Powder diet exacerbates oropharyngeal candidiasis in a mouse model.

This study reports on the influence of a powder diet in a mouse model of oropharyngeal candidiasis (OPC), a significant health concern caused primarily by Candida albicans. Despite identical nutritional composition, we found that a powdered diet significantly increased Candida burdens and oral lesions, and aggravated weight loss compared to a standard pelleted diet. High fungal burdens and severe oral lesions were accomplished within 48 hours after infection with only one dose of cortisone. Moreover, mice on a powder diet recovered a week after infection. Using a powder diet, we thus modified the cortisone OPC murine model in a way that simplifies the infection process, enhances reproducibility, and facilitates studies investigating both pathogenesis and recovery processes. Our findings also underscore the pivotal role of the physical form of the diet in the progression and severity of oral Candida infection in this model. Future research should investigate this relationship further to broaden our understanding of the underlying mechanisms, potentially leading to novel prevention strategies and improved disease management.IMPORTANCEOropharyngeal candidiasis (OPC) is a multifactorial disease and a significant health concern. We found that the physical form of the diet plays a critical role in the severity and progression of OPC. We developed a modified cortisone OPC murine model that facilitates studies investigating pathogenesis and recovery processes.

Mathematical modeling of the Candida albicans yeast to hyphal transition reveals novel control strategies.

Candida albicans, an opportunistic fungal pathogen, is a significant cause of human infections, particularly in immunocompromised individuals. Phenotypic plasticity between two morphological phenotypes, yeast and hyphae, is a key mechanism by which C. albicans can thrive in many microenvironments and cause disease in the host. Understanding the decision points and key driver genes controlling this important transition and how these genes respond to different environmental signals is critical to understanding how C. albicans causes infections in the host. Here we build and analyze a Boolean dynamical model of the C. albicans yeast to hyphal transition, integrating multiple environmental factors and regulatory mechanisms. We validate the model by a systematic comparison to prior experiments, which led to agreement in 17 out of 22 cases. The discrepancies motivate alternative hypotheses that are testable by follow-up experiments. Analysis of this model revealed two time-constrained windows of opportunity that must be met for the complete transition from the yeast to hyphal phenotype, as well as control strategies that can robustly prevent this transition. We experimentally validate two of these control predictions in C. albicans strains lacking the transcription factor UME6 and the histone deacetylase HDA1, respectively. This model will serve as a strong base from which to develop a systems biology understanding of C. albicans morphogenesis.

Risky Business: Oropharyngeal Bacteria Linked to Risk for Invasive Fungal Infection.

This work combines a clinical investigation with a mouse model of fungal infection to study the role of bacterial microbiota in fungal invasion. The investigators identified a dysbiosis in the oropharyngeal mucosa that was associated with a high risk for invasive infection in hematologic oncology patients. This study builds on our current understanding that the pathogenesis of fungal infections has to be studied in the context of a specific host background and a site-specific bacterial microbiota.

Candida albicans induces mucosal bacterial dysbiosis that promotes invasive infection.

Infectious complications are a common cause of morbidity and mortality in cancer patients undergoing chemotherapy due to increased risk of oral and gastrointestinal candidiasis, candidemia and septicemia. Interactions between C. albicans and endogenous mucosal bacteria are important in understanding the mechanisms of invasive infection. We published a mouse intravenous chemotherapy model that recapitulates oral and intestinal mucositis, and myelosuppression in patients receiving 5-fluorouracil. We used this model to study the influence of C. albicans on the mucosal bacterial microbiome and compared global community changes in the oral and intestinal mucosa of the same mice. We validated 16S rRNA gene sequencing data by qPCR, in situ hybridization and culture approaches. Mice receiving both 5Fu and C. albicans had an endogenous bacterial overgrowth on the oral but not the small intestinal mucosa. C. albicans infection was associated with loss of mucosal bacterial diversity in both sites with indigenous Stenotrophomonas, Alphaproteobacteria and Enterococcus species dominating the small intestinal, and Enterococcus species dominating the oral mucosa. Both immunosuppression and Candida infection contributed to changes in the oral microbiota. Enterococci isolated from mice with oropharyngeal candidiasis were implicated in degrading the epithelial junction protein E-cadherin and increasing the permeability of the oral epithelial barrier in vitro. Importantly, depletion of these organisms with antibiotics in vivo attenuated oral mucosal E-cadherin degradation and C. albicans invasion without affecting fungal burdens, indicating that bacterial community changes represent overt dysbiosis. Our studies demonstrate a complex interaction between C. albicans, the resident mucosal bacterial microbiota and the host environment in pathogenesis. We shed significant new light on the role of C. albicans in shaping resident bacterial communities and driving mucosal dysbiosis.

