Functional Redundancy in Candida auris Cell Surface Adhesins Crucial for Cell-Cell Interaction and Aggregation.

Candida auris is an emerging nosocomial fungal pathogen associated with life-threatening invasive disease due to its persistent colonization, high level of transmissibility and multi-drug resistance. Aggregative and non-aggregative growth phenotypes for C. auris strains with different biofilm forming abilities, drug susceptibilities and virulence characteristics have been described. Using comprehensive transcriptional analysis we identified key cell surface adhesins that were highly upregulated in the aggregative phenotype during in vitro and in vivo grown biofilms using a mouse model of catheter infection. Phenotypic and functional evaluations of generated null mutants demonstrated crucial roles for the adhesins Als5 and Scf1 in mediating cell-cell adherence, coaggregation and biofilm formation. While individual mutants were largely non-aggregative, in combination cells were able to co-adhere and aggregate, as directly demonstrated by measuring cell adhesion forces using single-cell atomic force spectroscopy. This co-adherence indicates their role as complementary adhesins, which despite their limited similarity, may function redundantly to promote cell-cell interaction and biofilm formation. Functional diversity of cell wall proteins may be a form of regulation that provides the aggregative phenotype of C. auris with flexibility and rapid adaptation to the environment, potentially impacting persistence and virulence.

Functional Redundancy in Candida auris Cell Surface Adhesins Crucial for Cell-Cell Interaction and Aggregation.

Candida auris is an emerging nosocomial fungal pathogen associated with life-threatening invasive disease due to its persistent colonization, high level of transmissibility and multi-drug resistance. Aggregative and non-aggregative growth phenotypes for C. auris strains with different biofilm forming abilities, drug susceptibilities and virulence characteristics have been described. Using comprehensive transcriptional analysis we identified key cell surface adhesins that were highly upregulated in the aggregative phenotype during in vitro and in vivo grown biofilms using a mouse model of catheter infection. Phenotypic and functional evaluations of generated null mutants demonstrated crucial roles for the adhesins Als5 and Scf1 in mediating cell-cell adherence, coaggregation and biofilm formation. While individual mutants were largely non-aggregative, in combination cells were able to co-adhere and aggregate, as directly demonstrated by measuring cell adhesion forces using single-cell atomic force spectroscopy. This co-adherence indicates their role as complementary adhesins, which despite their limited similarity, may function redundantly to promote cell-cell interaction and biofilm formation. Functional diversity of cell wall proteins may be a form of regulation that provides the aggregative phenotype of C. auris with flexibility and rapid adaptation to the environment, potentially impacting persistence and virulence.

A Longitudinal Metagenomic Comparative Analysis of Oral Microbiome Shifts in Patients Receiving Proton Radiation versus Photon Radiation for Head and Neck Cancer.

Introduction: Due to the radiation-sparing effects on salivary gland acini, changes in the composition of the oral microbiome may be a driver for improved outcomes in patients receiving proton radiation, with potentially worse outcomes in patients exposed to photon radiation therapy. To date, a head-to-head comparison of oral microbiome changes at a metagenomic level with longitudinal sampling has yet to be performed in these patient cohorts. Methods and Materials: To comparatively analyze oral microbiome shifts during head and neck radiation therapy, a prospective pilot cohort study was performed at the Maryland Proton Treatment Center and the University of Maryland Marlene and Stewart Greenebaum Comprehensive Cancer Center. A longitudinal metagenomic comparative analysis of oral microbiome shifts was performed at three time points (pre-radiation, during radiation, and immediately post-radiation). Head and neck cancer patients receiving proton radiation (n = 4) were compared to photon radiation (n = 4). Additional control groups included healthy age- and sex-matched controls (n = 5), head and neck cancer patients who never received radiation therapy (n = 8), and patients with oral inflammatory disease (n = 3). Results: Photon therapy patients presented with lower microbial alpha diversity at all timepoints, and there was a trend towards reduced species richness as compared with proton therapy. Healthy controls and proton patients exhibited overall higher and similar diversity. A more dysbiotic state was observed in patients receiving photon therapy as compared to proton therapy, in which oral microbial homeostasis was maintained. Mucositis was observed in 3/4 photon patients and was not observed in any proton patients during radiation therapy. The bacterial de novo pyrimidine biosynthesis pathway and the nitrate reduction V pathway were comparatively higher following photon exposure. These functional changes in bacterial metabolism may suggest that photon exposure produces a more permissive environment for the proliferation of pathogenic bacteria. Conclusion: Oral microbiome dysbiosis in patients receiving photon radiation may be associated with increased mucositis occurrence. Proton radiation therapy for head and neck cancer demonstrates a safer side effect profile in terms of oral complications, oral microbiome dysbiosis, and functional metabolic status.

