Antiviral action of a functionalized plastic surface against human coronaviruses.

Viruses may persist on solid surfaces for long periods, which may contribute to indirect transmission. Thus, it is imperative to develop functionalized surfaces that will lower the infectious viral load in everyday life. Here, we have tested a plastic surface functionalized with tall oil rosin against the seasonal human coronavirus OC43 as well as severe acute respiratory syndrome coronavirus 2. All tested non-functionalized plastic surfaces showed virus persistence up to 48 h. In contrast, the functionalized plastic showed good antiviral action already within 15 min of contact and excellent efficacy after 30 min over 90% humidity. Excellent antiviral effects were also observed at lower humidities of 20% and 40%. Despite the hydrophilic nature of the functionalized plastic, viruses did not adhere strongly to it. According to helium ion microscopy, viruses appeared flatter on the rosin-functionalized surface, but after flushing away from the rosin-functionalized surface, they showed no apparent structural changes when imaged by transmission electron microscopy of cryogenic or negatively stained specimens or by atomic force microscopy. Flushed viruses were able to bind to their host cell surface and enter endosomes, suggesting that the fusion with the endosomal membrane was halted. The eluted rosin from the functionalized surface demonstrated its ability to inactivate viruses, indicating that the antiviral efficacy relied on the active leaching of the antiviral substances, which acted on the viruses coming into contact. The rosin-functionalized plastic thus serves as a promising candidate as an antiviral surface for enveloped viruses.IMPORTANCEDuring seasonal and viral outbreaks, the implementation of antiviral plastics can serve as a proactive strategy to limit the spread of viruses from contaminated surfaces, complementing existing hygiene practices. In this study, we show the efficacy of a rosin-functionalized plastic surface that kills the viral infectivity of human coronaviruses within 15 min of contact time, irrespective of the humidity levels. In contrast, non-functionalized plastic surfaces retain viral infectivity for an extended period of up to 48 h. The transient attachment on the surface or the leached active components do not cause major structural changes in the virus or prevent receptor binding; instead, they effectively block viral infection at the endosomal stage.

Directed Assembly of Cellulose Nanocrystals in Their Native Solid-State Template of a Processed Fiber Cell Wall.

Nanoparticle assembly is intensely surveyed because of the numerous applications within fields such as catalysis, batteries, and biomedicine. Here, directed assembly of rod-like, biologically derived cellulose nanocrystals (CNCs) within the template of a processed cotton fiber cell wall, that is, the native origin of CNCs, is reported. It is a system where the assembly takes place in solid state simultaneously with the top-down formation of the CNCs via hydrolysis with HCl vapor. Upon hydrolysis, cellulose microfibrils in the fiber break down to CNCs that then pack together, resulting in reduced pore size distribution of the original fiber. The denser packing is demonstrated by N2 adsorption, water uptake, thermoporometry, and small-angle X-ray scattering, and hypothetically assigned to attractive van der Waals interactions between the CNCs.

A muramidase from Acremonium alcalophilum hydrolyse peptidoglycan found in the gastrointestinal tract of broiler chickens.

This study evaluates peptidoglycan hydrolysis by a microbial muramidase from the fungus Acremonium alcalophilum in vitro and in the gastrointestinal tract of broiler chickens. Peptidoglycan used for in vitro studies was derived from 5 gram-positive chicken gut isolate type strains. In vitro peptidoglycan hydrolysis was studied by three approaches: (a) helium ion microscopy to identify visual phenotypes of hydrolysis, (b) reducing end assay to quantify solubilization of peptidoglycan fragments, and (c) mass spectroscopy to estimate relative abundances of soluble substrates and reaction products. Visual effects of peptidoglycan hydrolysis could be observed by helium ion microscopy and the increase in abundance of soluble peptidoglycan due to hydrolysis was quantified by a reducing end assay. Mass spectroscopy confirmed the release of hydrolysis products and identified muropeptides from the five different peptidoglycan sources. Peptidoglycan hydrolysis in chicken crop, jejunum, and caecum samples was measured by quantifying the total and soluble muramic acid content. A significant increase in the proportion of the soluble muramic acid was observed in all three segments upon inclusion of the microbial muramidase in the diet.