Fungal diseases: Oral dysbiosis in susceptible hosts.

The oral cavity is colonized by a large number of microorganisms that are referred to collectively as the oral microbiota. These indigenous microorganisms have evolved in symbiotic relationships with the oral mucosal immune system and are involved in maintaining homeostasis in the oral cavity. Although Candida species are commonly found in the healthy oral cavity without causing infection, these fungi can become pathogenic. Recents advances indicate that the development of oral candidiasis is driven both by Candida albicans overgrowth in a dysbiotic microbiome and by disturbances in the host's immune system. Perturbation of the oral microbiota triggered by host-extrinsic (ie, medications), host-intrinsic (ie, host genetics), and microbiome-intrinsic (ie, microbial interactions) factors may increase the risk of oral candidiasis. In this review, we provide an overview of the oral mycobiome, with a particular focus on the interactions of Candida albicans with some of the most common oral bacteria and the oral mucosal immune system. Also, we present a summary of our current knowledge of the host-intrinsic and host-extrinsic factors that can predispose to oral candidiasis.

Biofilm Interactions of Candida albicans and Mitis Group Streptococci in a Titanium-Mucosal Interface Model.

Streptococci from the mitis group (represented mainly by Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis, and Streptococcus gordonii) form robust biofilms with Candida albicans in different experimental models. These microorganisms have been found in polymicrobial biofilms forming on titanium biomaterial surfaces in humans with peri-implant disease. The purpose of this work was to study mutualistic interactions in biofilms forming on titanium and their effect on the adjacent mucosa, using a relevant infection model. Single and mixed biofilms of C. albicans and each Streptococcus species were grown on titanium disks. Bacterial and fungal biovolume and biomass were quantified in these biofilms. Organotypic mucosal constructs were exposed to preformed titanium surface biofilms to test their effect on secretion of proinflammatory cytokines and cell damage. C. albicans promoted bacterial biofilms of all mitis Streptococcus species on titanium surfaces. This relationship was mutualistic since all bacterial species upregulated the efg1 hypha-associated gene in C. albicans Mixed biofilms caused increased tissue damage but did not increase proinflammatory cytokine responses compared to biofilms comprising Candida alone. Interestingly, spent culture medium from tissues exposed to titanium biofilms suppressed Candida growth on titanium surfaces.IMPORTANCE Our findings provide new insights into the cross-kingdom interaction between C. albicans and Streptococcus species representative of the mitis group. These microorganisms colonize titanium-based dental implant materials, but little is known about their ability to cause inflammation and damage of the adjacent mucosal tissues. Using an in vitro biomaterial-mucosal interface infection model, we showed that mixed biofilms of each species with C. albicans enhance tissue damage. One possible mechanism for this effect is the increased fungal hypha-associated virulence gene expression we observed in mixed biofilms with these species. Interestingly, we also found that the interaction of multispecies biofilms with organotypic mucosal surfaces led to the release of growth-suppressing mediators of Candida, which may represent a homeostatic defense mechanism of the oral mucosa against fungal overgrowth. Thus, our findings provide novel insights into biofilms on biomaterials that may play an important role in the pathogenesis of mucosal infections around titanium implants.

The Relationship of Candida albicans with the Oral Bacterial Microbiome in Health and Disease.

