Antimicrobial resistance is a global health threat, calling for new antibiotics. Good candidates could be peptide-based compounds that target special lipids that only exist in bacterial, but not in human cell membranes. These drugs kill pathogens without detectable resistance, which has generated considerable interest.

Here, using an integrative structural biology approach, we show that drugs that target special lipids in bacterial membranes use sophisticated supramolecular killing mechanisms^1-3.

1. Shukla, R. et al. Teixobactin kills bacteria by a two-pronged attack on the cell envelope. Nature 608, 390-396, doi:10.1038/s41586-022-05019-y (2022).
2. Shukla, R. et al. An antibiotic from an uncultured bacterium binds to an immutable target. Cell 186, 4059-4073.e4027, doi:10.1016/j.cell.2023.07.038 (2023).
3. Jekhmane, S. et al. Host defence peptide plectasin targets bacterial cell wall precursor lipid II by a calcium-sensitive supramolecular mechanism. Nature Microbiology 9, 1778-1791, doi:10.1038/s41564-024-01696-9 (2024).
4. Ntallis, C., Martin, N., Edwards, A., Weingarth, M. (2025) Nature Reviews Microbiology, Bacterial cell envelope-targeting antibiotics

Cystic fibrosis (CF)-associated lung infections caused by Pseudomonas aeruginosa and Staphylococcus aureus remain difficult to treat due to multidrug resistance and the redox instability of the pulmonary environment, which can impair antibiotic efficacy. In this study, we investigated alvinellacin (ALV), a disulfide-stabilized β-hairpin antimicrobial peptide derived from the deep-sea polychaete Alvinella pompejana, as a potential therapeutic agent naturally adapted to redox-fluctuating conditions. The antibacterial and antibiofilm activities of ALV were evaluated against multidrug-resistant clinical isolates under CF-like reducing conditions (6 mM DTT). Circular dichroism analysis showed that DTT did not alter the β-hairpin secondary structure of ALV, supporting its structural stability in CF-like environments. Mechanistic analyses included pore-forming assay, membrane interaction studies, scanning electron microscopy, lipid-binding assays, cytotoxicity testing, and resistance induction assays, while in vivo efficacy was assessed using the Galleria mellonella infection model. ALV demonstrated strong bactericidal activity that was maintained in the presence of NaCl or human serum. ALV did not induce bacterial resistance and effectively inhibited early-stage biofilm formation and disrupted preformed biofilms, including those of the clinical isolate, even under reducing conditions. The peptide showed selective permeabilization of bacterial membrane linked to its stronger affinity for bacterial membrane lipids and negligible interaction with host-like membranes, with no observed cytotoxicity. In vivo, ALV significantly improved survival in infected larvae. These findings highlight ALV as a promising redox-resilient antimicrobial candidate for treating multidrug-resistant CF lung infections.

Over the past decade, the antimicrobial pipeline has produced several new agents targeting multidrug-resistant Gram-negative pathogens; however, their impact has been limited by the rapid emergence of resistance and heterogeneous activity across resistance mechanisms. Metallo-β-lactamase-producing Enterobacterales and highly resistant Pseudomonas aeruginosa remain challenging targets. Parallel strategies, including microbiome modulation, bacteriophage therapy, and immunotherapeutic approaches, are under investigation and may complement antibiotic therapy, although robust clinical evidence is still evolving. RCT conducted to date have provided important insights but remain limited in their ability to inform optimal clinical management. These studies are typically small, heterogeneous, and primarily designed to support regulatory approval rather than to define treatment strategies for critical ill patients. Consequently, individuals with true “no-option” DTR infections are underrepresented, and the applicability of trial findings to real-world clinical decision-making remains constrained. Guideline development reflects these limitations, with recent recommendations incorporating new antimicrobial agents but often lacking prioritisation and adaptability to local epidemiology, diagnostic capacity, and antimicrobial availability. This has contributed to variability in implementation across regions and highlights the need for more dynamic, evidence-linked clinical algorithms. The presentation will discuss the need to fundamentally rethink clinical trial design. Traditional RCT are poorly suited to the complexity and heterogeneity of DTR infections. Adaptive platform trials offer a more efficient and ethically robust alternative. These designs allow multiple interventions to be evaluated simultaneously and support the continuous evolution of treatment strategies based on accumulating data. Successful advancement in this field will depend on improved data harmonisation, integration of real-world evidence, and the development of perpetual cohort-based platforms. Ultimately, addressing DTR Gram-negative infections requires not only new therapeutics but also a transformation in how clinical evidence is generated, moving from drug-centred evaluation to patient-centred optimisation of treatment strategies in highly vulnerable populations.

