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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. 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.

All serious fungal infections need appropriate antifungal therapy for successful patient outcome. Only a few classes of antifungal drugs are available, so the emergence of resistance to single drug classes and now multidrug resistance greatly hampers patient management. Azole resistance among Candida and Aspergillus species is one of the greatest challenges to clinical success, followed by echinocandin and multidrug resistance among some Candida species, especially Candida glabrata. The spread of agriculturally derived azole-resistant Aspergillus fumigatus and emerging threats such as multidrug resistant Candida auris are also alarming. The molecular mechanisms that cause drug resistance are naturally occurring in less susceptible species and are acquired in strains of susceptible organisms. Drug resistance mechanisms include altered drug-target interactions, reduced cellular drug concentrations mediated by drug efflux transporters, and permeability barriers associated with biofilms. Although C auris is inherently multidrug resistant, other strains typically develop resistance through stepwise selection of multiple drug-resistance mechanisms. Cellular stress induced by drug treatment promotes adaptation, which contributes to breakthrough resistance. Drug exposure also drives the emergence of resistance. An effective antifungal stewardship programme is essential to control drug resistance, and should incorporate rapid fungal diagnostics, therapeutic drug monitoring, and clinical intervention teams. The development of better diagnostic tools and strategies that allow targeted use of antifungals is essential to preserve drug effectiveness.

Background The objective of this study was to assess the cumulative incidence of invasive candidiasis (IC) in intensive care units (ICUs) in Europe. Methods A multinational, multicenter, retrospective study was conducted in 23 ICUs in 9 European countries, representing the first phase of the candidemia/intra-abdominal candidiasis in European ICU project (EUCANDICU). Results During the study period, 570 episodes of ICU-acquired IC were observed, with a cumulative incidence of 7.07 episodes per 1000 ICU admissions, with important between-center variability. Separated, non-mutually exclusive cumulative incidences of candidemia and IAC were 5.52 and 1.84 episodes per 1000 ICU admissions, respectively. Crude 30-day mortality was 42%. Age (odds ratio [OR] 1.04 per year, 95% CI 1.02–1.06, p  < 0.001), severe hepatic failure (OR 3.25, 95% 1.31–8.08, p 0.011), SOFA score at the onset of IC (OR 1.11 per point, 95% CI 1.04–1.17, p 0.001), and septic shock (OR 2.12, 95% CI 1.24–3.63, p 0.006) were associated with increased 30-day mortality in a secondary, exploratory analysis. Conclusions The cumulative incidence of IC in 23 European ICUs was 7.07 episodes per 1000 ICU admissions. Future in-depth analyses will allow explaining part of the observed between-center variability, with the ultimate aim of helping to improve local infection control and antifungal stewardship projects and interventions.

Aspergillus conida are ubiquitous in the environment, including freshwater, water for bathing, and in drinking water. Vulnerable patients and those suffering from allergic diseases are susceptible to aspergillosis. Avoidance of Aspergillus is of paramount importance. Potential outbreaks of aspergillosis in hospital facilities have been described where the water supply has been implicated. Little is known regarding the risk of exposure to Aspergillus in water. How does Aspergillus survive in water? This review explores the biofilm state of Aspergillus growth based on recent literature and suggests that biofilms are responsible for the persistence of Aspergillus in domestic and healthcare facilities’ water supplies.

Almost 380,000 new cases of oral cancer were reported worldwide in 2020. Oral squamous cell carcinoma (OSCC) accounts for 90% of all types of oral cancers. Emerging studies have shown association of Toll-like receptors (TLRs) in carcinogenesis. The present study aimed to investigate the expression levels and tissue localization of TRL1 to TRL10 and NF-κB between OSCC and healthy oral mucosa, as well as effect of Candida colonization in TRL expression in OSCC. Full thickness biopsies and microbial samples from 30 newly diagnosed primary OSCC patients and 26 health controls were collected. The expression of TLR1 to TLR10 and NF-κB was analyzed by immunohistochemistry. Microbial samples were collected from oral mucosa to detect Candida . OSCC epithelium showed lower staining intensity of TRL1, TRL2 TRL5, and TRL8 as compared to healthy controls. Similarly, staining intensity of TRL3, TRL4, TRL7, and TRL8 were significantly decreased in basement membrane (BM) zone. Likewise, OSCC endothelium showed lower staining intensity of TLR4, TLR7 and TLR8. Expression of NF-κB was significantly stronger in normal healthy tissue compared to OSCC sample. Positive correlation was found between the expression of NF-κB, TRL9 and TRL10 in basal layer of the infiltrative zone OSCC samples (P = 0.04 and P = 0.002, respectively). Significant increase in TRL4 was seen in BM zone of sample colonized with Candida (P = 0.01). According to the limited number of samples, our data indicates downregulation of TLRs and NF-κB in OSCC, and upregulation of TLR4 expression with presence of Candida.

