Background
Antimicrobial peptides (AMPs) represent promising candidates in the search for new antimicrobial agents. The increasing incidence of infections caused by multidrug-resistant (MDR) bacteria, together with the declining efficacy of existing antibiotics, highlights the urgent global need for novel therapeutics. CAP-18 is an α-helical peptide belonging to the cathelicidin family. Its enantiomer, D-CAP-18, also shows potential for drug development and optimization. Because linear peptides are often unstable in vivo, cyclization of both peptides was undertaken to improve stability. This study aimed to evaluate the antimicrobial activity of cyclic CAP-18 and cyclic D-CAP-18 against MDR bacteria.

Methods
Antimicrobial activity was assessed using broth microdilution assays to determine minimum inhibitory concentrations (MICs) of the cyclic peptides against American Type Culture Collection (ATCC) reference strains and a panel of well-characterized clinical MDR isolates, including extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing strains. Four MDR E. coli and five MDR K. pneumoniae clinical isolates were tested.

Results
Both cyclic CAP-18 and cyclic D-CAP-18 exhibited greater activity against Gram-negative than Gram-positive bacteria. MIC values for cyclic CAP-18 against E. coli ranged from 2 to 8 mg/L, while cyclic D-CAP-18 showed MICs of 2 to 4 mg/L. Against K. pneumoniae, cyclic CAP-18 demonstrated MICs of 4 to 32 mg/L, and cyclic D-CAP-18 showed MICs of 4 to 16 mg/L.

Conclusion
Cyclic CAP-18 and cyclic D-CAP-18 display promising antimicrobial activity against MDR Gram-negative bacteria, including both ATCC reference strains and clinical isolates. These preliminary findings support their potential as candidates for further antimicrobial drug development.

Escherichia coli remains the leading cause of urinary tract infections worldwide, while antimicrobial resistance (AMR) poses a growing public health threat, contributing to over 1.2 million deaths annually in 2019 and projected to cause 10 million deaths per year by 2050. Extensive and often inappropriate use of fluoroquinolones, particularly ciprofloxacin, has accelerated resistance development in E. coli, largely driven by target-site mutations and efflux pump overexpression, thereby severely limiting therapeutic options. Innovative strategies that enhance antibiotic efficacy are therefore urgently required to address this challenge.

In this study, the antibacterial activities of antimicrobial peptides (AMPs)—Batroxicidin (BatxC), Crotalicidin (Ctn), Latarcin-2a (Ltc-2a), and Tat-2—were evaluated individually and in combination with ciprofloxacin against ciprofloxacin-resistant clinical E. coli isolates. Minimum inhibitory concentrations (MICs) were determined using the broth microdilution method. Drug–drug interactions were assessed by checkerboard assays and interpreted using the fractional inhibitory concentration index (FICI) according to EUCAST/CLSI criteria. The contribution of efflux mechanisms was examined using the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Cytotoxicity was evaluated in HeLa cells using the MTT assay.

Ciprofloxacin combinations with BatxC, Ctn, and Ltc-2a resulted in fourfold reductions in ciprofloxacin MICs with additive interactions, whereas the ciprofloxacin–Tat-2 combination produced a 16-fold MIC reduction, consistent with partial synergy. Notably, triple combinations of ciprofloxacin, AMPs, and CCCP reduced ciprofloxacin MICs to <1 µg/mL (≥64-fold), highlighting efflux inhibition-mediated antibiotic resensitization. Cytotoxicity analysis showed that Ctn exhibited excellent biocompatibility, with ciprofloxacin–Ctn combinations maintaining ≥75% cell viability.

Overall, AMP-based combination therapy represents a promising, mechanism-driven strategy to restore ciprofloxacin activity against resistant E. coli. Further in vivo validation and mechanistic studies are required to support clinical translation.

Antimicrobial resistance (AMR) is an increasing threat according to the World Health Organization. Pseudomonas aeruginosa is a Gram-negative and opportunistic organism that develops multidrug resistance in several ways, which requires a better understanding of the mechanisms and new and effective solutions to overcome AMR. The transcriptome, a complete set of RNA molecules, provides information about gene expression, regulation, and function, leading to the translation of this information for disease diagnosis, treatment, and drug development and discovery. Integration of transcriptome data and a genome-scale metabolic model (GEM) of the organism is one of the useful strategies to identify and understand metabolic activities related to AMR. The discovery of reporter metabolites (RM) and their specific metabolic pathways may be a powerful method to study metabolic responses and cellular mechanisms of AMR under different conditions, leading to potential therapeutic targets or biomarkers. This study integrates transcriptomic data from 414 drug-resistant clinical isolates with a genome-scale metabolic model (GEM) of P. aeruginosa to identify metabolic adaptations under four antibiotic stresses: ceftazidime (CAZ), ciprofloxacin (CIP), meropenem (MEM), and tobramycin (TOB). Differential gene expression (DGE) analysis revealed largely drug-specific responses, with minimal overlap in differentially expressed genes (DEGs) across conditions. Using the Reporter Metabolite (RM) algorithm, metabolites central to the resistance phenotype were identified. Pathway enrichment analysis highlighted antibiotic-specific alterations in metabolic networks, many of which are associated with biofilm formation and virulence. Based on the findings, we propose a novel adjuvant therapy composed of condition-specific metabolites (propionic and acetic acids, L-inositol, glutamine, glutarate, fumarate, and melatonin) to enhance antibiotic efficacy and reduce resistance development. This systems biology approach provides a comprehensive framework for metabolic intervention in AMR pathogens.

