The choice of effective biocides used for routine hospital practice should consider the role of disinfectants in the maintenance and development of local resistome and how they might affect antibiotic resistance gene transfer within the hospital microbial population. Currently, there is little understanding of how different biocides contribute to eDNA release that may contribute to gene transfer and subsequent environmental retention. Here, we investigated how different biocides affect the release of eDNA from mature biofilms of two opportunistic model strains Pseudomonas aeruginosa ATCC 27853 (PA) and Staphylococcus aureus ATCC 25923 (SA) and contribute to the hospital resistome in the form of surface and water contaminants and dust particles. The effect of four groups of biocides, alcohols, hydrogen peroxide, quaternary ammonium compounds, and the polymeric biocide polyhexamethylene guanidine hydrochloride (PHMG-Cl), was evaluated using PA and SA biofilms. Most biocides, except for PHMG-Cl and 70% ethanol, caused substantial eDNA release, and PHMG-Cl was found to block biofilm development when used at concentrations of 0.5% and 0.1%. This might be associated with the formation of DNA–PHMG-Cl complexes as PHMG-Cl is predicted to bind to AT base pairs by molecular docking assays. PHMG-Cl was found to bind high-molecular DNA and plasmid DNA and continued to inactivate DNA on surfaces even after 4 weeks. PHMG-Cl also effectively inactivated biofilm-associated antibiotic resistance gene eDNA released by a pan-drug-resistant Klebsiella strain, which demonstrates the potential of a polymeric biocide as a new surface-active agent to combat the spread of antibiotic resistance in hospital settings.

Novel antibiotic combinations may act synergistically to inhibit the growth of multidrug-resistant bacterial pathogens but predicting which combination will be successful is difficult, and standard antimicrobial susceptibility testing may not identify important physiological differences between planktonic free-swimming and biofilm-protected surface-attached sessile cells. Using a nominally macrolide-resistant model Klebsiella pneumoniae strain (ATCC 10031) we demonstrate the effectiveness of several macrolides in inhibiting biofilm growth in multi-well plates, and the ability of azithromycin (AZM) to improve the effectiveness of the antibacterial last-agent-of-choice for K. pneumoniae infections, colistin methanesulfonate (CMS), against biofilms. This synergistic action was also seen in biofilm tests of several K. pneumoniae hospital isolates and could also be identified in polymyxin B disc-diffusion assays on azithromycin plates. Our work highlights the complexity of antimicrobial-resistance in bacterial pathogens and the need to test antibiotics with biofilm models where potential synergies might provide new therapeutic opportunities not seen in liquid culture or colony-based assays.

Collagen and its derivates are typically obtained by extracting them from fresh animal tissues. Lately, however, there has been an increased interest in obtaining collagen from other sources, such as waste material, because of the growing trend to replace synthetic materials with sustainable, natural counterparts in various industries, as well as to ensure a rational waste revalorization. In this paper, collagen was obtained from non-tanned waste of leather production, taken at different stages of the production process: limed pelt, delimed pelt, and fleshings. A stepwise extraction through acid hydrolysis in 0.5 M acetic acid and subsequent precipitation with NaCl lead to collagen-containing protein extracts. The highest collagen yield was achieved in extracts based on delimed pelt (2.3% m/m after a first extraction round, and an additional 1.4% m/m after the second round). Hyp/Hyl molar ratios of 10.91 in these extracts suggest the presence of type I collagen. Moreover, gels based on these collagen extracts promote adhesion and spreading of HEK293 cells, with cells grown on collagen from delimed pelt showing a larger nuclear and cell expansion than cells grown on traditional bovine tendon atelocollagen. This suggests that these collagen gels are promising natural biomedical carriers and could be used in a wide range of medical and cosmetic applications.