Candida albicans is an opportunistic pathogen colonizing the oropharyngeal, esophageal, and gastrointestinal mucosa in most healthy humans. In immunocompromised hosts, this fungal organism can cause mucosal candidiasis in these sites. C. albicans also causes fungemia, a serious consequence of cancer cytotoxic chemotherapy, which is thought to develop from fungal translocation through compromised mucosal barriers. Changes in endogenous bacterial population size or composition as well as changes in the host environment can transform fungal commensals into opportunistic pathogens in the upper and lower GI tract. Pioneering studies from our group have shown that a ubiquitous oral commensal of the mitis streptococcal group (Streptococcus oralis) has a mutualistic relationship with C. albicans, with C. albicans enabling streptococcal biofilm growth at mucosal sites, and S. oralis facilitating invasion of the oral and esophageal mucosa by C. albicans. In these studies, we used a cortisone-induced immunosuppression mouse model. More recently, the development of a novel mouse chemotherapy model has allowed us to examine the interactions of C. albicans with the endogenous bacterial microbiota in the oral and small intestinal mucosa, two sites adversely affected by cytotoxic chemotherapy. In this model, oral inoculation with C. albicans causes severe dysbiosis in the mucosal bacterial composition in both sites. We also found that antibiotic treatment ameliorates invasion of the oral mucosa but aggravates dissemination through the intestinal mucosa. In this chapter, we discuss work from our laboratory and others examining the relationships of C. albicans with oral bacteria and their role in mucosal homeostasis or disease.

Streptococcus oralis and Candida albicans Synergistically Activate µ-Calpain to Degrade E-cadherin From Oral Epithelial Junctions.

Streptococcus oralis forms robust mucosal biofilms with Candida albicans that have increased pathogenic potential. In this study, using oral epithelial cultures, organotypic oral mucosal constructs, and a mouse model of oral infection, we demonstrated that S. oralis augmented C. albicans invasion through epithelial junctions. C. albicans and S. oralis decreased epithelial E-cadherin levels by synergistically increasing µ-calpain, a proteolytic enzyme that targets E-cadherin. In the mouse coinfection model this was accompanied by increased fungal kidney dissemination. Coinfection with a secreted aspartyl protease (sap) mutant sap2456 and S. oralis increased µ-calpain and triggered mucosal invasion and systemic dissemination, suggesting that fungal protease activity is not required for invasion during coinfection. We conclude that C. albicans and S. oralis synergize to activate host enzymes that cleave epithelial junction proteins and increase fungal invasion.

Antifungal medications or disinfectants for denture stomatitis.

DATA SOURCES: The Cochrane database of systematic reviews, the Cochrane Central Register of Controlled Trials (CENTRAL), Medline and Embase databases and reference lists of identified articles were searched with no language restriction. STUDY SELECTION: Randomised controlled trials that compared the efficacy of antifungal medications with other treatments of denture-related erythematous stomatitis in adults wearing conventional acrylic removable complete dentures were included. Trials of seven days or fewer were excluded. DATA EXTRACTION AND SYNTHESIS: Study assessment and data extraction were carried out independently by at least two reviewers. Study quality was assessed using Cochrane methodology. Odds Ratios (OR) and 95% Confidence Interval (CI) were calculated to compare results across studies using a random effects model. RESULTS: Fourteen randomised controlled trials were included in the review, with eight studies contributing to the meta-analysis. No statistically significant difference between antifungal treatment and disinfection methods was found for both clinical and microbiological outcomes. Meta-analysis showed a statistically significant difference between an antifungal and a placebo for the microbiological outcome (OR=0.32; 95% CI: 0.12-0.89; Z=-2.2; p=0.028), favouring the antifungals. There was no statistically significant difference between antifungal and placebo for the clinical outcome (OR=0.2; 95% CI: 0.04-1.04; Z=-1.9; p=0.056). CONCLUSIONS: The findings of this review and meta-analysis suggest that disinfection methods could be considered as an adjunct or alternative to antifungal medications in the treatment of denture stomatitis.

High-Throughput Monitoring of Pathogenic Fungal Growth Using Whole Slide Imaging for Rapid Antifungal Susceptibility Assessment.

