isolation therefore remains unclear. The purpose of this study was to undertake a detailed characterisation of this dual-species interaction. We used standard biofilm characterisation methodologies alongside an RNASeq analysis to assess the response of C. albicans to E. faecalis. We evaluated the relevance of pH to dual-species biofilm interactions and demonstrated that E. faecalis rapidly and significantly impacted C. albicans morphogenesis and biofilm formation, which was mirrored by levels of gene expression. These transcripts were enriched in amino acids biosynthesis and metabolism pathways in co-cultures, a finding that guided our investigation into pH related mechanism. We were able to demonstrate the direct role of E. faecalis induced low pH, which inhibited C. albicans hyphal morphogenesis and biofilm formation. The results suggest that the anti-candidal effect of E. faecalis is not based solely on a single mechanism, instead it may involve various mechanisms, which collectively reflects the complexity of interaction between C. albicans and E. faecalis and impacts treatment outcomes.

Investigating the Transcriptome of Candida albicans in a Dual-Species Staphylococcus aureus Biofilm Model.

Candida albicans is an opportunistic pathogen found throughout multiple body sites and is frequently co-isolated from infections of the respiratory tract and oral cavity with Staphylococcus aureus. Herein we present the first report of the effects that S. aureus elicits on the C. albicans transcriptome. Dual-species biofilms containing S. aureus and C. albicans mutants defective in ALS3 or ECE1 were optimised and characterised, followed by transcriptional profiling of C. albicans by RNA-sequencing (RNA-seq). Altered phenotypes in C. albicans mutants revealed specific interaction profiles between fungus and bacteria. The major adhesion and virulence proteins Als3 and Ece1, respectively, were found to have substantial effects on the Candida transcriptome in early and mature biofilms. Despite this, deletion of ECE1 did not adversely affect biofilm formation or the ability of S. aureus to interact with C. albicans hyphae. Upregulated genes in dual-species biofilms corresponded to multiple gene ontology terms, including those attributed to virulence, biofilm formation and protein binding such as ACE2 and multiple heat-shock protein genes. This shows that S. aureus pushes C. albicans towards a more virulent genotype, helping us to understand the driving forces behind the increased severity of C. albicans-S. aureus infections.

Recurrent Vulvovaginal Candidiasis: a Dynamic Interkingdom Biofilm Disease of Candida and Lactobacillus.

Despite the strikingly high worldwide prevalence of vulvovaginal candidiasis (VVC), treatment options for recurrent VVC (RVVC) remain limited, with many women experiencing failed clinical treatment with frontline azoles. Further, the cause of onset and recurrence of disease is largely unknown, with few studies identifying potential mechanisms of treatment failure. This study aimed to assess a panel of clinical samples from healthy women and those with RVVC to investigate the influence of Candida, the vaginal microbiome, and how their interaction influences disease pathology. 16S rRNA sequencing characterized disease by a reduction in specific health-associated Lactobacillus species, such as Lactobacillus crispatus, coupled with an increase in Lactobacillus iners. In vitro analysis showed that Candida albicans clinical isolates are capable of heterogeneous biofilm formation, and we found the presence of hyphae and C. albicans aggregates in vaginal lavage fluid. Additionally, the ability of Lactobacillus to inhibit C. albicans biofilm formation and biofilm-related gene expression was demonstrated. Using RNA sequencing technology, we were able to identify a possible mechanism by which L. crispatus may contribute to re-establishing a healthy vaginal environment through amino acid acquisition from C. albicans. This study highlights the potential formation and impact of Candida biofilms in RVVC. Additionally, it suggests that RVVC is not entirely due to an arbitrary switch in C. albicans from commensal to pathogen and that understanding interactions between this yeast and vaginal Lactobacillus species may be crucial to elucidating the cause of RVVC and developing appropriate therapies. IMPORTANCE RVVC is a significant burden, both economically and for women's health, but its prevalence is poorly documented globally due to the levels of self-treatment. Identifying triggers for development and recurrence of VVC and the pathogenesis of the microbes involved could considerably improve prevention and treatment options for women with recurrent, azole-resistant cases. This study therefore aimed to examine the interkingdom dynamics from healthy women and those with RVVC using next-generation sequencing techniques and to further investigate the molecular interactions between C. albicans and L. crispatus in a relevant biofilm coculture system.

Filling the Void: An Optimized Polymicrobial Interkingdom Biofilm Model for Assessing Novel Antimicrobial Agents in Endodontic Infection.

