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The prohibition of antibiotics has led to extensive research and use of phytogenic feed additives. James Barrie Kirkpatrick described four subspecies of eucalyptus (family Myrtaceae), including Eucalýptus globulus, in 1974. The maximum concentrations of quercetin-3D-glycoside (1703.30 g/mL), astragalin (1737.82 g/mL), chlorogenic acid (342.14 g/mL), catechin (282.54 g/mL), rosmarinic acid (36.39 g/mL), and 3,4-dihydroxybenzoic acid (27.55 g/mL) were found in samples of ultrasonic extraction with ethyl alcohol (extraction module 1:5, temperature of 32 °C, an ultrasonic exposure time of 25 min). Antimicrobial activity was observed in all studied samples after 12 h of incubation (against gram-positive (Bacillus subtilis) and gram-negative (Pseudomonas aeruginosa) bacteria, as well as representatives of yeast fungi (Candida albicans)); a more pronounced antimicrobial effect (lysis zone) was observed after ultrasonic processing of extracts for 20 and 25 min. Bacillus subtilis, Pseudomonas aeruginosa, and Candida albicans had lysis areas of 10.0 mm (20 min extraction with ultrasonic treatment), 13.0 mm (20 min extraction without ultrasonic treatment), and 15.5 mm (25 min extraction with ultrasonic treatment), respectively. E. globulus was demonstrated to be a source of biologically active phenolic compounds with antibacterial and fungicidal activity. More research on the use of E. globulus in feed additives is required.

Silver nanoparticles (AgNPs) exhibit unusual biocidal properties thanks to which they find a wide range of applications in diverse fields of science and industry. Numerous research studies have been devoted to the bactericidal properties of AgNPs while less attention has been focused on their fungicidal activity. Our studies were therefore oriented toward determining the impact of AgNPs characterized by different physicochemical properties on Fusarium avenaceum and Fusarium equiseti . The main hypothesis assumed that the fungicidal properties of AgNPs characterized by comparable morphology can be shaped by stabilizing agent molecules adsorbed on nanoparticle surfaces. Two types of AgNPs were prepared by the reduction of silver ions with sodium borohydride (SB) in the presence of trisodium citrate (TC) or cysteamine hydrochloride (CH). Both types of AgNPs exhibited a quasi-spherical shape. Citrate-stabilized AgNPs (TCSB-AgNPs) of an average size of 15 ± 4 nm were negatively charged. Smaller (12 ± 4 nm), cysteamine-capped AgNPs (CHSB-AgNPs) were characterized by a positive surface charge and higher silver ion release profile. The phytopathogens were exposed to the AgNPs in three doses equal to 2.5, 5 and 10 mg L −1 over 24 and 240 h. Additionally, the impact of silver ions delivered in the form of silver nitrate and the stabilizing agents of AgNPs on the fungi was also investigated. The response of phytopathogens to these treatments was evaluated by determining mycelial growth, sporulation and changes in the cell morphology. The results of our studies showed that CHSB-AgNPs, especially at a concentration of 10 mg L −1 , strongly limited the vegetative mycelium growth of both species for short and long treatment times. The cell imaging revealed that CHSB-AgNPs damaged the conidia membranes and penetrated into the cells, while TCSB-AgNPs were deposited on their surface. The fungistatic (lethal) effect was demonstrated only for silver ions at the highest concentration for the F. equiseti species in the 240 h treatment. The number of spores of both Fusarium species was significantly reduced independently of the type of silver compounds used. Generally, it was found that the positively charged CHSB-AgNPs were more fungicidal than negatively charged TCSB-AgNPs. Thereby, it was established that the stabilizing agents of AgNPs and surface charge play a crucial role in the shaping of their fungicidal properties.

