The global healthcare community faces escalating threats from antimicrobial resistance (AMR); therefore, the need for novel, non-traditional antimicrobials has become increasingly urgent. Antimicrobial peptides (AMPs), naturally produced by a wide array of organisms, including marine invertebrates, offer a promising alternative to conventional antibiotics. AMPs typically act by disrupting microbial membranes, a mechanism less prone to resistance development. Mussels, being sessile filter feeders in pathogen-rich marine environments, have evolved expansive and diverse innate immune systems. The genomic diversity and adaptive resilience of Mytilus spp. remain underexploited resources for drug discovery, particularly in the search for new AMPs with clinical and ecological relevance.

The central objective of the project is to identify, functionally validate, and characterize novel AMPs from Mytilus genomes. The research plan integrates in silico genomic screening with experimental in vitro validation. Initially, genome assemblies and transcriptomic datasets from multiple Mytilus species will be analyzed to identify gene families coding for AMP-like sequences. The bioinformatics strategy includes sequence motif detection, gene clustering, evolutionary analysis of presence/absence variation, and structural modeling of predicted peptides. These tasks will be supported by national HPC resources provided through the PLGrid infrastructure, which allows high-throughput data processing, molecular simulations, and structural predictions. Experimentally, we will establish short-term primary cultures from Mytilus hemocytes and digestive gland cells. These cultures will serve as models to assess immune gene expression and AMP induction under controlled stimulation. Culture viability and responsiveness will be monitored using fluorescein diacetate (FDA) assays and quantitative PCR. Peptides derived from highly expressed or computationally prioritized genes will be purified either from conditioned media or synthesized chemically when necessary. Functional testing will involve comprehensive antimicrobial profiling against a library of multidrug-resistant strains.

Project No. 2025/57/B/NZ8/03838 funded by the National Science Centre (Poland).

β-lactams represent cornerstone antibiotics, but resistance towards them is widespread [1]. Of particular concern are β-lactam-resistant Gram-negative bacteria, for which alternative therapeutic options are extremely limited. To address this unmet medical need, we developed a natural product screening platform for searching antibiotic adjuvants to be used in combination with β-lactams against multidrug-resistant Gram-negative (MDR-GN) bacteria. To this end, we first selected five clinically-relevant models of MDR-GN characterized by β-lactam resistance (with MICs of ceftriaxone, meropenem, and/or ceftazidime–avibactam in the range 8-1024 µg/ml) and we identified the β-lactamases acting as resistance determinants (namely, CTX-M, KPC-3, KPC-31, VIM-1, and VIM-2). Then, we performed the high-throughput biological activity-guided screening of a microbial library comprising 39,000 crude extracts mainly obtained from actinomycetes’ and filamentous fungi’s culture broths. The screening assay was developed to select putative HITs with no or negligible direct antimicrobial activity, but capable of counteracting resistance mechanisms by inhibiting β-lactamase activity. Notably, >200 HITs capable of restoring the activity of β-lactams against the selected resistant isolates, even when the antibiotics were used at 1/4 or 1/8 the MIC value, were identified. The candidates were further subjected to activity-guided purification, resulting in the isolation of several compounds with the desired adjuvant activity, which underwent chemical and biological characterization for dereplication and novelty assessment. Preliminary findings suggested that the observed bioactivities were mostly associated to metal chelating agents. As further perspectives, an in-depth characterization of these compounds is being conducted, to better elucidate their mode of action through in vitro and in silico studies, to test their efficacy in conjunction with β-lactams against a broader panel of MDR-GN clinical isolates, and to assess their cytotoxicity on various eukaryotic cell models, ultimately paving the way for their possible preclinical development and prospective therapeutic application.

[1] GBD 2021 Antimicrobial Resistance Collaborators. (2024). The Lancet. 404(10459):1199-1226.

