Antimicrobial resistance (AMR) represents a major global health challenge requiring coordinated action across human and animal sectors. While antimicrobial use (AMU) in livestock has been extensively studied, prescribing and treatment practices in companion animals remain comparatively underexamined despite the potential for resistant organisms to circulate within shared household environments. Understanding the drivers of antimicrobial exposure in pets is therefore critical for advancing stewardship efforts.

This study evaluated patterns of AMU in dogs and identified behavioural factors influencing treatment decisions among veterinarians and dog owners in Northern Ireland. A behavioural economics framework was applied to explore how time-related decision preferences, particularly present bias, may contribute to antimicrobial demand.

Questionnaire data from 355 dog owners were analysed to characterise prescribing trends and determine predictors associated with antimicrobial exposure. Overall, 58% of dogs had received antimicrobials at least once (mean treatment duration 6 days, SD = 2.06). Amoxicillin-clavulanic acid and metronidazole were the most frequently reported agents, commonly prescribed for skin or wound infections (25%) and gastrointestinal conditions (18%). Logistic regression demonstrated higher odds of antimicrobial use among older dogs (≥8 years; β = 0.11, p = 0.002), insured animals (β = 0.61, p = 0.011), and owners exhibiting present-biased decision-making (β = 0.52, p = 0.048). Owner demographics and household characteristics were not significantly associated with AMU. Although familiarity with AMR was limited (26%), most respondents reported strong trust in veterinary guidance (87%).

These findings suggest that behavioural influences play an important role in antimicrobial exposure in companion animals. Stewardship strategies that align with short-term decision priorities such as emphasising active management plans, structured follow-up, and clear clinical thresholds may help reduce precautionary prescribing. Strengthening behaviourally informed communication within veterinary practice could support more judicious antimicrobial use and contribute to broader One Health AMR mitigation efforts.

The global increase in antibiotic use in food animals has raised awareness of the need to implement interventions to reduce antibiotic misuse. In Peru, small-scale farmers often have limited knowledge about the proper use of antibiotics and tend to use them incorrectly. Bottom-up interventions and participatory approaches can promote sustainable and long-lasting behavioral changes by valuing farmers’ expertise and increasing program acceptability. We aimed to evaluate if participatory interventions can reduce antibiotic misuse on small- and medium-scale laying hen farms from the Ica and Lima regions in Peru. Considering previous surveys and interviews with farmers from the same area, and similar published evidence, the intervention was designed in collaboration with farmers through a prioritization workshop. Prioritized practices included acidification of feed or water, disposal of animal feces, hand hygiene, facility disinfection, and the use of exclusive farm clothing and boots. Intervention farms received training, veterinary advice and a kit consisting of infographics, logbooks, and a container for disposing of antibiotic packaging. Egg production, feed consumption, health incidents and antibiotic use were recorded over a six-month period. A total of 27 intervention farms and 24 control farms were enrolled. During the monitoring period, four farms withdrew from the intervention group and five withdrew from the control group. At baseline, the use of antibiotics as growth promoters was reported by 86.9% (20/23) of intervention farms and 73.7% (14/19) of control farms. Over the intervention period, most practices improved among intervention farms, except for exclusive clothing and boots, while control farms remained largely unchanged. By the end of the study, antibiotic use as a growth promoter remained high (91.3% intervention, 84.2% control). Additionally, other antibiotic misuse—including prophylaxis, metaphylaxis, and treatment of non-bacterial episodes—remained similar between groups (69.6% intervention, 63.2% control), suggesting that participatory interventions alone were insufficient to reduce antibiotic misuse. Quantitative antibiotic use will be estimated using weight-based indicators.

Glycopeptide antibiotics (GPAs) (vancomycin, teicoplanin, oritavancin, telavancin, dalbavancin) are clinically used to treat severe infections caused by multi-drug-resistant (MDR) Gram-positive pathogens and Clostridioides difficile. Most of these antibiotics are natural products or semisynthetic derivatives of natural products, blocking bacterial cell wall biosynthesis by binding to the terminal D-Ala-D-Ala terminus of the peptidoglycan precursor [1]. Recently, genome mining has emerged as a powerful approach for the identification of novel glycopeptide scaffolds and their previously unrecognized producing organisms [2]. In this framework, we screened 600 genomes belonging to the Pseudonocardiales order and identified 18 biosynthetic gene clusters (BGCs) predicted to encode previously unknown GPAs [3,4]. One of these molecules, named kineomicins from its producer strain Actinokineospora auranticolor, was produced up to an exceptionally high production rate, exceeding 1 g/L in a benchtop bioreactor. The resulting antibiotic complex was microbiologically characterized and the structure of its main congener, KmcB, was elucidated by LC-MS, MS/MS, and NMR spectroscopy, revealing a unique peptide scaffold [4]. Biological and chemical profiling of kineomicins is ongoing, highlighting the potential of this new antibiotic. Parallel studies on two additional putative GPA-producing strains are further validating genome mining as a key strategy in the discovery and development of new GPAs to counteract AMR spread.