In the present study, a method for the synthesis of gelatin-stabilized copper oxide nanoparticles was developed. Synthesis was carried out by direct chemical precipitation. Copper sulfate, chloride, and acetate were used as precursors for the copper oxide synthesis. Gelatin was used as a stabilizer. It was found that the formation of monophase copper oxide II only occurred when copper acetate was used as a precursor. Our results showed that particles of the smallest diameter are formed in an aqueous medium (18 ± 6 nm), and those of th largest diameter—in an isobutanol medium (370 ± 131 nm). According to the photon correlation spectroscopy data, copper oxide nanoparticles synthesized in an aqueous medium were highly stable and had a monomodal size distribution with an average hydrodynamic radius of 61 nm. The study of the pH effect on the colloidal stability of copper oxide nanoparticles showed that the sample was stable in the pH range of 6.8 to 11.98. A possible mechanism for the pH influence on the stability of copper oxide nanoparticles is described. The effect of the ionic strength of the solution on the stability of the CuO nanoparticles sol was also studied, and the results showed that Ca 2+ ions had the greatest effect on the sample stability. IR spectroscopy showed that the interaction of CuO nanoparticles with gelatin occurred through the hydroxyl group. It was found that CuO nanoparticles stabilized with gelatin have a fungicidal activity at concentration equivalent 2.5 · 10 −3  mol/L and as a material for food nanopackaging can provide an increase in the shelf life of products on the example of strawberries and tomatoes. We investigated the possibility of using methylcellulose films modified with CuO nanoparticles for packaging and storage of hard cheese “Holland”. The distribution of CuO nanoparticles in the methylcellulose film was uniform. We found that methylcellulose films modified with CuO nanoparticles inhibited the growth and development of QMAFAM, coliforms, yeast and mold in experimental cheese sa mples. Our research has shown that during the cheese storage in thermostat at 35 ± 1 °C for 7 days, CuO nanoparticles migrated to the product from the film. Nevertheless, it is worth noting that the maximum change in the concentration of copper in the experimental samples was only 0.12 µg/mg, which is not a toxic concentration. In general, the small value of migration of CuO nanoparticles confirms the high stability of the developed preparation. Our results indicated that the CuO nanoparticles stabilized with gelatin have a high potential for use in food packaging – both as an independent nanofilm and as part of other packaging materials.

The abuse of antibiotics has led to the emergence of multidrug-resistant microbial pathogens, presenting a pressing challenge in global healthcare. Membrane-disrupting antimicrobial peptides (AMPs) combat so-called superbugs via mechanisms different than conventional antibiotics and have good application prospects in medicine, agriculture, and the food industry. However, the mechanism-of-action of AMPs has not been fully characterized at the cellular level due to a lack of high-resolution imaging technologies that can capture cellular-membrane disruption events in the hydrated state. Previously, we reported PepD2M, a de novo-designed AMP with potent and wide-spectrum bactericidal and fungicidal activity. In this study, we use cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM) to directly visualize the pepD2M-induced disruption of the outer and inner membranes of the Gram-negative bacterium Escherichia coli , and compared with a well-known pore-forming peptide, melittin. Our high-resolution cryo-ET images reveal how pepD2M disrupts the E. coli membrane using a carpet/detergent-like mechanism. Our studies reveal the direct membrane-disrupting consequence of AMPs on the bacterial membrane by cryo-ET, and this information provides critical insights into the mechanisms of this class of antimicrobial agents. Antimicrobial peptide mechanism of membrane disruption have not been fully characterized at the cellular level. Here, authors use cryo-electron tomography and AFM to directly visualize the disruption of the outer and inner membranes of Escherichia coli by a de novo-designed peptide.

Fungi are very common infectious pathogens, which may cause invasive and potentially life-threatening infections. However, the efficacy of antifungal medications remains limited. Herein, a Cu 2+ -phenolic nanoflower is designed to combat fungal infections by combining cuproptosis and cell wall digestion. Firstly, protocatechuic acid (PA)-Cu 2+ (PC) nanopetals are prepared by coordination interaction. Lywallzyme (Lyw) is then added to induce the self-assembly of PC to form Lyw loaded PC (PCW) nanoflowers. PCW nanoflowers can effectively adhere to fungal surface and Lyw can digest fungal cell walls to facilitate Cu 2+ to penetrate into fungal interior, thereby exerting a synergistic fungicidal effect. PCW nanoflowers exhibit excellent fungicidal activity even in protein-rich and high-salt conditions, where dissociative Cu 2+ completely loses fungicidal activity. Transcriptome sequencing analysis reveals that PCW can lead to fungal cuproptosis. The in vivo fungicidal effect of PCW nanoflowers is confirmed on a murine skin fungal infection model and a murine fungal keratitis model. Fungal infections can be debilitating and life-threatening. Here, the authors report a fungicidal strategy by the combination of cell wall digestion and cuproptosis using lywallzyme-assembled copper-phenolic nanoflowers demonstrating synergistic effects in fungal infection and fungal keratitis models.

Amphotericin B (AmB) is a prototypical small molecule natural product that can form ion channels in living eukaryotic cells and has remained refractory to microbial resistance despite extensive clinical utilization in the treatment of life-threatening fungal infections for more than half a century. It is now widely accepted that AmB kills yeast primarily via channel-mediated membrane permeabilization. Enabled by the iterative cross-coupling-based synthesis of a functional group deficient derivative of this natural product, we have discovered that channel formation is not required for potent fungicidal activity. Alternatively, AmB primarily kills yeast by simply binding ergosterol, a lipid that is vital for many aspects of yeast cell physiology. Membrane permeabilization via channel formation represents a second complementary mechanism that further increases drug potency and the rate of yeast killing. Collectively, these findings (i) reveal that the binding of a physiologically important microbial lipid is a powerful and clinically validated antimicrobial strategy that may be inherently refractory to resistance, (ii) illuminate a more straightforward path to an improved therapeutic index for this clinically vital but also highly toxic antifungal agent, and (iii) suggest that the capacity for AmB to form proteinlike ion channels might be separable from its cytocidal effects.