Early and adequate empiric antibiotic therapy is essential in the treatment of pneumonia–sepsis, influencing clinical outcomes. This retrospective before–after study aimed to appraise the impact of local ASP (written guidelines and antibiotic restriction) on antibiotic (AB) use and clinical outcomes in patients requiring intensive care due to pneumonia–sepsis. This single-center retrospective observational study was conducted in the ICU of a pulmonology department in Hungary. Data collection for the pre-intervention period occurred from January 2018 to May 2022, while that for the ASP period was from June 2022 to March 2024. The patients admitted to the ICU with pneumonia–sepsis were mainly men (58/101, 57.4% and 84/128, 65.6%); the need for ICU increased with age, and most of the patients belonged to 65+ age group in both study phases (68.3% and 58.6%). The majority of the patients had four or more comorbidities (57.4% and 40.6%). In-hospital mortality was relatively high (42.6% and 41.4%), with most of the patients already losing their lives in the ICU (33/43, 76.7% and 37/53, 69.8%). A significant increase in guideline-adherent agent selection (34.5%) and the use of combination therapy (35.0%) was observed, while the use of fluoroquinolones decreased significantly (-31.1%). In the after period, a significant decrease in the number of patients using restricted ABs (-53.3%) was observed. In one-third of these cases (10/34, 29.4% and 16/40, 40%), 2–4 MDR pathogens were detected simultaneously, resulting in a significant increase in direct costs (10.5%). The use of inappropriate AB therapy was relatively low in the presence of MDRs in both phases (5.9% and 15%). In the ASP period, guideline adherence was associated with slightly better clinical outcomes (30-day mortality: -0.8% and LOS: -22.6%). Interrupted time series analysis (ITS) indicates a decrease in the trend of AB use in the ICU (from 0.16 /30 days to -0.22 /30 days). ASP implementation in the ICU resulted in a significant improvement in the appropriate use of ABs, and guideline adherence led to slightly better clinical outcomes. ASP may hold promise in improving AMR with a sustained long-term effect.

INTRODUCTION: Carbapenemase-producing organisms (CPOs) pose a major global health threat. According to EARS-Net, Greece has one of Europe’s highest CPO-incidences in healthcare-associated infections, endemic for KPC, NDM, VIM, and OXA-48-like-carrying CPOs, but not for IMP. This study was conducted on metagenomes recovered from intensive dairy farms in Greece and harbored bla_IMP-determinants to explore their genetic context and dynamics underlying bla_IMP-dissemination, adopting a One Health-related approach.

METHODS: Short reads and contigs were comparatively analyzed using BLASTn, MEGA, and Easyfig software; assembled with MEGAHIT; and mapped to references (MZ816709.1, AJ550807.1) using Burrows-Wheeler-Aligner and SAMtools.

RESULTS: Analyses revealed that bla_IMP-associated reads were identical or highly similar to bla_IMP-13. Numerous reads and contigs covered the entire length of a Tn402-like transposon, including transposase (tnpA/R/M, tniA/B) and mercury-resistance (merRTPCADE) genes and a class-1 integron harboring bla_IMP-13 variant, aac(6′), aadA1, qacEdelta1, and sul1, but lacking qacG2 genes.

DISCUSSION/CONCLUSIONS: Tn402-like transposons as carriers of class-1 integrons harboring bla_IMP-13 variants have mainly been restricted to isolates from human clinical settings, promoted by the intensive use of carbapenems in these environments. Since carbapenems are banned from use in food-producing animals, it is surprising to note this highly mobile genetic element to be recovered from dairy cattle feces and environments. This suggests that any associated horizontal transfer could introduce bla_IMP-13 variants into novel hosts, with the human–agricultural interface being a critical hotspot for spillovers. Given that bla_IMP has not yet been reported in human clinical settings in Greece, moving personnel from areas with high bla_IMP-13-carrying CPO incidence could be a potential source, creating secondary reservoirs and forming a cycle that changes traditional epidemiological boundaries. This is highly concerning, as widely used veterinary antibiotics could provide co-selection pressure, favoring the spread of bla_IMP-13-variant-carrying CPOs. Our findings highlight the need for proactive metagenomic surveillance and strengthened monitoring strategies.

Antibiotic resistance is a global problem that requires different strategies, including new types of antibiotics. Methicillin-resistant Staphylococcus aureus (MRSA) and drug-resistant Pseudomonas aeruginosa are involved in skin infections, ventilator-associated pneumonia, and bacteremia. This poster reports an improved peptide antibiotic for treating such challenging infections. We designed a minimal peptide verine previously by combining LL-37 membrane-targeting elements with our peptide design concept for systemic use (PNAS 2020;117:19446-19454). Here we advanced verine based on the findings from the antimicrobial peptide database version 6 (APD6; Nucleic Acids Res. 2026;54:D363-D374). Aksarbin, the improved peptide, showed a favorable in vitro cytotoxicity profile to six cell lines comparable to daptomycin. In vivo, the peptide exhibited a single maximum tolerated dose exceeding 100 mg/kg and demonstrated no toxicity to mice following one week of intravenous injection at the treatment dose of 10 mg/kg. Aksarbin was potent against gentamicin, piperacillin, ceftazidime, colistin-resistant clinical strains, New Delhi Metallo-β-lactamase (NAM) strains, gram-positive MRSA, and extended-spectrum beta-lactamase (ESBL) gram-negative Escherichia coli, P. aeruginosa, and Klebsiella pneumoniae strains. It also showed antibiofilm capability and suppressed bacterial virulence factors based on transcriptomic and proteomic studies. In addition, bacteria did not develop resistance to aksarbin after multiple passages experiment. Significantly, aksarbin demonstrated topical efficacy in S. aureus USA300 infected biofilm wounds, and systemic efficacy in P. aeruginosa-infected bacteremia and overnight infected pneumonia murine models. Combined, these results underscore the advance in designing antimicrobial peptides with systemic efficacy and the therapeutic potential of aksarbin in combating drug-resistant pathogens, especially difficult-to-kill gram-negative bacteria.