Invasive fungal infections are a major health threat with high morbidity and mortality, highlighting the urgent need for rapid diagnostic tools to detect antifungal resistance. Traditional culture-based antifungal susceptibility testing (AFST) methods often fall short due to their lengthy process. In our previous research, we developed a whole-slide imaging (WSI) technique for the high-throughput assessment of bacterial antibiotic resistance. Building on this foundation, this study expands the application of WSI by adapting it for rapid AFST through high-throughput monitoring of the growth of hundreds of individual fungi. Due to the distinct "budding" growth patterns of fungi, we developed a unique approach that utilizes specific cell number change to determine fungi replication, instead of cell area change used for bacteria in our previous study, to accurately determine the growth rates of individual fungal cells. This method not only accelerates the determination of antifungal resistance by directly observing individual fungal cell growth, but also yields accurate results. Employing Candida albicans as a representative model organism, reliable minimum inhibitory concentration (MIC) of fluconazole inhibiting 100% cells of Candida albicans (denoted as MIC100) was obtained within 3h using the developed method, while the modified broth dilution method required 72h for the similar reliable result. In addition, our approach was effectively utilized to test blood culture samples directly, eliminating the need to separate the fungi from whole blood samples spiked with Candida albicans. These features indicate the developed method holds great potential serving as a general tool in rapid antifungal susceptibility testing and MIC determination.

Oral candidiasis: pathogenesis, clinical presentation, diagnosis and treatment strategies.

Oral candidiasis is a clinical fungal infection that is the most common opportunistic infection affecting the human oral cavity. This article reviews the pathogenesis, clinical presentations, diagnosis and treatmentstrategies for oral candidiasis.

Candida albicans biofilms do not trigger reactive oxygen species and evade neutrophil killing.

Neutrophils are found within Candida albicans biofilms in vivo and could play a crucial role in clearing the pathogen from biofilms forming on catheters and mucosal surfaces. Our goal was to compare the antimicrobial activity of neutrophils against developing and mature C. albicans biofilms and identify biofilm-specific properties mediating resistance to immune cells. Antibiofilm activity was measured with the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)2H-tetrazolium-5-carboxanilide assay and a molecular Candida viability assay. Reactive oxygen species generation was assessed by measuring fluorescence of 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester in preloaded neutrophils. We found that mature biofilms were resistant to leukocytic killing and did not trigger reactive oxygen species, even though neutrophils retained their viability and functional activation potential. Beta-glucans found in the extracellular matrix negatively affected antibiofilm activities. We conclude that these polymers act as a decoy mechanism to prevent neutrophil activation and that this represents an important innate immune evasion mechanism of C. albicans biofilms.

A prebiotic diet modulates the oral microbiome composition and results in the attenuation of oropharyngeal candidiasis in mice.

Oral bacteria can influence the ability of Candida albicans to cause oropharyngeal candidiasis (OPC). We recently reported that a Lactobacillus johnsonii-enriched oral microbiota reduced C. albicans virulence in an immunosuppressed OPC mouse model. As a follow-up, in this work, we aimed to enrich the resident oral Lactobacillus communities with a prebiotic diet to further assess their effect on the severity of OPC. We tested the effect of a prebiotic xylo-oligosaccharides (XOS)-enriched diet in the oral global bacterial composition and severity of OPC. We assessed changes in the oral microbiome composition via 16S-rRNA gene high-throughput sequencing, validated by qPCR. The impact of the prebiotic diet on Candida infection was assessed by quantifying changes in oral fungal and bacterial biomass and scoring tongue lesions. Contrary to expectations, oral Lactobacillus communities were not enriched by the XOS-supplemented diet. Yet, XOS modulated the oral microbiome composition, increasing Bifidobacterium abundance and reducing enterococci and staphylococci. In the OPC model, the XOS diet attenuated Candida virulence and bacterial dysbiosis, increasing lactobacilli and reducing enterococci on the oral mucosa. We conclude that XOS attenuates Candida virulence by promoting a bacterial microbiome structure more resilient to Candida infection. IMPORTANCE This is the first study on the effects of a prebiotic diet on the oral mucosal bacterial microbiome and an oropharyngeal candidiasis (OPC) mouse model. We found that xylo-oligosaccharides change the oral bacterial community composition and attenuate OPC. Our results contribute to the understanding of the impact of the oral bacterial communities on Candida virulence.

Corrected and Republished from: "Understanding Lactobacillus paracasei and Streptococcus oralis Biofilm Interactions through Agent-Based Modeling".

