Liposome destruction by hydrodynamic cavitation in comparison to chemical, physical and mechanical treatments

Liposomes are widely applied in research, diagnostics, medicine and in industry. In this study we show for the first time the effect of hydrodynamic cavitation on liposome stability and compare it to the effect of well described chemical, physical and mechanical treatments. Fluorescein loaded giant 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid vesicles were treated with hydrodynamic cavitation as promising method in inactivation of biological samples. Hydrodynamic treatment was compared to various chemical, physical and mechanical stressors such as ionic strength and osmolarity agents (glucose, Na+, Ca2+, and Fe3+), free radicals, shear stresses (pipetting, vortex mixing, rotational shear stress), high pressure, electroporation, centrifugation, surface active agents (Triton X-100, ethanol), microwave irradiation, heating, freezing-thawing, ultrasound (ultrasonic bath, sonotrode). The fluorescence intensity of individual fluorescein loaded lipid vesicles was measured with confocal laser microscopy. The distribution of lipid vesicle size, vesicle fluorescence intensity, and the number of fluorescein loaded vesicles was determined before and after treatment with different stressors. The different environmental stressors were ranked in order of their relative effect on liposome fluorescein release. Of all tested chemical, physical and mechanical treatments for stability of lipid vesicles, the most detrimental effect on vesicles stability had hydrodynamic cavitation, vortex mixing with glass beads and ultrasound. Here we showed, for the first time that hydrodynamic cavitation was among the most effective physico-chemical treatments in destroying lipid vesicles. This work provides a benchmark for lipid vesicle robustness to a variety of different physico-chemical and mechanical parameters important in lipid vesicle preparation and application

Viscoelastic response of Escherichia coli biofilms to genetically altered expression of extracellular matrix components

How viscoelastic properties of the extracellular matrix affect the various biological functions conferred by biofilms is an important question in microbiology. In this study viscoelastic response of Escherichia coli biofilms to genetically altered expression of extracellular matrix components was studied. Biofilms of the wild type E. coli MG1655 and its mutant strains producing different amounts of extracellular matrix components (curli, colonic acid, poly-β-1,6-N-acetyl-D-glucosamine) were used to examine the viscoelastic behavior of biofilms grown at solid -atmosphere interface. The results suggest that the presence of curli proteins dominates biofilm mechanical behavior. The rheological data indicate that cohesive energy of the biofilm was highest in the wild type strain. The results demonstrate the importance of extracellular matrix composition for biofilm mechanical properties. We propose that by genetically altering the expression of extracellular matrix polymers bacteria are able to modulate the mechanical properties of their local environment in accordance with bulk environmental conditions

Acetan and acetan-like polysaccharides

Bacteria produce a variety of multifunctional polysaccharides, including structural, intracellular, and extracellular polysaccharides. They are attractive for the industrial sector due to their natural origin, sustainability, biodegradability, low toxicity, stability, unique viscoelastic properties, stable cost, and supply. When incorporated into different matrices, they may control emulsification, stabilization, crystallization, water release, and encapsulation. Acetan is an important extracellular water-soluble polysaccharide produced mainly by bacterial species of the genera Komagataeibacter and Acetobacter. Since its original description in Komagataeibacter xylinus, acetan-like polysaccharides have also been described in other species of acetic acid bacteria. Our knowledge on chemical composition of different acetan-like polysaccharides, their viscoelasticity, and the genetic basis for their production has expanded during the last years. Here, we review data on acetan biosynthesis, its molecular structure, genetic organization, and mechanical properties. In addition, we have performed an extended bioinformatic analysis on acetan-like polysaccharide genetic clusters in the genomes of Komagataeibacter and Acetobacter species. The analysis revealed for the first time a second acetan-like polysaccharide genetic cluster, that is widespread in both genera. All species of the Komagataeibacter possess at least one acetan genetic cluster, while it is present in only one third of the Acetobacter species surveyed

Peptide signaling without feedback in signal production operates as a true quorum sensing communication system in Bacillus subtilis

