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Multidrug-resistant Gram-negative bacteria are a growing clinical threat, driving the search for alternative antimicrobial strategies, such as antimicrobial peptide (AMP)- based materials. However, the rational design of such systems remains constrained by simplified membrane models that neglect critical components of the Gram-negative envelope, such as lipopolysaccharides and cardiolipin, and fail to capture its dual-membrane architecture. This work establishes a materials-oriented experimental framework for constructing membrane-mimetic oligolamellar structures that actively integrate the human AMP LL-37. These hierarchically organized assemblies emulate the compositional and structural complexity of the Gram-negative inner and outer membranes and have the potential to serve as tunable soft-matter platforms for the delivery of AMPs. Combining small-angle X-ray scattering, electron microscopy, electrophoretic mobility analysis, and coarse-grained molecular dynamics simulations, we show that LL-37 interacts strongly with cardiolipin, driving phase transitions from multilamellar vesicles to nanoscale assemblies, followed by membrane stacking. This restructuring phenomenon is unlikely to occur in conventional single-bilayer systems. In the presence of lipopolysaccharides, polysaccharide side chains modulate but do not suppress this transition, revealing a lipid-specific reorganisation mechanism relevant to the design of AMP-based materials targeting Gram-negative bacteria. These results deepen mechanistic understanding of AMP-membrane interactions and establish design principles for peptide-integrated soft materials with programmable structural responses. The presented platform further enables the development of antimicrobial biointerfaces through targeted membrane remodeling.

Multidrug-resistant Gram-negative bacteria are a growing clinical threat, driving the search for alternative antimicrobial strategies, such as antimicrobial peptide (AMP)- based materials. However, the rational design of such systems remains constrained by simplified membrane models that neglect critical components of the Gram-negative envelope, such as lipopolysaccharides and cardiolipin, and fail to capture its dual-membrane architecture. This work establishes a materials-oriented experimental framework for constructing membrane-mimetic oligolamellar structures that actively integrate the human AMP LL-37. These hierarchically organized assemblies emulate the compositional and structural complexity of the Gram-negative inner and outer membranes and have the potential to serve as tunable soft-matter platforms for the delivery of AMPs. Combining small-angle X-ray scattering, electron microscopy, electrophoretic mobility analysis, and coarse-grained molecular dynamics simulations, we show that LL-37 interacts strongly with cardiolipin, driving phase transitions from multilamellar vesicles to nanoscale assemblies, followed by membrane stacking. This restructuring phenomenon is unlikely to occur in conventional single-bilayer systems. In the presence of lipopolysaccharides, polysaccharide side chains modulate but do not suppress this transition, revealing a lipid-specific reorganisation mechanism relevant to the design of AMP-based materials targeting Gram-negative bacteria. These results deepen mechanistic understanding of AMP-membrane interactions and establish design principles for peptide-integrated soft materials with programmable structural responses. The presented platform further enables the development of antimicrobial biointerfaces through targeted membrane remodeling.

Catheter-associated urinary tract infections (CAUTI) are complex infections often involving multi-species bacteria. Escherichia coli is frequently an early coloniser. Subsequent colonisation by Pseudomonas aeruginosa and coexistence mechanisms between the two strains within urethral catheters is not yet fully understood. In this study, metabolic adaptations between co-isolated clinical E. coli and P. aeruginosa strains were investigated. It was found that P. aeruginosa outgrew E. coli in artificial urine medium (AUM), whereas E. coli dominated in culture broth such as Iso-sensitest. No evidence of direct antagonism was observed. Metabolite analyses revealed distinct metabolite patterns indicating cross-feeding and metabolic adaptations. In AUM, stress-response metabolites were elevated. Additionally, E. coli appeared to experience Fe-limitation in AUM, while the same was not observed for P. aeruginosa. The results highlight the influence of nutrient conditions on processes within mixed biofilms.

Introduction: Bacterial colonization of medical devices is promoting hospital-acquired infections leading to worsening patient outcomes and high costs for society. Sequential bacterial colonization of surfaces may provide altered conditions that benefit pathogens.