Pseudomonas fluorescens SBW25 is a model soil- and plant-associated bacterium capable of forming a variety of air–liquid interface biofilms in experimental microcosms and on plant surfaces. Previous investigations have shown that cellulose is the primary structural matrix component in the robust and well-attached Wrinkly Spreader biofilm, as well as in the fragile Viscous Mass biofilm. Here, we demonstrate that both biofilms include extracellular DNA (eDNA) which can be visualized using confocal laser scanning microscopy (CLSM), quantified by absorbance measurements, and degraded by DNase I treatment. This eDNA plays an important role in cell attachment and biofilm development. However, exogenous high-molecular-weight DNA appears to decrease the strength and attachment levels of mature Wrinkly Spreader biofilms, whereas low-molecular-weight DNA appears to have little effect. Further investigation with CLSM using an amyloid-specific fluorophore suggests that the Wrinkly Spreader biofilm might also include Fap fibers, which might be involved in attachment and contribute to biofilm strength. The robust nature of the Wrinkly Spreader biofilm also allowed us, using MALDI-TOF mass spectrometry, to identify matrix-associated proteins unable to diffuse out of the structure, as well as membrane vesicles which had a different protein profile compared to the matrix-associated proteins. CLSM and DNase I treatment suggest that some vesicles were also associated with eDNA. These findings add to our understanding of the matrix components in this model pseudomonad, and, as found in other biofilms, biofilm-specific products and material from lysed cells contribute to these structures through a range of complex interactions.

Conflict can have devastating effects on the development and spread of antimicrobial resistance. In Ukraine, early data post-injury are limited. We aim to explore extensively drug-resistant (XDR) Gram negative phenotypes and genotypes for infections arising early following conflict-associated wounds in Ukraine. Carbapenem-resistant infections following conflict-associated wounds in Ukraine (February–May 2024) underwent extended antimicrobial susceptibility testing (AST) for 19 antimicrobial agents using 2025 European Committee for Antimicrobial Susceptibility Testing breakpoints. Carbapenemase genes were identified using a novel multiplex molecular resistance assay. Infections arising in the first seven days versus those arising after seven days since injury were compared by logistic regression. Significance was set at p < 0.05. 100 isolates were tested (53, 53.0% Klebsiella pneumoniae; 16, 16.0% other Enterobacterales; 18, 18.0% Acinetobacter baumanii and 13; 13.0% Pseudomonas aeruginosa). Gentamicin (p = 0.0046) and colistin (p = 0.049) resistance were higher in infections arising later. Overall, resistance rates for amikacin (74/100, 74.0%), cefiderocol (44/100, 44.0%) and ceftazidime-avibactam (26/79, 67.1%) were observed. Prevalent resistance genes included NDM + OXA-48-like (24/100, 24.0%), NDM-only (24/100, 24.0%) and KPC (9/100, 9%). Others included OXA 23-like/51-like, IMP and/or mcr1. Earlier infection isolates had a higher burden of carbapenemases/isolate (p = 0.006). Extensively drug-resistant infections were observed early post-injury in Ukraine, with some trend to further resistance in those arising later in the patient pathway. A diverse presence of carbapenemase genes amid XDR Gram negative phenotypes highlights the importance of early screening for mechanisms of resistance in this setting. This study was funded by the 10.13039/501100000632 Healthcare Infection Society and CW+ Charity.

Staphylococcus aureus (S. aureus) is a causative agent of many hospital- and community-acquired infections with the tendency to develop resistance to all known antibiotics. Therefore, the development of novel antistaphylococcal agents is of urgent need. Sortase A is considered a promising molecular target for the development of antistaphylococcal agents. The main aim of this study was to identify novel sortase A inhibitors. In order to find novel antistaphylococcal agents, we performed phenotypic screening of a library containing 15512 compounds against S. aureus ATCC43300. The molecular docking of hits was performed using the DOCK program and 10 compounds were selected for in vitro enzymatic activity inhibition assay. Two inhibitors were identified, N,N-diethyl-N′-(5-nitro-2-(quinazolin-2-yl)phenyl)propane-1,3-diamine (1) and acridin-9-yl-(1H-benzoimidazol-5-yl)-amine (2), which decrease sortase A activity with IC₅₀ values of 160.3 µM and 207.01 µM, respectively. It was found that compounds 1 and 2 possess antibacterial activity toward 29 tested multidrug resistant S. aureus strains with MIC values ranging from 78.12 to 312.5 mg/L. These compounds can be used for further structural optimization and biological research.