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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.

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.

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.

Abstract 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.

Abstract Objective Silver nanoparticles (AgNPs) can be difficult or expensive to obtain or synthesize for laboratories in resource-limited facilities. The purpose of this work was to optimize a synthesis method for a fast, facile, and cost-effective synthesis of AgNPs with antimicrobial activity, which can be readily implemented in non-specialized facilities and laboratories. Results The optimized method uses a rather simple and rapid chemical reduction process that involves the addition of a polyvinylpyrrolidone solution to a warmed silver nitrate solution under constant vigorous stirring, immediately followed by the addition of sodium borohydride. The total synthesis time is less than 15 min. The obtained AgNPs exhibit an aspect ratio close to 1, with an average size of 6.18 ± 5 nm. AgNPs displayed potent antimicrobial activity, with Minimal Inhibitory Concentration values of ≤ 4 µg mL−1 for Staphylococcus aureus and ≤ 2 µg mL−1 for Candida albicans. The resulting method is robust and highly reproducible, as demonstrated by the characterization of AgNPs from different rounds of syntheses and their antimicrobial activity.

The canonical model of biofilm formation begins with the attachment and growth of microbial cells on a surface. While these in vitro

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 10‑15 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.

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

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.

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.

Tuberculosis produces two clinical manifestations: active and latent (non-apparent) disease. The latter is estimated to affect one-third of the world population and constitutes a source of continued transmission should the disease emerge from its hidden state (reactivation). Methods to diagnose latent TB have been evolving and aim to detect the disease in people who are truly infected with M. tuberculosis, versus those where other mycobacteria, or even other pathologies not related to TB, are present. The current use of proteomic and transcriptomic approaches may lead to improved detection methods in the coming years.

ABSTRACT The opportunistic pathogenic fungus Candida albicans can cause devastating infections in immunocompromised patients. Its ability to undergo a morphogenetic transition from yeast to filamentous forms allows it to penetrate tissues and damage tissues, and the expression of genes associated with a number of pathogenetic mechanisms is also coordinately regulated with the yeast-to-hypha conversion. Therefore, it is widely considered that filamentation represents one of the main virulence factors of C. albicans. We have previously identified N-[3-(allyloxy)-phenyl]-4-methoxybenzamide (compound 9029936) as the lead compound in a series of small-molecule inhibitors of C. albicans filamentation and characterized its activity both in vitro and in vivo. This compound appears to be a promising candidate for the development of alternative antivirulence strategies for the treatment of C. albicans infections. In this study, we performed RNA sequencing analysis of samples obtained from C. albicans cells grown under filament-inducing conditions in the presence or absence of this compound. Overall, treatment with compound 9029936 resulted in 618 upregulated and 702 downregulated genes. Not surprisingly, some of the most downregulated genes included well-characterized genes associated with filamentation and virulence such as SAP5, ECE1 (candidalysin), and ALS3, as well as genes that impact metal chelation and utilization. Gene ontology analysis revealed an overrepresentation of cell adhesion, iron transport, filamentation, biofilm formation, and pathogenesis processes among the genes downregulated during treatment with this leading compound. Interestingly, the top upregulated genes suggested an enhancement of vesicular transport pathways, particularly those involving SNARE interactions. IMPORTANCE These results from whole-genome transcriptional profiling provide further insights into the biological activity and mode of action of a small-molecule inhibitor of C. albicans filamentation. This information will assist in the development of novel antivirulence strategies against C. albicans infections.

