Antimicrobial resistance poses a growing global challenge, necessitating the development of novel antibacterial strategies. One promising approach is based on short amino acid motifs that are significantly underrepresented or absent in bacterial proteomes (URS).

Using a computational strategy developed in our laboratory, Navon et al. (PNAS 2016) identified URSs by detecting patterns missing from proteomic datasets. These sequences are often species-specific and, in some cases, completely absent from bacterial proteomes. Introduction of a URSs can lead to translational arrest and even cell death. These sequences are not URSs in the human proteome, suggesting potential use as antibiotics.

Two URSs shown to induce translational inhibition in E. coli are CMY and CMYW. Based on these findings, we proposed two implementation strategies: delivery of synthetic URS-containing peptides or expression of URS-encoding sequences.

Chemically synthesized peptides, including CMY- and CMYW-based sequences, were applied to Escherichia coli and Salmonella cultures. These peptides exhibited limited antibacterial activity, likely due to restricted membrane permeability associated with their hydrophobic nature. To overcome this limitation, the peptides were conjugated to cell-penetrating peptides (CPPs), resulting in a substantial improvement in antibacterial activity. Optimized peptides, including RLLRRCMYW, exhibited MIC values in the range of 8–16 µM, comparable to those of conventional antibiotics, due to improved cellular uptake and stability. Notably, CMYW-based peptides showed enhanced activity relative to CMY, consistent with stronger ribosomal stalling and their absence from bacterial proteomes.

In addition, heterologous expression of URS-encoding sequences inhibited bacterial growth, supporting that URS motifs impair growth through disruption of translation.

Structural determination of ribosome–URSpep complexes (Tarabeh et al., in preparation) provides insight into their molecular interactions with the ribosome.

Our current work focuses on further optimizing peptide permeability and stability to enhance intracellular targeting of the ribosome and maximize antibacterial activity.

Background: Microbial keratitis (infection of the cornea, the clear membrane overlying the pupil and coloured iris in the eye) is often caused by Pseudomonas aeruginosa, especially in contact lens wearers. Commonly used antibiotics are failing against this bacterium as it is becoming increasingly resistant to antimicrobials. This study was conducted to isolate and characterize bacteriophages (phages) that could be used to design a phage cocktail that is active against multi-drug resistant (MDR) ocular P. aeruginosa isolates.

Methods: Raw untreated sewage water was used for isolation and purification of phages following the Phage-on-Tap protocol. Phage confirmation, enumeration and identification were performed by spot assay, double layer plaque assay and transmission electron microscopy (TEM), respectively. Lytic activity of phages was determined by host range evaluation against thirteen P. aeruginosa isolates that had previously been characterised as MDR and resistant to another older set of phages. Further characterization of new phages used one step growth curves and thermal stability.

Results: Four phages were isolated and purified. These phages produced high titres of phage, approximately 10¹⁰ plaqe forming untis (pfu)/ml. TEM analysis revealed that the phages belong to two families, Podoviridae and Siphoviridae. All phages were stable at temperatures from 4°C to 50°C. Host range via spot assays showed that Sullo-1, Sullo-2, Sullo-3 and Sullo-4 had lytic activity against 77%, 69%, 55% and 55% of P. aeruginosa strains. The latent period was around ~ 20 minutes for Sullo-1, Sullo-2 and Sullo-3 and 40 minutes for Sullo-4. The burst size of Sullo-1, Sullo-2, Sullo-3 and Sullo-4 was 38, 140, 246 and 73 pfu/infected cell, respectively.

Conclusions: The newly isolated phages had efficient lytic activity against ocular P. aeruginosa isolates and could be distinguished in terms of morphology, host range, thermal stability, burst size and latent period.

Pseudomonas aeruginosa (Pae) is a major opportunistic human pathogen, classified by the WHO in 2024 as a high-priority antibiotic resistant threat. It causes severe infections in immunocompromised individuals and patients with cystic fibrosis, largely due to a combination of intrinsic and acquired resistance mechanisms. CRISPR interference (CRISPRi) enables programmable, transcriptional repression of target genes and is well-suited for genome-wide interrogation of gene-drug interactions, allowing identification of essential pathways that become particularly vulnerable under antibiotic pressure. In CRISPRi, a single guide RNA (sgRNA) directs a catalytically inactive Cas9 protein (dCas9) to a target sequence adjacent to a PAM site, blocking RNA polymerase binding or elongation.