Novel Remineralizing and Antibiofilm Low-Shrinkage-Stress Nanocomposites to Inhibit Salivary Biofilms and Protect Tooth Structures.

Recurrent caries remain a persistent concern, often linked to microleakage and a lack of bioactivity in contemporary dental composites. Our study aims to address this issue by developing a low-shrinkage-stress nanocomposite with antibiofilm and remineralization capabilities, thus countering the progression of recurrent caries. In the present study, we formulated low-shrinkage-stress nanocomposites by combining triethylene glycol divinylbenzyl ether and urethane dimethacrylate, incorporating dimethylaminododecyl methacrylate (DMADDM), along with nanoparticles of calcium fluoride (nCaF2) and nanoparticles of amorphous calcium phosphate (NACP). The biofilm viability, biofilm metabolic activity, lactic acid production, and ion release were evaluated. The novel formulations containing 3% DMADDM exhibited a potent antibiofilm activity, exhibiting a 4-log reduction in the human salivary biofilm CFUs compared to controls (p < 0.001). Additionally, significant reductions were observed in biofilm biomass and lactic acid (p < 0.05). By integrating both 10% NACP and 10% nCaF2 into one formulation, efficient ion release was achieved, yielding concentrations of 3.02 ± 0.21 mmol/L for Ca, 0.5 ± 0.05 mmol/L for P, and 0.37 ± 0.01 mmol/L for F ions. The innovative mixture of DMADDM, NACP, and nCaF2 displayed strong antibiofilm effects on salivary biofilm while concomitantly releasing a significant amount of remineralizing ions. This nanocomposite is a promising dental material with antibiofilm and remineralization capacities, with the potential to reduce polymerization-related microleakage and recurrent caries.

Novel Bioactive Nanocomposites Containing Calcium Fluoride and Calcium Phosphate with Antibacterial and Low-Shrinkage-Stress Capabilities to Inhibit Dental Caries.

OBJECTIVES: Composites are commonly used for tooth restorations, but recurrent caries often lead to restoration failures due to polymerization shrinkage-stress-induced marginal leakage. The aims of this research were to: (1) develop novel low-shrinkage-stress (L.S.S.) nanocomposites containing dimethylaminododecyl methacrylate (DMADDM) with nanoparticles of calcium fluoride (nCaF2) or amorphous calcium phosphate (NACP) for remineralization; (2) investigate antibacterial and cytocompatibility properties. METHODS: Nanocomposites were made by mixing triethylene glycol divinylbenzyl ether with urethane dimethacrylate containing 3% DMADDM, 20% nCaF2, and 20% NACP. Flexural strength, elastic modulus, antibacterial properties against Streptococcus mutans biofilms, and cytotoxicity against human gingival fibroblasts and dental pulp stem cells were tested. RESULTS: Nanocomposites with DMADDM and nCaF2 or NACP had flexural strengths matching commercial composite control without bioactivity. The new nanocomposite provided potent antibacterial properties, reducing biofilm CFU by 6 logs, and reducing lactic acid synthesis and metabolic function of biofilms by 90%, compared to controls (p < 0.05). The new nanocomposites produced excellent cell viability matching commercial control (p > 0.05). CONCLUSIONS: Bioactive L.S.S. antibacterial nanocomposites with nCaF2 and NACP had excellent bioactivity without compromising mechanical and cytocompatible properties. The new nanocomposites are promising for a wide range of dental restorations by improving marginal integrity by reducing shrinkage stress, defending tooth structures, and minimizing cariogenic biofilms.

Denture stomatitis-An interdisciplinary clinical review.