ABSTRACT Candidiasis affects a wide variety of immunocompromised and medically compromised patients. Candida albicans, a major human fungal pathogen, accounts for about 50% of all cases, while the remainder are caused by the less pathogenic non-albicans Candida species (NACS). These species are believed to be less pathogenic, in part, because they do not filament as readily or robustly as C. albicans, although definitive evidence is lacking. To address this question, we used strains for two NACS, Candida tropicalis and Candida parapsilosis, which were genetically engineered to constitutively express the key transcriptional regulator UME6 and drive strong filamentation both in vitro and during infection in vivo. Unexpectedly, both strains showed a dramatic reduction in organ fungal burden in response to UME6 expression. Consistent with these findings, we observed that a C. tropicalis hyperfilamentous mutant was significantly reduced and a filamentation-defective mutant was slightly increased for organ fungal burden. Comprehensive immune profiling generally did not reveal any significant changes in the host response to UME6 expression in the NACS that could explain the increased clearance of infection. Interestingly, whole-genome transcriptional profiling indicated that while genes important for filamentation were induced by UME6 expression in C. tropicalis and C. parapsilosis, other genes involved in a variety of processes important for pathogenesis were strongly downregulated. These findings suggest that there are fundamental evolutionary differences in the relationship between morphology and pathogenicity among Candida species and that NACS do not necessarily possess the same virulence properties as C. albicans. IMPORTANCE Many immunocompromised individuals, including HIV/AIDS and cancer patients, are susceptible to candidiasis. About half of all cases are caused by the major fungal pathogen Candida albicans, whereas the remainder are due to less pathogenic non-albicans Candida species (NACS). Generation of filamentous cells represents a major virulence property of C. albicans, and the NACS are believed to be less pathogenic, in part, because they do not filament as well as C. albicans does. To address this question, we determined the pathogenicity of two NACS strains that have been genetically engineered to promote filamentation during infection. Surprisingly, these strains showed a dramatic reduction in pathogenicity. The host immune response did not appear to be affected. However, unlike C. albicans, filamentation of the NACS was associated with downregulation of several genes important for pathogenicity processes. Our results suggest that there are fundamental evolutionary differences in the relationship between filamentation and pathogenesis in NACS compared to C. albicans.

ABSTRACT Fungal and bacterial populations coexist in the oral cavity, frequently forming mixed-species biofilms that complicate treatment against polymicrobial infections. However, despite relevance to oral health, the bidirectional interactions between these microbial populations are poorly understood. In this study, we aimed to elucidate the mechanisms underlying the interactions between the fungal species Candida albicans and the bacterial species Streptococcus gordonii as they coexist in mixed-species biofilms. Specifically, the interactions of different C. albicans mutant strains deficient in filamentation (efg1Δ/Δ and brg1Δ/Δ), adhesive interactions (als3Δ/Δ and bcr1Δ/Δ), and production of matrix exopolymeric substances (EPS) (kre5Δ/Δ, mnn9Δ/Δ, rlm1Δ/Δ, and zap1Δ/Δ) were evaluated with S. gordonii under different conditions mimicking the environment in the oral cavity. Interestingly, our results revealed that growth of the biofilm-deficient C. albicans als3Δ/Δ and bcr1Δ/Δ mutant strains in synthetic saliva or with S. gordonii restored their biofilm-forming ability. Moreover, challenging previous observations indicating an important role of morphogenetic conversions in the interactions between these two species, our results indicated a highly synergistic interaction between S. gordonii and the C. albicans filamentation-deficient efg1Δ/Δ and brg1Δ/Δ deletion mutants, which was particularly noticeable when the mixed biofilms were grown in synthetic saliva. Importantly, dual-species biofilms were found to exhibit increase in antimicrobial resistance, indicating that components of the fungal exopolymeric material confer protection to streptococcal cells against antibacterial treatment. Collectively, these findings unravel a high degree of complexity in the interactions between C. albicans and S. gordonii in mixed-species biofilms, which may impact homeostasis in the oral cavity. IMPORTANCE Microbial communities have a great impact in health and disease. C. albicans interacts with multiple microorganisms in the oral cavity, frequently forming polymicrobial biofilms. We report on the synergistic interactions between C. albicans and the Gram-positive bacterium S. gordonii, for which we have examined the different contributions of adhesive interactions, filamentation, and the extracellular matrix to the formation of dual-species biofilms. Our results demonstrate that growth in the presence of the bacterium can restore the biofilm-forming ability of different C. albicans mutant strains with defects in adhesion and filamentation. The mixed-species biofilms also show high levels of resistance to antibacterial and antifungal antibiotics, and our results indicate that the fungal biofilm matrix protects bacterial cells within these mixed-species biofilms. Our observations add to a growing body of evidence indicating a high level of complexity in the reciprocal interactions and consortial behavior of fungal/bacterial biofilms.

