Photoacoustic removal of Enterococcus faecalis biofilms from titanium surface with an Er:YAG laser using super short pulses.

Biofilms that grow on implant surfaces pose a great risk and challenge for the dental implant survival. In this work, we have applied Er:YAG photoacoustic irrigation using super short pulses (Er:YAG-SSP) to remove biofilms from the titanium surfaces in the non-contact mode. Mature Enterococcus faecalis biofilms were treated with saline solution, chlorhexidine, and hydrogen peroxide, or photoacoustically with Er:YAG-SSP for 10 or 60 s. The number of total and viable bacteria as well as biofilm surface coverage was determined prior and after different treatments. Er:YAG-SSP photoacoustic treatment significantly increases the biofilm removal rate compared to saline or chemically treated biofilms. Up to 92% of biofilm-covered surface can be cleaned in non-contact mode during 10 s without the use of abrasives or chemicals. In addition, Er:YAG-SSP photoacoustic irrigation significantly decreases the number of viable bacteria that remained on the titanium surface. Within the limitations of the present in vitro model, the ER:YAG-SSP seems to constitute an efficient therapeutic option for quick debridement and decontamination of titanium implants without using abrasives or chemicals.

A Monte Carlo study of giant vesicle morphologies in nonequilibrium environments.

It is known that giant vesicles undergo dynamic morphological changes when exposed to a detergent. The solubilization process may take multiple pathways. In this work, we identify lipid vesicle shape dynamics before the solubilization of 1,2-dioleoyl-sn-glycero-3-phosphocholine giant vesicles with Triton X-100 (TR) detergent. The violent lipid vesicle dynamics was observed with laser confocal scanning microscopy and was qualitatively explained via a numerical simulation. A three-dimensional Monte Carlo scheme was constructed that emulated the nonequilibrium conditions at the beginning stages of solubilization, accounting for a gradual addition of TR detergent molecules into the lipid bilayers. We suggest that the main driving factor for morphology change in lipid vesicles is the associative tendency of the TR molecules, which induces spontaneous curvature of the detergent inclusions, an intrinsic consequence of their molecular shape. The majority of the observed lipid vesicle shapes in the experiments were found to correspond very well to the numerically calculated shapes in the phase space of possible solutions. The results give an insight into the early stages of lipid vesicle solubilization by amphiphilic molecules, which is nonequilibrium in nature and very difficult to study.

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

How the viscoelastic properties of the extracellular matrix affect the various biological functions conferred by biofilms is an important question in microbiology. In this study, the viscoelastic response of Escherichia coli biofilms to the 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, colanic acid, and poly-ß-1,6-N-acetyl-d-glucosamine) were used to examine the viscoelastic behavior of biofilms grown at the solid-atmosphere interface. The results suggest that the presence of curli proteins dominates biofilm mechanical behavior. The rheological data indicate that the cohesive energy of the biofilm was the 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.

Structure and Dynamics of a Model Polymer Mixture Mimicking a Levan-Based Bacterial Biofilm of Bacillus subtilis.

In this paper, we report on the structure and dynamics of biologically important model polymer mixtures that mimic the extracellular polymeric matrix in native biofilm of Bacillus subtilis. This biofilm is rich in nonionic polysaccharide levan, but also contains other biopolymers such as DNA and proteins in small concentrations. Aiming to identify the contribution of each component to the formation of the biofilm, our investigations encompassed dynamic rheology, small-angle X-ray scattering, dynamic light scattering, microscopy, densitometry, and sound velocity measurements. As it turned out, this very powerful combination of techniques is able to provide solid results on the dynamical and structural aspects of the microbiologically and chemically complex biofilm formations. Macroscopic rheological measurements revealed that the addition of DNA to levan solution increased the viscosity, pseudoplasticity, and elasticity of the system. The addition of protein contributed similarly, but also increased the rigidity of the system. This confirms that the presence of minor biofilm components is essential for biofilm formation. DNA and proteins appear to confine levan molecules within their supramolecular structure and, in this way, restrict the role of levan to merely a filling agent. These findings were complemented by small-angle X-ray scattering data, which provided insight into the structure on a molecular scale. One of the essential goals of this work was to compare the structural properties of the native biofilm and synthetic biofilm mixture.