Here, we performed a genome-wide CRISPRi screen in the P. aeruginosa PAO1 reference strain, using an sgRNA pooled library exposed to sub-inhibitory concentrations of 12 antibiotics commonly used against Pae: aminoglycosides, β-lactams (including carbapenems), fluoroquinolones, and polymyxins. The analysis identified essential gene-drug interactions and pinpointed pathways (e.g., cell wall synthesis, cell division, fatty acids and lipopolysaccharide biosynthesis) whose repression altered antibiotic susceptibility. These genes represent potential therapeutic targets or adjuvant candidates to enhance existing antimicrobial efficacy while reducing toxicity associated with high drug doses. Overall, this platform provides a robust approach for investigating essential genes and their role in resistance, offering a promising strategy to identify pathogen vulnerabilities.

This work is supported by the Polish National Science Centre grant 2021/43/D/NZ2/02151.

Over the past 20 years, the study of antimicrobial resistance mechanisms has evolved from a largely descriptive discipline into a predictive science. The SENTRY Antimicrobial Surveillance Program—which collects and characterizes approximately 30,000 clinical isolates annually from around the world—has helped define the modern understanding of resistance epidemiology and directly supported the development and positioning of many antibacterial agents approved in the last 15 years.

A major inflection occurred around 2010, when the development of β-lactam/β-lactamase inhibitor combinations began to require precise differentiation of activity against specific resistance mechanisms. This shift transformed the field, driving a move from retrospective observation to systematic, prospective surveillance integrated with advanced molecular characterization.

In this presentation I will discuss how the genetic characterization studies performed using the SENTRY Program platform aided the differentiation of antimicrobial agents and provided support for clinical decisions. Ultimately, these insights underscore a fundamental shift in antimicrobial therapy—from choosing drugs based on organisms to selecting them based on resistance mechanisms.

The UNGA on AMR in September 2024 provided a seminal moment to recapture the momentum generated in 2015 but sadly, due to a number of reasons it did not resonate. The diminished funding to the UN has curbed the ambition of most WHO/FAO AMR programs, and the discontinued funding of the Fleming Fund by the UK GOV signals a shift in international and international priorities. Whilst bacteria are increasingly capable of communicating with each other, the human race seems to lack such unity to produce tractable solutions - the AMR solutions are known and tractable but as a global medico-scientific community we lack vision and belief to come together to seriously address AMR. The amount spent on AMR is approx. X1000 less than that spent on arms/defence, yet the number of deaths due to AMR is 100-fold to that of international conflict – and thus, our priorities are not aligned with the risk. Ironically, and tragically, international conflict further worsens the local issues of AMR infection. Climate change and environmental pollution with plastics also exacerbate the AMR problem. The antibiotic pipeline is challenged by relentlessly increasing levels of AMR which cuts across all UN SDGs and yet, still remains largely invisible to the general public. The successful arrest of global AMR is not a knowledge-gap or logistical issue, but a financial issue – we know what the solutions are! My lecture will attempt to bring all these factors into play but also discuss possible solutions and the pivotal role that outside players will play in leading on the world-stage to tackle global AMR.

Author: Professor Dr. Patrice Nordmann

Keywords: AMR; β-lactams; combined resistance; multidrug resistance

Session 4: Conventional and Novel Approaches in the Discovery of New Antimicrobial Agents

Title: Emerging Antibiotic Resistances Worldwide

Abstract:

Clinically significant multidrug resistance is increasingly reported, particularly among enterobacterial species (e.g., Escherichia coli, Klebsiella pneumoniae, Enterobacter spp.), as well as Pseudomonas aeruginosa and Acinetobacter baumannii.

The antibiotic resistance traits that are already widespread in these Gram-negative bacteria are mainly due to extended-spectrum β-lactamase (ESBL) and carbapenemase production. ESBLs confer resistance to all β-lactams, with the exception of cephamycins and carbapenems, whereas carbapenemases confer resistance to virtually all β-lactams, including carbapenems.

Although novel β-lactams have recently been introduced, such as cefiderocol, ceftazidime-avibactam, meropenem-vaborbactam, imipenem-relebactam, and aztreonam-avibactam, emerging resistance to these agents has already been observed.

In addition, combined resistance to other antibiotic classes, potentially leading to pandrug resistance, may be associated with virulence traits. This recent development may further complicate the future management of infections associated with multidrug-resistant bacteria.

Antimicrobial resistance (AMR) is driven by the global dissemination of carbapenemase-producing Enterobacterales, compromising last-resort antibiotics and challenging clinical management. Within a One Health framework, carbapenemases represent key molecular markers to investigate transmission dynamics across human, animal, food, and environmental compartments. This presentation integrates current evidence on the occurrence and spread of carbapenemase producers along the food chain, including food-producing animals, retail products, and processing environments. Mechanistic insights into persistence and dissemination remain limited but may involve biofilm formation and horizontal gene transfer. Despite advances in surveillance, critical limitations remain, including inconsistent detection methodologies, underrepresentation of specific resistance mechanisms, and insufficient integration of genomic data for source attribution. Recent environmental monitoring initiatives expand surveillance capacity but frequently lack defined thresholds for intervention and translational pathways to control. By focusing on carbapenemases, this talk evaluates the extent to which current surveillance frameworks inform effective mitigation strategies, emphasizing the need for integrated, risk-based approaches with clear translational impact across the One Health continuum.