Antibiotic resistance is a major global issue requiring coordinated action from researchers, industry, and healthcare systems. Affecting human, animal, and environmental health, it is now addressed through the “One Health” approach. The World Health Organization has identified priority pathogens capable of developing multidrug resistance and emphasizes the urgent need for new treatments, regulatory strategies, and diagnostic tools.

Rapid and accessible detection methods are essential, particularly for low-resource settings. Currently, the antibiogram remains the standard method but requires 48–72 hours, limiting timely decision-making. Faster alternatives are therefore needed.

Among resistance mechanisms, efflux pumps—identified since the 1980s—play a key role. These systems expel toxic compounds, including antibiotics and antiseptics, preventing them from reaching effective intracellular concentrations. Their overproduction is frequently observed in multidrug-resistant bacteria, making them an important target for detection. Identifying strains with hyperactive efflux can improve therapeutic choices and help limit the spread of resistance.

Based on the ability of efflux pumps to transport diverse molecules, we investigated fluorescent phenazinium derivatives as potential substrates. Compounds synthesized by Olivier Siri’s team were evaluated by comparing their accumulation in bacterial strains with varying efflux activity. This approach led to the identification of a molecule capable of distinguishing between normal, inactive, and overproducing efflux systems.

After optimization, the method was tested on around 100 Staphylococcus aureus isolates previously characterized by conventional techniques, achieving over 80% accuracy. It was also successfully applied to other Gram-positive bacteria. With slight modifications, the method was extended to Gram-negative species such as Escherichia coli and Klebsiella, with further validation ongoing.

This test is rapid, cost-effective, and easy to perform, delivering results within hours and saving up to 48 hours compared to standard methods. It has been patented and is currently marketed as Colorflux®.

The increasing prevalence of antibiotic-resistant bacteria highlights the urgent need for alternative antimicrobial agents derived from natural sources. In this study, we investigated the organ-specific phytochemical composition and antibacterial potential of hydroethanolic extracts obtained from flowers, leaves, and stems of Ebenus pinnata Aiton, an endemic Moroccan species. Comprehensive metabolite profiling was performed using UHPLC–ESI–MS, while total phenolic (TPC), flavonoid (TFC), and proanthocyanidin (TPrC) contents were quantified spectrophotometrically. Antibacterial activity was evaluated using agar diffusion, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) assays against representative Gram-positive and Gram-negative bacteria.

UHPLC–ESI–MS analysis revealed pronounced organ-specific chemical signatures. Flowers were enriched in flavanols, particularly epigallocatechin, alongside flavonoids and phenolic glycosides. Leaves were dominated by glycosylated flavonoids and hydroxycinnamic acids, whereas stems accumulated diverse phenolic acids, proanthocyanidins, and triterpenoids, including ursolic acid. Quantitative assays confirmed significant inter-organ variability, with stems exhibiting the highest TPC, TFC, and TPrC values, followed by leaves and flowers. Antibacterial assays demonstrated a clear selectivity toward Gram-positive bacteria. Flower and stem extracts showed the strongest activity, particularly against Micrococcus luteus and Staphylococcus aureus, with low MIC values (2.60–5.20 mg/mL) and bactericidal effects (MBC/MIC ≤ 2.50). In contrast, Gram-negative strains were markedly less susceptible.

Antibacterial efficacy correlated more strongly with the qualitative composition of phenolic constituents, especially flavanols and proanthocyanidins, than with total phenolic abundance. These findings highlight the importance of organ-specific phytochemical allocation in shaping antimicrobial activity and position E. pinnata, particularly its floral and stem tissues, as a promising source of bioactive phenolics with potential relevance for the development of plant-derived antibacterial agents.