Viscoelastic properties of levan-DNA mixtures important in microbial biofilm formation as determined by micro- and macrorheology.

We studied the viscoelastic properties of homogeneous and inhomogeneous levan-DNA mixtures using optical tweezers and a rotational rheometer. Levan and DNA are important components of the extracellular matrix of bacterial biofilms. Their viscoelastic properties influence the mechanical as well as molecular-transport properties of biofilm. Both macro- and microrheology measurements in homogeneous levan-DNA mixtures revealed pseudoplastic behavior. When the concentration of DNA reached a critical value, levan started to aggregate, forming clusters of a few microns in size. Microrheology using optical tweezers enabled us to measure local viscoelastic properties within the clusters as well as in the DNA phase surrounding the levan aggregates. In phase-separated levan-DNA mixtures, the results of macro- and microrheology differed significantly. The local viscosity and elasticity of levan increased, whereas the local viscosity of DNA decreased. On the other hand, the results of bulk viscosity measurements suggest that levan clusters do not interact strongly with DNA. Upon treatment with DNase, levan aggregates dispersed. These results demonstrate the advantages of microrheological measurements compared to bulk viscoelastic measurements when the materials under investigation are complex and inhomogeneous, as is often the case in biological samples.

Evaluating SAXS Results on Aqueous Solutions of Various Bacterial Levan utilizing the String-of-Beads Model.

Polysaccharide levan is a homopolymer of fructose and is an important component of plants, yeast, fungi and some bacterial biofilms. In this paper we report on the structural properties of aqueous solutions of bacterial levan utilizing smallangle X-ray scattering and light microscopy. In addition to commercially available levan isolated from Zymomonas mobilis and Erwinia herbicola, we also studied levan isolated and purified from the biofilm of Bacillus subtilis. The smallangle X-ray scattering data were analyzed by the string-of-beads model that revealed qualitative differences in the structure of levan molecules. Levan can be represented as a semi-flexible chain that interacts intra- and inter-molecularly and therefore forms various suprastructures on larger size scales. Increasing the concentration of levan makes the levan structure more compact, which was observed on the nano as well as on the micro scale. The structures with most homogeneously distributed polymer local density were found in B. subtilis levan solutions.

Structure and dynamics of a polysaccharide matrix: aqueous solutions of bacterial levan.

The polysaccharide levan is a homopolymer of fructose and appears in nature as an important structural component of some bacterial biofilms. This paper reports the structural and dynamic properties of aqueous solutions of levan of various origin obtained from dynamic rheological, small-angle X-ray scattering, static and dynamic light scattering, as well as density and sound velocity measurements, determination of polymer branching after per-O-methylation, and microscopy. Besides samples of commercially available levan from Zymomonas mobilis and Erwinia herbicola, we also isolated, purified, and studied a levan sample from the biofilm of Bacillus subtilis. The results of dynamic rheological and light scattering measurements revealed very interesting viscoelastic properties of levan solutions even at very low polymer concentrations. The findings were complemented by small-angle X-ray scattering data that revealed some important differences in the structure of the aqueous levan solutions at the molecular level. Besides presenting detailed dynamic and structural results on the polysaccharide systems of various levans, one of the essential goals of this work was to point out the level of structural information that may be obtained for such polymer systems by combining basic physicochemical, rheological, and various light scattering techniques.

Mechanical constraints to unbound expansion of B. subtilis on semi-solid surfaces.