PURPOSE: An interdisciplinary clinical review on denture stomatitis formulated by experts from prosthodontics, oral medicine, and oral microbiology is presented. The review outlines the etiopathogenesis, clinical features, and management strategies of denture stomatitis from an interdisciplinary perspective. MATERIALS AND METHODS: English-language articles relating to the definition, incidence, gender distribution, geographical distribution, etiology, pathogenesis, symptoms, signs, treatment, and prognosis of denture stomatitis were retrieved via electronic literature search. Relevant articles were summarized for this manuscript with a view toward providing pragmatic information. RESULTS: Denture stomatitis represents a very common, multifactorial infectious, inflammatory, and hyperplastic condition which is primarily caused by poor oral hygiene, poor denture hygiene, and full-time; mainly night-time denture wear, bringing about the emergence of advanced Candida-containing polymicrobial biofilms in close proximity to the host's mucosal tissues. Denture stomatitis shares clinically relevant associations with dental caries, periodontitis, median rhomboid glossitis, angular cheilitis, aspirational pneumonia, and associated mortality. CONCLUSIONS: Effective, long-term treatment of denture stomatitis relies upon sustained patient-driven behavioral change which should focus on daily prosthesis-level cleaning and disinfection, removal of dentures at night, every night, engagement with professional denture maintenance, and when required, denture replacement. Antifungal medications offer limited benefits outside of short-term use due to the emergence of antifungal resistance. For frail, older, medically compromised, and nursing home populations, treatment of denture stomatitis lowers the risk of aspirational pneumonia and associated mortality.

Long-Term Post-COVID-19 Associated Oral Inflammatory Sequelae.

The oral cavity remains an underappreciated site for SARS-CoV-2 infection despite the myriad oral conditions observed in COVID-19 patients. Recently, replicating SARS-CoV-2 was found inside salivary epithelial cells resulting in inflammation and atrophy of salivary glands. Saliva possesses healing properties crucial for maintaining the health of the oral mucosa. Specifically, salivary antimicrobial peptides, most notable, histatin-5 exclusively produced in salivary glands, plays a vital role in innate immunity against colonizing microbial species. The demonstration of SARS-CoV-2 destruction of gland tissue where histatin-5 is produced strongly indicate that histatin-5 production is compromised due to COVID-19. Here we present a case of a patient presenting with unexplained chronic oral dysesthesia and dysgeusia post-recovery from COVID-19. To explore potential physiological mechanisms behind the symptoms, we comparatively analyzed saliva samples from the patient and matched healthy subject for histatin-5 and key cytokines. Findings demonstrated significantly reduced histatin-5 levels in patient's saliva and activation of the Th17 inflammatory pathway. As histatin-5 exhibits potent activity against the opportunistic oral pathogen Candida albicans, we evaluated saliva potency against C. albicans ex vivo. Compared to control, patient saliva exhibited significantly reduced anti-candidal efficacy. Although speculative, based on history and salivary analysis we hypothesize that salivary histatin-5 production may be compromised due to SARS-CoV-2 mediated salivary gland destruction. With the current lack of emphasis on implications of COVID-19 on oral health, this report may provide lacking mechanistic insights that may lead to reassessment of risks for oral opportunistic infections and mucosal inflammatory processes in acutely-ill and recovered COVID-19 patients.

The oral mycobiome: Oral epithelial dysplasia and oral squamous cell carcinoma.

Fungi, a diverse group of eukaryotic organisms, play distinct roles in health and disease. Recent advances in the field of mycobiology have enabled the characterization of the "human mycobiome." The human mycobiome has extensively been studied in various disease models. However, to date, the role of the oral mycobiome in oral carcinogenesis has yet to be elucidated. Candida albicans, the most common oral colonizer, has been speculated to display tumorigenic effects; however, the literature lacks consistent documentation from mechanistic studies on whether oral mycobiota act as drivers, facilitators, or passive colonizers of oral premalignancy and cancer. This review article provides an overview of existing hypothesis-driven mechanistic models that outline the complex interplay between the oral mycobiome and oral epithelial dysplasia as well as their potential clinical implications.

Application of proper orthogonal decomposition for evaluation of coherent structures and energy contents in microbial biofilms.