This study aims to identify and characterize, through in silico approaches, potential inhibitors of serine- and metallo-β-lactamases, enzymes directly associated with antimicrobial resistance (AMR). The ChEMBL database was screened to select molecules with reported bioactivity against TEM-1 (PDB ID: 1ERM) and KPC-2 (PDB ID: 6D15), both class A enzymes, and BcII (PDB ID: 2M5D), a class B enzyme. Based on IC₅₀ values, 200 molecules were selected for TEM-1, 89 for KPC-2, and 100 for BcII. The protonation states of the selected compounds at pH 7.4 were determined using MarvinSketch. Three-dimensional structures were built in Discovery Studio Visualizer and optimized using the semiempirical PM7 method implemented in MOPAC. Protocol validation was carried out by redocking the co-crystallized ligands, followed by molecular docking simulations of the selected compounds. Docking results were analyzed using dbCICA, which correlates binding affinity with interaction patterns in the enzyme active site. Post-processing of docking data enabled the identification of intermolecular interaction patterns that may assist the rational design of novel inhibitors. The most active ligands established multiple hydrogen bonds with conserved catalytic residues, including SER70, SER130, GLU166, ASN170, and LYS73. In contrast, less active compounds exhibited fewer interactions with these key residues. The dbCICA models showed limited predictive performance, with low coefficients of determination (r² = 0.24 for TEM-1, 0.37 for KPC-2, and 0.29 for BcII). Among the evaluated residues, GLU166 was consistently identified as relevant for both class A enzymes. Despite the modest statistical performance, the findings indicate that potent inhibitors tend to interact with essential catalytic residues, providing mechanistic insights. These interaction signatures will guide pharmacophore modeling and ZINC-based virtual screening, followed by in vitro validation to identify β-lactamase inhibitors as antibiotic adjuvants against AMR.

Bacterial infections pose a significant threat to global health, especially with the growing resistance to antibiotics. Among critical priority pathogens, Pseudomonas species are of particular concern due to their intrinsic resistance mechanisms and capacity to acquire novel resistance genes. Despite their clinical importance, relatively few studies have characterized antimicrobial resistance genes (ARGs) in environmental Pseudomonas strains, especially those encoding β-lactamases.

In this study, we analyzed a collection of 56 bacterial genomes isolated under carbapenem antibiotic selection from municipal and hospital wastewater systems across Ontario. Whole-genome sequencing revealed that 33 of these isolates belonged to 11 Pseudomonas species; further annotation in CARD and ResFinder databases identified the presence of known ARGs across all isolates. However, most of the carbapenem-resistant isolates did not show observable β-lactamases or carbapenemase gene determinants. To investigate the nature of the antibiotic resistance, we constructed cosmid-based genomic libraries using DNA extracted from highly resistant Pseudomonas isolates. After confirming carbapenem resistance, the positive clones were sequenced using Oxford Nanopore Technology. Further analysis showed a group of clones with overlapping regions when mapped to the reference genome, containing several uncharacterized coding sequences. Through functional screening of these clones, subcloning and Tn5 mutagenesis, we aim to identify and characterize novel resistance genes, with particular emphasis on uncharacterized β-lactamase and carbapenemase activities. This approach not only supports the detection of ARGs that may be overlooked by sequence-based methods but also enables the characterization of their phenotypic effects and potential clinical implications of ARGs found in non-clinical settings. Furthermore, it emphasizes the importance of monitoring wastewater as a reservoir system for emerging resistance threats.

