Invasive fungal infections are a growing global health threat, driven by the limited availability of antifungal therapies and the rapid spread of multidrug-resistant yeasts. This study explored the effects of N-terminal lipidation on Trem-4, a decapeptide derived from the antimicrobial peptide Trematocine-HSK, to enhance its antifungal potential. Trem-4 was modified with caprylic (C8) and myristic (C14) fatty acid chains, generating two lipopeptides: Cap-T4 and Myr-T4. Biophysical analyses, including fluorescence-based assays and circular dichroism, showed that lipidation significantly improved peptide–membrane interactions compared to the non-lipidated form. The two derivatives displayed distinct behaviors, with differences in aggregation and membrane selectivity depending on acyl chain length. Antifungal activity was assessed against 60 clinically characterized yeast isolates, including Candida spp., Candidozyma haemuli complex, and Cryptococcus neoformans, under both planktonic and biofilm conditions. Both lipopeptides exhibited broad-spectrum activity, against clinically relevant species, with conserved activity against antifungal-resistant strains. Importantly, both compounds remained effective against mature biofilms, causing metabolic disruption and structural damage. Cap-T4 demonstrated consistent activity across species, while Myr-T4 showed enhanced potency against selected isolates, particularly C. auris. Biocompatibility evaluation revealed a concentration-dependent cytotoxicity and hemolysis, more pronounced for Myr-T4. However, both compounds were well tolerated in vivo in the Galleria mellonella model. Overall, Cap-T4 emerged as the most promising candidate for further preclinical development. These findings support N-terminal lipidation as an effective strategy to improve the antifungal efficacy of short antimicrobial peptides and highlight its potential for developing new treatments against drug-resistant fungal pathogens.

Essential oils (EOs) are widely investigated for antimicrobial activity, yet minimum inhibitory concentrations (MICs) lack cross-study comparability due to the absence of a harmonized reference assay. Here, we present a protocol to determine MICs of EOs against aerobic bacterial pathogens using a broth microdilution technique. We describe steps for incorporating EO dispersion using Tween 80 and preparing a standardized bacterial inoculum. We detail procedures for establishing two-fold dilution series in 96-well plates and visually determining the MIC endpoint.

Oral coinfections involving herpes simplex virus (HSV) and Candida albicans can potentially interact and exacerbate each other. Starting from bibliographical investigation, this study aimed to examine the effectiveness of some essential oils (EOs), and their commercial formulations, both against C. albicans and HSV-1, identifying their antimicrobial, anti-inflammatory and allergenic potential. A preliminary review examined essential oils' efficacy against HSV-1. Broth microdilution tested 14 EOs, a commercial formulation (LA), and a homemade one (MIX) against three fungal strains. The quality of LA, MIX and single EOs was assessed by Solid Phase Microextraction (SPME) sampling coupled with gas chromatography-mass spectrometry (GC-MS) analysis. To assess the allergenic activity of MIX, LA, and single EOs a Basophil Activation Test (BAT) was standardized. ELISA tests were done to evaluate the anti-inflammatory activity. The bibliographic search highlighted seven EOs active against HSV-1. Four EOs showing strong antifungal activity were blended, following IFRA lip-application limits, to create a formulation (MIX) for comparison with a commercial herpes treatment (LA). Formulations were active against HSV-1, able to modulate the expression of pro (TNF-αLA = -29.7% and TNF-αMIX = -33.6%) and anti-inflammatory (IL-1β LA = -50.0% and IL-1β MIX = -25.0%) cytokines and no allergenic. MIX reinforces target cells and blocks viral entry, while LA also limits intracellular replication. EO-based formulations show promise for managing HSV-1 and Candida co-infections, offering antiviral, antifungal, and anti-inflammatory effects. BAT results indicate no basophil activation at tested concentrations, supporting their safety.