The rising threat of antibiotic-resistant Staphylococcus aureus necessitates novel therapeutic strategies. This study elucidates the dual mechanism by which the NSAID flufenamic acid (FFA) enhances bacterial killing by immune cells. We identified the quorum-sensing AgrAC two-component system as a key target of FFA in S. aureus. Through promoter-reporter assays, EMSA, and mutagenesis, FFA was shown to bind the response regulator AgrA at a novel site (E27) within its regulatory domain, inhibiting virulence gene expression (e.g., α-toxin). This makes FFA the first reported inhibitor targeting this AgrA domain, distinct from existing DNA-binding domain inhibitors.

Concurrently, FFA’s immunomodulatory action is essential for its efficacy. In macrophages, FFA inhibited the NLRP3 inflammasome, a process critical for promoting phagosome-mitochondria colocalization and subsequent bactericidal reactive oxygen species (ROS) generation. This NLRP3-dependent mechanism was validated in vivo, where FFA lost its effect in nlrp3-KO mice. While exhibiting minimal direct antibacterial activity (MIC >400 µM), FFA acted synergistically with gentamicin, significantly reducing bacterial loads in vivo.

Thus, FFA represents a promising anti-virulence agent that uniquely combines suppression of bacterial pathogenicity via AgrA inhibition with potentiation of host innate immunity via NLRP3 blockade. This dual, host-pathogen targeting strategy minimizes selective pressure for resistance and supports the therapeutic potential of repurposing FFA, particularly in topical or combination regimens, against staphylococcal infections.

Introduction: The macrolide-resistant strains of Streptococcus pneumoniae are the latest additions to the WHO's List of Priority Antibiotic-Resistant Bacteria, responsible for over 3 million deaths annually, of which 300,000 are in children under 5 years of age. One of the most promising novel targets for antibacterial drug development is mRNA. The present study employs a rational framework based on four criteria related to mRNA distribution, function, and metabolism, and identifies them as suitable targets for antibacterial drug discovery. It focuses on seven of them, which are found in the S. pneumoniae genome: the RNase P, FMN, TPP, PreQ1-II, Purine, ykoK leader, and Glycine riboswitches.

Methods: The selection of targets and the subsequent rational design of ASOs targeting the macrolide-resistant S. pneumoniae are based on bioinformatics and genomic studies, including analyses of international databases, Clustal X multiple alignments, selection of appropriate motifs, BLAST searches, and biochemical pathway analyses.

Results: The mRNAs found in S. pneumoniae are grouped into four categories: most suitable, very suitable, suitable, and not suitable. Most suitable riboswitches (FMN, TPP, Purine, and PreQ₁₎ regulate essential metabolites, with no alternative biosynthetic pathways or transport. Very suitable riboswitches (ykoK leader) control the critical metabolite’s biosynthesis and transport. Suitable riboswitches (Glycine) have alternative biosynthetic pathways and do not control their transport. Five different antisense oligonucleotides (ASO) have been designed to target them.

Conclusions: Gene expression is regulated at the transcriptional or translational level. Using mRNA, we develop innovative strategies to design ASOs that inhibit S. pneumoniae growth. Our proprietary protocols for suitability and ASO design enable us to develop novel ASOs (with precise nucleotide sequences for specific binding, modifications that enhance stability and induce RNase H activity, and attached cell-penetrating peptides) targeted to the most suitable mRNAs, with potential as effective antimicrobial therapeutics with growth-inhibitory effects.

Antibiotic resistance is widely recognized as one of the most pressing global health challenges, resulting from the rapid and uncontrolled spread of multidrug-resistant bacterial and fungal pathogens. The progressive loss of effectiveness of conventional antibiotics highlights the urgent need for alternative therapeutic strategies. In this scenario, antimicrobial peptides (AMPs) have attracted increasing attention as promising candidates for the treatment of infectious diseases, due to their broad-spectrum activity, diverse mechanisms of action, and reduced likelihood of inducing resistance compared to conventional antibiotics.

