Many pathogens involved in human infection have rapidly increased their antibiotic resistance, reducing the effectiveness of therapies in recent decades. Most of them can form biofilms and effective drugs are not available to treat these formations. Natural products could represent an efficient solution in discovering and developing new drugs to overcome antimicrobial resistance and treat biofilm-related infections. In this study, 20 secondary metabolites produced by pathogenic fungi of forest plants and belonging to diverse classes of naturally occurring compounds were evaluated for the first time against clinical isolates of antibiotic-resistant Gram-negative and Gram-positive bacteria. epi-Epoformin, sphaeropsidone, and sphaeropsidin A showed antimicrobial activity on all test strains. In particular, sphaeropsidin A was effective at low concentrations with Minimum Inhibitory Concentration (MIC) values ranging from 6.25 μg/mL to 12.5 μg/mL against all reference and clinical test strains. Furthermore, sphaeropsidin A at sub-inhibitory concentrations decreased methicillin-resistant S. aureus (MRSA) and P. aeruginosa biofilm formation, as quantified by crystal violet staining. Interestingly, mixtures of sphaeropsidin A and epi-epoformin have shown antimicrobial synergistic effects with a concomitant reduction of cytotoxicity against human immortalized keratinocytes. Our data show that sphaeropsidin A and epi-epoformin possess promising antimicrobial properties.

The increasing incidence of periprosthetic joint infections (PJIs) has led to a growing interest in developing strategies to prevent and treat this severe complication. The surgical site’s application of antiseptic solutions to eliminate contaminating bacteria and eradicate the bacterial biofilm has been increasing over time. Even though it has been proven that combining antimicrobials could enhance their activities and help overcome acquired microbial resistance related to the topical use of antibiotics, the toxicity of integrated solutions is not well described. This study aimed to evaluate the cytotoxicity of solutions containing povidone–iodine (PI) and hydrogen peroxide (H${}_2$O${}_2$), alone or in combination, after 1.3 and 5 min of exposure. Chondrocytes, tenocytes, and fibroblast-like synoviocytes were used for cytotoxicity analysis. Trypan blue stain (0.4% in PBS) was applied to evaluate the dead cells. All solutions tested showed a progressive increase in toxicity as exposure time increased except for PI at 0.3%, which exhibited the lowest toxicity. The combined solutions reported a reduced cellular killing at 3 and 5 min than H${}_2$O${}_2$ at equal concentrations, similar results to PI solutions.

Over the years, the increasing acquisition of antibiotic resistance genes has led to the emergence of highly resistant bacterial strains and the loss of standard antibiotics’ efficacy, including β-lactam/β-lactamase inhibitor combinations and the last line carbapenems. Klebsiella pneumoniae is considered one of the major exponents of a group of multidrug-resistant ESKAPE pathogens responsible for serious healthcare-associated infections. In this study, we proved the antimicrobial activity of two analogues of Temporin L against twenty carbapenemase-producing K. pneumoniae clinical isolates. According to the antibiotic susceptibility assay, all the K. pneumoniae strains were resistant to at least one other class of antibiotics, in addition to beta-lactams. Peptides 1B and C showed activity on all test strains, but the lipidated analogue C expressed the greater antimicrobial properties, with MIC values ranging from 6.25 to 25 µM. Furthermore, the peptide C showed bactericidal activity at MIC values. The results clearly highlight the great potential of antimicrobial peptides both as a new treatment option for difficult-to-treat infections and as a new strategy of drug-resistance control.

The increasing resistance of fungi to conventional antifungal drugs has prompted worldwide the search for new compounds. In this work, we investigated the antifungal properties of acylated Temporin L derivatives, Pent-1B and Dec-1B, against Candida albicans, including the multidrug-resistant strains. Acylated peptides resulted to be active both on reference and clinical strains with MIC values ranging from 6.5 to 26 µM, and they did not show cytotoxicity on human keratinocytes. In addition, we also observed a synergistic or additive effect with voriconazole for peptides Dec-1B and Pent-1B through the checkerboard assay on voriconazole-resistant Candida strains. Moreover, fluorescence-based assays, NMR spectroscopy, and confocal microscopy elucidated a potential membrane-active mechanism, consisting of an initial electrostatic interaction of acylated peptides with fungal membrane, followed by aggregation and insertion into the lipid bilayer and causing membrane perturbation probably through a carpeting effect. Graphical abstract

Corynebacterium striatum (C. striatum) is an emerging multidrug-resistant (MDR) pathogen associated with nosocomial infections. In this scenario, we screened the antimicrobial activity of the anthelmintic drugs doramectin, moxidectin, selamectin and niclosamide against 20 C. striatum MDR clinical isolates. Among these, niclosamide was the best performing drug against C. striatum. Niclosamide cytotoxicity was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay on immortalized human keratinocyte cells (HaCaT). After 20 h of treatment, the recorded 50% cytotoxic concentration (CC₅₀) was 2.56 μg/mL. The antibacterial efficacy was determined via disc diffusion, broth microdilution method and time-killing. Against C. striatum, niclosamide induced a growth inhibitory area of 22 mm and the minimum inhibitory concentration that inhibits 90% of bacteria (MIC₉₀) was 0.39 μg/mL, exhibiting bactericidal action. The biofilm biomass eradicating action was investigated through crystal violet (CV), MTT and confocal laser scanning microscopy (CLSM). Niclosamide affected the biofilm viability in a dose-dependent manner and degraded biomass by 55 and 49% at 0.39 μg/mL and 0.19 μg/mL. CLSM images confirmed the biofilm biomass degradation, showing a drastic reduction in cell viability. This study could promote the drug-repurposing of the anthelmintic FDA-approved niclosamide as a therapeutic agent to counteract the C. striatum MDR infections.