IMPORTANCE: How bacterial cells colonize new territory is a problem of fundamental microbiological and biophysical interest and is key to the emergence of several phenomena of biological, ecological, and medical relevance. Here, we demonstrate how bacteria stuck in a colony of finite size can resume exploration of new territory by aquaplaning and how they fine tune biofilm viscoelasticity to surface material properties that allows them differential mobility. We show how changing local interfacial forces and colony viscosity results in a plethora of bacterial morphologies on surfaces with different physical and mechanical properties.

Biofilm removal from Difficult-to-Reach places via secondary cavitation within a constrained geometry mimicking a Periodontal/Peri-Implant pocket.

Biofilm removal from the apical region of the periodontal or peri-implant pocket, which is very difficult to achieve with mechanical instruments, is a major unresolved issue in dentistry. Here, we propose the use of photoacoustically induced streaming and secondary cavitation to achieve superior cleaning efficacy in the apical region of the periodontal and peri-implant pocket. We have used a prefabricated narrow wedge system that mimics the consistency of periodontal and peri-implant pockets of both healthy and severely inflamed tissue. We studied the effect of single-pulse modality Er:YAG on Pseudomonas aeruginosa biofilm removal. We used different laser energies, fiber-tip positions, and laser treatment durations. The cleaning process was monitored in real-time with a high-speed camera after each individual laser pulse application. The obtained results suggest that biofilm cleaning efficacy in a difficult-to-reach place in healthy model tissue is directly related to the onset of secondary cavitation bubble formation, which correlates with a significant improvement of biofilm removal from the apical region of the periodontal or peri-implant pocket. In comparison to the healthy tissue model, the laser energy in inflamed tissue model had to be increased to obtain comparable biofilm cleaning efficacy. The advantage of photoacoustic cavitation compared to other methods is that laser-induced cavitation can trigger secondary cavitation at large distances from the point of laser application, which in principle allows biofilm removal at distant locations not reachable with a laser fiber tip or other mechanical instruments.

eDNA Provides a Scaffold for Autoaggregation of B. subtilis in Bacterioplankton Suspension.

The self-binding of bacterial cells, or autoaggregation, is, together with surface colonization, one of the first steps in the formation of a mature biofilm. In this work, the autoaggregation of B. subtilis in dilute bacterial suspensions was studied. The dynamics of cell lysis, eDNA release, and bacterial autoaggregate assembly were determined and related to the spatial autocorrelation of bacterial cells in dilute planktonic bacterial suspensions. The non-random distribution of cells was associated with an eDNA network, which stabilized the initial bacterial cell-cell aggregates. Upon the addition of DNase I, the aggregates were dispersed. The release of eDNA during cell lysis allows for the entrapment of bacterial drifters at a radius several times the size of the dying bacteria. The size of bacterial aggregates increased from 2 to about 100 µm in diameter in dilute bacterial suspensions. The results suggest that B. subtilis cells form previously unnoticed continuum of autoaggregate structures during planktonic growth.

Biofilm Removal from In Vitro Narrow Geometries Using Single and Dual Pulse Er:YAG Laser Photoacoustic Irrigation.

The disinfection and removal of biofilm from titanium dental implants remains a great challenge in oral medicine. Here we present results of novel photoacoustic irrigation laser modalities for biofilm removal in model geometries mimicking the peri-implant pocket. The efficacy of single pulse (Er:YAG-SSP) and dual pulse (Er:YAG-AutoSWEEPS) photoacoustic irrigation modalities were determined for Enterococcus faecalis biofilm decontamination from titanium surfaces in narrow cylindrical and square gap geometries. The density of bacteria as well as the number of live bacteria were determined prior and after different photoacoustic treatments. Both SSP and AutoSWEEPS photoacoustic irrigation techniques removed at least 92% of biofilm bacteria during the 10 s photoacoustic treatment. The effectiveness of cleaning was better in the narrow square gap geometry compared to the cylindrical geometry. The dual pulse Er:YAG-AutoSWEEPS photoacoustic irrigation showed better results compared to SSP modality. No chemical adjuvants were needed to boost the effectiveness of the photoacoustic irrigation in the saline solution. The results imply that photoacoustic irrigation is an efficient cleaning method for debridement and decontamination in narrow geometries and should be considered as a new therapeutic option for the treatment of peri-implant diseases.