The global rise in antibiotic-resistant bacterial infections necessitates innovative strategies beyond conventional antibiotics. Phage-derived depolymerases selectively degrade bacterial capsules and biofilms, weakening pathogen defenses and enhancing immune clearance. This approach offers a highly specific and resistance-sparing intervention. Our study aims to determine whether phage depolymerases can enhance the efficacy of phage therapy, potentially serving as a novel anti-virulence strategy for bacterial infections. A panel of seven lytic Acinetobacter baumannii phages, isolated from local sewage samples, and six recombinant depolymerases were employed. The effects of depolymerases on each phage's host range and bacterial growth inhibition were assessed using spot-testing assays and bacterial growth curves. Synergy assays demonstrated that combining varying concentrations (0.8–50 ng/mL) of depolymerases with 10²–10⁸ PFU/mL of phage significantly inhibited the growth of several A. baumannii strains. This combination outperformed high doses of either phage or depolymerase alone, as shown by area under the curve analysis, achieving up to 85% growth inhibition compared to the controls. Notably, certain phage–depolymerase combinations led to a remarkable six-log increase in the phage endpoint titer. Efficiency of plating studies further showed that depolymerases expanded the phage host range, enabling broader therapeutic applications without the need for phage adaptation or engineering. To our knowledge, this is the first study to show that phage depolymerases enhance both the efficacy and host range of therapeutic phages. The promises and challenges of exploring phage depolymerases as a strategy to combat antimicrobial resistance, particularly against bacteria that produce protective capsules or biofilms, will be discussed.

Introduction
The role of the environment in the dissemination of antimicrobial resistance is increasingly recognized within the One Health framework. The airborne transmission route of antibiotic resistance genes (ARGs) is particularly significant since ARG-carrying bioaerosols could travel long distances and remain in the atmosphere for a long period of time. In addition, mobile genetic elements (MGEs) can promote the spread of ARGs in the environment.

Objective
This study aimed to characterize the seasonal dynamics of the airborne resistome and mobilome across diverse environments in the Belgrade metropolitan area, Serbia.

Methods
Outdoor air samples were collected at several urban, suburban, and rural locations across four seasons. Airborne DNA was subjected to shotgun metagenomic sequencing (Illumina NovaSeq X Plus), followed by bioinformatic analysis. Resistance genes were annotated using MEGARes, while integrons, insertion sequences (ISs), and plasmids were identified using Integrall, ISfinder, and PLSDB, respectively.

Findings
Shotgun metagenomic analysis revealed pronounced seasonal variation in the composition and abundance of the airborne resistome across all sampled environments. ARGs dominated the resistome across all seasons, followed by metal resistance genes (MRGs) and multi-compound resistance genes (MCRGs). Resistome abundance and gene richness increased progressively from spring to winter, accompanied by a higher number of season-specific resistance genes during autumn and winter. In addition, the airborne mobilome was dominated by integron- and plasmid-associated sequences, while ISs accounted for a smaller proportion of the total mobilome. Total MGE abundance exhibited a modest seasonal trend, with winter tending to show higher levels than other seasons. MGE diversity and the number of season-specific elements increased toward winter, driven mainly by plasmid-derived sequences.

Conclusion
Our results demonstrate that air represents a dynamic environmental reservoir and potential dissemination pathway for antimicrobial resistance across the Belgrade metropolitan area, emphasizing the importance of incorporating airborne environments into One Health surveillance strategies.

Antimicrobial resistance (AMR) is increasingly recognised as a significant health challenge, driven not only by the clinical use of antibiotics but also by the dissemination of antibiotic residues and resistance genes (ARGs) through wastewater systems and natural ecosystems. Spatially explicit exposure models, such as ePiE, quantify antibiotic concentrations in surface waters; however, current environmental AMR risk assessment methods rely on simplified assumptions about microbial community responses. Existing approaches, such as the 8-day SELECT bioassay, typically infer selection from growth inhibition thresholds without explicitly capturing differential growth dynamics between susceptible and resistant bacteria, offering limited mechanistic insight into how AMR selection emerges under realistic environmental exposure scenarios.