Methods: In this study we have investigated the interactions between two pairs of clinical isolates collected from patients that were on mechanical ventilation. Two patients were first colonized by Staphylococcus aureus and thereafter Pseudomonas aeruginosa settled. The two P. aeruginosa isolates were weak colonizers in monoculture. We investigated two hypotheses: (1) S. aureus preconditions material surfaces, facilitating adhesion of later colonizers. (2) S. aureus provides an altered nutrient environment promoting the growth and settlement of other bacteria.

Results: Surface preconditioning did not seem to enhance colonization of P. aeruginosa. However, bacterial growth, biofilm formation, ratio of colony forming units, and metabolic profiles were influenced by co-cultivation. The effects varied depending on nutrient content in the medium.

Discussion: In general, co-cultures appeared to benefit clinical isolates to a higher degree, compared to reference strains. The results indicate that differences in airway microenvironment between patients may have a large effect on the infection process and which pathogens that persist.

Environmental bacterial biofilms play many roles in the ecosystem including cycling of nutrients and serving as food for grazing organisms. Their function is linked to their microbial and chemical composition that may be altered by several parameters including environmental stressors. This manuscript presents a well-characterized model system of four bacterial isolates from a small Swedish river: Pseudomonas sp., Sphingomonas sp., Rhizobium sp. and Pararhizobium sp. Microbiological and chemical phenotypes were investigated including cell and biofilm morphology, as well as biochemical composition in absence and presence of the drug trimethoprim. Vibrational spectroscopy, cryo-X-ray photoelectron spectroscopy and confocal optical microscopy were applied to investigate and characterize monocultures and cocultures. The chemical characterization showed variation of the energy storage substance polyhydroxyalkanoates as well as polysaccharides between isolates and drug exposures. Spatial heterogeneities were observed using Raman microspectroscopy where Sphingomonas sp. cells, formed small clusters, inside the four species consortium, an organization that appeared to protect this isolate during exposure to trimethoprim.

Polymer brushes exhibit unique structural and dynamic properties compared to free polymers due to their confined and tethered nature. While coarse-grained (CG) models for free polymers are well-established in the literature, their direct application to polymer brushes is limited. This is because brushes demonstrate distinct conformational behaviors, scaling laws, and responses to environmental stimuli that may not be accurately captured by models developed for free polymers. We have systematically evaluated chemically specific CG parameters within the MARTINI v3 force field for methyl-methacrylate-based polymer brushes. We have found that the most CG parameter assignments led to an excessive coiling of the brush chains and a strong dependence of the calculated swelling coefficient SC with the water model used (RW, SW, and TW). We traced these issues to an imbalance between polymer-polymer and polymer-water interactions. The revised parameter set accurately reproduced experimental swelling coefficients for methyl-methacrylate-based brushes with varying grafting densities, pH responsiveness, charge, and hydrophilicity, namely, poly(dimethylaminoethyl methacrylate) (pDMAEMA), poly((2-methacryloyloxy)-ethyl trimethylammonium chloride) (pMETAC), poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)] ammonium hydroxide (pMEDSAH), and poly(3-sulfopropyl methacrylate) (pSPMA). Furthermore, these parameters mitigated brush chain hypercoiling and dependence on the water model, ensuring better reproducibility of experimental data and alignment with theoretical brush models.

This study explores the virulence mechanisms of Vibrio cholerae, with a particular emphasis on HapA, a zinc metalloprotease delivered via outer membrane vesicles (OMVs). The findings reveal that OMV-associated HapA disrupts the integrity of tight and adherens junctions in intestinal epithelial cell models more effectively than its purified counterpart, suggesting that association with OMVs substantially potentiates the pathogenic effects of HapA. The study further details the uptake of V. cholerae OMVs by epithelial cells, as well as their targeted degradation of key junctional proteins, including claudin, ZO-1, and ?-catenin. These results highlight the critical role of OMV-associated HapA in compromising epithelial barrier function. Additionally, the use of spheroids and intestinal organoids in our experiments provides deeper insight into bacterial pathogenesis, offering valuable information for the development of targeted therapeutic strategies.