There is a growing realization that endodontic infections are often polymicrobial, and may contain Candida spp. Despite this understanding, the development of new endodontic irrigants and models of pathogenesis remains limited to mono-species biofilm models and is bacterially focused. The purpose of this study was to develop and optimize an interkingdom biofilm model of endodontic infection and use this to test suitable anti-biofilm actives. Biofilms containing Streptococcus gordonii, Fusobacterium nucleatum, Porphyromonas gingivalis, and Candida albicans were established from ontological analysis. Biofilms were optimized in different media and atmospheric conditions, prior to quantification and imaging, and subsequently treated with chlorhexidine, EDTA, and chitosan. These studies demonstrated that either media supplemented with serum were equally optimal for biofilm growth, which were dominated by S. gordonii, followed by C. albicans. Assessment of antimicrobial activity showed significant effectiveness of each antimicrobial, irrespective of serum. Chitosan was most effective (3 log reduction), and preferentially targeted C. albicans in both biofilm treatment and inhibition models. Chitosan was similarly effective at preventing biofilm growth on a dentine substrate. This study has shown that a reproducible and robust complex interkingdom model, which when tested with the antifungal chitosan, supports the notion of C. albicans as a key structural component.

Candida albicans as an Essential "Keystone" Component within Polymicrobial Oral Biofilm Models?

Background: Existing standardized biofilm assays focus on simple mono-species or bacterial-only models. Incorporating Candida albicans into complex biofilm models can offer a more appropriate and relevant polymicrobial biofilm for the development of oral health products. Aims: This study aimed to assess the importance of interkingdom interactions in polymicrobial oral biofilm systems with or without C. albicans, and test how these models respond to oral therapeutic challenges in vitro. Materials and Methods: Polymicrobial biofilms (two models containing 5 and 10 bacterial species, respectively) were created in parallel in the presence and absence of C. albicans and challenged using clinically relevant antimicrobials. The metabolic profiles and biomasses of these complex biofilms were estimated using resazurin dye and crystal violet stain, respectively. Quantitative PCR was utilized to assess compositional changes in microbial load. Additional assays, for measurements of pH and lactate, were included to monitor fluctuations in virulence "biomarkers." Results: An increased level of metabolic activity and biomass in the presence of C. albicans was observed. Bacterial load was increased by more than a factor of 10 in the presence of C. albicans. Assays showed inclusion of C. albicans impacted the biofilm virulence profiles. C. albicans did not affect the biofilms' responses to the short-term incubations with different treatments. Conclusions: The interkingdom biofilms described herein are structurally robust and exhibit all the hallmarks of a reproducible model. To our knowledge, these data are the first to test the hypothesis that yeasts may act as potential "keystone" components of oral biofilms.

Candida auris Phenotypic Heterogeneity Determines Pathogenicity In Vitro.

Candida auris is an enigmatic yeast that provides substantial global risk in health care facilities and intensive care units. A unique phenotype exhibited by certain isolates of C. auris is their ability to form small clusters of cells known as aggregates, which have been to a limited extent described in the context of pathogenic traits. In this study, we screened several nonaggregative and aggregative C. auris isolates for biofilm formation, where we observed a level of heterogeneity among the different phenotypes. Next, we utilized an RNA sequencing approach to investigate the transcriptional responses during biofilm formation of a nonaggregative and aggregative isolate of the initial pool. Observations from these analyses indicate unique transcriptional profiles in the two isolates, with several genes identified relating to proteins involved in adhesion and invasion of the host in other fungal species. From these findings, we investigated for the first time the fungal recognition and inflammatory responses of a three-dimensional skin epithelial model to these isolates. In these models, a wound was induced to mimic a portal of entry for C. auris We show that both phenotypes elicited minimal response in the model minus induction of the wound, yet in the wounded tissue, both phenotypes induced a greater response, with the aggregative isolate more proinflammatory. This capacity of aggregative C. auris biofilms to generate such responses in the wounded skin highlights how this opportunistic yeast is a high risk within the intensive care environment where susceptible patients have multiple indwelling lines.IMPORTANCECandida auris has recently emerged as an important cause of concern within health care environments due to its ability to persist and tolerate commonly used antiseptics and disinfectants, particularly when attached to a surface (biofilms). This yeast is able to colonize and subsequently infect patients, particularly those that are critically ill or immunosuppressed, which may result in death. We have undertaken analysis on two different phenotypic types of this yeast, using molecular and immunological tools to determine whether either of these has a greater ability to cause serious infections. We describe that both isolates exhibit largely different transcriptional profiles during biofilm development. Finally, we show that the inability to form small aggregates (or clusters) of cells has an adverse effect on the organism's immunostimulatory properties, suggesting that the nonaggregative phenotype may exhibit a certain level of immune evasion.