Bacterial quorum sensing (QS) is based on signal molecules (SM), which increase in concentration with cell density. At critical SM concentration, a variety of adaptive genes sharply change their expression from basic level to maximum level. In general, this sharp transition, a hallmark of true QS, requires an SM dependent positive feedback loop, where SM enhances its own production. Some communication systems, like the peptide SM-based ComQXPA communication system of Bacillus subtilis, do not have this feedback loop and we do not understand how and if the sharp transition in gene expression is achieved. Based on experiments and mathematical modeling, we observed that the SM peptide ComX encodes the information about cell density, specific cell growth rate, and even oxygen concentration, which ensure power-law increase in SM production. This enables together with the cooperative response to SM (ComX) a sharp transition in gene expression level and this without the SM dependent feedback loop. Due to its ultra-sensitive nature, the ComQXPA can operate at SM concentrations that are 100–1000 times lower than typically found in other QS systems, thereby substantially reducing the total metabolic cost of otherwise expensive ComX peptide

Elucidation of the AI-2 communication system in the food-borne pathogen Campylobacter jejuni by whole-cell-based biosensor quantification

The food-borne pathogen Campylobacter jejuni produces autoinducer-2 (AI-2) as an interspecies signalling molecule. AI-2 can trigger enhanced colonisation and biofilm formation, and this poses a serious risk to public health. To date, this communication system of C. jejuni is only partially understood, as detection and quantifi- cation of such autoinducer signalling molecules in complex media is hard to achieve. We have developed a whole-cell Vibrio harveyi-based biosensor assay to accurately quantify and follow production of AI-2 by C. jejuni 81–176 in a defined growth medium and in a model food system. Several V. harveyi strains were tested, but the most sensitive bioluminescent response to C. jejuni AI-2 was achieved with V. harveyi MM30, likely due to its ability to self-amplify the response to AI-2. The AI-2 concentrations measured by this biosensor were confirmed using an independent analytical method, HPLC-FLD, which we introduced for Campylobacter analytics for the first time. The AI-2 concentration produced by C. jejuni 81–176 in the model food system was ~5-fold that in the defined growth medium, at the same cell density. Together with the linear increments in AI-2 concentrations with cell density, this suggests that in C. jejuni, AI-2 represents a metabolic by-product rather than a true quorum- sensing molecule. This biosensor method is highly sensitive, as shown by the reduction in the limit of detection (by a factor of 100) compared to HPLC-FLD, and it enables quantification of AI-2 in complex matrices, such as food, which will help to improve the quality and safety of food production

Sequestration of polystyrene microplastics by jellyfish mucus

The worldwide microplastics pollution is a serious environmental and health problem that is currently not effectively mitigated. In this work we tested jellyfish mucus as a new bioflocculent material capable of sequestration of polystyrene microplastics in aqueous environments. Mucus material was collected from different jellyfish species and was used to trap fluorescently tagged polystyrene microspheres. The efficiency of removal was tested using varying concentrations of microplastics and mucus. The interaction between the microplastics and mucus was determined by viscosity measurements and confocal laser scanning microscopy. Different mucus preparation methods were also tested: freshly prepared, mechanically sheared, freeze-thawed, freeze-dried, and hydrolyzed mucus. The results demonstrate that jellyfish mucus can efficiently sequester polystyrene microplastics particles from the suspension. The fraction of the removed microplastics was highest with freshly prepared mucus and decreased with freeze-thawing and freeze-drying. The mucus ability to sequester microplastics was completely lost in the hydrolyzed mucus. The results imply that the intact jellyfish mucus has the potential to be used as a biopolymer capable of removing microplastics material

Kin discrimination modifies strain distribution, spatial segregation and incorporation of extracellular matrix polysaccharide mutants of Bacillus subtilis strains into mixed floating biofilms