Silver nanoparticles offer a possible means of fighting antibacterial resistance. Most of their antibacterial properties are attributed to their silver ions. In the present work, we study the actions of positively charged silver nanoparticles against both methicillin-sensitive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus. We use aberration-corrected transmission electron microscopy to examine the bactericidal effects of silver nanoparticles and the ultrastructural changes in bacteria that are induced by silver nanoparticles. The study revealed that our 1 nm average size silver nanoparticles induced thinning and permeabilization of the cell wall, destabilization of the peptidoglycan layer, and subsequent leakage of intracellular content, causing bacterial cell lysis. We hypothesize that positively charged silver nanoparticles bind to the negatively charged polyanionic backbones of teichoic acids and the related cell wall glycopolymers of bacteria as a first target, consequently stressing the structure and permeability of the cell wall. This hypothesis provides a major mechanism to explain the antibacterial effects of silver nanoparticles on Staphylococcus aureus. Future research should focus on defining the related molecular mechanisms and their importance to the antimicrobial activity of silver nanoparticles.

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.

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.

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.

The current emerging COVID-19 pandemic has caused a global impact on every major aspect of our societies. It is known that SARS-Cov-2 can endure harsh environmental conditions for up to 72 h, which may contribute to its rapid spread. Therefore, effective containment strategies, such as sanitizing, are critical. Nanotechnology can represent an alternative to reduce the COVID-19 spread, particularly in critical areas, such as healthcare facilities and public places. Nanotechnology-based products are effective at inhibiting different pathogens, including viruses, regardless of their drug-resistant profile, biological structure, or physiology. Although there are several approved nanotechnology-based antiviral products, this work aims to highlight the use of nanomaterials as sanitizers for the prevention of the spread of mainly SARS-Cov-2. It has been widely demonstrated that nanomaterials are an alternative for sanitizing surfaces to inactivate the virus. Also, antimicrobial nanomaterials can reduce the risk of secondary microbial infections on COVID-19 patients, as they inhibit the bacteria and fungi that can contaminate healthcare-related facilities. Finally, cost-effective, easy-to-synthesize antiviral nanomaterials could reduce the burden of the COVID-19 on challenging environments and in developing countries.

Biofilm-associated Pseudomonas aeruginosa infections remain a significant clinical challenge since the conventional antibiotic treatment or combination therapies are largely ineffective; and new approaches are needed. To circumvent the major challenges associated with discovery of new antimicrobials, we have screened a library of compounds that are commercially available and approved by the FDA (Prestwick Chemical Library) against P. aeruginosa for effective antimicrobial and anti-biofilm activity. A preliminary screen of the Prestwick Chemical Library alone did not yield any repositionable candidates, but in a screen of combinations with a fixed sub-inhibitory concentration of the antibiotic colistin we observed 10 drugs whose bacterial inhibiting activity was reproducibly enhanced, seven of which were enhanced by more than 50%. We performed checkerboard assays of these seven drugs in combination with colistin against planktonic cells, and analysis of their interactions over the complete combination matrix using the Zero Interaction Potency (ZIP) model revealed interactions that varied from highly synergistic to completely antagonistic. Of these, five combinations that showed synergism were down-selected and tested against preformed biofilms of P. aeruginosa. Two of the five combinations were active against preformed biofilms of both laboratory and clinical strain of P. aeruginosa, resulting in a 2-log reduction in culturable cells. In summary, we have identified synergistic combinations of five commercially available, FDA-approved drugs and colistin that show antimicrobial activity against planktonic P. aeruginosa (Clomiphene Citrate, Mitoxantrone Dihydrochloride, Methyl Benzethonium Chloride, Benzethonium Chloride, and Auranofin) as well as two combinations (Auranofin and Clomiphene Citrate) with colistin that show antibiofilm activity.

