Candidalysin: An unlikely aide for fungal gut commensalism.

Fungi colonize the mammalian gastrointestinal (GI) tract and can adopt both commensal and opportunistic lifestyles. In a recent issue of Nature, Liang et al. unraveled the complex interplay between Candida morphotypes and the gut bacterial microbiota and described a key role for candidalysin in gut colonization.1.

High-Throughput Screening of the Repurposing Hub Library to Identify Drugs with Novel Inhibitory Activity against Candida albicans and Candida auris Biofilms.

Candidiasis is one of the most frequent nosocomial infections affecting an increasing number of at-risk patients. Candida albicans remains the most frequent causative agent of candidiasis, but, in the last decade, C. auris has emerged as a formidable multi-drug-resistant pathogen. Both species are fully capable of forming biofilms, which contribute to resistance, increasing the urgency for new effective antifungal therapies. Repurposing existing drugs could significantly accelerate the development of novel therapies against candidiasis. Here, we have screened the Repurposing Hub library from the Broad Institute, containing over 6000 compounds, in search for inhibitors of C. albicans and C. auris biofilm formation. The primary screen identified 57 initial hits against C. albicans and 33 against C. auris. Confirmatory concentration-dependent assays were used to validate the activity of the initial hits and, at the same time, establish their anti-biofilm potency. Based on these results, ebselen, temsirolimus, and compound BAY 11-7082 emerged as the leading repositionable compounds. Subsequent experiments established their spectrum of antifungal activity against yeasts and filamentous fungi. In addition, their in vivo activity was examined in the murine models of hematogenously disseminated C. albicans and C. auris infections. Although promising, further in vitro and in vivo studies are needed to confirm their potential use for the therapy of candidiasis and possibly other fungal infections.

Development of an Imaging Flow Cytometry Method for Fungal Cytological Profiling and Its Potential Application in Antifungal Drug Development.

Automated imaging techniques have been in increasing demand for the more advanced analysis and efficient characterization of cellular phenotypes. The success of the image-based profiling method hinges on assays that can rapidly and simultaneously capture a wide range of phenotypic features. We have developed an automated image acquisition method for fungal cytological profiling (FCP) using an imaging flow cytometer that can objectively measure over 250 features of a single fungal cell. Fungal cells were labeled with calcofluor white and FM4-64FX, which bind to the cell wall and lipophilic membrane, respectively. Images of single cells were analyzed using IDEAS® software. We first acquired FCPs of fungal cells treated with fluconazole, amphotericin B, and caspofungin, each with a distinct mode of action, to establish FCP databases of profiles associated with specific antifungal treatment. Once fully established, we investigated the potential application of this technique as a screening methodology to identify compounds with novel antifungal activity against Candida albicans and Cryptococcus neoformans. Altogether, we have developed a rapid, powerful, and novel image-profiling method for the phenotypic characterization of fungal cells, also with potential applications in antifungal drug development.

Screening the medicine for malaria venture's Pandemic Response Box to identify novel inhibitors of Candida albicans and Candida auris biofilm formation.

Candida spp. are opportunistic yeasts capable of forming biofilms, which contribute to resistance, increasing the urgency for new effective antifungal therapies. Repurposing existing drugs could significantly accelerate the development of novel therapies against candidiasis. We screened the Pandemic Response Box containing 400 diverse drug-like molecules active against bacteria, viruses or fungi, for inhibitors of Candida albicans and Candida auris biofilm formation. Initial hits were identified based on the demonstration of >70% inhibitory activity. Dose-response assays were used to confirm the antifungal activity of initial hits and establish their potency. The spectrum of antifungal activity of the leading compounds was determined against a panel of medically important fungi, and the in vivo activity of the leading repositionable agent was evaluated in murine models of C. albicans and C. auris systemic candidiasis. The primary screening identified 20 hit compounds, and their antifungal activity and potency against C. albicans and C. auris were validated using dose-response measurements. From these experiments, the rapalog everolimus, emerged as the leading repositionable candidate. Everolimus displayed potent antifungal activity against different Candida spp., but more moderate levels of activity against filamentous fungi. Treatment with everolimus increased survival of mice infected with C. albicans, but not those with C. auris. The screening of the Pandemic Response Box resulted in the identification of several drugs with novel antifungal activity, with everolimus emerging as the main repositionable candidate. Further in vitro and in vivo studies are needed to confirm its potential therapeutic use.

