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Aspergillus and Fusarium are two economically important genera of fungi. They cause significant yield losses and contamination of crops with mycotoxins. In this study we aimed to evaluate the impact of 2,4‐diacetylphloroglucinol (2,4‐DAPG) on Aspergillus and Fusarium fungi. It is hypothesized that two fungal genera, which have different ecological strategies, react differently to stress caused by a secondary metabolite produced by rhizosphere Pseudomonas species. We found that 2,4‐DAPG was able to reduce biofilm formation of Aspergillus and Fusarium, as reflected in biomass and its chemical composition. Furthermore, subinhibitory concentrations of 2,4‐DAPG increased the levels of ergosterol and polysaccharides (α‐ and β‐glucans, chitin) in the cell membrane and cell wall of Aspergillus, while decreasing them in Fusarium. 2,4‐DAPG altered the production of secondary metabolites, especially mycotoxins and extracellular proteases. The production of ochratoxin A was decreased in A. ochraceus, and T‐2 toxin and zearalenone, on the contrary, were increased in F. culmorum and F. sporotrichioides, respectively. Thus, using 2,4‐DAPG we demonstrated that the ecological role of fungi determines their reaction to antibiotic substances produced by the plant microbiome. Our data contributes to understanding the molecular mechanisms behind symbiotic relationships in natural communities, which are mediated by the biosynthesis of antibiotics. [ABSTRACT FROM AUTHOR]

Fungal contamination in drinking water has garnered considerable attention over the past few decades, especially considering the detrimental consequences of pathogenic fungal species on both human and animal health. The formation of biofilms by certain species is a considerable factor contributing to the emergence of severe fungal infections. This research was designed to isolate and identify fungi, particularly those capable of forming biofilms from 150 samples of drinking water sourced from various locations. The isolated fungal species were tested for them in vitro biofilm formation using a microtitration plate method and the crystal violet assay was applied to quantify the established biofilms. The effectiveness of three disinfectants, namely ozone, chlorine, and hydrogen peroxide, in preventing the formation of biofilms by the most isolated fungal species was monitored. The findings indicated that Aspergillus species were the most prevalent in drinking water, comprising 63.33% (95/150) of the total number of fungal species identified. Aspergillus fumigatus and Aspergillus flavus were identified as the primary contributors to biofilm formation in drinking water distribution systems with prevalence rates of 41.00 and 34.00%, respectively, among all Aspergillus species. The outcomes of the in vitro studies demonstrated that the ozone disinfectant exhibited promising results in inhibiting fungal biofilms compared to chlorine and hydrogen peroxide. In conclusion, these findings provided valuable insights for water distribution authorities to develop effective regimens for controlling biofilm-forming fungal species using suitable antifungal biofilm disinfectants. [ABSTRACT FROM AUTHOR]

Candida species cause life-threatening infections with high morbidity and mortality rates and their resistance to conventional therapy is closely linked to biofilm formation. Thus, the development of new approaches to study Candida biofilms and the identification of novel therapeutic strategies could yield improved clinical outcomes. In the current study, we have set up an impedance-based in vitro system to study Candida spp. biofilms in real-time and to evaluate their sensitivity to two conventional antifungal groups used in clinical practice - azoles and echinocandins. Both fluconazole and voriconazole were unable to inhibit biofilm formation in most strains tested, while echinocandins showed biofilm inhibitory capacity at relatively low concentrations (starting from 0.625 mg/L). However, assays performed on 24 h Candida albicans and C. glabrata biofilms revealed that micafungin and caspofungin failed to eradicate mature biofilms at all tested concentrations, evidencing that once formed, Candida spp. biofilms are extremely difficult to eliminate using currently available antifungals. We then evaluated the antifungal and anti-biofilm effect of andrographolide, a natural compound isolated from the plant Andrographis paniculata with known antibiofilm activity on Gram-positive and Gram-negative bacteria. Optical density measures, impedance evaluation, CFU counts, and electron microscopy data showed that andrographolide strongly inhibits planktonic Candida spp. growth and halts Candida spp. biofilm formation in a dose-dependent manner in all tested strains. Moreover, andrographolide was capable of eliminating mature biofilms and viable cell numbers by up to 99.9% in the C. albicans and C. glabrata strains tested, suggesting its potential as a new approach to treat multi-resistant Candida spp. biofilm-related infections.

Biofilm formation is an important microbial strategy for fungal pathogens, such as Fusarium, Aspergillus, and Candida, to establish keratitis in patients wearing soft contact lenses. Despite the well-documented 2006 outbreak of Fusarium keratitis that eventually led to the withdrawal of the Bausch & Lomb multipurpose lens care solution ReNu with MoistureLoc ("MoistureLoc") from the global market, contact lens care systems and solutions currently available on the market do not specifically target fungal biofilms. This is partially due to the lack of recognition and understanding of important roles that fungal biofilms play in contact lens associated fungal keratitis (CLAFK). This review aims to reemphasize the link between fungal biofilms and CLAFK, and deepen our comprehension of its importance in pathogenesis and persistence of this medical device-related infection. [ABSTRACT FROM AUTHOR]

