Bacteriophage-based applications have a renaissance today, increasingly marking their use in industry, medicine, food processing, biotechnology, and more. However, phages are considered resistant to various harsh environmental conditions; besides, they are characterized by high intra-group variability. Phage-related contaminations may therefore pose new challenges in the future due to the wider use of phages in industry and health care. Therefore, in this review, we summarize the current knowledge of bacteriophage disinfection methods, as well as highlight new technologies and approaches. We discuss the need for systematic solutions to improve bacteriophage control, taking into account their structural and environmental diversity.

Urinary tract infections are one of the most frequent bacterial diseases worldwide. UPECs are the most prominent group of bacterial strains among pathogens responsible for prompting such infections. As a group, these extra-intestinal infection-causing bacteria have developed specific features that allow them to sustain and develop in their inhabited niche of the urinary tract. In this study, we examined 118 UPEC isolates to determine their genetic background and antibiotic resistance. Moreover, we investigated correlations of these characteristics with the ability to form biofilm and to induce a general stress response. We showed that this strain collection expressed unique UPEC attributes, with the highest representation of FimH, SitA, Aer, and Sfa factors (100%, 92.5%, 75%, and 70%, respectively). According to CRA (Congo red agar) analysis, the strains particularly predisposed to biofilm formation represented 32.5% of the isolates. Those biofilm forming strains presented a significant ability to accumulate multi-resistance traits. Most notably, these strains presented a puzzling metabolic phenotype—they showed elevated basal levels of (p)ppGpp in the planktonic phase and simultaneously exhibited a shorter generation time when compared to non-biofilm-forming strains. Moreover, our virulence analysis showed these phenotypes to be crucial for the development of severe infections in the Galleria mellonella model.

Enterohemorrhagic Escherichia coli (EHEC) is a common pathotype of E. coli that causes numerous outbreaks of foodborne illnesses. EHEC is a zoonotic pathogen that is transmitted from animals to humans. Ruminants, particularly cattle, are considered important reservoirs for virulent EHEC strains. Humans can become infected with EHEC through the consumption of contaminated food and water or through direct contact with infected animals or humans. E. coli O157:H7 is one of the most commonly reported causes of foodborne illnesses in developed countries. The formation of attaching and effacing (A/E) lesions on the intestinal epithelium, combined with Shiga toxin production, is a hallmark of EHEC infection and can lead to lethal hemolytic–uremic syndrome (HUS). For the phage-dependent regulation of Shiga toxin production, antibiotic treatment is contraindicated, as it may exacerbate toxin production, limiting therapeutic options to supportive care. In response to this challenge and the growing threat of antibiotic resistance, phytochemicals have emerged as promising antivirulence agents. These plant-derived compounds target bacterial virulence mechanisms without promoting resistance. Therefore, the aim of this study is to summarize the recent knowledge on the use of phytochemicals targeting EHEC. We focused on the molecular basis of their action, targeting the principal virulence determinants of EHEC.

Single-molecule techniques are highly sensitive tools that can reveal reaction intermediates often obscured in experiments involving large ensembles of molecules. Therefore, they provide unprecedented information on the mechanisms that control biomolecular reactions. Currently, one of the most significant single-molecule assays is Magnetic Tweezers (MT), which probes enzymatic reactions at high spatio-temporal resolutions on tens, if not hundreds, of molecules simultaneously. For high-resolution MT experiments, a short double-stranded DNA molecule (less than 2,000 base pairs) is typically attached between a micron-sized superparamagnetic bead and a surface. The fabrication of such a substrate is key for successful single-molecule assays, and several papers have discussed the possibility of improving the fabrication of short DNA constructs. However, reported yields are usually low and require additional time-consuming purification steps (e.g., gel purification). In this paper, we propose the use of a Golden Gate Assembly assay that allows for the production of DNA constructs within minutes (starting from PCR products). We discuss how relevant parameters may affect the yield and offer single-molecule experimentalists a simple yet robust approach to fabricate DNA constructs.

Purpose: Biofilm-related infections, especially those associated with medical devices like catheters, pose significant clinical challenges due to their resistance to conventional treatments. This study investigates a green chemistry-based approach to synthesize silver nanoparticles (AgNPs) stabilized with trans-cinnamaldehyde (t-CA) and evaluates their potential for combating microbial biofilms and based on novel mechanism of action. Methods: Silver nanoparticles (t-CA-AgNPs) were synthesized using t-CA as both a reducing and stabilizing agent. The NPs were then thoroughly characterized using UV-Vis spectroscopy, X-ray diffraction (XRD), electron microscopy (TEM, SEM, STEM), and dynamic light scattering (DLS). We evaluated its antimicrobial potential against the most prevalence biofilm-forming pathogens including Pseudomonas aeruginosa, Escherichia coli and Candida albicans using minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) assays. Moreover, we investigated the mechanism of action of t-CA-AgNPs underlying biofilm inhibition. Biofilm formation and structure were verified by SEM imagining. Results: DLS analysis confirmed that t-CA-AgNPs had an average particle diameter of 2.5 nm, coupled with a notably negative zeta potential (− 45 mV), indicative of good colloidal stability. t-CA-AgNPs displayed potent antimicrobial properties, with MIC values ranging from 26 to 412 μg/mL and MBC values from 103 to 825 μg/mL. Biofilm formation inhibitory properties reached 88.74% of inhibition for P. aeruginosa and 70.60% for E. coli. Moreover, we found potent metal ion-chelating capabilities, importantly, in binding and reducing ferrous ions, the crucial factor of biofilm formation. Furthermore, t-CA-AgNPs substantially impaired biofilm development on catheter surfaces, underscoring their robust antibiofilm potential. Conclusion: Presented here t-CA-AgNPs exhibit significant antimicrobial and antibiofilm activity. By effectively targeting critical elements in biofilm formation, such as ferrous ions, coupled with antimicrobial potential of both active compounds, these green-synthesized NPs have potential applications in significantly improving the safety and effectiveness of medical devices. However, further studies are needed to ensure their efficacy in clinical use.

Antimicrobial resistance in Escherichia coli remains a critical threat in both human and veterinary medicine, demanding innovative strategies beyond traditional antibiotics. Here, we reveal that trans‑cinnamaldehyde (t‑CA), a natural phenylpropanoid, not only inhibits bacterial growth but also actively reshapes transcriptional programs through the RelA‑mediated stringent response. Using RNA‑seq, we demonstrate that sub‑inhibitory t‑CA (0.25 ×MIC) triggers broad transcriptional remodelling, suppressing key virulence pathways such as iron acquisition, quorum sensing, and acid resistance - effects that were not pronounced in a relA-deficient background. Consistently, t-CA induced RelA-dependent accumulation of the stringent response alarmone, (p)ppGpp, linking stress signalling with repression of virulence determinants. Functionally, t-CA impaired extracellular matrix production and significantly reduced biofilm biomass and metabolic activity. In host-relevant assays, t-CA showed moderate cytotoxicity in mammalian cell lines and attenuated LPS-driven inflammatory cytokine output in Peripheral Blood Mononuclear Cells (PBMCs) model. Finally, post-infection administration of t-CA improved survival in the Galleria mellonella infection model. Together, these results identify stringent response signalling as a key axis underlying t-CA-driven virulence attenuation and support t-CA as a candidate antivirulence adjuvant for combating difficult-to-treat E. coli infections.