Antifungal therapy of Candida biofilms: Past, present and future.

Virtually all Candida species linked to clinical candidiasis are capable of forming highly resistant biofilms on different types of surfaces, which poses an additional significant threat and further complicates therapy of these infections. There is a scarcity of antifungal agents, and their effectiveness, particularly against biofilms, is limited. Here we provide a historical perspective on antifungal agents and therapy of Candida biofilms. As we reflect upon the past, consider the present, and look towards the future of antifungal therapy of Candida biofilms, we believe that there are reasons to remain optimistic, and that the major challenges of Candida biofilm therapy can be conquered within a reasonable timeframe.

A 384-Well Microtiter Plate Model for Candida Biofilm Formation and Its Application to High-Throughput Screening.

Candidiasis, infections caused by Candida spp., represents one of the most common nosocomial infections afflicting an expanding number of compromised patients. Antifungal therapeutic options are few and show limited efficacy. Moreover, biofilm formation is frequently associated with different manifestations of candidiasis and further complicates therapy. Thus, there is an urgent need for new effective therapeutic agents, particularly those with anti-biofilm activity. Here we describe the development of a novel, simple, fast, economical, and highly reproducible 384-well microtiter plate model for the formation of both Candida albicans and Candida auris biofilms and its application in high-throughput screening (HTS) techniques.

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.

A Facile High-Throughput Model of Surface-Independent Staphylococcus aureus Biofilms by Spontaneous Aggregation.

Many microbes in their natural habitats are found in biofilm ecosystems attached to surfaces and not as free-floating (planktonic) organisms. Furthermore, it is estimated that nearly 80% of human infections are associated with biofilms. Biofilms are traditionally defined as three-dimensional, structured microbial communities that are attached to a surface and encased in a matrix of exopolymeric material. While this view of biofilm largely arises from in vitro studies under static or flow conditions, in vivo observations have indicated that this view of biofilms is essentially true only for foreign-body infections on catheters or implants where biofilms are attached to the biomaterial. In mucosal infections such as chronic wounds or cystic fibrosis or joint infections, biofilms can be found unattached to a surface and as three-dimensional aggregates. In this work, we describe a high-throughput model of aggregate biofilms of methicillin-resistant Staphylococcus aureus (MRSA) using 96-well plate hanging-drop technology. We show that MRSA forms surface-independent biofilms, distinct from surface-attached biofilms, that are rich in exopolymeric proteins, polysaccharides, and extracellular DNA (eDNA), express biofilm-related genes, and exhibit heightened antibiotic resistance. We also show that the surface-independent biofilms of clinical isolates of MRSA from cystic fibrosis and central catheter-related infections demonstrate morphological differences. Overall, our results show that biofilms can form by spontaneous aggregation without attachment to a surface, and this new in vitro system can model surface-independent biofilms that may more closely mimic the corresponding physiological niche during infection.IMPORTANCE The canonical model of biofilm formation begins with the attachment and growth of microbial cells on a surface. While these in vitro models reasonably mimic biofilms formed on foreign bodies such as catheters and implants, this is not the case for biofilms formed in cystic fibrosis and chronic wound infections, which appear to present as aggregates not attached to a surface. The hanging-drop model of biofilms of methicillin-resistant Staphylococcus aureus (MRSA), the major causative organism of skin and soft tissue infections, shows that these biofilms display morphological and antibiotic response patterns that are distinct from those of their surface-attached counterparts, and biofilm growth is consistent with their in vivo location. The simplicity and throughput of this model enable adoption to investigate other single or polymicrobial biofilms in a physiologically relevant setting.