Resolving Bio-Nano Interactions of E. coli Bacteria-Dragonfly Wing Interface with Helium Ion and 3D-Structured Illumination Microscopy to Understand Bacterial Death on Nanotopography.

Obtaining a comprehensive understanding of the bactericidal mechanisms of natural nanotextured surfaces is crucial for the development of fabricated nanotextured surfaces with efficient bactericidal activity. However, the scale, nature, and speed of bacteria-nanotextured surface interactions make the characterization of the interaction a challenging task. There are currently several different opinions regarding the possible mechanisms by which bacterial membrane damage occurs upon interacting with nanotextured surfaces. Advanced imaging methods could clarify this by enabling visualization of the interaction. Charged particle microscopes can achieve the required nanoscale resolution but are limited to dry samples. In contrast, light-based methods enable the characterization of living (hydrated) samples but are limited by the resolution achievable. Here we utilized both helium ion microscopy (HIM) and 3D structured illumination microscopy (3D-SIM) techniques to understand the interaction of Gram-negative bacterial membranes with nanopillars such as those found on dragonfly wings. Helium ion microscopy enables cutting and imaging at nanoscale resolution, while 3D-SIM is a super-resolution optical microscopy technique that allows visualization of live, unfixed bacteria at ∼100 nm resolution. Upon bacteria-nanopillar interaction, the energy stored due to the bending of natural nanopillars was estimated and compared with fabricated vertically aligned carbon nanotubes. With the same deflection, shorter dragonfly wing nanopillars store slightly higher energy compared to carbon nanotubes. This indicates that fabricated surfaces may achieve similar bactericidal efficiency as dragonfly wings. This study reports in situ characterization of bacteria-nanopillar interactions in real-time close to its natural state. These microscopic approaches will help further understanding of bacterial membrane interactions with nanotextured surfaces and the bactericidal mechanisms of nanotopographies so that more efficient bactericidal nanotextured surfaces can be designed and fabricated, and their bacteria-nanotopography interactions can be assessed in situ.

Microencapsulation of Enteric Bacteriophages in a pH-Responsive Solid Oral Dosage Formulation Using a Scalable Membrane Emulsification Process.

A scalable low-shear membrane emulsification process was used to produce microencapsulated Escherichia coli-phages in a solid oral dosage form. Uniform pH-responsive composite microparticles (mean size ~100 µm) composed of Eudragit® S100 and alginate were produced. The internal microstructure of the gelled microcapsules was studied using ion-milling and imaging, which showed that the microparticles had a solid internal core. The microencapsulation process significantly protected phages upon prolonged exposure to a simulated gastric acidic environment. Encapsulated phages that had been pre-exposed to simulated gastric acid were added to actively growing bacterial cells using in vitro cell cultures and were found to be effective in killing E. coli. Encapsulated phages were also shown to be effective in killing actively growing E. coli in the presence of human epithelial cells. Confocal microscopy images showed that the morphology of encapsulated phage-treated epithelial cells was considerably better than controls without phage treatment. The encapsulated phages were stable during refrigerated storage over a four-week period. The process of membrane emulsification is highly scalable and is a promising route to produce industrial quantities of pH-responsive oral solid dosage forms suitable for delivering high titres of viable phages to the gastrointestinal tract.

Microencapsulation of Salmonella-Specific Bacteriophage Felix O1 Using Spray-Drying in a pH-Responsive Formulation and Direct Compression Tableting of Powders into a Solid Oral Dosage Form.

The treatment of enteric bacterial infections using oral bacteriophage therapy can be challenging since the harsh acidic stomach environment renders phages inactive during transit through the gastrointestinal tract. Solid oral dosage forms allowing site-specific gastrointestinal delivery of high doses of phages, e.g., using a pH or enzymatic trigger, would be a game changer for the nascent industry trying to demonstrate the efficacy of phages, including engineered phages for gut microbiome modulation in expensive clinical trials. Spray-drying is a scalable, low-cost process for producing pharmaceutical agents in dry powder form. Encapsulation of a model Salmonella-specific phage (Myoviridae phage Felix O1) was carried out using the process of spray-drying, employing a commercially available Eudragit S100® pH-responsive anionic copolymer composed of methyl methacrylate-co-methacrylic acid formulated with trehalose. Formulation and processing conditions were optimised to improve the survival of phages during spray-drying, and their subsequent protection upon exposure to simulated gastric acidity was demonstrated. Addition of trehalose to the formulation was shown to protect phages from elevated temperatures and desiccation encountered during spray-drying. Direct compression of spray-dried encapsulated phages into tablets was shown to significantly improve phage protection upon exposure to simulated gastric fluid. The results reported here demonstrate the significant potential of spray-dried pH-responsive formulations for oral delivery of bacteriophages targeting gastrointestinal applications.