As common commensals residing on mucosal tissues, Lactobacillus species are known to promote health, while some Streptococcus species act to enhance the pathogenicity of other organisms in those environments. In this study we used a combination of in vitro imaging of live biofilms and computational modeling to explore biofilm interactions between Streptococcus oralis, an accessory pathogen in oral candidiasis, and Lactobacillus paracasei, an organism with known probiotic properties. A computational agent-based model was created where the two species interact only by competing for space, oxygen, and glucose. Quantification of bacterial growth in live biofilms indicated that S. oralis biomass and cell numbers were much lower than predicted by the model. Two subsequent models were then created to examine more complex interactions between these species, one where L. paracasei secretes a surfactant and another where L. paracasei secretes an inhibitor of S. oralis growth. We observed that the growth of S. oralis could be affected by both mechanisms. Further biofilm experiments support the hypothesis that L. paracasei may secrete an inhibitor of S. oralis growth, although they do not exclude that a surfactant could also be involved. This contribution shows how agent-based modeling and experiments can be used in synergy to address multiple-species biofilm interactions, with important roles in mucosal health and disease. IMPORTANCE We previously discovered a role of the oral commensal Streptococcus oralis as an accessory pathogen. S. oralis increases the virulence of Candida albicans infections in murine oral candidiasis and epithelial cell models through mechanisms which promote the formation of tissue-damaging biofilms. Lactobacillus species have known inhibitory effects on biofilm formation of many microbes, including Streptococcus species. Agent-based modeling has great advantages as a means of exploring multifaceted relationships between organisms in complex environments such as biofilms. Here, we used an iterative collaborative process between experimentation and modeling to reveal aspects of the mostly unexplored relationship between S. oralis and L. paracasei in biofilm growth. The inhibitory nature of L. paracasei on S. oralis in biofilms may be exploited as a means of preventing or alleviating mucosal fungal infections.

Whole-Genome Sequencing of Lactobacillus johnsonii MT4, a Novel Strain Isolated from the Oral Cavity of C57BL/6 Mice.

Lactobacillus johnsonii strain MT4, isolated from the oral cavity of C57BL/6 mice, elicits antimicrobial activity against disease-associated microorganisms. Short-read sequencing of the whole genome revealed a single genome of 1,883,026 bp, with a GC content of 34.4%, and no plasmids.

Synergistic interaction between Candida albicans and commensal oral streptococci in a novel in vitro mucosal model.

Candida albicans is a commensal colonizer of the gastrointestinal tract of humans, where it coexists with highly diverse bacterial communities. It is not clear whether this interaction limits or promotes the potential of C. albicans to become an opportunistic pathogen. Here we investigate the interaction between C. albicans and three species of streptococci from the viridans group, which are ubiquitous and abundant oral commensal bacteria. The ability of C. albicans to form biofilms with Streptococcus oralis, Streptococcus sanguinis, or Streptococcus gordonii was investigated using flow cell devices that allow abiotic biofilm formation under salivary flow. In addition, we designed a novel flow cell system that allows mucosal biofilm formation under conditions that mimic the environment in the oral and esophageal mucosae. It was observed that C. albicans and streptococci formed a synergistic partnership where C. albicans promoted the ability of streptococci to form biofilms on abiotic surfaces or on the surface of an oral mucosa analogue. The increased ability of streptococci to form biofilms in the presence of C. albicans could not be explained by a growth-stimulatory effect since the streptococci were unaffected in their growth in planktonic coculture with C. albicans. Conversely, the presence of streptococci increased the ability of C. albicans to invade organotypic models of the oral and esophageal mucosae under conditions of salivary flow. Moreover, characterization of mucosal invasion by the biofilm microorganisms suggested that the esophageal mucosa is more permissive to invasion than the oral mucosa. In summary, C. albicans and commensal oral streptococci display a synergistic interaction with implications for the pathogenic potential of C. albicans in the upper gastrointestinal tract.

Host cell invasion and virulence mediated by Candida albicans Ssa1.