Biofilms are major contributors to delayed wound healing and there is a need for clinically relevant experimental models to assess theranostics. Microorganisms release volatile organic compounds (VOCs) and the ability to identify these in infected cutaneous wounds could lead to efficient non-invasive diagnosis. The aims here were to develop and assess bacterial biofilm formation and identify their VOC profiles in an in vitro model and validate in human ex vivo incisional and excisional cutaneous wound models. Biofilm development was assessed using multiple microscopy techniques with biofilm-forming deficient controls and quantified using metabolic and biomass assays; and VOC production measured by gas chromatography-mass spectrometry. The production of most VOCs was affected by biofilm development and model used. Some VOCs were specific either for planktonic or biofilm growth. The relative abundance of some VOCs was significantly increased or decreased by biofilm growth phase (P < 0.05). Some Staphylococcus aureus and Pseudomonas aeruginosa VOCs correlated with biofilm metabolic activity and biomass (R ≤ −0.5; ≥0.5). We present for the first time bacterial biofilm formation in human ex vivo cutaneous wound models and their specific VOC profiles. These models provide a vehicle for human skin-relevant biofilm studies and VOC detection has potential clinical translatability in efficient non-invasive diagnosis of wound infection.

The fungal diagnostic landscape is evolving; whereas previously, traditional culture-based methods dominated, most invasive fungal disease is now diagnosed with non-culture-based tests, including direct microscopy, antigen, antibody, and molecular assays, supported by histopathology and radiology. Access to and turnaround time of diagnostic tests and their clinical implementation varies across the UK. The British Society for Medical Mycology convened an expert group to update its 2015 best practice guidance. Guidance on histopathology and radiology investigations remain unchanged from the 2015 standards of care. For each mycological test, we include test-specific commentaries and accompanying tables, with expected turnaround times (sample collection to reporting). Based on recent evidence, new or stronger recommendations include use of Pneumocystis PCR; 1,3-β-D-glucan testing for suspected invasive candidiasis and Pneumocystis pneumonia; higher volume respiratory sample cultures; Aspergillus antigen or antibody-based testing in expanded clinically vulnerable populations, including patients in intensive care units and patients with chronic respiratory disease (including asthma); use of Candida PCR and Mucorales PCR in specific contexts; pan-fungal PCR and DNA sequencing for fungal identification from positive microscopy or histopathology specimens; and inclusion of posaconazole and isavuconazole in therapeutic antifungal monitoring recommendations. We discuss integration of diagnostic tests with antifungal stewardship and common clinical scenarios. Although recommendations focus on adults and UK practice, use in paediatric populations and worldwide applicability is discussed. Recommendations are presented as auditable standards to facilitate implementation and quality improvement measures. An emphasis on integration of combined diagnostics with antifungal stewardship and clinical pathways extends guideline relevance beyond microbiology laboratories to clinicians investigating patients with multimorbidity and suspected fungal disease within increasingly complex health-care systems.

Background Urogenital atrophy (UA) is a common treatment-limiting side effect of endocrine therapies. Topical estrogen is effective but systemic absorption may counter aromatase inhibitor efficacy. Numerous complementary approaches are marketed for use in UA without rigorous testing of their estrogenicity. We tested multiple essential oils in cancer cell growth and estrogen reporter assays in vitro and assessed clinical outcomes with the essential oil pessaries (EOPs) in breast cancer survivors with UA. Methods Effects on cell growth were tested in hormone-dependent (MCF-7) and -independent (MDA-MB-231) cell lines using the sulforhodamine-B assay. An estrogen response element (ERE) luciferase reporter assay was used to assess estrogenicity directly. Antifungal activity against two common pathogenic yeasts was assessed using standard microdilution methods. EOPs were offered to breast cancer survivors with symptomatic UA and the service evaluated using serial questionnaires. Results Two essential oils, Cymbopogon martinii and Pelargonium graveolens, demonstrated marked estrogenicity, stimulating ER+ cell growth and ERE-luciferase reporter activity to levels seen with premenopausal estradiol concentrations. Additional oils were screened for estrogenicity and Lavandula angustifolia and Chamaemelum nobile identified as non/minimally estrogenic. The antifungal activity of this combination of oils was confirmed. A second cohort of breast cancer survivors with UA received the second generation EOP with comparable improvement in symptom scores suggesting that estrogenicity may not be required for optimal therapy of UA. Conclusion Certain essential oils demonstrate profound estrogenicity and caution should be exercised before their use in breast cancer survivors. Our minimally estrogenic pessary will be formally tested in clinical trials.