Fungi represent a very important cause of microbial eye infections, especially in tropical and developing countries, as they could cause sight-threating disease, such as keratitis and ocular candidiasis, resulting in irreversible vision loss. Candida species are among the most frequent microorganisms associated with fungal infection. Although Candida albicans is still the most frequently detected organism among Candida subspecies, an important increase in non-albicans species has been reported. Mycotic infections often represent an important diagnostic-clinical problem due to the difficulties in performing the diagnosis and a therapeutic problem due to the limited availability of commercial drugs and the difficult penetration of antifungals into ocular tissues. The ability to form biofilms is another feature that makes Candida a dangerous pathogen. In this review, a summary of the state-of-the-art panorama about candida ocular pathology, diagnosis, and treatment has been conducted. Moreover, we also focused on new prospective natural compounds, including nanoparticles, micelles, and nanocarriers, as promising drug delivery systems to better cure ocular fungal and biofilm-related infections. The effect of the drug combination has also been examined from the perspective of increasing efficacy and improving the course of infections caused by Candida which are difficult to fight. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance is a major public health concern worldwide. Albeit to a lesser extent than bacteria, fungi are also becoming increasingly resistant to antifungal drugs. Moreover, due to the small number of antifungal classes, therapy options are limited, complicating the clinical management of mycoses. In this view, antimicrobial peptides (AMPs) are a potential alternative to conventional drugs. Among these, Proline-rich antimicrobial peptides (PrAMPs), almost exclusively of animal origins, are of particular interest due to their peculiar mode of action. In this study, a search for new arginine- and proline-rich peptides from plants has been carried out with a bioinformatic approach by sequence alignment and antimicrobial prediction tools. Two peptide candidates were tested against planktonic cells and biofilms of Candida albicans and Candida glabrata strains, including resistant isolates. These peptides showed similar potent activity, with half-maximal effective concentration values in the micromolar range. In addition, some structural and functional features, revealing peculiar mechanistic behaviors, were investigated. [ABSTRACT FROM AUTHOR]

Biofilm formation is an important microbial strategy for fungal pathogens, such as Fusarium, Aspergillus, and Candida, to establish keratitis in patients wearing soft contact lenses. Despite the well-documented 2006 outbreak of Fusarium keratitis that eventually led to the withdrawal of the Bausch & Lomb multipurpose lens care solution ReNu with MoistureLoc (“MoistureLoc”) from the global market, contact lens care systems and solutions currently available on the market do not specifically target fungal biofilms. This is partially due to the lack of recognition and understanding of important roles that fungal biofilms play in contact lens associated fungal keratitis (CLAFK). This review aims to reemphasize the link between fungal biofilms and CLAFK, and deepen our comprehension of its importance in pathogenesis and persistence of this medical device-related infection.

[Display omitted] Fungal nail infection (Onychomycosis) often requires prolonged treatment and is associated with a high risk of resistance to treatment. Here in this contribution, we introduce a novel approach to enhance penetration and antifungal activity of the antifungal drug griseofulvin (GF). Solid dispersions were prepared with hydroxypropyl methylcellulose acetate succinate (HPMCAS) and combined with surfactant (either sodium dodecyl sulphate (SDS), dodecyl trimethylammonium bromide (DTAB), or Pluronic F127) using mechanochemical activation. The prepared powders were then suspended with spray-dried silica-coated silver nanoparticles and applied onto infected bovine hooves to assess permeability and antifungal activity. The results showed that the prepared nanosuspensions significantly suppressed fungal activity causing disruption of fungal biofilms. Raman mapping showed enhanced permeation while dynamic vapor sorption (DVS), and particle size measurements showed varied effects depending on the type of surfactant and milling conditions. The prepared nanosuspensions displayed enhanced solubility of the poorly soluble drug reaching approximately 1.2 mg/mL. The results showed that the dispersions that contained DTAB displayed maximum efficacy while the inclusion of colloidal silver did not seem to significantly improve the antifungal activity compared to other formulations. [ABSTRACT FROM AUTHOR]

Fungi represent a very important cause of microbial eye infections, especially in tropical and developing countries, as they could cause sight-threating disease, such as keratitis and ocular candidiasis, resulting in irreversible vision loss. Candida species are among the most frequent microorganisms associated with fungal infection. Although Candida albicans is still the most frequently detected organism among Candida subspecies, an important increase in non-albicans species has been reported. Mycotic infections often represent an important diagnostic-clinical problem due to the difficulties in performing the diagnosis and a therapeutic problem due to the limited availability of commercial drugs and the difficult penetration of antifungals into ocular tissues. The ability to form biofilms is another feature that makes Candida a dangerous pathogen. In this review, a summary of the state-of-the-art panorama about candida ocular pathology, diagnosis, and treatment has been conducted. Moreover, we also focused on new prospective natural compounds, including nanoparticles, micelles, and nanocarriers, as promising drug delivery systems to better cure ocular fungal and biofilm-related infections. The effect of the drug combination has also been examined from the perspective of increasing efficacy and improving the course of infections caused by Candida which are difficult to fight.