[1] Marcone et al. 2018 Biotechnol Adv. 36(2):534-554. [2] Yushchuk et al. 2021. ACS Chem Biol. 16(5):915-928. [3] Andreo-Vidal et al. 2021. Antibiotics (Basel). 10(12):1533. [4] Yushchuk et al. 2025. Commun Chem. 8(1):134.

Introduction

Antimicrobial resistance (AMR) is a global health crisis and one of the top ten public health threats according to the WHO. In recent decades, few antibiotics have reached the market to tackle infections caused by multidrug-resistant (MDR) bacteria. Thus, new antimicrobials are needed.

This project evaluates the activity of PLP-3, a synthetic antimicrobial peptide designed in silico as a Protegrin-1 analogue, against a panel of MDR pathogens.

Methods

The antibacterial activity of PLP-3 was evaluated by determining the Minimum Inhibitory Concentration (MIC) against a clinical collection of MDR strains, including Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium. Time–kill curves (TKCs) were performed to assess bactericidal activity, and checkerboard assays were used to evaluate synergies with conventional antibiotics. The Minimum Biofilm Inhibitory Concentration (MBIC) and Minimum Biofilm Eradication Concentration (MBEC) were also determined. Finally, PLP-3-resistant mutants were generated and sequenced to elucidate the mechanism of action.

Results

PLP-3 MIC₅₀ and MIC₉₀ ranged between 1-4mg/L and 1-16mg/L across all Gram-negative and Gram-positive strains tested, respectively. TKC confirmed that PLP-3 is bactericidal at concentrations ≥ 2xMIC after 24h, with viable counts falling below the detection limit. Checkerboard assays showed that most antibiotic combinations were additive, achieving synergy with meropenem against a clinical strain of A. baumannii. As for PLP-3 antibiofilm activity, both MBIC₅₀ and MBEC₅₀ ranged between 4-32mg/L and 64->64mg/L for Gram-negative strains, respectively, and 1-16mg/L and 16->64mg/L for Gram-positive strains. Regarding resistant mutants, variant calling analysis indicated that the prevalent mechanism appears to involve a reduction in surface negative charge to minimize electrostatic interactions with PLP-3.

Conclusion

PLP-3 shows broad-spectrum bactericidal activity against MDR bacteria for planktonic and biofilm-associated cells, which is a promising candidate for the development of new antimicrobials to combat AMR.

The rising threat of antimicrobial resistance makes novel therapeutics necessary. In this study, we designed nature-inspired antimicrobial peptides (AMPs) and evaluated their biological potency and safety profile. The synthesized peptides exhibited exceptional antibacterial and antifungal activity, characterized by low minimum inhibitory concentration (MIC range: 0.5-2 µg/mL), values across all standard ATCC strains tested (E. coli, S. aureus, P. aureginosa and C.albicans). Meanwhile, cytotoxicity assays confirmed their selectivity as they displayed no significant toxic effects on mammalian cells. Building on this favorable safety index, the peptides also demonstrated high stability against proteolytic degradation, overcoming a common limitation of natural AMPs. To elucidate their mode of action, we employed molecular modelling to predict peptide-membrane interactions. These studies confirmed that the peptides compromise bacterial membrane integrity alongside membrane permeability assays. This finding was visually corroborated by scanning and transmission electron microscopy (SEM/TEM), which revealed irreversible structural damage and cell lysis. Furthermore, we evaluated kinetics of resistance development by exposing E. coli to serial passages of the peptides versus gentamicin. While the bacteria rapidly developed high-level resistance to gentamicin, no resistance was observed against our peptide candidates. These results show that the designed AMPs are potent and safe and offer a robust solution to resistance development. Their unique membrane-targeting mechanism, combined with a high safety profile and an inability to induce rapid resistance, positions them as promising candidates for future clinical development against multidrug-resistant infections.