This study investigates the potential of novel synthetic AMPs inspired by natural peptide scaffolds and developed through advanced rational design approaches. Natural peptides from a Mediterranean medical plant Charybdis pancration (Steinh.) Speta were used as starting templates for the design of multiple modified peptide sequences employing in silico tools based on artificial intelligence and deep-learning models. These computational strategies enabled the optimization of key physicochemical properties associated with antimicrobial and antibiofilm activity.

The designed peptides were subsequently evaluated using a combination of in silico analyses and in vitro tests to validate their predicted MICs against relevant Gram-negative pathogens (Acinetobacter baumannii and Pseudomonas aeruginosa) and Gram-positive pathogens (antibiotic-resistant Staphylococcus aureus). In particular, the in silico analysis predicted MIC values against Pseudomonas aeruginosa ranging from 48.8 to 10.9 µg/mL.

Overall, this work highlights the value of integrating natural peptide sources with artificial intelligence-based design methodologies for the discovery of new antimicrobial candidates. Such an approach may help to expand the current antimicrobial pipeline and provide innovative solutions to combat the growing threat of antibiotic resistance.

Introduction: Marine fungi are an underexplored reservoir of bioactive secondary metabolites with significant potential for the development of new therapeutic agents to combat infectious diseases and antimicrobial resistance.

Broad objectives: In this work, the antimicrobial properties of ten marine fungal strains Trichoderma afroharzianum (HP58), Penicillium citrinum (HP26), Talaromyces austrocalifornicus (HPα8), Penicillium rubens (HP10), Tamaricicola sp. (PN38), Phaeosphaeriaceae sp. (PN33), Clonostachys rosea (IG119), Trichoderma sp. (HP31), Talaromyces catalonicus (HP25), and Fusarium clavus (HP54) were investigated using crude extracts obtained from cultures grown on different media.

Methods: Chloroform and butanol extracts from cultures grown on malt extract medium (MEA) were evaluated against Gram-positive and Gram-negative bacterial reference strains exhibiting antibiotic resistance, as well as against the yeast Candida albicans. Antimicrobial activity was assessed through determination of the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum fungicidal concentration (MFC) and antibiofilm activity.

Results: The preliminary in vitro results identified strains Penicillium citrinum (HP26), Talaromyces austrocalifornicus (HPα8), Penicillium rubens (HP10)and Clonostachys rosea IG119 as promising producers of antimicrobial secondary metabolites. In particular, Clonostachys rosea IG119 showed an interesting ability to inhibit biofim formation of Staphylococcus aureus ATCC 25923 with a Biofilm Inhibition Concentration 50% (BIC₅₀) of 22 μg/ml.

Conclusions: Optimization of cultivation parameters could support the isolation of bioactive compounds and enable their detailed chemical and pharmacological characterization. Overall, these findings indicate that marine fungi deserve to be considered an excellent source of antimicrobials.

Background: Biofilm formation by multidrug resistant (MDR) Pseudomonas aeruginosa contributes to increased morbidity and mortality in patients with pulmonary diseases. SET-M33L is a synthetic tetra-branched peptide that previously showed antimicrobial activity on standard strains of MDR gram-negative bacteria and improved stability in biological fluids than linear versions. PEGylated version of the peptide, SET-M33L-PEG, further displayed enhanced resistance to P. aeruginosa elastase. Therefore, we investigated these two antimicrobial peptides (AMPs) for their antimicrobial and antibiofilm activity against 10 selected P. aeruginosa clinical isolates, including MDR strains.

Methods: The effect of AMPs on outer membrane (OM) integrity was investigated by measuring N-Phenyl-1-naphthylamine (NPN) uptake. Their minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), minimum biofilm inhibitory concentrations (MBICs) were evaluated with gold standard methodologies. Effect on pre-formed biofilm was evaluated measuring bacterial viability assessed by resazurin dye assay. Conventional antimicrobial compounds tobramycin, ceftazidime, and polymyxin B were used as comparator controls.

Results: Dose-dependent increase of NPN fluorescence intensity after treatment with AMPs indicated a disruption of OM integrity in all tested strains. MICs and MBCs values of the AMPs were 7 to 100 times lower than for tobramycin, and 10 to 300 times lower than for ceftazidime, for the respective MDR strains. AMPs MBICs values ranged from 0.3 μM to 21.8 μM, while tobramycin presented the highest MBIC values among the compounds, ranging between 2.1 μM and 273.8 μM. With increasing concentrations of AMPs, a reduction in cell viability of 50% to 100% was observed in pre-formed biofilms. Fractional inhibitory concentration (FIC) indices showed an additive effect (0.5 < FBC < 1), while fractional bactericidal concentration (FBC) indices showed synergistic effects (FBC < 0.5) for most isolates when the AMPs were combined with tobramycin or ceftazidime.