Screening the CALIBR ReFRAME Library in Search for Inhibitors of Candida auris Biofilm Formation.

Candida auris is an emerging yeast which, since its first isolation about a decade ago, has spread rapidly and triggered major infectious outbreaks in health care facilities around the world. C. auris strains often display resistance to clinically-used antifungal agents, contributing to high mortality rates. Thus, there is an urgent need for new antifungals to contain the spread of this emerging multi-drug resistant pathogen and to improve patient outcomes. However, the timeline for the development of a new antifungal agent typically exceeds 10­15 years. Thus, repurposing of current drugs could significantly accelerate the development and eventual deployment of novel therapies for the treatment of C. auris infections. Toward this end, in this study we have profiled a library of known drugs encompassing approximately 12,000 clinical-stage or FDA-approved small molecules in search for known molecules with antifungal activity against C. auris; more specifically, those capable of inhibiting C. auris biofilm formation. From this library, 100 compounds displaying antifungal activity were identified in the initial screen, including 26 compounds for which a dose-response relationship with biofilm-inhibitory activity against C. auris could be confirmed. Of these, five were identified as the most interesting potential repositionable candidates. Due to their known pharmacological and human safety profiles, identification of such compounds should allow for their accelerated preclinical and clinical development for the treatment of C. auris infections.

Bismuth nanoparticles obtained by a facile synthesis method exhibit antimicrobial activity against Staphylococcus aureus and Candida albicans.

BACKGROUND: Bismuth compounds are known for their activity against multiple microorganisms; yet, the antibiotic properties of bismuth nanoparticles (BiNPs) remain poorly explored. The objective of this work is to further the research of BiNPs for nanomedicine-related applications. Stable Polyvinylpyrrolidone (PVP)-coated BiNPs were produced by a chemical reduction process, in less than 30 min. RESULTS: We produced stable, small, spheroid PVP-coated BiNPs with a crystalline organization. The PVP-BiNPs showed potent antibacterial activity against the pathogenic bacterium Staphylococcus aureus and antifungal activity against the opportunistic pathogenic yeast Candida albicans, both under planktonic and biofilm growing conditions. CONCLUSIONS: Our results indicate that BiNPs represent promising antimicrobial nanomaterials, and this facile synthetic method may allow for further investigation of their activity against a variety of pathogenic microorganisms.

Inhibition of Mixed Biofilms of Candida albicans and Methicillin-Resistant Staphylococcus aureus by Positively Charged Silver Nanoparticles and Functionalized Silicone Elastomers.

Both bacterial and fungal organisms display the ability to form biofilms; however, mixed bacterial/fungal biofilms are particularly difficult to control and eradicate. The opportunistic microbial pathogens Candida albicans and Staphylococcus aureus are among the most frequent causative agents of healthcare-acquired infections, and are often co-isolated forming mixed biofilms, especially from contaminated catheters. These mixed species biofilms display a high level of antibiotic resistance; thus, these infections are challenging to treat resulting in excess morbidity and mortality. In the absence of effective conventional antibiotic treatments, nanotechnology-based approaches represent a promising alternative for the treatment of highly recalcitrant polymicrobial biofilm infections. Our group has previously reported on the activity of pure positively charged silver nanoparticles synthesized by a novel microwave technique against single-species biofilms of C. albicans and S. aureus. Here, we have expanded our observations to demonstrate that that silver nanoparticles display dose-dependent activity against dual-species C. albicans/S. aureus biofilms. Moreover, the same nanoparticles were used to functionalize catheter materials, leading to the effective inhibition of the mixed fungal/bacterial biofilms. Overall, our results indicate the potent activity of silver nanoparticles against these cross-kingdom biofilms. More studies are warranted to examine the ability of functionalized catheters in the prevention of catheter-related bloodstream infections.

Silver Nanoantibiotics Display Strong Antifungal Activity Against the Emergent Multidrug-Resistant Yeast Candida auris Under Both Planktonic and Biofilm Growing Conditions.