Microbial biofilms are structured communities of surface-associated microbial populations embedded in a matrix of extracellular polysaccharides that provide protection for biofilm cells. Among the wide plethora of microbial species adept at forming biofilms, the fungal pathogen Candida albicans (C. albicans), is one of the most notable. C. albicans biofilm development occurs in a series of sequential steps over a period of 24 h. Various quantitative and microscopic methods are available for the monitoring and evaluation of biofilms, including several innovative real-time methods for the evaluation of the cell-to-cell dynamics occurring during biofilm formation. These methods utilize biosensors which capture electrical, acoustic, and reflectance signals in bacterial populations (Li et al., 2021; Li et al., 2020; Kim et al., 2021; Paredes et al., 2021; Reipa et al., 2021). Additionally, machine learning, deep learning and other computational approaches have progressively been incorporated in the field of microbiology (Qu et al., 2019; Goodswen et al., 2021; Zhang et al., 2021; Ghannam and Techtmann, 2021; Rani et al., 2021; Berg et al., 2019) including some studies in biofilms (Buetti-Dinh et al., 2019; Srivastava et al., 2020; Hartmann et al., 2021; Dimauro et al., 2021) but given the potential of machine and deep learning, this niche is in large need of collaborative work between microbiology and engineering or physics experts to propel machine learning to a higher level. Therefore, whilst promising advances have been made, there is an urgent need for extensive development to take place to study and comprehend the complex interaction of microbial pathogens during biofilm formation. Specifically, there is a lot left to be understood about biofilm energy kinetics, and who the active microbial populations are. We infer that biofilm formation is an extremely diverse phenomenon and that each microorganism exerts different pathways to form a biofilm. Thus, we reasoned on the need for a model that would allow us to study the energy kinetics during C. albicans biofilm development. Modal decomposition techniques (MDTs) commonly used in fluid mechanics are gaining popularity outside their original field and might help decipher some of the dynamically relevant structures of biofilm formation. MDTs permit the identification of coherent structures in fluids and have been used in complex applications of information obtained during a particular time-lapse. A common MDT, Proper Orthogonal Decomposition (POD), can be used in reduced order modelling and machine learning applications. POD allows decomposition of a physical field influenced by different variables that may affect its physical properties. We aimed to evaluate the applicability of this technique in the analysis of energy kinetics during microbial biofilm formation, more specifically C. albicans biofilms. Using POD, we were able to easily distinguish visually distinct modes of growth of C. albicans cells in PBS and RPMI in terms of energy accumulation during the kinetic experiment. Comparing both PBS and RPMI, RPMI contains more energetic and dynamically relevant structures than PBS.

Therapeutic implications of C. albicans-S. aureus mixed biofilm in a murine subcutaneous catheter model of polymicrobial infection.

Biofilm-associated polymicrobial infections tend to be challenging to treat. Candida albicans and Staphylococcus aureus are leading pathogens due to their ability to form biofilms on medical devices. However, the therapeutic implications of their interactions in a host is largely unexplored. In this study, we used a mouse subcutaneous catheter model for in vivo-grown polymicrobial biofilms to validate our in vitro findings on C. albicans-mediated enhanced S. aureus tolerance to vancomycin in vivo. Comparative assessment of S. aureus recovery from catheters with single- or mixed-species infection demonstrated failure of vancomycin against S. aureus in mice with co-infected catheters. To provide some mechanistic insights, RNA-seq analysis was performed on catheter biofilms to delineate transcriptional modulations during polymicrobial infections. C. albicans induced the activation of the S. aureus biofilm formation network via down-regulation of the lrg operon, repressor of autolysis, and up-regulation of the ica operon and production of polysaccharide intercellular adhesin (PIA), indicating an increase in eDNA production, and extracellular polysaccharide matrix, respectively. Interestingly, virulence factors important for disseminated infections, and superantigen-like proteins were down-regulated during mixed-species infection, whereas capsular polysaccharide genes were up-regulated, signifying a strategy favoring survival, persistence and host immune evasion. In vitro follow-up experiments using DNA enzymatic digestion, lrg operon mutant strains, and confocal scanning microscopy confirmed the role of C. albicans-mediated enhanced eDNA production in mixed-biofilms on S. aureus tolerance to vancomycin. Combined, these findings provide mechanistic insights into the therapeutic implications of interspecies interactions, underscoring the need for novel strategies to overcome limitations of current therapies.

Comparative Evaluations of the Pathogenesis of Candida auris Phenotypes and Candida albicans Using Clinically Relevant Murine Models of Infections.