With the rapid increase in the frequency of azole-resistant species, combination therapy appears to be a promising tool to augment the antifungal activity of azole drugs against resistant Candida species. Here, we report the effect of aprepitant, an antiemetic agent, on the antifungal activities of azole drugs against the multidrug-resistant Candida auris. Aprepitant reduced the minimum inhibitory concentration (MIC) of itraconazole in vitro, by up to eight-folds. Additionally, the aprepitant/itraconazole combination interfered significantly with the biofilm-forming ability of C. auris by 95 ± 0.13%, and significantly disrupted mature biofilms by 52 ± 0.83%, relative to the untreated control. In a Caenorhabditis elegans infection model, the aprepitant/itraconazole combination significantly prolonged the survival of infected nematodes by ~90% (five days post-infection) and reduced the fungal burden by ~92% relative to the untreated control. Further, this novel drug combination displayed broad-spectrum synergistic interactions against other medically important Candida species such as C. albicans, C. krusei, C. tropicalis, and C. parapsilosis (ƩFICI ranged from 0.08 to 0.31). Comparative transcriptomic profiling and mechanistic studies indicated aprepitant/itraconazole interferes significantly with metal ion homeostasis and compromises the ROS detoxification ability of C. auris. This study presents aprepitant as a novel, potent, and broad-spectrum azole chemosensitizing agent that warrants further investigation.

Thitinan Kitisin,1 Watcharamat Muangkaew,1 Sumate Ampawong,2 Passanesh Sukphopetch1 1Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; 2Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University, Bangkok, ThailandCorrespondence: Passanesh Sukphopetch Email natthanej.lup@mahidol.ac.thIntroduction: Central venous catheter (CVC) is a medical device that is used to administer medication for a long duration. Colonization by an emerging opportunistic pathogen Scedosporium apiospermum in the CVC lumen is frequently reported to cause severe complications in patients. Here, we describe the effect of fungal quorum-sensing molecule (QSM) known as tryptophol (TOH) to control S. apiospermum colonization in catheter tube lumens in both in vitro and in vivo models.Methods: Antifungal susceptibility of TOH against S. apiospermum was compared with voriconazole, and the colony diameter was determined on days 2, 4, and 6. Experimental catheterization rat model was conducted with pre-coating of TOH and voriconazole or an uncoated control and an infection with S. apiospermum. Biofilm formation on the catheter luminal surface was assessed using the scanning electron microscopy, crystal violet, and 2,3-bis(2-methoxy-4-ni-tro-5-sulfophenyl)-5-(phenylamino)-carbonyl-2H-tetra-zolium hydroxide (XTT) reduction assays. Brain and lung samples of catheterized rats were histopathologically assessed. Serum samples from catheterized rats were injected into Galleria mellonella larvae. Survival of catheterized rats and G. mellonella was determined.Results: TOH impeded the growth of S. apiospermum by reducing the colony diameter in a dose-dependent manner. TOH coating remarkably lessened S. apiospermum biofilm formation and fungal cell viability on the catheter luminal surface. Additionally, TOH coating lessens cerebral edema that is associated with abscess and invasive pulmonary damages due to S. apiospermum catheter-related infection. Furthermore, TOH coating also lessened the virulence of S. apiospermum in sera of experimental catheterized rats and extended the survival rate of larvae Galleria mellonella infection model.Conclusion: An alternative modification of catheter by coating with TOH is effective in preventing S. apiospermum colonization in vivo. Our study gives a new strategy to control catheter contamination and prevents nosocomial diseases due to S. apiospermum infection.Keywords: Scedosporium apiospermum, central venous catheter, quorum-sensing molecules, tryptophol, fungal biofilms, antifungal susceptibility, scedosporiosis, Galleria mellonella

Lichen substances have been first described in the 1870s, and around 10 000 compounds have been isolated and characterized. Most of them have been evaluated for their activity on planktonic microorganisms (bacteria and fungi). More recently, microorganisms colonizing the lichen thallus have been isolated and identified using DNA sequencing, giving access to a wide diversity of culturable microorganisms. The increasing research in lichen-associated microbiomes in recent years has emphasized a wide range of metabolites as a potential source of bioactive compounds. In parallel, humans are facing microbial resistance to conventional antimicrobial drugs. One of the reasons is the biofilm lifestyle of microorganisms. Indeed, the aggregation of microbial communities inside biofilms is now well known and characterized, and some possible ways to fight and destroy biofilms are identified (quorum sensing inhibitors, etc.). The present review aims to summarize the anti-biofilm potential of lichen metabolites and those from their associated microorganisms (bacteria and/or fungi). Are the metabolites isolated from lichens and their associated fungi displaying any anti-biofilm activity? This literature synthesis highlights the metabolites of interest as new anti-biofilm drugs and shows the lack of current biological research dealing with biofilm and lichen metabolites. Acetone and ethyl acetate extracts are the most studied sources of anti-biofilm agents. Only two lichen metabolites, usnic acid and evernic acid, have been evaluated both as antifungal and antibacterial biofilm compounds. Terpenoids from lichens are still poorly explored for this activity., (© 2024 The Author(s). Chemistry & Biodiversity published by Wiley‐VHCA AG.)