Water treatment by cavitation: Understanding it at a single bubble - bacterial cell level.

Cavitation is a potentially useful phenomenon accompanied by extreme conditions, which is one of the reasons for its increased use in a variety of applications, such as surface cleaning, enhanced chemistry, and water treatment. Yet, we are still not able to answer many fundamental questions related to efficacy and effectiveness of cavitation treatment, such as: "Can single bubbles destroy contaminants?" and "What precisely is the mechanism behind bubble's cleaning power?". For these reasons, the present paper addresses cavitation as a tool for eradication and removal of wall-bound bacteria at a fundamental level of a single microbubble and a bacterial cell. We present a method to study bubble-bacteria interaction on a nano- to microscale resolution in both space and time. The method allows for accurate and fast positioning of a single microbubble above the individual wall-bound bacterial cell with optical tweezers and triggering of a violent microscale cavitation event, which either results in mechanical removal or destruction of the bacterial cell. Results on E. coli bacteria show that only cells in the immediate vicinity of the microbubble are affected, and that a very high likelihood of cell detachment and cell death exists for cells located directly under the center of a bubble. Further details behind near-wall microbubble dynamics are revealed by numerical simulations, which demonstrate that a water jet resulting from a near-wall bubble implosion is the primary mechanism of wall-bound cell damage. The results suggest that peak hydrodynamic forces as high as 0.8 µN and 1.2 µN are required to achieve consistent E. coli bacterial cell detachment or death with high frequency mechanical perturbations on a nano- to microsecond time scale. Understanding of the cavitation phenomenon at a fundamental level of a single bubble will enable further optimization of novel water treatment and surface cleaning technologies to provide more efficient and chemical-free processes.

Surfactin molecules with a cone-like structure promote the formation of membrane domains with negative spontaneous curvature and induce membrane invaginations.

Surfactin uniquely influences lipid bilayer structure by initially inducing membrane invaginations before solubilization. In this study, we exposed DOPC giant vesicles to various surfactin concentrations at different temperatures and observed surfactin-induced membrane invaginations by using differential interference contrast and confocal laser fluorescence microscopy. These invaginations were stable at room temperature but not at higher temperatures. Surfactin molecules induce membrane nanodomains with negative spontaneous curvature and membrane invaginations despite their intrinsic conical shape and intrinsic positive curvature. Considering the experimentally observed capacity of surfactin to fluidize lipid acyl chains and induce partial dehydration of lipid headgroups, we propose that the resulting surfactin-lipid complexes exhibit a net negative spontaneous curvature. We further conducted 3D numerical Monte Carlo (MC) simulations to investigate the behaviour of vesicles containing negative curvature nanodomains within their membrane at varying temperatures. MC simulations demonstrated strong agreement with experimental results, revealing that invaginations are preferentially formed at low temperatures, while being less pronounced at elevated temperatures. Our findings go beyond the expectations of the Israelachvili molecular shape and packing concepts analysis. These concepts do not take into account the influence of specific interactions between neighboring molecules on the inherent shapes of molecules and their arrangement within curved membrane nanodomains. Our work contributes to a more comprehensive understanding of the complex factors governing vesicle morphology and membrane organization and provides insight into the role of detergent-lipid interactions in modulating vesicle morphology.

The evolution of cavitation in narrow soft-solid wedge geometry mimicking periodontal and peri-implant pockets.

