Biofilm formation is an important virulence factor for pathogenic fungi. Both yeasts and filamentous fungi can adhere to biotic and abiotic surfaces, developing into highly organized communities that are resistant to antimicrobials and environmental conditions. In recent years, new genera of fungi have been correlated with biofilm formation. However, Candida biofilms remain the most widely studied from the morphological and molecular perspectives. Biofilms formed by yeast and filamentous fungi present differences, and studies of polymicrobial communities have become increasingly important. A key feature of resistance is the extracellular matrix, which covers and protects biofilm cells from the surrounding environment. Furthermore, to achieve cell–cell communication, microorganisms secrete quorum-sensing molecules that control their biological activities and behaviors and play a role in fungal resistance and pathogenicity. Several in vitro techniques have been developed to study fungal biofilms, from colorimetric methods to omics approaches that aim to identify new therapeutic strategies by developing new compounds to combat these microbial communities as well as new diagnostic tools to identify these complex formations in vivo. In this review, recent advances related to pathogenic fungal biofilms are addressed.

Polymicrobial biofilms adhere to surfaces and enhance pathogen resistance to conventional treatments, significantly contributing to chronic infections in the respiratory tract, oral cavity, chronic wounds, and on medical devices. This review examines antimicrobial peptides (AMPs) as a promising alternative to traditional antibiotics for treating biofilm‐associated infections. AMPs, which can be produced as part of the innate immune response or synthesized therapeutically, have broad‐spectrum antimicrobial activity, often disrupting microbial cell membranes and causing cell death. Many specifically target negatively charged bacterial membranes, unlike host cell membranes. Research shows AMPs effectively inhibit and disrupt polymicrobial biofilms and can enhance conventional antibiotics' efficacy. Preclinical and clinical research is advancing, with animal studies and clinical trials showing promise against multidrug‐resistant bacteria and fungi. Numerous patents indicate increasing interest in AMPs. However, challenges such as peptide stability, potential cytotoxicity, and high production costs must be addressed. Ongoing research focuses on optimizing AMP structures, enhancing stability, and developing cost‐effective production methods. In summary, AMPs offer a novel approach to combating biofilm‐associated infections, with their unique mechanisms and synergistic potential with existing antibiotics positioning them as promising candidates for future treatments.

Aim: This study aimed to evaluate the activity of 2′-hydroxychalcone-loaded in nanoemulsion (NLS + 2′chalc), the cytotoxic effect and toxicity against Paracoccidioides brasiliensis and Paracoccidio... Graphical abstract

Dodecyl protocatechuate (dodecyl) is a derivative of protocatechuic acid (3,4-dihydroxybenzoic acid) that possesses anti-oxidant and antifungal properties. Nanostructured lipid systems (NLS) can potentiate the action of many antifungal agents, reducing the required dose and side effects by improving their activity. This work aimed to evaluate dodecyl protocatechuate loaded into a nanostructured lipid system (NLS+dodecyl) as a strategy for the treatment of Paracoccidioides brasiliensis and Paracoccidioides lutzii in vitro. Antifungal activity against P. brasiliensis and P. lutzii was evaluated using the microdilution technique. NLS+dodecyl showed high antifungal activity with a minimum inhibitory concentration (MIC) ranging from 0.06 to 0.03 μg/mL; 4 to 16-fold higher than that of free dodecyl. NLS+dodecyl was able to inhibit fungal adhesion of the extracellular artificial matrix proteins (laminin and fibronectin), resulting in 82.4% and 81% inhibition, respectively, an increase of 8% to 17% compared with free dodecyl. These findings corroborate previous results demonstrating 65% and 74% inhibition of fungal adhesion in pulmonary fibroblast cells by dodecyl and NLS+dodecyl, respectively, representing a 9% increase in inhibition for NLS+dodecyl. Subsequently, cytotoxicity was evaluated using the 0.4% sulforhodamine B assay. NLS+dodecyl did not exhibit cytotoxicity in MRC5 (human pneumocyte) and HepG2 (human hepatic carcinoma) cells, thus increasing the selectivity index (SI) for NLS+dodecyl. In addition, cytotoxicity was evaluated in vivo using the Caenorhabditis elegans model; neither dodecyl nor NLS+dodecyl exhibited any cytotoxic effects. Taken together, these results suggest that NLS can be used as a strategy to improve the activity of dodecyl against P. brasiliensis and P. lutzii because it improves antifungal activity, increases the inhibition of fungal adhesion in lung cells and the extracellular matrix in vitro, and does not exhibit any toxicity both in vitro and in vivo.

Co-infection of Mycobacterium tuberculosis and Paracoccidioides brasiliensis, present in 20% in Latin America, is a public health problem due to a lack of adequate diagnosis. These microorganisms are capable of forming biofilms, mainly in immunocompromised patients, which can lead to death due to the lack of effective treatment for both diseases. The present research aims to show for the first time the formation of mixed biofilms of M. tuberculosis and P. brasiliensis (Pb18) in vitro, as well as to evaluate the action of 3’hydroxychalcone (3’chalc) -loaded nanoemulsion (NE) (NE3’chalc) against monospecies and mixed biofilms, the formation of mixed biofilms of M. tuberculosis H37Rv (ATCC 27294), 40Rv (clinical strains) and P. brasiliensis (Pb18) (ATCC 32069), and the first condition of formation (H37Rv +Pb18) and (40Rv + Pb18) and second condition of formation (Pb18 + H37Rv) with 45 days of total formation time under both conditions. The results of mixed biofilms (H37Rv + Pb18) and (40Rv + Pb18), showed an organized network of M. tuberculosis bacilli in which P. brasiliensis yeasts are connected with a highly extracellular polysaccharide matrix. The (Pb18 + H37Rv) showed a dense biofilm with an apparent predominance of P. brasiliensis and fragments of M. tuberculosis. PCR assays confirmed the presence of the microorganisms involved in this formation. The characterization of NE and NE3’chalc displayed sizes from 145.00 ± 1.05 and 151.25 ± 0.60, a polydispersity index (PDI) from 0.20± 0.01 to 0.16± 0.01, and zeta potential -58.20 ± 0.92 mV and -56.10 ± 0.71 mV, respectively. The atomic force microscopy (AFM) results showed lamellar structures characteristic of NE. The minimum inhibitory concentration (MIC) values of 3’hidroxychalcone (3’chalc) range from 0.97- 7.8 µg/mL and NE3’chalc from 0.24 - 3.9 µg/mL improved the antibacterial activity when compared with 3’chalc-free, no cytotoxicity. Antibiofilm assays proved the efficacy of 3’chalc-free incorporation in NE. These findings contribute to a greater understanding of the formation of M. tuberculosis and P. brasiliensis in the mixed biofilm. In addition, the findings present a new possible NE3’chalc treatment alternative for the mixed biofilms of these microorganisms, with a high degree of relevance due to the lack of other treatments for these comorbidities.