Second-generation antimicrobial lipopeptides are considered very promising tools to combat the spread of systemic fungal infections caused by fungi multidrug resistance and biofilm-associated infections. The present work reports a study on the lipopeptide Myr-B, derived from myristoylation of the natural antimicrobial peptide Chionodracine. The aim was to exploit its therapeutic potential by developing a suitable procedure for its encapsulation in liposomes, with a view to overcoming its in vivo instability and tendency to aggregation. A systematic investigation allowed to select two optimal liposome formulations, based on dimyristoylphosphatidylcholine, having chains of the same length of the lipopetide, and cholesterol and/or cholesteryl-hemisuccinate. Thin-film hydration and lipid-cake preparation methods were explored, and chemical-physical characterization was integrated with molecular dynamics simulations. The lipid-cake method proved to be the optimal approach, yielding monodisperse, stable liposomes with high encapsulation efficiency. Encapsulation in both liposome formulations markedly enhanced antifungal activity, lowering minimum inhibitory concentrations more than ten-fold against Candida albicans and approximately four-fold against Candida tropicalis compared to free Myr-B. LIVE/DEAD imaging also confirmed a strong reduction in biofilm formation. Liposomes displayed minimal haemolysis, low cytotoxicity toward human fibroblasts, and good in vivo tolerability in Galleria mellonella. Notably, specific interactions between cholesteryl-hemisuccinate and the peptide involved a peculiar liposome structure and a slower release from the liposome. Overall, both Myr-B-loaded liposomes produced by the lipid cake protocol significantly potentiates MYR-B efficacy while maintaining a favourable safety profile. These formulations are not universally applicable to all lipopeptides; however, the underlying criteria presented in this work are.

Background/Objectives: In recent years, antimicrobial resistance has become a major threat to global health, and scientific research aiming to identify new therapeutic resources is a priority. Essential oils (EOs), obtained from spices belonging to the culinary tradition, like Carum carvi essential oil (CC-EO), are of great interest for their antimicrobial activity, but the methods used to evaluate their efficacy need to be standardized. The aims of this work were to evaluate the following: (i) the best microbiological in vitro test; (ii) the best surfactant; and (iii) the best microbiological target of CC-EO and its method of administration. Methods: CC-EO quality was evaluated using gas chromatography–mass spectrometry. Antimicrobial susceptibility testing with drugs currently in use was performed. Antimicrobial effectiveness against 70 clinical strains belonging to S. aureus, E. coli, E. faecalis, K. pneumoniae, P. aeruginosa, S. pyogenes, and C. albicans was evaluated. Two microbial tests (broth microdilution tests and disk diffusion), generally used in routine clinical practice, were compared. To choose the best vehicle, Tween80, DMSO, and ethanol were evaluated. The antimicrobial efficacy of vapors was assessed using a microatmosphere test. Results: The broth microdilution test is confirmed as the best in evaluating the antimicrobial activity of EOs. The most suitable EOs vehicle for antimicrobial testing was Tween80. CC-EO and its vapors were effective against GRAM+ and C. albicans strains, both sensible and resistant, and ineffective against GRAM−. Conclusions: In the future, it may be possible to include CC-EO in topical or spray formulations for the treatment of GRAM+ and C. albicans infections.

Drug-resistant fungal pathogens pose an increasing threat to human health. Nanoparticles are promising tools for treating and limiting fungal resistance due to their ability to attack microorganisms via multiple mechanisms. In this work, hydroxyapatite (HA) nanoparticles were synthesized and functionalized with copper by ionic exchange at different solution concentrations (from 0.01 to 0.2 M). The physico-chemical properties of the samples were studied using low-temperature N2 adsorption volumetry, elemental analysis, X-ray diffraction, electron microscopy and IR spectroscopy of adsorbed CO. All the obtained HA particles were poorly crystalline, elongated in the c-axis direction, and had a high specific surface area (ca. 200 m2/g). Copper was incorporated into HA surface layers with a Cu2+ doping content proportional to the initial concentration, reaching a maximum value of 14 %wt. The antifungal activity of the samples was tested against drug-resistant clinical isolates of Cryptococcus neoformans and several Candida species strains (C. parapsilosis, C. krusei, C. tropicalis, C. albicans, C. glabrata, C. auris). Minimal inhibitory concentrations and fungal growth curves were determined. Cytocompatibility evaluation showed that both undoped and Cu-doped HA samples were not toxic to mammalian cells. The Cu- containing HA samples demonstrated potential as effective and safe antifungal agents with wide-spectrum activity, representing a promising candidate for therapeutic approaches against diverse fungal infections.