Chitosan Ameliorates Candida auris Virulence in a Galleria mellonella Infection Model.

Candida auris has emerged as a multidrug-resistant nosocomial pathogen over the last decade. Outbreaks of the organism in health care facilities have resulted in life-threatening invasive candidiasis in over 40 countries worldwide. Resistance by C. auris to conventional antifungal drugs such as fluconazole and amphotericin B means that alternative therapeutics must be explored. As such, this study served to investigate the efficacy of a naturally derived polysaccharide called chitosan against aggregative (Agg) and nonaggregative (non-Agg) isolates of C. aurisin vitro and in vivo. In vitro results indicated that chitosan was effective against planktonic and sessile forms of Agg and non-Agg C. auris In a Galleria mellonella model to assess C. auris virulence, chitosan treatment was shown to ameliorate killing effects of both C. auris phenotypes (NCPF 8973 and NCPF 8978, respectively) in vivo Specifically, chitosan reduced the fungal load and increased survival rates of infected Galleria, while treatment alone was nontoxic to the larvae. Finally, chitosan treatment appeared to induce a stress-like gene expression response in NCPF 8973 in the larvae likely arising from a protective response by the organism to resist antifungal activity of the compound. Taken together, results from this study demonstrate that naturally derived compounds such as chitosan may be useful alternatives to conventional antifungals against C. auris.

Development of a high throughput and low cost model for the study of semi-dry biofilms.

The persistence of microorganisms as biofilms on dry surfaces resistant to the usual terminal cleaning methods may pose an additional risk of transmission of infections. In this study, the Centre for Disease Control (CDC) dry biofilm model (DBM) was adapted into a microtiter plate format (Model 1) and replicated to create a novel in vitro model that replicates conditions commonly encountered in the healthcare environment (Model 2). Biofilms of Staphylococcus aureus grown in the two models were comparable to the biofilms of the CDC DBM in terms of recovered log10 CFU well-1. Assessment of the antimicrobial tolerance of biofilms grown in the two models showed Model 2 a better model for biofilm formation. Confirmation of the biofilms' phenotype with an extracellular matrix deficient S. aureus suggested stress tolerance through a non-matrix defined mechanism in microorganisms. This study highlights the importance of conditions maintained in bacterial growth as they affect biofilm phenotype and behaviour.

Modelling of pH-dependence to develop a strategy for stabilising mAbs at acidic steps in production.

Engineered proteins are increasingly being required to function or pass through environmental stresses for which the underlying protein has not evolved. A major example in health are antibody therapeutics, where a low pH step is used for purification and viral inactivation. In order to develop a computational model for analysis of pH-stability, predictions are compared with experimental data for the relative pH-sensitivities of antibody domains. The model is then applied to proteases that have evolved to be functional in an acid environment, showing a clear signature for low pH-dependence of stability in the neutral to acidic pH region, largely through reduction of salt-bridges. Interestingly, an extensively acidic protein surface can maintain contribution to structural stabilisation at acidic pH through replacement of basic sidechains with polar, hydrogen-bonding groups. These observations form a design principle for engineering acid-stable proteins.

Candida auris: A Decade of Understanding of an Enigmatic Pathogenic Yeast.

Candida auris is an enigmatic yeast that continues to stimulate interest within the mycology community due its rapid and simultaneous emergence of distinct clades. In the last decade, almost 400 manuscripts have contributed to our understanding of this pathogenic yeast. With dynamic epidemiology, elevated resistance levels and an indication of conserved and unique pathogenic traits, it is unsurprising that it continues to cause clinical concern. This mini-review aims to summarise some of the key attributes of his remarkable pathogenic yeast.

Combined Antifungal Resistance and Biofilm Tolerance: the Global Threat of Candida auris.

The enigmatic yeast Candida auris has emerged over the last decade and rapidly penetrated our consciousness. The global threat from this multidrug-resistant yeast has generated a call to arms from within the medical mycology community. Over the past decade, our understanding of how this yeast has spread globally, its clinical importance, and how it tolerates and resists antifungal agents has expanded. This review highlights the clinical importance of antifungal resistance in C. auris and explores our current understanding of the mechanisms associated with azole, polyene, and echinocandin resistance. We also discuss the impact of phenotypic tolerance, with particular emphasis on biofilm-mediated resistance, and present new pipelines of antifungal drugs that promise new hope in the management of C. auris infection.