Introduction

Wastewater-based epidemiology (WBE) relies on the assumption that antibiotics and antimicrobial resistance (AMR) markers remain stable during sewer transport, allowing wastewater measurements to reflect community-level antibiotic consumption and resistance trends. However, evidence suggests that in-sewer processes, largely linked to biofilm activity, can cause antibiotic degradation and may affect the distribution of antimicrobial resistance genes (ARGs). This creates uncertainty as to whether wastewater signals represent population-level trends or whether they are influenced by in-sewer changes. To address this, we investigated the stability and behaviour of 11 antibiotics and 54 ARGs in laboratory-scale sewer pipes (operated for > 12 months prior to sampling), using influent wastewater from the North-East of Scotland. Clarithromycin and its associated resistance gene ermB_1 were examined in detail.

Methods

Four pipes (clay and plastic, with and without biofilms) were operated in triplicate. Wastewater was sampled over 24 hours without disturbing biofilms. Antibiotics were quantified using direct injection UPLC-MS/MS, while ARG behaviour was assessed using SmartChip high-throughput qPCR.

Results

Biofilm pipes showed greater antibiotic degradation than biofilm-free controls, with clarithromycin degradation reaching 59–65% over 24 hours compared to 20–23% in controls, confirming a biofilm-driven effect on degradation. This suggests that estimation of community antibiotic usage using WBE may require corrections for in-sewer losses. Despite this, the abundance of ermB_1 did not increase in the wastewater. Instead, biofilm systems exhibited equal or lower ermB_1 levels than biofilm-free pipes, with plastic biofilm pipes showing both the highest antibiotic degradation and the lowest ARG signal. This indicates that tracing ARG abundance using WBE may be better suited to assessing long-term trends rather than short-term changes.

Conclusions

These findings demonstrate that sewer biofilms influence antibiotic stability without necessarily promoting increased ARG abundance in wastewater. This supports more robust interpretation of WBE data across multiple antibiotics and resistance genes, improving confidence in wastewater-based AMR surveillance.

Helicobacter pylori is a major human pathogen and a type I carcinogen, the transmission dynamics of which remain incompletely understood. From a One Health perspective, the interaction between environmental reservoirs, human hosts, and antimicrobial resistance represents a major concern. The Bogotá River basin, impacted by pollution and used extensively by communities, constitutes an ecological interface for pathogen circulation and genetic exchange.

Objective: The aim of this study was to characterize the environmental and clinical molecular profiles of H. pylori in the Bogotá River basin, focusing on virulence genotypes and clarithromycin resistance, and to assess the potential role of contaminated water in shaping bacterial diversity and antimicrobial susceptibility.

Methods: Fifty-one water samples from different sections of the Bogotá River and 24 gastric biopsies from exposed residents were analyzed. Detection of H. pylori was performed by PCR, targeting the vacA gene. Virulence genotyping included vacA alleles (s, m, i), cagA, and glmM. Clarithromycin resistance was evaluated by sequencing the 23S rRNA gene to identify resistance-associated mutations.

Results: H. pylori DNA was detected in 41.2% of river water samples and 50% of gastric biopsies, demonstrating dissemination. River samples displayed highly heterogeneous virulence genotypes, dominated by glmM(+) cagA(–) vacA s1m1 (28.6%) and s1m1i1 (19%). No water sample harbored the cagA gene, and viable bacteria could not be cultured, suggesting persistence primarily in non-culturable but potentially infectious states. Critically, all environmental samples exhibited wild-type 23S rRNA sequences, indicating absence of clarithromycin resistance. In contrast, 25% of clinical isolates carried the A2143G mutation, which is a key determinant of clarithromycin resistance.

Conclusions: These findings support a One Health model in which the aquatic environment acts as a reservoir of H. pylori genetic material that may contribute to bacterial evolution and antimicrobial resistance in human hosts through natural transformation. The clear divergence in clarithromycin susceptibility between environmental and clinical samples underscores the role of human-associated selective pressures.

Our project investigates the transmission dynamics of antimicrobial resistance (AMR) across the food chain, focusing on the dissemination of antibiotic resistance genes (ARGs) and resistant bacteria. We integrate whole metagenome sequencing with culture-based approaches to systematically characterize ARG reservoirs in environmental and food matrices and to evaluate potential connectivity between compartments.