In this study, we aim to bridge this gap by integrating experimental and modelling approaches. First, we will adapt an in vitro growth inhibition bioassay into a co-culture competition assay that simultaneously exposes fluorescently labelled wild-type and ciprofloxacin-resistant Escherichia coli strains to antibiotics. By quantifying shifts in relative abundance, this assay will provide a direct and time-efficient measure of AMR selection pressure. Second, we will employ data from both current and adapted assays to develop mechanistic in silico models describing antibiotic-dependent growth inhibition for susceptible and resistant strains. These models will generate validated rules for bacterial growth and competition, supporting the development of agent-based simulations of environmental microbial communities.

Together, this work will establish an experimentally grounded and mechanistically informed framework for evaluating AMR selection in environmental contexts, enabling more robust prospective risk assessments for antibiotics and supporting stronger environmental protection strategies.

In this work, the hierarchical cluster analysis (HCA) of time series is proposed for monitoring antimicrobial resistance (AMR). The purpose was to develop a new mathematical tool or new applications that could allow the identification of pairs of bacteria/antimicrobial agents that require early intervention or monitoring.

Time series data of resistant bacteria/antimicrobial agent pairs was obtained from the public website of ECDC in 2023.

Variables: Resistant isolate percentage values in Portugal from 2012 to 2021 were obtained. Methods: HCA was performed directly on the time series data using the standard Affinity coefficient and Euclidean distance. The complete linkage was used as the aggregation criteria. Results: Enterococcus faecium|Aminopenicillins presents very high resistance (87,96% +/- 3,12 [min-83,66/max-94.44] and Escherichia coli |Carbapenems presenting the lowest resistance values (0,18% +/-0,17 [min-0,02/max-0,53]; this is quite curious, since the WHO considers this pair as a critical priority. The HCA with the (1-standard Affinity coefficient) distance showed three different clusters. The first cluster includes K. pneumoniae |Carbapenems and E. coli |Carbapenems. The third cluster includes E. faecium|Vancomycin, E. faecium| Gentamicin, Enterococcus faecalis|Vancomycin, Acinetobacter spp.|Aminoglycosides, Fluoroquinolones, and Carbapenems. The second cluster is a heterogeneous group showing some clusters not analyzed here. The first cluster includes two Enterobacteria considered Critical Priority by the WHO. These bacteria can produce several resistance mechanisms, namely carbapenemase enzymes. The third cluster includes Acinetobacter spp. with all antimicrobial agents studied, including Carbapenems, Enterococcus with Vancomycin and Gentamicin. Conclusions: This was the first time that the Affinity coefficient and Euclidean distance were applied to the AMR profile. The standard Affinity coefficient (Silhouette coefficient’s (0.8)) presented to be more suitable than Euclidean distance (Silhouette coefficient’s value (0.4)) for identifying patterns and trends in resistance values in AMR data because it was shown to be more robust in capturing similar behaviors in resistance values over time and a better cluster compactness and separability.

Antibacterial resistance is rising globally, with particular concern involving multidrug-resistant Gram-negative pathogens listed in the WHO Global Priority List (2024), i. e. carbapenem-resistant and third-generation cephalosporin-resistant Escherichia coli and Klebsiella pneumoniae and also carbapenem-resistant Pseudomonas aeruginosa. In this study, we present a dual strategy to reduce polymyxin B and colistin toxicity while maintaining efficacy in the murine model of infection. We used a disulfide-based chemical modification (soft-drug approach) alongside a combination therapy with a clinically approved compound. We will present in vitro microbiological testing (MIC against MDR gram negative bacteria including colistin-resistant strains such as K. pneumoniae ST147), in vivo pharmacokinetic data, acute toxicity and safety evaluation (nephrotoxicity and biomarkers KIM-1, clusterin, NGAL, THF-α) and efficacy in clinically relevant murine infection sepsis models. Taken together, the combination approach increased the tolerated dose of polymyxins by up to fivefold in mice, from 20 to 100 mg/kg subcutaneously and from 4 to 20 mg/kg intravenously, while maintaining antibacterial efficacy.

(https://jpiamr.eu/projects/muryxin/)