We have previously identified a small molecule compound, N-[3-(allyloxy)-phenyl]-4-methoxybenzamide (9029936), that exerts potent inhibitory activity against filamentation and biofilm formation by the Candida albicans SC5314 strain and represents a lead candidate for the development of anti-virulence approaches against C. albicans infections. Here we present data from a series of experiments to further characterize its in vitro activity and drug-like characteristics. We demonstrate the activity of this compound against a panel of C. albicans clinical isolates, including several displaying resistance to current antifungals; as well as against a set of C. albicans gain of function strains in key transcriptional regulators of antifungal drug resistance. The compound also inhibits filamentation and biofilm formation in the closely related species C. dubliniensis, but not C. glabrata or C. tropicalis. Combinatorial studies reveal the potential of compound 9029936 to be used together with currently available conventional antifungals. Results of serial passage experiments indicate that repeated exposure to this compound does not elicit resistance. Viability staining of C. albicans in the presence of high concentrations of compound 9029936 confirms that the compound is not toxic to fungal cells, and cytological staining using image flow cytometry analysis reveals that treatment with the lead compound affects hyphal length, with additional effects on cell wall and integrity of the membrane system. In vitro pharmacological profiling provides further evidence that the lead compound displays a safe profile, underscoring its excellent “drug-like” characteristics. Altogether these results confirm the potential of this compound to be further developed as a true anti-virulence agent for the treatment of C. albicans infections, including those refractory to treatment with conventional antifungal agents.

ABSTRACT Candida albicans remains the main etiologic agent of candidiasis, the most common fungal infection and now the third most frequent infection in U.S. hospitals. The scarcity of antifungal agents and their limited efficacy contribute to the unacceptably high morbidity and mortality rates associated with these infections. The yeast-to-hypha transition represents the main virulence factor associated with the pathogenesis of C. albicans infections. In addition, filamentation is pivotal for robust biofilm development, which represents another major virulence factor for candidiasis and further complicates treatment. Targeting pathogenic mechanisms rather than growth represents an attractive yet clinically unexploited approach in the development of novel antifungal agents. Here, we performed large-scale phenotypic screening assays with 30,000 drug-like small-molecule compounds within ChemBridge’s DIVERSet chemical library in order to identify small-molecule inhibitors of C. albicans filamentation, and our efforts led to the identification of a novel series of bioactive compounds with a common biaryl amide core structure. The leading compound of this series, N-[3-(allyloxy)-phenyl]-4-methoxybenzamide, was able to prevent filamentation under all liquid and solid medium conditions tested, suggesting that it impacts a common core component of the cellular machinery that mediates hypha formation under different environmental conditions. In addition to filamentation, this compound also inhibited C. albicans biofilm formation. This leading compound also demonstrated in vivo activity in clinically relevant murine models of invasive and oral candidiasis. Overall, our results indicate that compounds within this series represent promising candidates for the development of novel anti-virulence approaches to combat C. albicans infections. IMPORTANCE Since fungi are eukaryotes, there is a limited number of fungus-specific targets and, as a result, the antifungal arsenal is exceedingly small. Furthermore, the efficacy of antifungal treatment is compromised by toxicity and development of resistance. As a consequence, fungal infections carry high morbidity and mortality rates, and there is an urgent but unmet need for novel antifungal agents. One appealing strategy for antifungal drug development is to target pathogenetic mechanisms associated with infection. In Candida albicans, one of the most common pathogenic fungi, morphogenetic transitions between yeast cells and filamentous hyphae represent a key virulence factor associated with the ability of fungal cells to invade tissues, cause damage, and form biofilms. Here, we describe and characterize a novel small-molecule compound capable of inhibiting C. albicans filamentation both in vitro and in vivo; as such, this compound represents a leading candidate for the development of anti-virulence therapies against candidiasis.