Candida albicans Ssa1 and Ssa2 are members of the HSP70 family of heat shock proteins that are expressed on the cell surface and function as receptors for antimicrobial peptides such as histatins. We investigated the role of Ssa1 and Ssa2 in mediating pathogenic host cell interactions and virulence. A C. albicans ssa1Δ/Δ mutant had attenuated virulence in murine models of disseminated and oropharyngeal candidiasis, whereas an ssa2Δ/Δ mutant did not. In vitro studies revealed that the ssa1Δ/Δ mutant caused markedly less damage to endothelial cells and oral epithelial cell lines. Also, the ssa1Δ/Δ mutant had defective binding to endothelial cell N-cadherin and epithelial cell E-cadherin, receptors that mediate host cell endocytosis of C. albicans. As a result, this mutant had impaired capacity to induce its own endocytosis by endothelial cells and oral epithelial cells. Latex beads coated with recombinant Ssa1 were avidly endocytosed by both endothelial cells and oral epithelial cells, demonstrating that Ssa1 is sufficient to induce host cell endocytosis. These results indicate that Ssa1 is a novel invasin that binds to host cell cadherins, induces host cell endocytosis, and is critical for C. albicans to cause maximal damage to host cells and induce disseminated and oropharyngeal disease.

Probiotics for Oral Candidiasis: Critical Appraisal of the Evidence and a Path Forward.

Oropharyngeal Candidiasis (OPC) is a mucosal fungal infection that is prevalent among patients with compromised immunity. The success of probiotics in treating chronic diseases with a microbial etiology component at other mucosal sites (i.e., gastro-intestinal, genitourinary and alveolar mucosae) has inspired research into the use of probiotics in the treatment of OPC. A growing body of research in vitro and in animal models indicates that some probiotic species and strains have inhibitory activities against Candida albicans growth, morphological switching, and biofilm formation. However, recent review and meta-analysis studies reveal a dearth of human randomized, controlled clinical trials on the efficacy of probiotics to treat or prevent OPC, while the majority of these have not based their selection of probiotic strains or the type of administration on sound pre-clinical evidence. In this mini-review, we assess the state of the field, outline some of the difficulties in translating lab results to clinical efficacy, and make recommendations for future research needed in order to move the field forward.

Insights From the Lactobacillus johnsonii Genome Suggest the Production of Metabolites With Antibiofilm Activity Against the Pathobiont Candida albicans.

Lactobacillus johnsonii is a probiotic bacterial species with broad antimicrobial properties; however, its antimicrobial activities against the pathobiont Candida albicans are underexplored. The aim of this study was to study the interactions of L. johnsonii with C. albicans and explore mechanisms of bacterial anti-fungal activities based on bacterial genomic characterization coupled with experimental data. We isolated an L. johnsonii strain (MT4) from the oral cavity of mice and characterized its effect on C. albicans growth in the planktonic and biofilm states. We also identified key genetic and phenotypic traits that may be associated with a growth inhibitory activity exhibited against C. albicans. We found that L. johnsonii MT4 displays pH-dependent and pH-independent antagonistic interactions against C. albicans, resulting in inhibition of C. albicans planktonic growth and biofilm formation. This antagonism is influenced by nutrient availability and the production of soluble metabolites with anticandidal activity.

A quantitative real-time RT-PCR assay for mature C. albicans biofilms.

BACKGROUND: Fungal biofilms are more resistant to anti-fungal drugs than organisms in planktonic form. Traditionally, susceptibility of biofilms to anti-fungal agents has been measured using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide (XTT) assay, which measures the ability of metabolically active cells to convert tetrazolium dyes into colored formazan derivatives. However, this assay has limitations when applied to high C. albicans cell densities because substrate concentration and solubility are limiting factors in the reaction. Because mature biofilms are composed of high cell density populations we sought to develop a quantitative real-time RT-PCR assay (qRT-PCR) that could accurately assess mature biofilm changes in response to a wide variety of anti-fungal agents, including host immune cells. RESULTS: The XTT and qRT-PCR assays were in good agreement when biofilm changes were measured in planktonic cultures or in early biofilms which contain lower cell densities. However, the real-time qRT-PCR assay could also accurately quantify small-medium size changes in mature biofilms caused by mechanical biomass reduction, antifungal drugs or immune effector cells, that were not accurately quantifiable with the XTT assay. CONCLUSIONS: We conclude that the qRT-PCR assay is more accurate than the XTT assay when measuring small-medium size effects of anti-fungal agents against mature biofilms. This assay is also more appropriate when mature biofilm susceptibility to anti-fungal agents is tested on complex biological surfaces, such as organotypic cultures.