The newly emerged Candida species Candida auris is associated with an exponential rise in life-threatening invasive disease in health care facilities worldwide. Unlike other species, C. auris exhibits a high level of transmissibility, multidrug resistance, and persistence in the environment, yet little is known about its pathogenesis largely due to limited data from animal models. Based on in vitro biofilm evaluations and confocal laser scanning microscopy, C. auris phenotypes with different biofilm-forming abilities were identified, indicating potential clinical implications. Using clinically relevant murine models of implanted catheter, oral, and intraperitoneal infections, we comparatively evaluated the host site-specific pathogenic potential of C. auris phenotypes and Candida albicans Based on the results of microbial recovery and scanning electron microscopy analysis of explanted catheters, compared to C. albicans, C. auris more avidly adhered and formed biofilms on catheters. However, although C. auris adhered to oral tissue ex vivo, unlike C. albicans, it failed to colonize the oral cavity in vivo, as demonstrated by microbial recovery and tissue histopathology analysis. In contrast, recovery from peritoneal lavage fluid and kidneys during time course experiments demonstrated that C. auris persisted longer in the peritoneal cavity and kidneys. Although there were clear niche-specific differences in pathogenic features between C. auris and C. albicans, no significant differences were noted between the C. auris phenotypes in vivo The combined findings highlight unique niche-specific pathogenic traits for C. auris warranting further investigations. Understanding the factors contributing to the rise of C. auris as a nosocomial pathogen is critical for controlling the spread of this species.IMPORTANCE The newly emerged Candida species C. auris has been associated with an exponential rise in invasive disease in health care facilities worldwide with a mortality rate approaching 60%. C. auris exhibits a high level of transmissibility, multidrug resistance, and persistence in hospital environments, yet little is known about its pathogenesis largely due to limited data from animal studies. We used clinically relevant murine models of infection to comparatively evaluate the host niche-specific pathogenic potential of C. auris and C. albicans Findings demonstrated that C. auris adheres more avidly, forming robust biofilms on catheters implanted in mice. However, although C. auris adhered to oral tissue ex vivo, it failed to colonize the oral cavity in vivo In contrast, in the intraperitoneal infection model, C. auris persisted longer in the peritoneal cavity and kidneys. Understanding the host-pathogen factors contributing to the rise of C. auris as a nosocomial pathogen is critical for controlling the spread of this species.

Candida auris: a fungus with identity crisis.

Candida auris is a new fungal species that has puzzlingly and simultaneously emerged on five continents. Since its identification in 2009, the scientific community has witnessed an exponential emergence of infection episodes and outbreaks in healthcare facilities world-wide. Candida auris exhibits several concerning features compared to other related Candida species, including persistent colonization of skin and nosocomial surfaces, ability to resist common disinfectants and to spread rapidly among patients. Resistance to multiple drug classes and misidentification by available laboratory identification systems has complicated clinical management, and outcomes of infection have generally been poor with mortality rates approaching 68%. Currently, the origins of C. auris are unclear, and therefore, it is impossible to determine whether environmental and climactic changes were contributing factors in its recent emergence as a pathogen. Nevertheless, a robust response involving rapid diagnostics, prompt interventions and implementation of precautions, are paramount in curtailing the spread of  infections by this fungal species. Importantly, there is a pressing need for the development of new antifungal drugs. In this article, we present a brief overview highlighting some of the important aspects of C. auris epidemiology, pathogenesis and its puzzling global emergence.

The Role of Candida albicans Secreted Polysaccharides in Augmenting Streptococcus mutans Adherence and Mixed Biofilm Formation: In vitro and in vivo Studies.

The oral cavity is a complex environment harboring diverse microbial species that often co-exist within biofilms formed on oral surfaces. Within a biofilm, inter-species interactions can be synergistic in that the presence of one organism generates a niche for another enhancing colonization. Among these species are the opportunistic fungal pathogen Candida albicans and the bacterial species Streptococcus mutans, the etiologic agents of oral candidiasis and dental caries, respectively. Recent studies have reported enhanced prevalence of C. albicans in children with caries indicating potential clinical implications for this fungal-bacterial interaction. In this study, we aimed to specifically elucidate the role of C. albicans-derived polysaccharide biofilm matrix components in augmenting S. mutans colonization and mixed biofilm formation. Comparative evaluations of single and mixed species biofilms demonstrated significantly enhanced S. mutans retention in mixed biofilms with C. albicans. Further, S. mutans single species biofilms were enhanced upon exogenous supplementation with purified matrix material derived from C. albicans biofilms. Similarly, growth in C. albicans cell-free spent biofilm culture media enhanced S. mutans single species biofilm formation, however, the observed increase in S. mutans biofilms was significantly affected upon enzymatic digestion of polysaccharides in spent media, identifying C. albicans secreted polysaccharides as a key factor in mediating mixed biofilm formation. The enhanced S. mutans biofilms mediated by the various C. albicans effectors was also demonstrated using confocal laser scanning microscopy. Importantly, a clinically relevant mouse model of oral co-infection was adapted to demonstrate the C. albicans-mediated enhanced S. mutans colonization in a host. Analyses of harvested tissue and scanning electron microscopy demonstrated significantly higher S. mutans retention on teeth and tongues of co-infected mice compared to mice infected only with S. mutans. Collectively, the findings from this study strongly indicate that the secretion of polysacharides from C. albicans in the oral environment may impact the development of S. mutans biofilms, ultimately increasing dental caries and, therefore, Candida oral colonization should be considered as a factor in evaluating the risk of caries.