Dermatophytosis is a prevalent fungal infection and public health burden, majorly caused by the attack of zoophilic fungi genera of Trichophyton and Microsporum. Among them, T. mentagrophytes and M. canis are the dominating pathogens that cause dermatophytosis in humans. Though anti-fungal treatments are available, the widespread drug resistance and minimal efficacy of conventional therapies cause recurring infections. In addition, prolonged anti-fungal medications induce several systemic side effects, including hepatotoxicity and leucopenia. The anti-dermatophytic formulation of biocompatible essential oil components (EOCs) is attractive due to their highly potent anti-dermatophytic action. Herein, two EOCs, Eugenol (EU) and Isoeugenol (IU), incorporated emulsion hydrogel (EOCs-EHG) synthesized from hydroxypropylmethyl cellulose and poly(ethylene glycol) methyl ether methacrylate. The cytocompatibility of the hydrogels is confirmed by treating them with fibroblast and keratinocyte cell lines. The EOCs-EHG demonstrated pH and temperature-responsive sustained release of entrapped EOCs and inhibited fungal spore germination. T. mentagrophytes and M. canis biofilms are eradicated at a minimal inhibitory concentration of 2 µg mL -1 each of EU and IU. The in vivo anti-dermatophytic activity of EOCs-EHG is confirmed in dermatophyte-infected Wistar albino rat models. The topical application of EOCs-EHG demonstrated complete infection eradication and facilitated skin regeneration, emphasizing the therapeutic potential of EOCs-EHG against dermatophytosis., (© 2024 Wiley‐VCH GmbH.)

Approximately four million people contract fungal infections every year in Brazil, primarily caused by Aspergillus spp. The ability of these fungi to form biofilms in tissues and medical devices complicates treatment and contributes to high rates of morbidity and mortality in immunocompromised patients. Psd2 is a pea defensin of 5.4 kDa that possesses good antifungal activity against planktonic cells of representative pathogenic fungi. Its function depends on interactions with membrane and cell wall lipid components such as glucosylceramide and ergosterol. In the present study, we characterized Aspergillus nidulans biofilm formation and determined the effect of Psd2 on A. nidulans biofilms. After 4 hours, A. nidulans conidia adhered to polystyrene surfaces and formed a robust extracellular matrix-producing biofilm at 24 h, increasing thickness until 48 h Psd2 inhibited A. nidulans biofilm formation in a dose-dependent manner. Most notably, at 10 μM Psd2 inhibited 50% of biofilm viability and biomass and 40% of extracellular matrix production. Psd2 significantly decreased the colonized surface area by the biofilm and changed its level of organization, causing a shortening of length and diameter of hyphae and inhibition of conidiophore formation. This activity against A. nidulans biofilm suggests a potential use of Psd2 as a prototype to design new antifungal agents to prevent biofilm formation by A. nidulans and related species.

Antimicrobial resistance is a major public health concern worldwide. Albeit to a lesser extent than bacteria, fungi are also becoming increasingly resistant to antifungal drugs. Moreover, due to the small number of antifungal classes, therapy options are limited, complicating the clinical management of mycoses. In this view, antimicrobial peptides (AMPs) are a potential alternative to conventional drugs. Among these, Proline-rich antimicrobial peptides (PrAMPs), almost exclusively of animal origins, are of particular interest due to their peculiar mode of action. In this study, a search for new arginine- and proline-rich peptides from plants has been carried out with a bioinformatic approach by sequence alignment and antimicrobial prediction tools. Two peptide candidates were tested against planktonic cells and biofilms of Candida albicans and Candida glabrata strains, including resistant isolates. These peptides showed similar potent activity, with half-maximal effective concentration values in the micromolar range. In addition, some structural and functional features, revealing peculiar mechanistic behaviors, were investigated.

Fungal biofilms, such as Candida albicans biofilms, are capable of surviving in hostile environments owing to their remarkable ability to adhere to surfaces and their tolerance to chemical interventions. Currently, therapeutic treatment options are few, making these biofilm-based infections problematic particularly due to their great tolerance to conventional antimicrobial drugs, thus causing serious health and economic problems. Therefore, the development of new drugs and antibiofilm specific therapies for the prevention and treatment of antifungal to eradicate biofilms are needed. This study was aimed at carrying out a patent review analysis to identify the innovation trends, and to explore the latest antifungal drugs and the specific therapeutic strategies available for the treatment of fungal biofilms. The present patent review was carried out using the Espacenet database, using the key words "biofilm and antifungal," from 2002 to December 2019. Through this review, it was possible to identify that most of the patent contents refer to new synthetic drugs derived from natural products and associations thereof with existing antifungal drugs. Methods and biomaterials for the treatment and prevention of fungal biofilms, mainly for C. albicans biofilms, which is the most isolated and studied fungal species, were also disclosed. The lack of scientific and technical information on the biofilm eradication subject is remarkable and further confirmed by the small number of patents identified in this survey. [ABSTRACT FROM AUTHOR]

A strong enhancement in the antifungal activity of amphotericin B (AmpB) encapsulated into shellac nanoparticles (NPs) surface functionalized with protease is reported. These AmpB‐loaded shellac NPs are fabricated by pH‐induced nucleation of aqueous solutions of shellac and AmpB in the presence of Poloxamer 407 (P407) as a steric stabilizer. The AmpB‐loaded shellac NPs are surface coated with the cationic protease Alcalase 2.4L FG. The performance of the AmpB‐encapsulated NPs against Candida albicans is evaluated. The AmpB‐loaded shellac NPs show a remarkable boost of their antifungal action compared to free AmpB when applied to C. albicans in both planktonic and biofilm forms. The surface functionalization with a cationic protease allows the NPs to adhere to the fungal cell walls, delivering AmpB directly to their membranes. Additionally, the hydrolyzing activity of the protease coating degrades the biofilm matrix, thus increasing the effectiveness of the encapsulated AmpB compared to free AmpB at the same concentration. The protease‐coated AmpB‐loaded shellac NPs show no greater toxicity to human adult keratinocyte cells (HaCaT) compared to the free AmpB. These AmpB nanocarriers demonstrat increased efficacy against C. albicans and can be potentially used to treat fungal biofilm infection in the clinic, for example, in recalcitrant chronic wounds.

