Protecting the Antibacterial Coating of Urinal Catheters for Improving Safety.

Catheter-associated urinary tract infections (CAUTI) are among the most common bacterial infections associated with prolonged hospitalization and increased healthcare expenditures. Despite recent advances in the prevention and treatment of these infections, there are still many challenges remaining, among them the creation of a durable catheter coating, which prevents bacterial biofilm formation. The current work reports on a method of protecting medical tubing endowed with antibiofilm properties. Silicone catheters coated sonochemically with ZnO nanoparticles (NPs) demonstrated excellent antibiofilm effects. Toward approval by the European Medicines Agency, it was realized that the ZnO coating would not withstand the regulatory requirements of avoiding dissolution for 14 days in artificial urine examination. Namely, after exposure to urine for 14 days, the coating amount was reduced by 90%. Additional coatings with either carbon or silica maintained antibiofilm activity against Staphylococcus aureus while resisting dissolution in artificial urine for 14 days (C- or SiO2-protected catheters exhibited only 29% reduction). HR-SEM images of the protected catheters indicate the presence of the ZnO coating as well as the protective layer. Antibiofilm activity of all catheters was evaluated both before and after exposure to artificial urine. It was shown that before artificial urine exposure, all coated catheters showed high antibiofilm properties compared to the uncoated control. Exposure of ZnO-coated catheters, without the protective layer, to artificial urine had a significant effect exhibited by the decrease in antibiofilm activity by almost 2 orders of magnitude, compared to unexposed catheters. Toxicity studies performed using a reconstructed human epidermis demonstrated the safety of the improved coating. Exposure of the epidermis to ZnO catheter extracts in artificial urine affects tissue viability compared with control samples, which was not observed in the case of ZnO NPs coating with SiO2 or C. We suggest that silica and carbon coatings confer some protection against zinc ions release, improving ZnO coating safety.

Synthesis and Characterization of Durable Antibiofilm and Antiviral Silane-Phosphonium Thin Coatings for Medical and Agricultural Applications.

Pathogens such as bacteria and viruses cause disease in a range of hosts, from humans to plants. Bacterial biofilms, communities of bacteria, e.g., Staphylococcus aureusand Escherichia coli, attached to the surface, create a protective layer that enhances their survival in harsh environments and resistance to antibiotics and the host's immune system. Biofilms are commonly associated with food spoilage and chronic infections, posing challenges for treatment and prevention. Tomato brown rugose fruit virus (ToBRFV), a newly discovered tobamovirus, infects tomato plants, causing unique symptoms on the fruit, posing a risk for tomato production. The present study focuses on the effectiveness of silane-phosphonium thin coatings on polymeric films, e.g., polypropylene. Phosphonium has significant antibacterial activity and is less susceptible to antibacterial resistance, making it a safer alternative with a reduced environmental impact. We successfully synthesized silane-phosphonium monomers as confirmed by 31P NMR and mass spectrometry. The chemical composition, thickness, morphology, and wetting properties of the coatings were tested by Fourier-transform infrared spectroscopy with attenuated total reflectance, focused ion beam, atomic force microscopy, environmental scanning electron microscope, and contact angle (CA) measurements. The antibiofilm and antibacterial activities of the coatings were tested against S. aureus and E. coli, while the antiviral activity was evaluated against ToBRFV. The significant antibiofilm and antiviral activity suggests applications in various fields including medicine, agriculture, and the food industry.

ZnO Quantum Photoinitiators as an All-in-One Solution for Multifunctional Photopolymer Nanocomposites.

Nanocomposites are constructed from a matrix material combined with dispersed nanosized filler particles. Such a combination yields a powerful ability to tailor the desired mechanical, optical, electrical, thermodynamic, and antimicrobial material properties. Colloidal semiconductor nanocrystals (SCNCs) are exciting potential fillers, as they display size-, shape-, and composition-controlled properties and are easily embedded in diverse matrices. Here we present their role as quantum photoinitiators (QPIs) in acrylate-based polymer, where they act as a catalytic radical initiator and endow the system with mechanical, photocatalytic, and antimicrobial properties. By utilizing ZnO nanorods (NRs) as QPIs, we were able to increase the tensile strength and elongation at break of poly(ethylene glycol) diacrylate (PEGDA) hydrogels by up to 85%, unlike the use of the same ZnO NRs acting merely as fillers. Simultaneously, we endowed the PEGDA hydrogels with post-polymerization photocatalytic and antimicrobial activities and showed their ability to decompose methylene blue and significantly eradicate antibiotic-resistant bacteria and viral pathogens. Moreover, we demonstrate two fabrication showcase methods, traditional molding and digital light processing printing, that can yield hydrogels with complex architectures. These results position SCNC-based systems as promising candidates to act as all-in-one photoinitiators and fillers in nanocomposites for diverse biomedical applications, where specific and purpose-oriented characteristics are required.