Candida auris is an emergent multidrug-resistant pathogenic yeast with an unprecedented ability for a fungal organism to easily spread between patients in clinical settings, leading to major outbreaks in healthcare facilities. The formation of biofilms by C. auris contributes to infection and its environmental persistence. Most antifungals and sanitizing procedures are not effective against C. auris, but antimicrobial nanomaterials could represent a viable alternative to combat the infections caused by this emerging pathogen. We have previously described an easy and inexpensive method to synthesize silver nanoparticles (AgNPs) in non-specialized laboratories. Here, we have assessed the antimicrobial activity of the resulting AgNPs on C. auris planktonic and biofilm growth phases. AgNPs displayed a strong antimicrobial activity against all the stages of all C. auris strains tested, representative of four different clades. Under planktonic conditions, minimal inhibitory concentration (MIC) values of AgNPs against the different strains were <0.5 µg ml-1; whereas calculated IC50 values for inhibition of biofilms formation were <2 µg ml-1 for all, but one of the C. auris strains tested. AgNPs were also active against preformed biofilms formed by all different C. auris strains, with IC50 values ranging from 1.2 to 6.2 µg ml-1. Overall, our results indicate potent activity of AgNPs against strains of C. auris, both under planktonic and biofilm growing conditions, and indicate that AgNPs may contribute to the control of infections caused by this emerging nosocomial threat.

Bismuth Nanoantibiotics Display Anticandidal Activity and Disrupt the Biofilm and Cell Morphology of the Emergent Pathogenic Yeast Candida auris.

Candida auris is an emergent multidrug-resistant pathogenic yeast, which forms biofilms resistant to antifungals, sanitizing procedures, and harsh environmental conditions. Antimicrobial nanomaterials represent an alternative to reduce the spread of pathogens-including yeasts-regardless of their drug-resistant profile. Here we have assessed the antimicrobial activity of easy-to-synthesize bismuth nanoparticles (BiNPs) against the emergent multidrug-resistant yeast Candida auris, under both planktonic and biofilm growing conditions. Additionally, we have examined the effect of these BiNPs on cell morphology and biofilm structure. Under planktonic conditions, BiNPs MIC values ranged from 1 to 4 µg mL-1 against multiple C. auris strains tested, including representatives of all different clades. Regarding the inhibition of biofilm formation, the calculated BiNPs IC50 values ranged from 5.1 to 113.1 µg mL-1. Scanning electron microscopy (SEM) observations indicated that BiNPs disrupted the C. auris cell morphology and the structure of the biofilms. In conclusion, BiNPs displayed strong antifungal activity against all strains of C. auris under planktonic conditions, but moderate activity against biofilm growth. BiNPs may potentially contribute to reducing the spread of C. auris strains at healthcare facilities, as sanitizers and future potential treatments. More research on the antimicrobial activity of BiNPs is warranted.

Current Antimycotics, New Prospects, and Future Approaches to Antifungal Therapy.

Fungal infections represent an increasing threat to a growing number of immune- and medically compromised patients. Fungi are eukaryotic organisms and, as such, there is a limited number of selective targets that can be exploited for antifungal drug development. This has also resulted in a very restricted number of antifungal drugs that are clinically available for the treatment of invasive fungal infections at the present time-polyenes, azoles, echinocandins, and flucytosine. Moreover, the utility of available antifungals is limited by toxicity, drug interactions and the emergence of resistance, which contribute to high morbidity and mortality rates. This review will present a brief summary on the landscape of current antifungals and those at different stages of clinical development. We will also briefly touch upon potential new targets and opportunities for novel antifungal strategies to combat the threat of fungal infections.

Screening Repurposing Libraries for Identification of Drugs with Novel Antifungal Activity.