Infectious diseases caused by pathogenic bacteria, fungi, and viruses pose a global health threat, aggravated by antimicrobial resistance (AMR). This study explores the antimicrobial efficacy of two nanostructured composite coatings -aluminum-doped zinc oxide with silver (AZO-Ag) and titanium dioxide with silver (TiO2-Ag)- on polyester substrates, against high-priority pathogens: susceptible and resistant Klebsiella pneumoniae and Staphylococcus aureus bacterial strains, Candida albicans clinical fungal strain, and H1N1 influenza virus. A multimodal analytical approach, encompassing SEM, live/dead fluorescence assays, CFU counts, RT-qPCR, and immunofluorescence, supported by ROS quantification, is adopted to evaluate antimicrobial activity and contribution of indirect and direct-contact mechanisms. Both coatings exhibit robust antimicrobial effects especially through indirect mechanisms, with TiO2-Ag generally more effective than AZO-Ag through indirect interactions while AZO-Ag more effective in some direct-contact scenarios. Bacterial reduction up to approximate to 99% and viral inactivation of 98% are observed. Cytotoxicity assays reveal some decline in fibroblast viability, underscoring the coatings' potential mainly for non-clinical applications, like packaging or high-touch surfaces. Moreover, the study emphasizes limitations in using single antimicrobial tests and necessity of combining diverse methodologies to comprehensively assess performance and mechanisms of action, finally proposing strategic guidelines for selecting appropriate antimicrobial materials and evaluation techniques tailored to specific application contexts.

Candida biofilms play a critical role in clinical settings, contributing to persistent and device-associated infections and conferring resistance to antifungal agents, particularly in immunocompromised or hospitalized patients. Biofilm formation varies among Candida species, including C. albicans and non-albicans species, such as C. glabrata, C. tropicalis, C. parapsilosis, and C. auris, due to species-specific transcriptional networks that regulate modes of biofilm development, extracellular matrix composition, and metabolic reprogramming. These differences influence biofilm responses to treatment and the severity of infections, which can be further complicated in polymicrobial biofilms that modulate colonization and virulence. Understanding the mechanisms driving biofilm formation and interspecies interactions is essential for developing effective therapies and requires appropriate experimental models. Available models range from simplified in vitro systems to more complex ex vivo and in vivo approaches. Static in vitro models remain widely used due to their simplicity and reproducibility, but they poorly mimic physiological conditions and require careful standardization. Ex vivo tissue models offer a balance between practicality and biological relevance, enabling the study of biofilm physiology, host–microbe interactions and immune responses. In vivo models, primarily in mice, remain the gold standard for testing antifungal therapies, while alternative systems such as Galleria mellonella larvae provide simpler, cost-effective approaches. Advanced in vitro platforms, including organ-on-chip systems, bridge the gap between simplified tests and physiological relevance by simulating fluid dynamics, tissue architecture, and immune complexity. This review aims to examine Candida biofilms across species, highlighting differences in structural diversity and clinical implications, and to provide a guide to the most widely used experimental models supporting studies on Candida biofilm biology for the development of new therapeutic targets or drug testing.

In this paper Juniperus phoenicea L. leaves essential oil (EO) and hydrolate (Hy) were analyzed, for the first time, by gas-chromatography/mass spectrometry (GC/MS) and headspace-gas chromatography-mass spectrometry (HS-GC-MS) respectively, with the aim to describe their chemical volatile profile. The antimicrobial tests carried out highlighted that the Hy did not show antimicrobial activity on all the sixteen tested strains, while the EO was able to exert a slight inhibitory activity against all S. aureus strains and both inhibitory and cytocidal activity against the E. faecium strains. Noteworthy was the ability to inhibit proinflammatory cytokines by both matrices and, in particular, by the Hy. Furthermore, the study of the antioxidant efficacy of the EO and the Hy carried out on Peripheral Blood Mononuclear Cells (PBMC) and on S. aureus strains, showed that these were able to neutralize the production of ROS in the presence of an oxidizing stimulus. This activity was longer using the EO than the Hy.