PrrT/A, a Pseudomonas aeruginosa Bacterial Encoded Toxin-Antitoxin System Involved in Prophage Regulation and Biofilm Formation.

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.

An Efficient, Counter-Selection-Based Method for Prophage Curing in Pseudomonas aeruginosa 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.

Characterization of PfiT/PfiA toxin-antitoxin system of Pseudomonas aeruginosa that affects cell elongation and prophage induction.

Toxin-antitoxin (TA) systems are small genetic modules usually consisting of two elements-a toxin and an antitoxin. The abundance of TA systems among various bacterial strains may indicate an important evolutionary role. Pseudomonas aeruginosa, which can be found in a variety of niches in nature, is an opportunistic pathogen for various hosts. While P. aeruginosa strains are very versatile and diverse, only a few TA systems were characterized in this species. Here, we describe a newly characterized TA system in P. aeruginosa that is encoded within the filamentous Pf4 prophage. This system, named PfiT/PfiA, is a homologue of the ParE/YefM TA system. It is a type II TA system, in which the antitoxin is a protein that binds the toxic protein and eliminates the toxic effect. PfiT/PfiA carries several typical type II characteristics. Specifically, it constitutes two small genes expressed in a single operon, PfiT inhibits growth and PfiA eliminates this effect, PfiA binds PfiT, and PfiT expression results in elongated cells. Finally, we assigned a novel function to this TA system, where an imbalance between PfiT and PfiA, favouring the toxin, resulted in cell elongation and an increase in virion production.

SawR a new regulator controlling pyomelanin synthesis in Pseudomonas aeruginosa.

Surface Acoustic Waves (SAW) were previously shown to inhibit biofilm formation, increase bacterial susceptibility to antibiotic treatment and alter the transcription pattern of Pseudomonas aeruginosa. Here we characterize one gene, sawR (PA3133), that is highly overexpressed when P. aeruginosa is exposed to SAW. SawR is a putative transcription factor belonging to the TetR regulator family. When overexpressed sawR causes numerous phenotypes, including the accumulation of a brown pigment which we identified as pyomelanin. In this study we describe how sawR regulates pyomelanin synthesis. We show that sawR down-regulates the expression levels of hmgA and this causes the accumulation of homogentisic acid which in turn undergoes oxidation and polymerization to pyomelanin. Using bioinformatics, we were able to identify a specific amino acid, arginine 23, which is found within the sawR DNA binding domain and is crucial for its regulatory activity. Our results indicate that sawR does not affect any other genes in the phenylalanine/tyrosine metabolic pathway and its repressive ability on hmgA is not mediated by the hmgA repressor PA2010 (i.e. hmgR). Taken together, our results shed light on the regulatory cascade controlling pyomelanin synthesis and uncover yet another unknown regulator involved in its regulation.

Molecular computing with plant cell phenotype serving as quality controlled output.

The ability of autonomous biomolecular computing devices to interact directly with biological systems and even with living organisms without any interface represents their main advantage over the electronic computers. This study shows that the expression of fluorescent proteins in live plant cells can be utilized as a highly accurate visual output of DNA-based computing. Each of the two possible outputs of a 2-symbol 2-state finite automaton was represented here by either green or cyan fluorescence in eukaryotic cells. The automata were programmed by the choice of several molecules from a library of 8 transition molecules, each containing a recognition site for a type II endonuclease. Two enzymes, endonuclease and a DNA ligase, as well as ATP, represented the hardware. Each input molecule, in the form of a dsDNA, included a string of symbols, 6 bp each, and a 6 bp terminator. The two detection molecules were also dsDNA, each containing a 4-base sticky end, complementary to the appropriately restricted terminator and a gene encoding for a different fluorescent protein. Computation was carried out by mixing all components in a homogeneous solution, leading to autonomous processing of the input molecule via repetitive cycles of digestion, hybridization, and ligation. The output processing procedure involved the creation of a circular dsDNA that contained the gene of either green fluorescent protein or cyan fluorescent protein. Insertion of these plasmids into onion cells by particle bombardment resulted in either green fluorescent or cyan fluorescent live cells as phenotypical output signals. The plasmid formation was an important step because it served as a quality control gate that transformed a rather noisy output into a clean signal. This process of noise elimination allowed for clean and flawless outputs with high fidelity and zero noise.