In periodontology and implantology, laser-induced cavitation has not yet been used to treat biofilm-related problems. In this study we have checked how soft tissue affects the evolution of cavitation in a wedge model representing periodontal and peri-implant pocket geometry. One side of the wedge model was composed of PDMS mimicking soft periodontal or peri-implant biological tissue, the other side was composed of glass mimicking hard tooth root or implant surface, which allowed observations of the cavitation dynamics with an ultrafast camera. Different laser pulse modalities, PDMS stiffness, and irrigants were tested for their effect on the evolution of cavitation in the narrow wedge geometry. The PDMS stiffness varied in a range that corresponds to severely inflamed, moderately inflamed, or healthy gingival tissue as determined by a panel of dentists. The results imply that deformation of the soft boundary has a major effect on the Er:YAG laser-induced cavitation. The softer the boundary, the less effective the cavitation. We show that in a stiffer gingival tissues model, photoacoustic energy can be guided and focused at the tip of the wedge model, where it enables generation of secondary cavitation and more effective microstreaming. The secondary cavitation was absent in severely inflamed gingival model tissue, but could be induced with a dual-pulse AutoSWEEPS laser modality. This should in principle increase cleaning efficiency in the narrow geometries such as those found in the periodontal and peri-implant pockets and may lead to more predictable treatment outcomes.

Bacterial cell wall material properties determine E. coli resistance to sonolysis.

The applications of bacterial sonolysis in industrial settings are plagued by the lack of the knowledge of the exact mechanism of action of sonication on bacterial cells, variable effectiveness of cavitation on bacteria, and inconsistent data of its efficiency. In this study we have systematically changed material properties of E. coli cells to probe the effect of different cell wall layers on bacterial resistance to ultrasonic irradiation (20 kHz, output power 6,73 W, horn type, 3 mm probe tip diameter, 1 ml sample volume). We have determined the rates of sonolysis decay for bacteria with compromised major capsular polymers, disrupted outer membrane, compromised peptidoglycan layer, spheroplasts, giant spheroplasts, and in bacteria with different cell physiology. The non-growing bacteria were 5-fold more resistant to sonolysis than growing bacteria. The most important bacterial cell wall structure that determined the outcome during sonication was peptidoglycan. If peptidoglycan was remodelled, weakened, or absent the cavitation was very efficient. Cells with removed peptidoglycan had sonolysis resistance equal to lipid vesicles and were extremely sensitive to sonolysis. The results suggest that bacterial physiological state as well as cell wall architecture are major determinants that influence the outcome of bacterial sonolysis.

Phi 6 Bacteriophage Inactivation by Metal Salts, Metal Powders, and Metal Surfaces.

The interaction of phages with abiotic environmental surfaces is usually an understudied field of phage ecology. In this study, we investigated the virucidal potential of different metal salts, metal and ceramic powders doped with Ag and Cu ions, and newly fabricated ceramic and metal surfaces against Phi6 bacteriophage. The new materials were fabricated by spark plasma sintering (SPS) and/or selective laser melting (SLM) techniques and had different surface free energies and infiltration features. We show that inactivation of Phi6 in solutions with Ag and Cu ions can be as effective as inactivation by pH, temperature, or UV. Adding powder to Ag and Cu ion solutions decreased their virucidal effect. The newly fabricated ceramic and metal surfaces showed very good virucidal activity. In particular, 45%TiO2 + 5%Ag + 45%ZrO2 + 5%Cu, in addition to virus adhesion, showed virucidal and infiltration properties. The results indicate that more than 99.99% of viruses deposited on the new ceramic surface were inactivated or irreversibly attached to it.

High virucidal potential of novel ceramic-metal composites fabricated via hybrid selective laser melting and spark plasma sintering routes.