Microorganisms in nature form multicellular groups called biofilms. In biofilms, bacteria embedded in the extracellular matrix (ECM) interact intensely due to their proximity. Most studies have investigated genetically homogeneous biofilms, leaving a gap in knowledge on genetically heterogeneous biofilms. Recent insights show that a Gram-positive model bacterium, Bacillus subtilis, discriminates between strains of high (kin) and low (nonkin) genetic similarity, reflected in merging (kin) and boundaries (nonkin) between swarms. However, it is unclear how kinship between interacting strains affects their fitness, the genotype assortment, and incorporation of the mutant lacking the main structural ECM polysaccharide (EpsA-O) into floating biofilms (pellicles). We cultivate Bacillus subtilis strains as mixtures of isogenic, kin and nonkin strain combinations in the biofilm-promoting medium (MSgg) in static conditions, allowing them to form pellicles. We show that in nonkin pellicles, the dominant strain strongly reduced the frequency of the other strain. Segregation of nonkin mixtures in pellicles increased and invasion of nonkin EpsA-O-deficient mutants into pellicles decreased compared to kin and isogenic floating biofilms. Kin and isogenic strains had comparable relative frequencies in pellicles and showed more homogenous cell mixing. Overall, our results emphasize kin discrimination as a social behavior that shapes strain distribution, spatial segregation and ECM mutant ability to incorporate into genetically heterogenous biofilms of B. subtili

Surfactin facilitates horizontal gene transfer in Bacillus subtilis

Genetic competence for the uptake and integration of extracellular DNA is a key process in horizontal gene transfer (HGT), one of the most powerful forces driving the evolution of bacteria. In several species, development of genetic competence is coupled with cell lysis. Using Bacillus subtilis as a model bacterium, we studied the role of surfactin, a powerful biosurfactant and antimicrobial lipopeptide, in genetic transformation. We showed that surfactin itself promotes cell lysis and DNA release, thereby promoting HGT. These results, therefore, provide evidence for a fundamental mechanism involved in HGT and significantly increase our understanding of the spreading of antibiotic resistance genes and diversification of microbial communities in the environment

Antibiofilm potential of Lavandula preparations against Campylobacter jejuni

New approaches for the control of Campylobacter jejuni biofilms in the food industry are being studied intensively. Natural products are promising alternative antimicrobial substances to control biofilm production, with particular emphasis on plant extracts. Dried flowers of Lavandula angustifolia were used to produce LEO, LEF, and LEW. The chemical compositions determined for these Lavandula preparations included seven major compounds that were selected for further testing. These were tested against C. jejuni, for biofilm degradation and removal. Next-generation sequencing was used to study the molecular mechanisms underlying LEO actions against C. jejuni adhesion and motility. Analysis of LEO revealed 1,8-cineol, linalool and linalyl acetate as the main components. For LEF and LEW, the main components were phenolic acid glycosides, with flavonoids rarely present. The minimal inhibitory concentrations of the Lavandula preparations and pure compounds against C. jejuni ranged from 0.2 mg/mL to 1 mg/mL. LEO showed the strongest biofilm degradation. The reduction of C. jejuni adhesion was ≥1 log10 CFU/mL, which satisfies European Food Safety Authority recommendations. Lavandula preparations reduced C. jejuni motility by almost 50%, which consequently can impact upon biofilm formation. These data are in line with the transcriptome analysis of C. jejuni, which indicated that LEO down-regulated genes important for biofilm formation. LEW also showed good antibacterial and anti-biofilm effects, particularly against adhesion and motility mechanisms. This defines an innovative approach using alternative strategies and novel targets to combat bacterial biofilm formation, and hence the potential to develop new effective agents with biofilm-degrading activities

Systems view of Bacillus subtilis pellicle development

In this study, we link pellicle development at the water–air interface with the vertical distribution and viability of the individual B. subtilis PS-216 cells throughout the water column. Real-time interfacial rheology and time-lapse confocal laser scanning microscopy were combined to correlate mechanical properties with morphological changes (aggregation status, filament formation, pellicle thickness, spore formation) of the growing pellicle. Six key events were identified in B. subtilis pellicle formation that are accompanied by a major change in viscoelastic and morphology behaviour of the pellicle. The results imply that pellicle development is a multifaceted response to a changing environment induced by bacterial growth that causes population redistribution within the model system, reduction of the viable habitat to the water–air interface, cell development, and morphogenesis. The outcome is a build-up of mechanical stress supporting structure that eventually, due to nutrient deprivation, reaches the finite thickness. After prolonged incubation, the formed pellicle collapses, which correlates with the spore releasing process. The pellicle loses the ability to support mechanical stress, which marks the end of the pellicle life cycle and entry of the system into the dormant state