The global diffusion of antibiotic resistance poses a severe threat to public health. Addressing antibiotic-resistant infections requires innovative approaches, such as antibacterial nanostructured surfaces (ANSs). These surfaces, featuring ordered arrays of nanostructures, exhibit the ability to kill bacteria upon contact. However, most currently developed ANSs utilize bioinert materials, lacking bioactivity crucial for promoting tissue regeneration, particularly in the context of bone infections. This study introduces ANSs composed of bioactive calcium phosphate nanocrystals. Two distinct ANSs were created through a biomineralization-inspired growth of amorphous calcium phosphate (ACP) precursors. The ANSs demonstrated efficient antibacterial properties against both Gram-negative (P. aeruginosa) and Gram-positive (S. aureus) antibiotic resistant bacteria, with up to 75 % mortality in adhered bacteria after only 4 h of contact. Notably, the ANS featuring thinner and less oriented nano-needles exhibited superior efficacy attributed to simultaneous membrane rupturing and oxidative stress induction. Moreover, the ANSs facilitate the proliferation of mammalian cells, enhancing adhesion, spreading, and reducing oxidative stress. The ANSs displayed also significant bioactivity towards human mesenchymal stem cells, promoting colonization and inducing osteogenic differentiation. Specifically, the ANS with thicker and more ordered nano-needles demonstrated heightened effects. In conclusion, ANSs introduced in this work have the potential to serve as foundation for developing bone graft materials capable of eradicate site infections while concurrently stimulating bone regeneration. Statement of significance Nanostructured surfaces with antibacterial properties through a mechano-bactericidal mechanism have shown significant potential in fighting antibiotic resistance. However, these surfaces have not been fabricated with bioactive materials necessary for developing devices that are both antibacterial and able to stimulate tissue regeneration. This study demonstrates the feasibility of creating nanostructured surfaces of ordered calcium phosphate nano-needles through a biomineralization-inspired growth. These surfaces exhibit dual functionality, serving as effective bactericidal agents against Gram-negative and Gram-positive antibiotic-resistant bacteria while also promoting the proliferation of mammalian cells and inducing osteogenic differentiation of human mesenchymal stem cells. Consequently, this approach holds promise in the context of bone infections, introducing innovative nanostructured surfaces that could be utilized in the development of antimicrobial and osteogenic grafts.

IntroductionThis study investigated the interaction with membrane mimetic systems (LUVs), bacterial membranes, the CD spectra, and the bactericidal activity of two designed trematocine mutants, named Trem-HK and Trem-HSK. Mutants were constructed from the scaffold of Trematocine (Trem), a natural 22-amino acid AMP from the Antarctic fish Trematomus bernacchii, aiming to increase their positive charge.MethodsThe selectivity of the designed AMPs towards bacterial membranes was improved compared to Trematocine, verified by their interaction with different LUVs and their membranolytic activity. Additionally, their α-helical conformation was not influenced by the amino acid substitutions. Our findings revealed a significant enhancement in antibacterial efficacy against ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae family) pathogens for both Trem-HK and Trem-HSK.ResultsFirstly, we showed that the selectivity of the two new designed AMPs towards bacterial membranes was greatly improved compared to Trematocine, verifying their interaction with different LUVs and their membranolytic activity. We determined that their α-helical conformation was not influenced by the amino acid substitutions. We characterized the tested bacterial collection for resistance traits to different classes of antibiotics. The minimum inhibitory and bactericidal concentration (MIC and MBC) values of the ESKAPE collection were reduced by up to 80% compared to Trematocine. The bactericidal concentrations of Trematocine mutants showed important membranolytic action, evident by scanning electron microscopy, on all tested species. We further evaluated the cytotoxicity and hemolytic activity of the mutants. At 2.5 μM concentration, both mutants demonstrated low cytotoxicity and hemolysis, indicating selectivity towards bacterial cells. However, these effects increased at higher concentrations.DiscussionAssessment of in vivo toxicity using the Galleria mellonella model revealed no adverse effects in larvae treated with both mutants, even at concentrations up to 20 times higher than the lowest MIC observed for Acinetobacter baumannii, suggesting a high potential safety profile for the mutants. This study highlights the significant improvement in antibacterial efficacy achieved by increasing the positive charge of Trem-HK and Trem-HSK. This improvement was reached at the cost of reduced biocompatibility. Further research is necessary to optimize the balance between efficacy and safety for these promising AMPs.

The overuse of antimicrobials in livestock farming has led to the development of resistant bacteria and the spread of antibiotic-resistant genes (ARGs) among animals. When manure containing these antibiotics is applied to agricultural fields, it creates a selective pressure that promotes the acquisition of ARGs by bacteria, primarily through horizontal gene transfer. Most research on ARGs focuses on their role in clinical antibiotic resistance and their transfer from environmental sources to bacteria associated with humans, such as Escherichia coli. The study investigates the spread of antibiotic-resistant genes (ARGs) through class 1 integrons in 27 Escherichia coli strains from pig manure. It focuses on six common ARGs (ermB, cmlA, floR, qnrS, tetA, and TEM) and the class 1 integron gene, assessing their prevalence in manure samples from three pig farms. The study found correlations and anticorrelations among these genes, indicating a predisposition of the integron in spreading certain ARGs. Specifically, cmlA and tetA genes were positively correlated with each other and negatively with int1, suggesting they are not transferred via Int1. Farm B had the highest int1 counts and a higher abundance of the TEM gene, but lower levels of cmlA and tetA genes. The results underscore the complexity of predicting ARG spread in agricultural environments and the associated health risks to humans through the food chain. The study's results offer valuable insights into the antibiotic-resistant genes (ARGs) profile in swine livestock, potentially aiding in the development of methods to trace ARGs in the environment.