Fungal organisms are ubiquitous in nature, and progress of modern medicine is creating an expanding number of severely compromised patients susceptible to a variety of opportunistic fungal infections. These infections are difficult to diagnose and treat, leading to high mortality rates. The limited antifungal arsenal, the toxicity of current antifungal drugs, the development of resistance, and the emergence of new multidrug-resistant fungi, all highlight the urgent need for new antifungal agents. Unfortunately, the development of a novel antifungal is a rather long and expensive proposition, and no new classes of antifungal agents have reached the market in the last 2 decades. Drug repurposing, or finding new indications for old drugs, represents a promising alternative pathway to drug development that is particularly appealing within the academic environment. In the last few years, there has been a growing interest in repurposing approaches in the antifungal arena, with multiple groups of investigators having performed screenings of different repurposing libraries against different pathogenic fungi in search for drugs with previously unrecognized antifungal effects. Overall, these repurposing efforts may lead to the fast deployment of drugs with novel antifungal activity, which can rapidly bring benefits to patients, while at the same time reducing health care costs.

FDA Approved Drug Library Screening Identifies Robenidine as a Repositionable Antifungal.

Due to the increasing prevalence of pathogenic fungal infections, the emergence of antifungal resistant clinical isolates worldwide, and the limited arsenal of available antifungals, developing new antifungal strategies is imperative. In this study, we screened a library of 1068 FDA-approved drugs to identify hits that exhibit broad-spectrum antifungal activity. Robenidine, an anticoccidial agent which has been widely used to treat coccidian infections of poultry and rabbits, was identified in this screen. Physiological concentration of robenidine (8 µM) was able to significantly inhibit yeast cell growth, filamentation and biofilm formation of Candida albicans - the most extensively studied human fungal pathogen. Moreover, we observed a broad-spectrum antifungal activity of this compound against fluconazole resistant clinical isolates of C. albicans, as well as a wide range of other clinically relevant fungal pathogens. Intriguingly, robenidine-treated C. albicans cells were hypersensitive to diverse cell wall stressors, and analysis of the cell wall structure by transmission electron microscopy (TEM) showed that the cell wall was severely damaged by robenidine, implying that this compound may target the cell wall integrity signaling pathway. Indeed, upon robenidine treatment, we found a dose dependent increase in the phosphorylation of the cell wall integrity marker Mkc1, which was decreased after prolonged exposure. Finally, we provide evidence by RNA-seq and qPCR that Rlm1, the downstream transcription factor of Mkc1, may represent a potential target of robenidine. Therefore, our data suggest that robenidine, a FDA approved anti-coccidiosis drug, displays a promising and broadly effective antifungal strategy, and represents a potentially repositionable candidate for the treatment of fungal infections.

Fast, facile synthesis method for BAL-mediated PVP-bismuth nanoparticles.

Bismuth is a water-insoluble non-toxic metallic element used in a wide array of pharmaceutical products, cosmetics, and catalysts, among others. Yet, the research regarding the use of bismuth nanoparticles (BiNPs) for antimicrobial treatments is scarce. Most of the current protocols for synthesizing BiNPs suitable for medical uses cannot be easily replicated in non-specialized laboratories. The objective of this work is to provide a fast, facile and economical method for synthesizing BiNPs. Bismuth nanoparticles were synthesized by a chemical reduction process, in less than 1 h, in a heated alkaline glycine solution; by the chelation and reduction of the bismuth (III) ions using dimercaptopropanol (BAL) and sodium borohydride respectively, and then coated and stabilized by polyvinylpyrrolidone (PVP). The resulting PVP-BiNPs were characterized by UV-Vis spectrophotometry and transmission electron microscopy (TEM). • We describe a simple, rapid and inexpensive method for the synthesis of bismuth nanoparticles. • This method allows synthesizing small nanoparticles with an aspect ratio close to one. • Bismuth nanoparticles have antimicrobial properties, this easy-to-replicate protocol may further the research on bismuth nanoparticles for biomedical applications.

CARD9-Associated Dectin-1 and Dectin-2 Are Required for Protective Immunity of a Multivalent Vaccine against Coccidioides posadasii Infection.

