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Researchers have been investigating the physical and morphological properties of biodegradable polymer and copolymer films, blending them with other chemicals to solve challenges in medical, industrial, and eco-environmental fields. The present study introduces a novel, straightforward method for preparing biodegradable hydrogels based on polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) for medical applications. The resulting PVA/PVP-based hydrogel uniquely combines the water absorbency, biocompatibility, and biodegradability of the polymer composite. For hygiene products and medical uses, such as wound healing, hydrogen peroxide (HP) was encapsulated in the PVA/PVP hydrogels for controlled release application. Incorporating PVP into PVA significantly enhances the hydrogel water absorbency and improves the mechanical properties. However, to mitigate the disadvantage of high water absorbency which could result in undesired early dissolution, efforts were made to increase the water resistance and the mechanical characteristics of these hydrogels using freeze–thaw (F/T) cycles and chemical crosslinking PVA chains with trisodium trimetaphosphate (STMP). The resulting hydrogels serve as environmentally friendly bio-based polymer blends, broadening their applications in medical and industrial products. The structural and morphological properties of the hydrogel were characterized using Fourier transform infrared spectroscopy (FTIR), environmental scanning electron microscope analysis (E-SEM), and water-swelling tests. The HP controlled release rate was evaluated through kinetic release experiments using the ex vivo skin model. The antibacterial activity of the hydrogel films was examined on four medically relevant bacteria: Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa, with an adapted disk diffusion assay. Using this assay, we also evaluated the antibacterial effect of the hydrogel films over the course of days, demonstrating the HP controlled release from these hydrogels. These findings support further in vivo investigation into controlled HP release systems for improved wound-healing outcomes.

Pseudomonas aeruginosa is one of the leading nosocomial opportunistic pathogens causing acute and chronic infections. Among its main virulent factors is the Type III secretion system (T3SS) which enhances disease severity by delivering effectors to the host in a highly regulated manner. Despite its importance for virulence, only six T3SS-dependent effectors have been discovered so far. Previously, we identified two new potential effectors using a machine-learning algorithm approach. Here we demonstrate that one of these effectors, PemB, is indeed virulent. Using a live Caenorhabditis elegans infection model, we demonstrate this effector damages the integrity of the intestine barrier leading to the death of the host. Implementing a high-throughput assay using Saccharomyces cerevisiae, we identified several candidate proteins that interact with PemB. One of them, EFT1, has an ortholog in C. elegans (eef-2) and is also an essential gene and a well-known target utilized by different pathogens to induce toxicity to the worm. Accordingly, we found that by silencing the eef-2 gene in C. elegans, PemB could no longer induce its toxic effect. The current study further uncovers the complex machinery assisting P. aeruginosa virulence and may provide novel insight how to manage infection associated with this hard-to-treat pathogen.

ABSTRACT Toxin-antitoxin (TA) systems are genetic modules that consist of a stable protein-toxin and an unstable antitoxin that neutralizes the toxic effect. In type II TA systems, the antitoxin is a protein that inhibits the toxin by direct binding. Type II TA systems, whose roles and functions are under intensive study, are highly distributed among bacterial chromosomes. Here, we identified and characterized a novel type II TA system PrrT/A encoded in the chromosome of the clinical isolate 39016 of the opportunistic pathogen Pseudomonas aeruginosa. We have shown that the PrrT/A system exhibits classical type II TA characteristics and novel regulatory properties. Following deletion of the prrA antitoxin, we discovered that the system is involved in a range of processes including (i) biofilm and motility, (ii) reduced prophage induction and bacteriophage production, and (iii) increased fitness for aminoglycosides. Taken together, these results highlight the importance of this toxin-antitoxin system to key physiological traits in P. aeruginosa. IMPORTANCE The functions attributed to bacterial TA systems are controversial and remain largely unknown. Our study suggests new insights into the potential functions of bacterial TA systems. We reveal that a chromosome-encoded TA system can regulate biofilm and motility, antibiotic resistance, prophage gene expression, and phage production. The latter presents a thus far unreported function of bacterial TA systems. In addition, with the emergence of antimicrobial-resistant bacteria, especially with the rising of P. aeruginosa resistant strains, the investigation of TA systems is critical as it may account for potential new targets against the resistant strains.

Prophages are bacteriophages in the lysogenic state, where the viral genome is inserted within the bacterial chromosome. They contribute to strain genetic variability and can influence bacterial phenotypes. Prophages are highly abundant among the strains of the opportunistic pathogen Pseudomonas aeruginosa and were shown to confer specific traits that can promote strain pathogenicity. The main difficulty of studying those regions is the lack of a simple prophage-curing method for P. aeruginosa strains. In this study, we developed a novel, targeted-curing approach for prophages in P. aeruginosa. In the first step, we tagged the prophage for curing with an ampicillin resistance cassette (ampR) and further used this strain for the sacB counter-selection marker’s temporal insertion into the prophage region. The sucrose counter-selection resulted in different variants when the prophage-cured mutant is the sole variant that lost the ampR cassette. Next, we validated the targeted-curing with local PCR amplification and Whole Genome Sequencing. The application of the strategy resulted in high efficiency both for curing the Pf4 prophage of the laboratory wild-type (WT) strain PAO1 and for PR2 prophage from the clinical, hard to genetically manipulate, 39016 strain. We believe this method can support the research and growing interest in prophage biology in P. aeruginosa as well as additional Gram-negative bacteria.