Pseudomonas aeruginosa colonizes the sputum of most adult cystic fibrosis patients, forming difficult-to-eradicate biofilms in which bacteria are protected in their self-produced extracellular polymeric substance (EPS) matrices. EPS provide biofilms with viscoelastic properties, causing time-dependent relaxation after stress-induced deformation, according to multiple characteristic time constants. These time constants reflect different biofilm (matrix) components. Since the viscoelasticity of biofilms has been related to antimicrobial penetration but not yet bacterial killing, this study aims to relate killing of P. aeruginosa, in its biofilm mode of growth, by three antimicrobials to biofilm viscoelasticity. P. aeruginosa biofilms were grown for 18 h in a constant-depth film fermenter, with mucin-containing artificial sputum medium (ASM+), artificial sputum medium without mucin (ASM−), or Luria-Bertani (LB) broth; this yielded 100-μm-thick biofilms that differed in their amounts of matrix environmental DNA (eDNA) and polysaccharides. Low-load compression testing, followed by three-element Maxwell analyses, showed that the fastest relaxation component, associated with unbound water, was most important in LB-medium-grown biofilms. Slower components due to water with dissolved polysaccharides, insoluble polysaccharides, and eDNA were most important in the relaxation of ASM+-grown biofilms. ASM−-grown biofilms showed intermediate stress relaxation. P. aeruginosa in LB-medium-grown biofilms was killed most by exposure to tobramycin, colistin, or an antimicrobial peptide, while ASM+ provided the most protective matrix, with less water and most insoluble polysaccharides and eDNA. In conclusion, stress relaxation of P. aeruginosa biofilms grown in different media revealed differences in matrix composition that, within the constraints of the antimicrobials and growth media applied, correlated with the matrix protection offered against different antimicrobials.

An important limitation in cell therapy for the regeneration of tissue is the initial lack of oxygen. After implantation of large 3D cell-seeded structures, cells die rather than contribute to tissue regenerating. Here we've tested oxygen-releasing materials to improve cell survival and growth after implantation. Calcium peroxide (CaO2) in a polymer matrix was used as source of oxygen. Two polymers were tested in order to slow down and extend the period of oxygen release, poly(D,L-lactic acid) and poly(lactic-co-glycolic acid). Compared to CaO2 particles, both releasing systems showed an initially higher and shorter oxygen release. Human mesenchymal stromal cells cultured on casted films of these oxygen-releasing composites required catalase to proliferate, indicating the production of cytotoxic hydrogen peroxide as intermediate. Poly(D,L-lactic acid) and poly(lactic-co-glycolic acid) are less suited for slowly oxygen-releasing materials. Catalase was able to reduce the cytotoxic effect of H2O2.

Optical-coherence-tomography (OCT) is a non-destructive tool for biofilm imaging, not requiring staining, and used to measure biofilm thickness and putative comparison of biofilm structure based on signal intensity distributions in OCT-images. Quantitative comparison of biofilm signal intensities in OCT-images, is difficult due to the auto-scaling applied in OCT-instruments to ensure optimal quality of individual images. Here, we developed a method to eliminate the influence of auto-scaling in order to allow quantitative comparison of biofilm densities in different images. Auto- and re-scaled signal intensities could be qualitatively interpreted in line with biofilm characteristics for single and multi-species biofilms of different strains and species (cocci and rod-shaped organisms), demonstrating qualitative validity of auto- and re-scaling analyses. However, specific features of pseudomonas and oral multi-species biofilms were more prominently expressed after re-scaling. Quantitative validation was obtained by relating average auto- and re-scaled signal intensities across biofilm images with volumetric-bacterial-densities in biofilms, independently obtained using enumeration of bacterial numbers per unit biofilm volume. The signal intensities in auto-scaled biofilm images did not significantly relate with volumetric-bacterial-densities, whereas re-scaled intensities in images of biofilms of widely different strains and species increased linearly with independently determined volumetric-bacterial-densities in the biofilms. Herewith, the proposed re-scaling of signal intensity distributions in OCT-images significantly enhances the possibilities of biofilm imaging using OCT.

Multidrug resistant bacterial infections threaten to become the number one cause of death by the year 2050. Development of antimicrobial dendritic polymers is considered promising as an alternative infection control strategy. For antimicrobial dendritic polymers to effectively kill bacteria residing in infectious biofilms, they have to penetrate and accumulate deep into biofilms. Biofilms are often recalcitrant to antimicrobial penetration and accumulation. Therefore, this work aims to determine the role of compact dendrons with different peripheral composition in their penetration into Pseudomonas aeruginosa biofilms. Red fluorescently labeled dendrons with pH-responsive NH3+ peripheral groups initially penetrated faster from a buffer suspension at pH 7.0 into the acidic environment of P. aeruginosa biofilms than dendrons with OH or COO– groups at their periphery. In addition, dendrons with NH3+ peripheral groups accumulated near the top of the biofilm due to electrostatic double-layer attraction with negatively charged biofilm components. However, accumulation of dendrons with OH and COO– peripheral groups was more evenly distributed across the depth of the biofilms than NH3+ composed dendrons and exceeded accumulation of NH3+ composed dendrons after 10 min of exposure. Unlike dendrons with NH3+ groups at their periphery, dendrons with OH or COO– peripheral groups, lacking strong electrostatic double-layer attraction with biofilm components, were largely washed-out during exposure to PBS without dendrons. Thus, penetration and accumulation of dendrons into biofilms is controlled by their peripheral composition through electrostatic double-layer interactions, which is an important finding for the further development of new antimicrobial or antimicrobial-carrying dendritic polymers.