ABSTRACT Current in vitro techniques for the culture of microorganisms, and particularly of delicate microbial biofilms, are still mostly limited to low-density plates and manual labor and are not amenable to automation and true high-throughput (HT) applications. We have developed a novel fully automated platform for the formation of mono- and polymicrobial biofilms of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans at the nanoscale level. The nBioChip is robotically printed, robustly handled, and scanned using a standard microarray reader. Using this technique, hundreds to thousands of identical nanobiofilms encapsulated in hydrogel spots were cultured on microscope slides. The spots can withstand the washing steps involved in screening assays. The miniaturized biofilms demonstrated characteristics similar to those displayed by conventionally formed macroscopic biofilms, including (i) three-dimensional architectural features, (ii) synthesis of exopolymeric matrix material, and (iii) elevated resistance to antibiotic treatment. On the basis of our results, the nBioChip can generate reliable high-throughput antimicrobial susceptibility testing (HT-AST) in 12 to 18 h. The chip serves as a proof-of-concept universal platform for high-throughput drug screening and other downstream applications and furthers understanding of microbial interactions in mixed-species communities at the nanoscale level. IMPORTANCE With an estimated 80% of infections being associated with a biofilm mode of growth and the ensuing recalcitrance of these biofilms with respect to conventional antibiotic treatment leading to high mortality rates, there is a dire and unmet need for the development of novel approaches to prevent, treat, and control these infections. Both bacteria and fungi are capable of forming biofilms that are inherently fragile and often polymicrobial in nature, which further complicates treatment. In this work, we showcase a nanobiofilm chip as a convenient platform for culturing several hundreds of mono- or polymicrobial biofilms and for susceptibility testing. This platform enables true ultra-high-throughput screening for antimicrobial drug discovery or diagnostics or for addressing fundamental issues in microbiology.

The human oral cavity is normally colonized by a wide range of microorganisms, including bacteria, fungi, Archaea, viruses, and protozoa. Within the different oral microenvironments these organisms are often found as part of highly organized microbial communities termed biofilms, which display consortial behavior. Formation and maintenance of these biofilms are highly dependent on the direct interactions between the different members of the microbiota, as well as on the released factors that influence the surrounding microbial populations. These complex biofilm dynamics influence oral health and disease. In the latest years there has been an increased recognition of the important role that interkingdom interactions, in particular those between fungi and bacteria, play within the oral cavity. Candida spp., and in particular C. albicans, are among the most important fungi colonizing the oral cavity of humans and have been found to participate in these complex microbial oral biofilms. C. albicans has been reported to interact with individual members of the oral bacterial microbiota, leading to either synergistic or antagonistic relationships. In this review we describe some of the better characterized interactions between Candida spp. and oral bacteria.

The oral cavity serves as a nutrient-rich haven for over 600 species of microorganisms. Although many are essential to maintaining the oral microbiota, some can cause oral infections such as caries, periodontitis, mucositis, and endodontic infections, and this is further exacerbated with dental implants. Most of these infections are mixed species in nature and associated with a biofilm mode of growth. Here, after optimization of different parameters including cell density, growth media, and incubation conditions, we have developed an in vitro model of C. albicans–S. gordonii mixed-species biofilms on titanium discs that is relevant to infections of peri-implant diseases. Our results indicate a synergistic effect for the development of biofilms when both microorganisms were seeded together, confirming the existence of beneficial, mutualistic cross-kingdom interactions for biofilm formation. The morphological and architectural features of these dual-species biofilms formed on titanium were determined using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Mixed biofilms formed on titanium discs showed a high level of resistance to combination therapy with antifungal and antibacterial drugs. This model can serve as a platform for further analyses of complex fungal/bacterial biofilms and can also be applied to screening of new drug candidates against mixed-species biofilms.