Sampling encompasses manure, agricultural soils prior to manure application, post-fertilization soils, crop products, meat products, and dairy fermented products. For each sample type, we perform taxonomic profiling and quantify total ARG abundance and diversity. Metagenome-assembled genomes (MAGs) are reconstructed to identify ARG-carrying bacterial hosts within complex microbial communities and to guide targeted cultivation strategies. In parallel, antibiotic-resistant bacteria are recovered from all matrices using selective and non-selective approaches. Isolates are subjected to phenotypic antibiotic susceptibility testing to establish resistance profiles, followed by whole genome sequencing for detailed characterization of ARG content, genomic context, and associated mobile genetic elements. Comparative genomic analyses are conducted to assess genetic relatedness among isolates and to explore potential links between environmental, primary production, and food-associated compartments.

Preliminary analyses indicate overlapping ARG patterns between manure and fertilized soils, and in some cases similar genotypic contexts in isolates recovered from different matrices. We have also achieved the genomic-guided recovery of enterobacteria with diverse ARGs. However, the primary emphasis of this work lies in the establishment of a robust, integrative methodological framework that combines metagenomics, culturomics, resistance phenotyping, and high-resolution genomics and creates a specific database to facilitate the analysis of our findings.

By applying this coordinated One Health approach, the study provides a comprehensive platform for investigating AMR distribution and resistance profiles across the food chain, supporting future risk assessment and mitigation strategies grounded in harmonized multi-compartment data generation.

Plasmids are self-replicating pieces of DNA that are particularly prominent in bacterial genomes. In addition to maintenance genes, plasmids typically carry cargo that allows cells to adapt to environmental challenges (such as exposure to antibiotics) or exploit new niches. The reason why these genes are kept in plasmids as opposed to integrating in the chromosome as not well understood, particularly considering the fitness cost of maintaining plasmids, a question known as "the plasmid paradox". Transient integration into the chromosome in the form of ICE (Integrative Conjugative Elements) appears to be part of the answer. Here we looked for other forms of plasmid integration. We collected 10,000+ complete genomes of E. coli from a NCBI. We identified MGEs associated with a plasmid origin of replication in the chromosome, with default parameters (95% id and 60% cov) and found 262 origins of replication in chromosomal sequence. Of these, 147 (56%) belonged to the IncQ incompatibility type and between 13 and 27 to IncF (5-10.3%). The high representation of IncQ origins of replication is remarkable. Unlike most plasmids, which replicate via a theta mode of replication, IncQ plasmids replicate by strand displacement, which includes replication initiation machinery for both strands, and limits plasmid size to less than 15 kb. We also found that, despite its size limitations, chromosome-integrated IncQ plasmids carry a significant load of antibiotic resistance genes. In the following cases: APH(3’)-Ia, sul2, APH(3’’)-lb, APH(6)-Id, catI, TEM-1, and CTX-M-1, integrated plasmids present between 5 and 7% of the total representation of the corresponding antibiotic resistance gene in the database. Thus, IncQ-mediated integration in the chromosome appears to facilitate the vertical transmission of a significant number of antibiotic resistance genes.

The rapid rise of antimicrobial resistance urges to develop new antimicrobial strategies that fit within a One Health perspective. Leaderless bacteriocins are attractive candidates, but because they are synthesized in their active form inside the cell, they raise a key question: how do producer bacteria protect themselves, and what does this mean for their safe use as antibiotic alternatives? To harness these molecules therapeutically, we need a clear picture of how they are produced and how immunity is ensured in the producing strain.

Enterocin DD14 (EntDD14) is a two-peptide leaderless bacteriocin produced by Enterococcus faecalis 14 that shows antibacterial, antiviral and immunomodulatory activities. Yet the molecular basis of self-immunity during active EntDD14 production has remained unclear. Here, we focused on DdD, a protein encoded within the EntDD14 biosynthetic cluster. In silico analyses suggested that DdD is a membrane-associated protein, and our genetic and functional data show that it is not required for EntDD14 production or export, but is crucial for self-immunity. Deleting ddD dramatically reduced resistance to both intracellularly produced and exogenously added EntDD14, whereas complementation with ddD restored full resistance.

By identifying DdD as a new and highly effective immunity factor, our work updates current models of leaderless bacteriocin biology and underline the importance of accessory proteins as protective “safety locks”, providing new insights into the defense mechanisms associated with leaderless bacteriocin production. These findings can be exploited to engineer safer bacteriocin-producing probiotics, limit off-target toxicity, supporting the responsible use of bacteriocins as antibiotic alternatives in One Health strategies against antimicrobial resistance.