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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

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

SARS-CoV-2 spread rapidly, causing millions of deaths across the globe. As a result, demand for medical supplies and personal protective equipment (PPE) surged and supplies dwindled. Separate entirely, hospital-acquired infections have become commonplace and challenging to treat. To explore the potential of novel sterilization techniques, this study evaluated the disinfection efficacy of Fathhome's ozone-based, dry-sanitizing device by dose and time response. Inactivation of human pathogens was tested on non-porous (plastic) surfaces. 95.42–100% inactivation was observed across all types of vegetative microorganisms and 27.36% inactivation of bacterial endospores tested, with no residual ozone detectable after completion. These results strongly support the hypothesis that Fathhome's commercial implementation of gas-based disinfection is suitable for rapid decontamination of a wide variety of pathogens on PPE and other industrially relevant materials.

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.

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.

ABSTRACT 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.

The unique symbiosis between a vertebrate salamander, Ambystoma maculatum, and unicellular green alga, Oophila amblystomatis, involves multiple modes of interaction. These include an ectosymbiotic interaction where the alga colonizes the egg capsule, and an intracellular interaction where the alga enters tissues and cells of the salamander. One common interaction in mutualist photosymbioses is the transfer of photosynthate from the algal symbiont to the host animal. In the A. maculatum–O. amblystomatis interaction, there is conflicting evidence regarding whether the algae in the egg capsule transfer chemical energy captured during photosynthesis to the developing salamander embryo. In experiments where we took care to separate the carbon fixation contributions of the salamander embryo and algal symbionts, we show that inorganic carbon fixed by A. maculatum embryos reaches 2% of the inorganic carbon fixed by O. amblystomatis algae within an egg capsule after 2 h in the light. After 2 h in the dark, inorganic carbon fixed by A. maculatum embryos is 800% of the carbon fixed by O. amblystomatis algae within an egg capsule. Using photosynthesis inhibitors, we show that A. maculatum embryos and O. amblystomatis algae compete for available inorganic carbon within the egg capsule environment. Our results confirm earlier studies suggesting a role of heterotrophic carbon fixation during vertebrate embryonic development. Our results also show that the considerable capacity of developing A. maculatum embryos for inorganic carbon fixation precludes our ability to distinguish any minor role of photosynthetically transferred carbon from algal symbionts to host salamanders using bicarbonate introduced to the egg system as a marker.

ABSTRACT 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. IMPORTANCE Candida 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.

Background: Evidence to support the role of Candida species in oral disease is limited. Often considered a commensal, this opportunistic yeast has been shown to play a role in denture related disease, though whether it is an active participant or innocent bystander remains to be determined. This study sought to understand the role of Candida species alongside the bacterial microbiome in a denture patient cohort, exploring how the microbiology of the denture was affected by oral hygiene practices. Materials and methods: In vitro denture cleansing studies were performed on a complex 9-species interkingdom denture biofilm model, with quantitative assessment of retained bacterial and fungal viable bioburdens. Patient hygiene measures were also collected from 131 patients, including OHIP, frequency of denture cleansing, oral hygiene measure and patient demographics. The bacterial microbiome was analysed from each patient, alongside quantitative PCR assessment of ITS (fungal) and 16S (bacterial) bioburden from denture, mucosa and intact dentition. Results: It was shown that following in vitro denture cleansing C. albicans were unresponsive to treatment, whereas bacterial biofilms could repopulate 100-fold, but were susceptible to subsequent treatment. Within the patient cohort, oral hygiene did not impact candidal or bacterial composition, nor diversity. The levels of Candida did not significantly influence the bacterial microbiome, though an observed gradient was suggestive of a microbial composition change in response to Candida load, indicating interkingdom interaction rather than an oral hygiene effect. Indeed, correlation analysis was able to show significant correlations between Candida species and key genera (Lactobacillus, Scardovia, Fusobacterium). Conclusions: Overall, this study has shown that the denture microbiome/mycobiome is relatively resilient to oral hygiene challenges, but that Candida species have potential interactions with key oral genera. These interactions may have a bearing on shaping community structure and a shift from health to disease when the opportunity arises.

ABSTRACT 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.

ABSTRACT Candida auris has emerged as a significant global nosocomial pathogen. This is primarily due to its antifungal resistance profile but also its capacity to form adherent biofilm communities on a range of clinically important substrates. While we have a comprehensive understanding of how other Candida species resist and respond to antifungal challenge within the sessile phenotype, our current understanding of C. auris biofilm-mediated resistance is lacking. In this study, we are the first to perform transcriptomic analysis of temporally developing C. auris biofilms, which were shown to exhibit phase- and antifungal class-dependent resistance profiles. A de novo transcriptome assembly was performed, where sequenced sample reads were assembled into an ~11.5-Mb transcriptome consisting of 5,848 genes. Differential expression (DE) analysis demonstrated that 791 and 464 genes were upregulated in biofilm formation and planktonic cells, respectively, with a minimum 2-fold change. Adhesin-related glycosylphosphatidylinositol (GPI)-anchored cell wall genes were upregulated at all time points of biofilm formation. As the biofilm developed into intermediate and mature stages, a number of genes encoding efflux pumps were upregulated, including ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporters. When we assessed efflux pump activity biochemically, biofilm efflux was greater than that of planktonic cells at 12 and 24 h. When these were inhibited, fluconazole sensitivity was enhanced 4- to 16-fold. This study demonstrates the importance of efflux-mediated resistance within complex C. auris communities and may explain the resistance of C. auris to a range of antimicrobial agents within the hospital environment. IMPORTANCE Fungal infections represent an important cause of human morbidity and mortality, particularly if the fungi adhere to and grow on both biological and inanimate surfaces as communities of cells (biofilms). Recently, a previously unrecognized yeast, Candida auris, has emerged globally that has led to widespread concern due to the difficulty in treating it with existing antifungal agents. Alarmingly, it is also able to grow as a biofilm that is highly resistant to antifungal agents, yet we are unclear about how it does this. Here, we used a molecular approach to investigate the genes that are important in causing the cells to be resistant within the biofilm. The work provides significant insights into the importance of efflux pumps, which actively pump out toxic antifungal drugs and therefore enhance fungal survival within a variety of harsh environments.