In this work, we combine selective laser melting (SLM) and spark plasma sintering (SPS) to fabricate new materials with high virucidal potential. Various bioactive disc-shaped ceramics, metal alloys, and composites were fabricated and tested against bacteriophage Phi6-a model system for RNA-enveloped viruses. We prepared silver-doped titanium dioxide (TiO2 + 2.5‒10% Ag), copper-doped titanium dioxide (TiO2 + 2.5‒10% Cu), Cu2NiSiCr, and Cu15Ni8Sn composite materials (metal lattices filled with ceramics). The virucidal tests of the ceramic and metal powders were performed in buffered suspensions, while the surfaces of the discs were tested by swabbing. The results show that the virus titer on the TiO2 + 10% Ag ceramic and CuNi2SiCr metal discs decreased by 4 logs after 15 min of exposure to the surfaces compared to the control ceramic and steel discs. We show that SLM 3D printed pre-alloyed CuNi2SiCr filled with bioactive TiO2 + 10% Ag nanopowders and sintered by the SPS process combines the simplicity of printing with the strength and virucidal properties of Ag and Cu materials. The proposed new virucidal materials were also used for the fabrication of prototype elevator buttons.

Thermal and Rheological Properties of Gluten-Free, Starch-Based Model Systems Modified by Hydrocolloids.

Obtaining good-quality gluten-free products represents a technological challenge; thus, it is important to understand how and why the addition of hydrocolloids influences the properties of starch-based products. To obtain insight into the physicochemical changes imparted by hydrocolloids on gluten-free dough, we prepared several suspensions with different corn starch/potato starch/hydroxpropyl methyl cellulose/xanthan gum/water ratios. Properties of the prepared samples were determined by differential scanning calorimetry and rheometry. Samples with different corn/potato starch ratios exhibited different thermal properties. Xanthan gum and HPMC (hydroxypropyl methyl cellulose) exhibited a strong influence on the rheological properties of the mixtures since they increased the viscosity and elasticity. HPMC and xanthan gum increased the temperature of starch gelatinization, as well as they increased the viscoelasticity of the starch model system. Although the two hydrocolloids affected the properties of starch mixtures in the same direction, the magnitude of their effects was different. Our results indicate that water availability, which plays a crucial role in the starch gelatinization process, could be modified by adding hydrocolloids such as, hydroxypropyl methyl cellulose and xanthan gum. By adding comparatively small amounts of the studied hydrocolloids to starch, one can achieve similar thermo-mechanical effects by the addition of gluten. Understanding these effects of hydrocolloids could contribute to the development of better quality gluten-free bread with optimized ingredient content.

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.

Asymmetric response of carbon metabolism at high and low salt stress in Vibrio sp. DSM14379.

Energy redistribution between growth and maintenance in salt-stressed cells is especially important for bacteria living in estuarine environments. In this study, Gram-negative bacterium Vibrio sp. DSM14379, isolated from the estuarine waters of the northern Adriatic Sea, was grown aerobically in a peptone-yeast extract medium with different salt concentrations (ranging from 0.3% to 10% (w/v) NaCl). Carbon flux through the central metabolic pathways was determined at low and high salt concentrations. At low salt concentrations, total endogenous respiration, dehydrogenase activity, and net intracellular adenosine triphosphate (ATP) concentration significantly increased, the phosphofructokinase and pyruvate kinase activity decreased, whereas glucose-6-phosphate dehydrogenase activity remained unchanged. The carrying capacity of bacterial culture decreased dramatically, indicating a severe metabolic imbalance at low salt concentrations. At high salt concentrations, carrying capacity decreased gradually. There was a large increase in glucose-6-phosphate dehydrogenase activity, which correlated with a 10-fold increase in concentration of osmoprotectant L-proline. There was no significant change of net intracellular ATP concentration, phosphofructokinase, or pyruvate kinase activity. The results indicate that Vibrio sp. DSM14379 central metabolic pathways respond to low and high salt concentrations asymmetrically; cells are better adapted to high salt concentrations. In addition, cells in the stationary phase can tolerate induced salt stress without a significant change in dehydrogenase activity or endogenous respiration for at least 1 h, but need to alter their macromolecular composition and carbon flux distribution for long-term survival.