The introduction of tissue engineering therapies for the repair of bone defects has been limited by poor survival of implanted cells. Because of the absence of a vascular network, the cells in a cell‐scaffold construct are not adequately supplied with oxygen and nutrients. Thus far, all but one strategies to solve this problem have failed. Fortunately, oxygen‐delivering biomaterials have shown promising results. In this study, composite microspheres comprising a poly(trimethylene carbonate) matrix and calcium peroxide particles (PTMC/CaO2) were prepared and assessed for their oxygen‐delivering capacity and potential cytotoxicity. PTMC/CaO2 composite microspheres were shown to release oxygen for several weeks. Oxygen release appeared to be dependent on the presence of cholesterol esterase in the medium. The microspheres were not cytotoxic and promoted mesenchymal stromal cell proliferation under hypoxic conditions in vitro. Copyright © 2016 John Wiley & Sons, Ltd.

Nosocomial infections are a major problem in medical health care. To solve this problem, a series of antimicrobial waterborne paints were prepared by using antimicrobial hyperbranched (HB) emulsifiers. The HB-emulsifiers were prepared by polymerizing AB2 monomers obtained in a one-step reaction of bis(hexamethylene)triamine and carbonyl biscaprolactam. The blocked isocyanate end groups (B groups) of the HB-polymer were utilized to introduce tertiary amino groups through the reaction with compounds comprising either a hydroxyl or a primary amino group and a tertiary amino group. Quaternization of the tertiary amines with 6 different alkyl bromides resulted in 12 amphiphilic cationic species. The 12 emulsifiers showed the successful inhibition and killing of 8 bacterial and 2 fungal strains. The killing efficacy increased with increasing alkyl chain length. The octyl-functionalized compound was chosen for suspension polymerizations because of the good compromise between killing and emulsifying properties. With this emulsifier, aqueous poly(methacrylate) suspensions were prepared, which were stable and had excellent killing properties.

SummaryIn real‐life situations, bacteria are often transmitted from biofilms growing on donor surfaces to receiver ones. Bacterial transmission is more complex than adhesion, involving bacterial detachment from donor and subsequent adhesion to receiver surfaces. Here, we describe a new device to study shear‐induced bacterial transmission from a (stainless steel) pipe to a (silicone rubber) tube and compare transmission of EPS‐producing and non‐EPS‐producing staphylococci. Transmission of an entire biofilm from the donor to the receiver tube did not occur, indicative of cohesive failure in the biofilm rather than of adhesive failure at the donor‐biofilm interface. Biofilm was gradually transmitted over an increasing length of receiver tube, occurring mostly to the first 50 cm of the receiver tube. Under high‐shearing velocity, transmission of non‐EPS‐producing bacteria to the second half decreased non‐linearly, likely due to rapid thinning of the lowly lubricious biofilm. Oppositely, transmission of EPS‐producing strains to the second tube half was not affected by higher shearing velocity due to the high lubricity and stress relaxation of the EPS‐rich biofilms, ensuring continued contact with the receiver. The non‐linear decrease of ongoing bacterial transmission under high‐shearing velocity is new and of relevance in for instance, high‐speed food slicers and food packaging.

Acetaminophen overdoses cause cell injury in the liver. It is widely accepted that liver toxicity is initiated by the reactive N-acetylpara-aminophenol (APAP) metabolite N-acetyl-p-benzoquinone imine (NAPQI), which first depletes glutathione and then irreversibly binds to mitochondrial proteins and nuclear DNA. As a consequence, mitochondrial respiration is inhibited, and DNA strands break. NAPQI also promotes the oxidative stress since glutathione is one of the main free-radical scavengers in the cell. However, so far it is unknown where exactly free radicals are generated. In this study, we used relaxometry, a novel technique that allows nanoscale magnetic resonance imaging detection of free radicals. The method is based on fluorescent nanodiamonds, which change their optical properties based on their magnetic surrounding. To achieve subcellular resolution, these nanodiamonds were targeted to cellular locations, that is, the cytoplasm, mitochondria, and the nucleus. Since relaxometry is sensitive to spin noise from radicals, we were able to measure the radical load in these different organelles. For the first time, we measured APAP-induced free-radical production in an organelle-specific manner, which helps predict and better understand cellular toxicity.