Candida tropicalis is one the most relevant biofilm-forming fungal species increasingly associated with invasive mucosal candidiasis worldwide. The amplified antifungal resistance supports the necessity for more effective and less toxic treatment, including the use of plant-derived natural products. Scopoletin, a natural coumarin, has shown antifungal properties against plant yeast pathogens. However, the antifungal activity of this coumarin against clinically relevant fungal species such as C. tropicalis remains to be established. Here, we investigated the potential antifungal properties and mechanisms of action of scopoletin against a multidrug-resistant C. tropicalis strain (ATCC 28707). First, scopoletin was isolated by high-performance liquid chromatography from Mitracarpus frigidus, a plant species (family Rubiaceae) distributed throughout South America. Next, scopoletin was tested on C. tropicalis cultivated for 48h in both planktonic and biofilm forms. Fungal planktonic growth inhibition was analyzed by evaluating minimal inhibitory concentration (MIC), time-kill kinetics and cell density whereas the mechanisms of action were investigated with nucleotide leakage, efflux pumps and sorbitol and ergosterol bioassays. Finally, the scopoletin ability to affect C. tropicalis biofilms was evaluated through spectrophotometric and whole slide imaging approaches. In all procedures, fluconazole was used as a positive control. MIC values for scopoletin and fluconazole were 50 and 250 μg/L respectively, thus demonstrating a fungistatic activity for scopoletin. Scopoletin induced a significant decrease of C. tropicalis growth curves and cell density (91.7% reduction) compared to the growth control. Its action was related to the fungal cell wall, affecting plasma membrane sterols. When associated with fluconazole, scopoletin led to inhibition of efflux pumps at the plasma membrane. Moreover, scopoletin not only inhibited the growth rate of preformed biofilms (68.2% inhibition at MIC value) but also significantly decreased the extent of biofilms growing on the surface of coverslips, preventing the formation of elongated fungal forms. Our data demonstrate, for the first time, that scopoletin act as an effective antifungal phytocompound against a multidrug-resistant strain of C. tropicalis with properties that affect both planktonic and biofilm forms of this pathogen. Thus, the present findings support additional studies for antifungal drug development based on plant isolated-scopoletin to treat candidiasis caused by C. tropicalis.

Fungal biofilms cause a major clinical problem with a shrinking armamentarium for treatment. Here, the design and synthesis of voriconazole‐inbuilt zinc 2‐methylimidazolates frameworks (V‐ZIF) is reported. Voriconazole is built in through coordination‐binding between zinc and voriconazole. These metal–organic‐frameworks with inbuilt voriconazole, reduce inadvertent voriconazole‐leakage, yield a zero‐order release kinetics of voriconazole, aid antifungal penetration in Candida albicans biofilms, and prevent Candida aggregation yielding better dispersal. Once accumulated in an acidic C. albicans biofilm, voriconazole dissociates from the metal–organic framework to cause membrane‐damage and killing of inhabiting fungi. Moreover, in a murine model, the V‐ZIFs eradicate open‐wound infections caused by C. albicans better than voriconazole in solution, with negligible side effects to the healthy tissues of major organs. Thus, V‐ZIFs may provide a welcome addition to the antifungal armamentarium currently available for the treatment of fungal biofilms. [ABSTRACT FROM AUTHOR]

Infections caused by invasive fungal biofilms have been widely associated with high morbidity and mortality rates, mainly due to the advent of antibiotic resistance. Moreover, fungal biofilms impose an additional challenge, leading to multidrug resistance. This fact, along with the contamination of medical devices and the limited number of effective antifungal agents available on the market, demonstrates the importance of finding novel drug candidates targeting pathogenic fungal cells and biofilms. In this context, an alternative strategy is the use of antifungal peptides (AFPs) against fungal biofilms. AFPs are considered a group of bioactive molecules with broad-spectrum activities and multiple mechanisms of action that have been widely used as template molecules for drug design strategies aiming at greater specificity and biological efficacy. Among the AFP classes most studied in the context of fungal biofilms, defensins, cathelicidins and histatins have been described. AFPs can also act by preventing the formation of fungal biofilms and eradicating preformed biofilms through mechanisms associated with cell wall perturbation, inhibition of planktonic fungal cells’ adhesion onto surfaces, gene regulation and generation of reactive oxygen species (ROS). Thus, considering the critical scenario imposed by fungal biofilms and associated infections and the application of AFPs as a possible treatment, this review will focus on the most effective AFPs described to date, with a core focus on antibiofilm peptides, as well as their efficacy in vivo, application on surfaces and proposed mechanisms of action.