Background. The 2015 Nobel Prize in Medicine, awarded for the discovery of artemisinin in Artemisia annua, reignited interest in aromatic plants, including Artemisia absinthium L. This article delves into the historical, ethnopharmacological and medicinal significance of A. absinthium, examining its bitter taste noted since ancient Greek times and its association with medicinal properties throughout history. Despite being banned in the 20th century due to perceived health risks; recent research has led to the reconsideration of A. absinthium’s potential applications. This study focuses on the prebiotic efficacy of essential oils (EOs) from two Artemisia species: A. absinthium and A. annua. Materials and methods. A broth microdilution test, growth curve test and in vivo models were used to study the impact of low doses (from 0.5% v/v to 0.00048 v/v) of Artemisia spp-EO on the three probiotic strains (Lactobacillus, Lactobacillus casei and Saccharomyces boulardii). Results. These essential oils, when used in minimal concentrations (lower than 0.06% v/v), are safe and exhibit prebiotic effects on major probiotic strains, supporting the traditional culinary use of Artemisia spp. Conclusion. This research opens avenues for potential applications in the food industry, emphasizing the need for further exploration into the prebiotic properties of Artemisia spp-EOs and their influence on the microbiota.

Background: Functionalized nanoparticles (NPs) represent a cutting edge in innovative clinical approaches, allowing for the delivery of selected compounds with higher specificity in a wider time frame. They also hold promise for novel theranostic applications that integrate both diagnostic and therapeutic functions. Pathogens are continuously evolving to try to escape the strategies designed to treat them. Objectives: In this work, we describe the development of a biotechnological device, Nano-Immuno-Probes (NIPs), for early detection and infections treatment. Human Herpes Simplex Virus 2 was chosen as model pathogen. Methods: NIPs consist of PLGA-PEG-Sulfone polymeric NPs conjugated to recombinant Fab antibody fragments targeting the viral glycoprotein G2. NIPs synthesis involved multiple steps and was validated through several techniques. Results: DLS analysis indicated an expected size increase with a good polydispersity index. Z-average and z-potential values were measured for PLGA-PEG-Bis-Sulfone NPs (86.6 ± 10.9 nm; –0.7 ± 0.3 mV) and NIPs (151 ± 10.4 nm; −5.1 ± 1.9 mV). SPR assays confirmed NIPs’ specificity for the glycoprotein G2, with an apparent KD of 1.03 ± 0.61 µM. NIPs exhibited no cytotoxic effects on VERO cells at 24 and 48 h. Conclusions: This in vitro study showed that NIPs effectively target HSV-2, suggesting the potential use of these nanodevices to deliver both contrast agents as well as therapeutic compounds.

In this work, Arthrospira platensis grown in Tuscany, Italy, was investigated using different analytical approaches to characterize its volatile and non-volatile chemical composition. The results showed the presence of a high number of volatile organic compounds (VOCs) such as hydrocarbons, furans, sulfides, alkanes, aldehydes, alcohols, ketones, esters and compounds belonging to other chemical classes such as fatty acids, alcohols and sugars. Furthermore, a proximal composition analysis was also performed to determine the protein, fat, carbohydrate and ash content. Total antioxidant capacity (TAC) determined by FRAP and ABTS•+ methods (5.96 mmol TE/g DW; 5.28 mmol Fe2+E/g DW, respectively), showed good reducing power and comparable free radical scavenging activity. The antibacterial power of spirulina-based alcoholic macerate (AM) was also evaluated against Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 060127853), Enterococcus faecalis (ATCC 29211), Klebsiella pneumoniae (ATCC 700603) and Candida albicans (ATCC 24433) and the obtained data have shown that it had no effect against pathogenic bacterial strains. On the contrary, at low concentrations, AM exerted a prebiotic effect on some probiotic strains such as L. casei if treated with AM concentrations ranging from 1.56% v/v and 3.12% v/v and L. rhamnosus if treated with AM concentrations lower than 0.78% v/v. In conclusion, this study highlighted how spirulina, based on the rich composition and its antioxidant and prebiotic effect, can represent a source of beneficial substances for human health.