Foodborne pathogens such as Listeria monocytogenes and Staphylococcus aureus can persist in food processing environments by forming resilient biofilms that resist standard sanitation methods and serve as long-term sources of contamination. Biofilm growth is often controlled by quorum sensing (QS), including the autoinducer-2 (AI-2) system, which orchestrates communication within and between bacterial species and promotes group behaviors. Targeting QS provides a promising, non-lethal approach for controlling biofilms. Lactic acid bacteria (LAB), commonly used in foods, are known for improving safety and quality, and recent evidence shows their metabolites can interfere with QS and prevent biofilm formation without harming free-floating bacteria, thus lowering the risk of resistance. In this study, we tested a sterile, pH-neutralized (pH 6.5) cell-free supernatant (CFS) from a Pediococcus acidilactici cheese isolate for its ability to prevent mixed-culture biofilm formation by three L. monocytogenes strains from poultry on stainless steel surfaces. This LAB strain was selected from a larger collection because of its ability to inhibit AI-2 signaling in a Vibrio harveyi bioluminescence reporter assay. During biofilm formation, the CFS (prepared in MRS broth) was added at 50% (v/v) to Brain Heart Infusion (BHI) broth and incubated at 37 °C for 48 h under nutrient-matched conditions with and without CFS. Treatment with P. acidilactici CFS reduced L. monocytogenes biofilm cell counts on stainless steel by approximately 99% (2 logs), while leaving planktonic bacterial populations unaffected. The antibiofilm effect remained stable after heat treatment (100 °C, 5 min), indicating the presence of heat-resistant metabolites. Ongoing work aims to identify these QS-interfering compounds and further examine their effects on AI-2 signaling and biofilm development. Our main goal is to develop environmentally safe, QS-based biocontrol methods that could preserve beneficial microbiota while controlling harmful biofilms in food environments.

Antimicrobial resistance (AMR) is a global problem that causes great concern to health and scientific authorities and compromises the use of antimicrobial agents in the treatment of infectious diseases. This problem needs to be addressed from the One Health perspective in which human, animal and environmental health are interconnected. Information about the relevance of the environment in this context has been increasingly reported in the last years. The group of Antimicrobial Resistance from de One Health perspective (OneHealth-UR) has analyzed AMR in different environmental niches as is the case of wastewater-treatment plants (WWTP) and wildlife animals, as storks and wild rabbits, among others. Two WWTPs of La Rioja have been followed (influent, effluent, sludges and receiving river at upstream and downstream points) using different technologies, with special emphasis in the dissemination of Extended-Spectrum-beta-Lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae isolates (ESBL-Ec/Kp) and carbapenemase-producing Enterobacterales (CP-E), and results will be shown as well as the comparative analysis with clinical isolates in the surrounding hospital. Moreover, the occurrence of critical mechanisms of resistance linked to ESBL-Ec/Kp and CP-E in wild birds or mecC-mediated methicillin-resistant Staphylococcus aureus in wild rabbits will be also reported through results obtained in our group in comparison with those of other groups. All these data will allow to increase our knowledge about the relevance of the environment as a crucial link in the study of AMR at global level, to adopt the adequate decisions to manage and control the AMR crisis.

The combination of amoxicillin (AMX) and clavulanic acid (CA) remains one of the most widely prescribed antibiotic therapies. However, its clinical efficacy is increasingly compromised by the emergence of bacterial resistance and persistence mechanisms, among both Gram-negative and Gram-positive pathogens.

To address this limitation, we developed a novel formulation combining AMX and CA with 1,8-cineole (CN), a natural compound well recognized in the pharmacopoeia for its broad pharmacological activities and favorable safety profile.

The obtained in vitro, in vivo and clinical trials demonstrate that this triple combination significantly enhances the antibacterial activity of AMX/CA against susceptible strains. Importantly, it also restores susceptibility in resistant and persistent bacterial populations.

In parallel, in silico investigations were conducted to elucidate the underlying mechanisms of action. Key bacterial targets were explored, including penicillin-binding proteins (PBPs), as well as molecular targets associated with bacterial persistence. These computational analyses suggest that AMX, CA, and CN can form multiple molecular complexes, including three binary combinations (AMX–CA, AMX–CN, and CA–CN) as well as a ternary complex (AMX–CA–CN). These entities may act simultaneously on bacterial targets and significantly reduce the probability of resistance emergence and bacterial survival in a persistent state.