Conclusion: SET-M33L and SET-M33L-PEG are promising antimicrobial agents against strong biofilm-forming P. aeruginosa, including MDR isolates.

Oxidation lagoons (OLs) are widely used wastewater treatment systems in rural and First Nation (FN) communities in Canada and are essential for protecting public health. Although designed to remove pollutants and pathogens, OLs may facilitate the exchange of mobile genetic elements and antibiotic resistance genes (ARGs), promoting antimicrobial resistance (AMR) among environmental bacteria and bacteriophages. This study investigated AMR dynamics by characterizing the resistome of bacterial isolates and metagenomic samples from an OL serving an FN community in Manitoba.

We combined wet- and dry-lab approaches to examine viable bacteria and bacterial and phage metagenomes across treatment stages. Thirty-five samples were collected between September 2022 and April 2023 from raw sewage (RS), lagoon (LG), a submerged attached growth reactor (SAGR), UV-disinfected effluent (EF), and an upstream control site (UPS) including surface water and sediment. Genomic and metagenomic DNA were sequenced using Oxford Nanopore Technology with de novo assembly. Viable isolates were enumerated, and bacterial and Escherichia coli counts were quantified using digital PCR.

Among 58 isolates, Aeromonas (50.87%) and Serratia (15.78%) predominated, with a lower representation of Pantoea, Escherichia, Lelliottia, Rahnella, Enterobacter, Buttiauxella, Acinetobacter, Yersinia, and Citrobacter. ARG analysis identified 32,559 ARG-associated elements, including 425 strict or perfect hits in 84.5% of isolates. Genes conferring resistance to carbapenems, β-lactams, macrolides, tetracyclines, and fluoroquinolones were detected. An Acinetobacter radioresistens isolate from UV-treated effluent carried blaOXA-23 and showed resistance to imipenem (64 µg/mL). Metagenomics (84 samples) revealed Caudoviricetes as the dominant viral class, with aminoglycoside, fluoroquinolone, and phenicol resistance genes most abundant. Gene copy numbers were highest at UPS and RS (1.48 and 1.10 × 10⁵ cells/mL). E. coli averaged 172.5 cells/mL in RS.

These findings demonstrate the persistence of clinically relevant bacteria and ARGs in treated wastewater, supporting the need for AMR surveillance in decentralized systems serving FN communities.

Objectives: The emergence of drug‑resistant fungal pathogens, such as Candida albicans (C. albicans), has intensified the global demand for antifungal agents with novel mechanisms of action. Conventional therapies, including azoles and polyenes, are increasingly limited by resistance and biofilm‑associated tolerance. Antimicrobial peptides (AMPs), naturally expressed in a variety of organisms as first-line defences against microbial infections, have emerged as promising alternatives due to their low propensity for resistance development, ability to disrupt microbial membranes, and inhibition of key virulence traits. While most current studies focus on peptides with antibacterial activity, relatively little is known about their antifungal properties. In this study, we investigated the antifungal and antivirulence activity of the AMP oreochromicin‑1 (oreoch-1), isolated from the gills of Nile tilapia (Oreochromis niloticus), against C. albicans.

Methods: An antifungal susceptibility assay was conducted to determine oreoch-1’s minimum inhibitory concentration (MIC), and its effect on growth kinetics was assessed. Hyphal formation was evaluated under filamentation-inducing conditions. Biofilm biomass reduction was quantified using crystal violet staining, and morphological changes were examined microscopically. The molecular responses to oreoch‑1 exposure were analysed by qRT-PCR for two virulence‑associated genes: HWP1 (adhesion) and ERG11 (ergosterol biosynthesis).

Results: Oreoch‑1 exhibited potent inhibitory activity with an MIC₉₀ of 25 μM, accompanied by reduced growth kinetics, a 67% reduction in biofilm formation, and an 85% reduction in hyphal formation. Microscopic analyses revealed pronounced morphological changes, including membrane disruption, surface deformation, and loss of structural integrity. Oreoch‑1 induced a clear dose‑dependent downregulation of both HWP1 and ERG11, with maximal suppression observed at MIC₉₀.

Conclusions: Collectively, these findings demonstrate that oreoch‑1 targets both structural and regulatory components of C. albicans pathogenicity. Its combined effects on growth inhibition, biofilm reduction, hyphal suppression, membrane disruption, and downregulation of virulence-associated genes highlight oreoch‑1 as a promising antifungal candidate for the management of C. albicans infections.