Infections caused by invasive fungal biofilms have been widely associated with high morbidity and mortality rates, mainly due to the advent of antibiotic resistance. Moreover, fungal biofilms impose an additional challenge, leading to multidrug resistance. This fact, along with the contamination of medical devices and the limited number of effective antifungal agents available on the market, demonstrates the importance of finding novel drug candidates targeting pathogenic fungal cells and biofilms. In this context, an alternative strategy is the use of antifungal peptides (AFPs) against fungal biofilms. AFPs are considered a group of bioactive molecules with broad-spectrum activities and multiple mechanisms of action that have been widely used as template molecules for drug design strategies aiming at greater specificity and biological efficacy. Among the AFP classes most studied in the context of fungal biofilms, defensins, cathelicidins and histatins have been described. AFPs can also act by preventing the formation of fungal biofilms and eradicating preformed biofilms through mechanisms associated with cell wall perturbation, inhibition of planktonic fungal cells' adhesion onto surfaces, gene regulation and generation of reactive oxygen species (ROS). Thus, considering the critical scenario imposed by fungal biofilms and associated infections and the application of AFPs as a possible treatment, this review will focus on the most effective AFPs described to date, with a core focus on antibiofilm peptides, as well as their efficacy in vivo , application on surfaces and proposed mechanisms of action. [ABSTRACT FROM AUTHOR]

Pseudallescheria/Scedosporium species are medically important fungi that are present in soil and human impacted areas and capable of causing a wide spectrum of diseases in humans. Although little is known about their pathogenesis, their growth process and infection routes are very similar to those of Aspergillus species, which grow as biofilms in invasive infections. All nine strains tested here displayed the ability to grow as biofilms in vitro and to produce a dense network of interconnected hyphae on both polystyrene and the surfaces of central venous catheters, but with different characteristics. Scedosporium boydii and S. aurantiacum clinical isolates were able to form biofilms faster than the corresponding environmental strains, as evidenced in kinetic assays for S. boydii and CLSM for S. aurantiacum. Biofilms formed by Pseudallescheria/Scedosporium species had significantly higher resistance to the class of antifungal azole than was observed in planktonic cells, indicating a protective role for this structure. In addition, the clinical S. aurantiacum isolate that formed the most robust biofilms was also more virulent in a larvae Galleria mellonella infection model, suggesting that the ability to form biofilms enhances virulence in Pseudallescheria/Scedosporium species.

The constraints and advantages in cellulolytic enzymes production by fungal biofilms for a consolidated bioconversion process were investigated during this study. The biofilm cultivations were carried out in reactors designed for consolidated bioprocessing Multispecies Biofilm Membrane reactors, (MBM) where an aerobic fungal biofilm produces the lignocellulolytic enzymes while a fermenting microorganism forms the fermentation product at anaerobic conditions. It was shown that although mycelial growth was limited in the MBM reactors compared to submerged cultivations, the secretion of cellulolytic enzymes per cell dry weight was higher. When Trichoderma reesei was used as the sole enzyme producer, cellobiose accumulated in the liquid medium as the result of the deficiency of β-glucosidase in the fungal secretome. To enhance β-glucosidase activity, T. reesei was co-cultivated with A. phoenicis which is a β-glucosidase overproducer. The two fungi formed a multispecies biofilm which produced a balanced cellulolytic cocktail for the saccharification of plant biomass. The mixed biofilm reached a 2.5 fold increase in β-glucosidase production, compared to the single T. reesei biofilm. The enzymatic systems of single and mixed biofilms were evaluated regarding their efficiency on cellulosic substrates degradation. Washed solids from steam pretreated beechwood, as well as microcrystalline cellulose were used as the substrates. The enzymatic system of the multispecies biofilm released four times more glucose than the enzymatic system of T. reesei alone from both substrates and hydrolyzed 78 and 60% of the cellulose content of washed solids from beechwood and microcrystalline cellulose, respectively. [ABSTRACT FROM AUTHOR]

Pseudallescheria/Scedosporium species are medically important fungi that are present in soil and human impacted areas and capable of causing a wide spectrum of diseases in humans. Although little is known about their pathogenesis, their growth process and infection routes are very similar to those of Aspergillus species, which grow as biofilms in invasive infections. All nine strains tested here displayed the ability to grow as biofilms in vitro and to produce a dense network of interconnected hyphae on both polystyrene and the surfaces of central venous catheters, but with different characteristics. Scedosporium boydii and S. aurantiacum clinical isolates were able to form biofilms faster than the corresponding environmental strains, as evidenced in kinetic assays for S. boydii and CLSM for S. aurantiacum. Biofilms formed by Pseudallescheria/Scedosporium species had significantly higher resistance to the class of antifungal azole than was observed in planktonic cells, indicating a protective role for this structure. In addition, the clinical S. aurantiacum isolate that formed the most robust biofilms was also more virulent in a larvae Galleria mellonella infection model, suggesting that the ability to form biofilms enhances virulence in Pseudallescheria/Scedosporium species. [ABSTRACT FROM AUTHOR]

White-rot fungi are efficient degraders of lignin whose extracellular enzymes have a potential to degrade organopollutants. In natural conditions these fungi enter into interactions with other organisms, which may affect their biodegradation capacity. The aim was to investigate the ability of Pleurotus ostreatus to form stable biofilms and to test the capacity of the fungus to degrade Remazol Brilliant Blue R in mixed cultures with bacteria. Bacterial counts were determined to see the behavior of the bacterium in the mixed culture with the fungus. In axenic conditions, the homogenized fungal mycelium was able to form an active biofilm which quickly degraded the dye. The addition of Pseudomonas fluorescens or Bacillus licheniformis bacteria at 10 6 CFU·mL − 1 did not affect the decolorization rate by 7-d-old fungal biofilms where the decolorization rate reached 90%. In contrast, when fragments of the fungal mycelium were used for inoculation to pre-formed biofilm of P . fluorescens , the biofilm was allowed to develop for one week's time, no decolorization of RBBR was observed and low activities of MnP and laccase were detected. The use of agar disks covered with fungal mycelium for the inoculation to pre-formed biofilm of P . fluorescens resulted in a fully developed biofilm that decolorized RBBR with similar efficiency as the pure P . ostreatus . The difference between the agar-disk- and homogenized-mycelium inoculated fungal biofilms was corroborated by the measurement of total fungal biofilm biomass that was 6-fold lower in the latter biofilm. Capability of the fungus to overcome the competition of the bacterial biofilm thus depended on the type of fungal growth centres, where intact hyphae were superior to the fragments of mycelium. A similar effect was not observed with the biofilms of B . licheniformis where the bacterial growth was less massive. The ability of P . ostreatus biofilms to resist massive bacterial stress was demonstrated. [ABSTRACT FROM AUTHOR]