With the emergence of antibiotic resistance, there is an increase in illness, death, and healthcare costs and it also poses a threat to medical procedures. Novel therapeutic agents with a multi-pronged effect are therefore urgently required so as to impede bacterial mutations that lead to antibiotic resistance strategies. Metallic nanoparticles, particularly those containing silver, have been showing promise to fulfil this role and these nanoscale powerhouses could be our answer. Green biochemical synthetic strategies coupled with traditional medicine have therefore gained popularity as it results in an eco-friendly strategy for nanoparticle synthesis and the added benefit of medicinal phytochemicals being incorporated into the nanoparticle thereby resulting in a potent antibacterial agent. This study therefore made use of the medicinal plant, Siphonochilus aethiopicus, which is known to contain antibiotic compounds for the synthesis of silver nanoparticles. These nanoparticles were found to have excellent broad-spectrum antibiotic activity when tested using the INT assay. When the silver nanoparticles were tested against Acinetobacter baumannii (ATCC 19606), concentrations of 50 µg/ml and higher resulted in more than 90% toxicity. Against Pseudomonas aeruginosa (ATCC 27853), more than 80% toxicity was achieved with 25 µg/ml of the silver nanoparticles and more than 90% toxicity resulted when concentrations of 50 µg/ml or higher were evaluated. These results were comparable to the positive control ciprofloxacin. The silver nanoparticles also displayed more than 90% toxicity against Enterococcus faecalis (ATCC 51299) at concentrations of 25 µg/ml and higher, comparable to the positive control ampicillin. Against Staphylococcus aureus (ATCC 43300), more than 90% toxicity was achieved at concentrations of 25 µg/ml and higher, and of note these results indicated that the silver nanoparticles performed slightly better than the positive control ciprofloxacin. These nanoparticles therefore show excellent potential to be further developed as novel antibiotics.

Most bacteria contain a peptidoglycan cell wall that encapsulates the bacterial cytoplasmic membrane, maintaining cell shape and integrity. While the cell wall is a major target for antibiotics, very few target the initial steps of peptidoglycan biosynthesis that occurs in the cytoplasm. This involves a series of Mur enzymes which catalyse the formation of the important intermediate UDP-MurNAc-pentapeptide. While the Mur enzymes are usually expressed as seperate proteins, in some bacteria they are synthesised as naturally ocurring chimeric proteins, such as MurC-Ddl in Chlamydial species. Unlike most chimeric proteins, the MurC and Ddl enzymes do not catalyse sequential reactions but may be linked due to allostery between the two enzymatic domains; the Ddl domain was inhibited by UDP-MurNAc and UDP-MurNAc-Ala, a substrate and product of MurC respectively, as well as an inhibitor of MurC. The structure of MurC-Ddl was resolved to 2.8 Å and revealed the MurC inhibitor bound to the MurC domain only. Additionally, MurC and Ddl could be fused in Chlamydia for stability since neither of these enzymatic domains could be expressed as isolated proteins that retained activity. Ddl in particular was completely unstable and may require the MurC domain to stabilise a large α-helix protruding from the protein that occurs at the C-terminus of Ddl. The clinical targeting of Mur ligases, such as MurC, remains under-developed and therefore these enzymes could represent good targets for the development of novel antibiotics. Since MurC and Ddl share a similar catalytic mechanism, MurC-Ddl could be amenable to multi-target inhibitors and the structure of MurC-Ddl may help direct the future development of novel inhibitors targeting this chimera. Furthermore, MurC inhibitors are likely to target other Mur ligases owing to their common structure and catalytic mechanism.

Introduction: In South Asia, the prevalence of Helicobacter pylori (H. pylori) infections may be as high as 60-80%, constituting a notable public health issue, with eradication strategies critical in reducing the H. pylori-associated disease burden. The aim of our study was to assess the knowledge, attitude and practices (KAP) regarding the screening, treatment, and follow-up of H. pylori-induced gastric ulcers among physicians in Lahore, Pakistan.

Methods: A self-administered, questionnaire-based cross-sectional study—including the development and validity assessment of a 25-item questionnaire—was carried out in two tertiary-care hospitals between January and May 2024. Statistical analyses (descriptive statistics, χ²-tests, binary logistic regression; 95% confidence interval [95%CI]) were carried out using SPSS 27.0. The study followed the STrengthening the Reporting of OBservational studies in Epidemiology (STROBE) guidelines.

Results: Among N=385 participants, 57.9% were male, 60.0% were aged between 25 and 34 years, 59.5% worked in a public hospital, and 55.3% had <5 years of working experience. A total of 32.9% had noted medical journals, while 27.0% reported online educational materials as their key sources of evidence-based information. Although 91.2% and 87.3% of physicians had good knowledge and attitude levels (≥50% score) pertaining to H. pylori-associated gastric ulcers, respectively, only 49.6% correctly identified H. pylori as being contagious. Participants aged 25–34 years (aOR: 0.217 [95% CI: 0.08-0.589]), who have <5 years of working experience (aOR: 0.328 [95% CI: 0.136-0.790]) and those working in public hospitals (aOR: 0.130 [95% CI: 0.048-0.352]) were less likely to show poor attitudes. A total of 76.5% made it routine to discuss the risk factors of H. pylori-induced ulcers with their patients, while 67.4% highlighted the importance of follow-up testing to confirm the eradication of H. pylori.

Conclusions: Inconsistent and empirical treatment approaches, and lack of routine screening and follow-up practices may further compromise eradication efforts, and contribute to the development of multidrug-resistance in H. pylori.