In natural environments, most microorganisms reside attached to a surface growing as a biofilm, which is a universal microbial strategy for their survival. As a result, several studies have focused on the role of the biofilm matrix (made of extracellular polymeric substances (EPS)) in tolerance to antimicrobials. However, few studies have focused directly on the characterization of EPS as a response to antibiotic stress. In this study, we analyzed the impact of trimethoprim (TMP) on the production and characteristics of EPS from four natural biofilm-producing river bacterial strains. Extraction and characterization of EPS were carried out at three concentrations of TMP. EPS properties were monitored using colorimetric tests, infrared spectroscopy and sugar composition analysis. For all strains, EPS quantity and chemistry changed starting at 0.1 mM TMP. The combined results suggest that environmental bacterial strains adapt their EPS production and chemical composition in response to antibiotic exposure. Bacteria may benefit from the change in EPS chemistry since it limits the penetration of TMP into the biofilm and thus protects the cells from the action of the antibiotic. Three main mechanisms are proposed: an increase in the proportion of (i) proteins and reactive functional groups, (ii) mannose and (iii) fatty acids. This study shows that EPS represents a key factor in antibiotic tolerance of bacteria via multiple mechanisms. Thus, this study broadens the discussion concerning antibiotic-resistance as it presents additional processes that may work in tune with genetically acquired resistance to enhance bacterial tolerance to antibiotics.

Cytolysin A (ClyA) is a pore-forming protein from a strongly silenced gene in non-pathogenic Escherichia coli, including typical commensal isolates in the intestinal microbiome of healthy mammalian hosts. Upon overproduction, ClyA-expressing bacteria display a cytolytic phenotype. However, it remains unclear whether sublytic amounts of native ClyA play a role in commensal E. coli-host interactions in vivo. Here, we show that sublytic amounts of ClyA are released via outer membrane vesicles (OMVs) and affect host cells in a remarkable manner. OMVs isolated from ClyA+ E. coli were internalised into cultured colon cancer cells. The OMV-associated ClyA caused reduced levels of cancer-activating proteins such as H3K27me3, CXCR4, STAT3 and MDM2 via the EZH2/H3K27me3/microRNA 622/CXCR4 signalling axis. Our results demonstrate that sublytic amounts of ClyA in OMVs from non-pathogenic E. coli can influence the stability of the EZH2 protein, reducing its activity in epigenetic regulation, causing elevated level of the tumour suppressor protein p53.

Introduction: Tuberculosis (TB) treatment typically involves a tailored combination of four antibiotics based on the drug resistance profile of the infecting strain. The increasing drug resistance of Mycobacterium tuberculosis (Mtb) requires the development of novel antibiotics to ensure effective treatment regimens. Gallium (Ga) is being explored as a repurposed drug against TB due to its ability to inhibit Mtb growth and disrupt iron metabolism. Given the potential interactions between Ga and established antibiotics, we investigated how a combination of Ga with levofloxacin (Lfx) or linezolid (Lzd) affects the growth and metabolome of a multidrug-resistant (MDR) Mtb clinical strain.

Methods: Mtb was cultured using a BACTEC 960 system with concentrations of Ga ranging from 125 to 1,000 μM and with 250 to 500 μM of Ga combined with 0.125 mg/L of Lfx or Lzd. For metabolome analysis, the antibacterials were used at concentrations that inhibited the growth of bacteria without causing cell death. Metabolites were extracted from Mtb cells and analyzed using chromatography-mass spectrometry.

Results: The MDR Mtb strain exhibited a dose-dependent response to Ga. Notably, the enhancement in growth inhibition was statistically significant for the Ga/Lfx combination compared to Ga alone, while no such significance was observed for Ga/Lzd. Moreover, exposure to Ga/Lfx or Ga/Lzd resulted in distinct metabolite profiles. Ga treatment increased the level of aconitate, fumarate, and glucose in the cells, suggesting the inhibition of iron-dependent aconitase and fumarate hydratase, as well as disruption of the pentose phosphate pathway. The levels of glucose, succinic acid, citric acid, and hexadecanoic acid followed a similar pattern in cells exposed to Ga and Ga/Lfx at 500 μM Ga but exhibited different trends at 250 μM Ga.

Discussion: In the presence of Lfx, the Mtb metabolome changes induced by Ga are more pronounced compared to those observed with Lzd. Lfx affects nucleic acids and transcription, which may enhance Ga-dependent growth inhibition by preventing the metabolic redirection that bacteria typically use to bypass iron-dependent enzymes.