Denture liners have physical properties that favour plaque accumulation and colonization by Candida species, irritating oral tissues and causing denture stomatitis. To isolate and determine the incidence of oral Candida species in dental prostheses, oral swabs were collected from the dental prostheses of 66 patients. All the strains were screened for their ability to form biofilms; both monospecies and dual-species combinations were tested. Candida albicans (63 %) was the most frequently isolated microorganism; Candida tropicalis (14 %), Candida glabrata (13 %), Candida rugosa (5 %), Candida parapsilosis (3 %), and Candida krusei (2 %) were also detected. The XTT assay showed that C. albicans SC5314 possessed a biofilm-forming ability significantly higher ( p < 0.001) than non- albicans Candida strains, after 6 h 37 °C. The total C. albicans CFU from a dual-species biofilm was less than the total CFU of a monospecies C. albicans biofilm. In contrast to the profuse hyphae verified in monospecies C. albicans biofilms, micrographies showed that the C. albicans /non- albicans Candida biofilms consisted of sparse yeast forms and profuse budding yeast cells that generated a network. These results suggested that C. albicans and the tested Candida species could co-exist in biofilms displaying apparent antagonism. The study provide the first description of C. albicans/C. rugosa mixed biofilm. [ABSTRACT FROM AUTHOR]

Biological removal of nitrogen and phosphorous from wastewater conventionally involves multiple processing steps to satisfy the differing oxygen requirements of the microbial species involved. In this work, simultaneous nitrification, denitrification, and phosphorous removal from synthetic wastewater were achieved by the fungus Neurospora discreta in a single-step, biofilm-based, aerobic process. The concentrations of carbon, nitrogen, and phosphorous in the synthetic wastewater were systematically varied to investigate their effects on nutrient removal rates and biofilm properties. Biofilm growth was significantly (p < 0.05) affected by carbon and nitrogen, but not by phosphorous concentration. Scanning electron microscopy revealed the effects of nutrients on biofilm microstructure, which in turn correlated with nutrient removal efficiencies. The carbohydrate and protein content in the biofilm matrix decreased with increasing carbon and nitrogen concentrations but increased with increasing phosphorous concentration in the wastewater. High removal efficiencies of carbon (96%), ammonium (86%), nitrate (100%), and phosphorus (82%) were achieved under varying nutrient conditions. Interestingly, decreasing the phosphorus concentration increased the nitrification and denitrification rates, and decreasing the nitrogen concentration increased the phosphorus removal rates significantly (p < 0.05). Correlations between biofilm properties and nutrient removal rates were also evaluated in this study. [Display omitted] • Single-step removal of nitrogen, phosphorous, and carbon using Neurospora discreta biofilms. • Nutrient concentrations affected biofilm properties and nutrient removal rates. • Higher nitrification/denitrification rates at lower phosphorus concentrations. • Higher phosphorus uptake rates at lower nitrogen concentrations. [ABSTRACT FROM AUTHOR]

Opportunistic infections caused by fungi can be a cause of death in immunocompromised individuals and nosocomial infections related to catheters and medical devices. In relation to the latter infection has been shown that fungi have a great ability to form biofilms on these materials, which favors its dissemination into the body. In addition, this type of organization facilitates the emergence of resistance to antifungal agents. For this reason, it is necessary to know the origin, development and control of biofilms, and the mechanisms of resistance and treatment options targeting this form of microbial organization. [ABSTRACT FROM AUTHOR]

Roots of the susceptible “JG-62” and resistant “WR-315” chickpeas (Cicer arietinum L.) were inoculated with a conidial suspension of Fusarium oxysporum f. sp. ciceris. Anatomical and biochemical studies were carried out in a time-course manner to elucidate the infection process and plant defence reactions. Scanning electron microscope images revealed fungal colonisation in the root hair region. Early occurrence of fungal biofilms associated with the infected “JG-62” root epidermis was also visualised. After 96 h of inoculation, a gradual accumulation of polysaccharide positive deposits was observed in the xylem vessels of the infected “JG-62” roots. Fungal mycelium was observed in the vessel lumen of infected “JG-62” after 22 days of inoculation. Due to fungal invasion during this period, some of the vessels also appeared collapsed in “JG-62”, whereas vessels in “WR-315” remained intact. The host plant defence responses specifically linked to the susceptible interactions were the induction of ascorbate peroxidase, guaiacol peroxidase and superoxide dismutase in roots and shoots. [ABSTRACT FROM PUBLISHER]