Healthy vaginal microbiota is dominated by Lactobacillus spp., which form a critical line of defence against pathogens, including Candida spp. The present study aims to identify vaginal lactobacilli exerting in vitro activity against Candida spp. and to characterize their antifungal mechanisms of action. Lactobacillus strains were isolated from vaginal swabs of healthy premenopausal women. The isolates were taxonomically identified to species level (L. crispatus B1-BC8, L. gasseri BC9-BC14 and L. vaginalis BC15-BC17) by sequencing the 16S rRNA genes. All strains produced hydrogen peroxide and lactate. Fungistatic and fungicidal activities against C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis and C. lusitaniae were evaluated by broth micro-dilution method. The broadest spectrum of activity was observed for L. crispatus BC1, BC4, BC5 and L. vaginalis BC15, demonstrating fungicidal activity against all isolates of C. albicans and C. lusitaniae. Metabolic profiles of lactobacilli supernatants were studied by 1H-NMR analysis. Metabolome was found to be correlated with both taxonomy and activity score. Exclusion, competition and displacement experiments were carried out to investigate the interference exerted by lactobacilli toward the yeast adhesion to HeLa cells. Most Lactobacillus strains significantly reduced C. albicans adhesion through all mechanisms. In particular, L. crispatus BC2, L. gasseri BC10 and L. gasseri BC11 appeared to be the most active strains in reducing pathogen adhesion, as their effects were mediated by both cells and supernatants. Inhibition of histone deacetylases was hypothesised to support the antifungal activity of vaginal lactobacilli. Our results are prerequisites for the development of new therapeutic agents based on probiotics for prophylaxis and adjuvant therapy of Candida infection.

Antimicrobial resistance is a matter of rising concern, especially in fungal diseases. Multiple reports all over the world are highlighting a worrisome increase in azole- and echinocandin-resistance among fungal pathogens, especially in Candida species, as reported in the recently published fungal pathogens priority list made by WHO. Despite continuous efforts and advances in infection control, development of new antifungal molecules, and research on molecular mechanisms of antifungal resistance made by the scientific community, trends in invasive fungal diseases and associated antifungal resistance are on the rise, hindering therapeutic options and clinical cures. In this context, in vitro susceptibility testing aimed at evaluating minimum inhibitory concentrations, is still a milestone in the management of fungal diseases. However, such testing is not the only type at a microbiologist’s disposal. There are other adjunctive in vitro tests aimed at evaluating fungicidal activity of antifungal molecules and also exploring tolerance to antifungals. This plethora of in vitro tests are still left behind and performed only for research purposes, but their role in the context of invasive fungal diseases associated with antifungal resistance might add resourceful information to the clinical management of patients. The aim of this review was therefore to revise and explore all other in vitro tests that could be potentially implemented in current clinical practice in resistant and difficult-to-treat cases.

Controlling toxigenic Fusarium graminearum (FG) is challenging. A bacterial strain (S76-3, identified as Bacillus amyloliquefaciens) that was isolated from diseased wheat spikes in the field displayed strong antifungal activity against FG. Reverse-phase high performance liquid chromatography and electrospray ionization mass spectrometry analyses revealed that S76-3 produced three classes of cyclic lipopeptides including iturin, plipastatin and surfactin. Each class consisted of several different molecules. The iturin and plipastatin fractions strongly inhibited FG; the surfactin fractions did not. The most abundant compound that had antagonistic activity from the iturin fraction was iturin A (m/z 1043.35); the most abundant active compound from the plipastatin fraction was plipastatin A (m/z 1463.90). These compounds were analyzed with collision-induced dissociation mass spectrometry. The two purified compounds displayed strong fungicidal activity, completely killing conidial spores at the minimal inhibitory concentration range of 50 µg/ml (iturin A) and 100 µg/ml (plipastatin A). Optical and fluorescence microscopy analyses revealed severe morphological changes in conidia and substantial distortions in FG hyphae treated with iturin A or plipastatin A. Iturin A caused leakage and/or inactivation of FG cellular contents and plipastatin A caused vacuolation. Time-lapse imaging of dynamic antagonistic processes illustrated that iturin A caused distortion and conglobation along hyphae and inhibited branch formation and growth, while plipastatin A caused conglobation in young hyphae and branch tips. Transmission electron microscopy analyses demonstrated that the cell walls of conidia and hyphae of iturin A and plipastatin A treated FG had large gaps and that their plasma membranes were severely damaged and separated from cell walls.