A majority of infections caused by Candida albicans—the most frequent fungal pathogen—are associated with biofilm formation. A salient feature of C. albicans biofilms is the presence of the biofilm matrix. This matrix is composed of exopolymeric materials secreted by sessile cells within the biofilm, in which all classes of macromolecules are represented, and provides protection against environmental challenges. In this review, we summarize the knowledge accumulated during the last two decades on the composition, structure, and function of the C. albicans biofilm matrix. Knowledge of the matrix components, its structure, and function will help pave the way to novel strategies to combat C. albicans biofilm infections.

ABSTRACT Among pathogenic fungi, Candida albicans is most frequently associated with biofilm formation, a lifestyle that is entirely different from the planktonic state. One of the distinguishing features of these biofilms is the presence of extracellular material, commonly referred to as the “biofilm matrix.” The fungal biofilm matrix embeds sessile cells within these communities and plays important structural and physiological functions, including antifungal drug resistance with important clinical repercussions. This matrix is mostly self-produced by the fungal cells themselves and is composed of different types of biopolymers. In C. albicans, the main components of the biofilm matrix are carbohydrates, proteins, lipids, and DNA, but many of them remain unidentified and/or poorly characterized. In their recent article, Zarnowski et al. [mBio 5(4):e01333-14, 2014, doi:10.1128/mBio.01333-14] used a variety of biochemical and state-of-the-art “omic” approaches (glycomics, proteomics, and lipidomics) to identify and characterize unique biopolymers present in the C. albicans biofilm matrix. Besides generating a true “encyclopedic” catalog of individual moieties from each of the different macromolecular categories, results also provide important insights into structural and functional aspects of the fungal biofilm matrix, particularly the interaction between different components and the contribution of multiple matrix constituents to biofilm antifungal drug resistance.

ABSTRACT Micro- and nanoscale technologies have radically transformed biological research from genomics to tissue engineering, with the relative exception of microbial cell culture, which is still largely performed in microtiter plates and petri dishes. Here, we present nanoscale culture of the opportunistic fungal pathogen Candida albicans on a microarray platform. The microarray consists of 1,200 individual cultures of 30 nl of C. albicans biofilms (“nano-biofilms”) encapsulated in an inert alginate matrix. We demonstrate that these nano-biofilms are similar to conventional macroscopic biofilms in their morphological, architectural, growth, and phenotypic characteristics. We also demonstrate that the nano-biofilm microarray is a robust and efficient tool for accelerating the drug discovery process: (i) combinatorial screening against a collection of 28 antifungal compounds in the presence of immunosuppressant FK506 (tacrolimus) identified six drugs that showed synergistic antifungal activity, and (ii) screening against the NCI challenge set small-molecule library identified three heretofore-unknown hits. This cell-based microarray platform allows for miniaturization of microbial cell culture and is fully compatible with other high-throughput screening technologies. IMPORTANCE Microorganisms are typically still grown in petri dishes, test tubes, and Erlenmeyer flasks in spite of the latest advances in miniaturization that have benefitted other allied research fields, including genomics and proteomics. Culturing microorganisms in small scale can be particularly valuable in cutting down time, cost, and reagent usage. This paper describes the development, characterization, and application of nanoscale culture of an opportunistic fungal pathogen, Candida albicans. Despite a more than 2,000-fold reduction in volume, the growth characteristics and drug response profiles obtained from the nanoscale cultures were comparable to the industry standards. The platform also enabled rapid identification of new drug candidates that were effective against C. albicans biofilms, which are a major cause of mortality in hospital-acquired infections.