Coccidioides species are fungal pathogens that can cause a widely varied clinical manifestation from mild pulmonary symptom to disseminated, life-threatening disease. We have previously created a subunit vaccine by encapsulating a recombinant coccidioidal Ag (rCpa1) in glucan-chitin particles (GCPs) as an adjuvant-delivery system. The GCP-rCpa1 vaccine has shown to elicit a mixed Th1 and Th17 response and confers protection against pulmonary coccidioidomycosis in mice. In this study, we further delineated the vaccine-induced protective mechanisms. Depletion of IL-17A in vaccinated C57BL/6 mice prior to challenge abrogated the protective efficacy of GCP-rCpa1 vaccine. Global transcriptome and Ingenuity Pathway Analysis of murine bone marrow-derived macrophages after exposure to this vaccine revealed the upregulation of proinflammatory cytokines (TNF-α, IL-6, and IL-1ß) that are associated with activation of C-type lectin receptors (CLR) Dectin-1- and Dectin-2-mediated CARD9 signaling pathway. The GCP formulation of rCpa1 bound soluble Dectin-1 and Dectin-2 and triggered ITAM signaling of corresponding CLR reporter cells. Furthermore, macrophages that were isolated from Dectin-1 -/-, Dectin-2 -/-, and CARD9 -/- mice significantly reduced production of inflammatory cytokines in response to the GCP-rCpa1 vaccine compared with those of wild-type mice. The GCP-rCpa1 vaccine had significantly reduced protective efficacy in Dectin-1 -/-, Dectin-2 -/-, and CARD9 -/- mice that showed decreased acquisition of Th cells in Coccidioides-infected lungs compared with vaccinated wild-type mice, especially Th17 cells. Collectively, we conclude that the GCP-rCpa1 vaccine stimulates a robust Th17 immunity against Coccidioides infection through activation of the CARD9-associated Dectin-1 and Dectin-2 signal pathways.

Inhibition of Candida auris Biofilm Formation on Medical and Environmental Surfaces by Silver Nanoparticles.

Candida auris is an emerging pathogenic fungus implicated in healthcare-associated outbreaks and causes bloodstream infections associated with high mortality rates. Biofilm formation represents one of the major pathogenetic traits associated with this microorganism. Unlike most other Candida species, C. auris has the ability to survive for weeks on different surfaces. Therefore, there is an urgent need to develop new effective control strategies to combat the threat of C. auris. Advances in nanotechnologies have emerged that carry significant potential impact against Candida biofilms. We obtained pure round silver nanoparticles (AgNPs) (1 to 3 nm in diameter) using a microwave-assisted synthetic approach. When tested against C. auris, our results indicated a potent inhibitory activity both on biofilm formation (half maximal inhibitory concentration (IC50) of 0.06 ppm) and against preformed biofilms (IC50 of 0.48 ppm). Scanning electron microscopy images of AgNP-treated biofilms showed cell wall damage mostly by disruption and distortion of the outer surface of the fungal cell wall. In subsequent experiments AgNPs were used to functionalize medical and environmental surfaces. Silicone elastomers functionalized with AgNPs demonstrated biofilm inhibition (>50%) at relatively low concentrations (2.3 to 0.28 ppm). Bandage dressings loaded with AgNPs inhibited growth of C. auris biofilms by more than 80% (2.3 to 0.017 ppm). Also, to demonstrate long-lasting protection, dressings loaded with AgNPs (0.036 ppm) were washed thoroughly with phosphate-buffered saline, maintaining protection against the C. auris growth from cycles 1 to 3 (>80% inhibition) and from cycles 4 to 6 (>50% inhibition). Our results demonstrate the dose-dependent activity of AgNPs against biofilms formed by C. auris on both medical (silicone elastomer) and environmental (bandage fibers) surfaces. The AgNPs-functionalized fibers retain the fungicidal effect even after repeated thorough washes. Overall these results point to the utility of silver nanoparticles to prevent and control infections caused by this emerging pathogenic fungus.