Background: Bacterial biofilms have been implicated in chronic rhinosinusitis (CRS). However, direct evidence in support of fungal biofilms in sinus disease is lacking in the literature. This study was designed to develop and characterize an in vitro Aspergillus fumigatus biofilm model on primary human sinonasal epithelial cell culture. Methods: Sinonasal biopsy specimens harvested during endoscopic sinus surgery of six CRS patients and three pituitary tumor (control) patients were cultured in Dulbecco's modified Eagle media (DMEM; Invitrogen)/Hams F12 airway media to encourage epithelial cell proliferation. Epithelial cells separated by immunomagnetic beads were seeded in tissue culture-treated Y-shaped microslides. At confluence the primary cultures were inoculated with A. fumigatus spores. Fungus was allowed to germinate and form biofilms under two in vitro conditions: (1) static (no flow through of media) and (2) continuous flow coculture (continuous flow movement of media). At regular intervals cocultures were stained with FUN-1, concanavalin A-alexa fluor 488, and examined by confocal scanning laser microscopy. Comstat software was used to assess biomass and thickness. Results: A. fumigatus formed three-dimensional biofilm structures with parallel-packed, cross-linked hyphae and channels/passages. Metabolically active hyphae showed orange-red fluorescing intravacuolar structures. Extracellular matrix (ECM) between/around the hyphae fluoresced intense green. A. fumigatus biofilms development occurred in five stages: (1) conidial attachment to epithelial cells, (2) hyphal proliferation, (3) ECM production, (4) hyphal parallel packing and cross-linking, and (5) channel/pores formation. Mature biofilms showed basal conidial, middle hyphal, and superficial ECM layers. Biofilms formed under flow conditions displayed more robust and faster growth kinetics when compared with that under static conditions, with a thick, stocky, wrinkly/undulating hyphal growth and extensive ECM production. The differences in biomass and average thickness of the cocultures under static and flow conditions were statistically significant after similar periods of incubation (p = 0.0002; p < 0.0001, respectively). Conclusion: To our knowledge, this is the first article of an in vitro model characterizing A. fumigatus biofilm formation using primary human sinonasal epithelium under different growth conditions. [ABSTRACT FROM AUTHOR]

Abstract: Biomass organization of Aspergillus niger biofilms and pellets stained with fluorescein isothiocyanate were analyzed by means of confocal laser scanning microscopy and detectable differences between both types of growth were found. Three-dimensional surface plot analysis of biofilm structure revealed interstitial voids and vertical growth compared with pellets. Growth was lower in biofilm and according to fluorescence profile obtained, biomass density increased at the surface (0–20μm). However, a decrease in fluorescence intensity was observed through optical sections of pellets even though growth was significantly higher than biofilms. Cryo scanning electron microscopy also showed structural differences. While biofilms showed a spatially ordered mycelium and well structured hyphal channels, pellets were characterized by an entangled and notoriously compacted mycelium. These findings revealed common structural characteristics between A. niger biofilms and those found in other microbial biofilms. Thus, biofilm microstructure may represent a key determinant of biofilm growth and physiology of filamentous fungi. [Copyright &y& Elsevier]

Persistent fungal infections caused by biofilms seriously endanger human health. In this study, a photosensitizer-polypeptide conjugate (PPa-cP) comprising a photosensitizer, pyropheophorbide a (PPa), and a cationic polypeptide (cP) is readily synthesized for effective antifungal and antibiofilm treatment. Compared with free PPa, the cationic PPa-cP shows enhanced binding ability to the negatively charged surface of Candida albicans (C. albicans) through electrostatic interactions. As a result, PPa-cP exhibits effective antifungal efficiency against both C. albicans and fluconazole-resistant C. albicans in vitro under light irradiation. The minimum inhibitory concentration (MIC) of PPa-cP for both C. albicans and fluconazole-resistant C. albicans is 1 µm. In addition, PPa-cP also shows improved penetration in a C. albicans biofilm, thus effectively eliminating the C. albicans biofilm by photodynamic effects. More importantly, PPa-cP demonstrats significantly enhanced therapeutic effects in a fluconazole-resistant C. albicans-infected rat model with minimal side effects. In conclusion, the current work presents an effective strategy to combat biofilm infections associated with biomedical equipment., (© 2022 Wiley-VCH GmbH.)

Infections caused by fungal species via their existence as biofilms on medical devices can cause organ damage via candidiasis and candidemia. Different Candida species like Candida albicans can pose a serious threat by resisting host's immune system and by developing drug resistance against existing antimycotic agents. Therefore, targeting of fungal membranes can be used as an alternative strategy to combat the fungal infections. Here, we present screening of different amphiphiles based on cholic acid against different Candida strains as these amphiphiles can act as potent membrane-targeting antimycotic agents. Structure-activity correlations, biochemical assays and electron microscopy studies showed that amphiphiles having 4 and 6 carbon chains are most potent, safe and can act on the fungal membranes. Candida albicans did not show emergence of drug resistance on repeated usage of these amphiphiles unlike fluconazole. We show that these amphiphiles can prevent the formation of biofilms and also have the ability to degrade preformed biofilms on different substrates including acrylic teeth. We further demonstrate that amphiphiles 4 and 6 can clear the Candida albicans wound infections and prevent the biofilm formation on indwelling devices in murine models. Therefore, amphiphiles derived from cholic acid and their coatings provide suitable alternatives for inhibiting the fungal infections.