Antibacterial Efficiency of Surface-Immobilized Flavobacterium-Infecting Bacteriophage.

Control of bacterial diseases by bacteriophages (phages) is gaining more interest due to increasing antibiotic resistance. This has led to technologies to attach phages on surfaces to form a biomaterial that can functionally display phages that interact with bacteria, to carry out successful infection cycles. Such a material could be applied in many environments where the target pathogens are expected. Although this approach has been applied successfully in a few studies already, the basis of the antibacterial effect by the immobilized phages is unclear, and the interpretation of the results depends on the study. Here, we studied the phage attachment density, their detachment rate, and infectivity on five different surfaces: silicon, amine-treated silicon, gold, carboxylate-treated gold, and cross-linker-activated carboxylate-treated gold. The density of attached phages varied between the different surfaces and was the highest on the cross-linker-activated carboxylate-treated gold. To understand whether the antibacterial effect is caused by the attached or the detached phages, the strength of the immobilization was analyzed by performing 3-12 washing steps. The detachment rates differed between the materials, with the amine-treated silicon surface generating the highest release of phages and maintaining the highest infectivity, even after extensive washing. However, covalent cross-linking seemed to interfere with the infectivity. Our results suggest that the detachment of the phages from the surface is a possible mechanism for the antibacterial effect. Furthermore, we introduce a measure of the infectivity by comparing the bacterial growth reductions produced by the phage-treated materials to the effect caused by a known number of free phages, resulting in a unit "effective PFU/surface area", a comparable standard between different studies.

Cellulose nanofibrils prepared by gentle drying methods reveal the limits of helium ion microscopy imaging.

TEMPO-oxidized cellulose nanofibrils (TCNFs) have unique properties, which can be utilised in many application fields from printed electronics to packaging. Visual characterisation of TCNFs has been commonly performed using Scanning Electron Microscopy (SEM). However, a novel imaging technique, Helium Ion Microscopy (HIM), offers benefits over SEM, including higher resolution and the possibility of imaging non-conductive samples uncoated. HIM has not been widely utilized so far, and in this study the capability of HIM for imaging of TCNFs was evaluated. Freeze drying and critical point drying (CPD) techniques were applied to preserve the open fibril structure of the gel-like TCNFs. Both drying methods worked well, but CPD performed better resulting in the specific surface area of 386 m2 g-1 when compared to 172 m2 g-1 and 42 m2 g-1 of freeze dried samples frozen in propane and nitrogen, respectively. HIM imaging of TCNFs was successful but high magnification imaging was challenging because the ion beam tended to degrade the TCNFs. The effect of the imaging parameters on the degradation was studied and an ion dose as low as 0.9 ion per nm2 was required to prevent the damage. This study points out the differences between the gentle drying methods of TCNFs and demonstrates beam damage during imaging like none previously reported with HIM. The results can be utilized in future studies of cellulose or other biological materials as there is a growing interest for both the HIM technique and bio-based materials.

Bacteriophage imaging: past, present and future.

The visualization of viral particles only became possible after the advent of the electron microscope. The first bacteriophage images were published in 1940 and were soon followed by many other publications that helped to elucidate the structure of the particles and their interaction with the bacterial hosts. As sample preparation improved and new technologies were developed, phage imaging became important approach to morphologically classify these viruses and helped to understand its importance in the biosphere. In this review we discuss the main milestones in phage imaging, how it affected our knowledge on these viruses and recent developments in the field.

Imaging Bacterial Colonies and Phage-Bacterium Interaction at Sub-Nanometer Resolution Using Helium-Ion Microscopy.

Imaging of microbial interactions has so far been based on well-established electron microscopy methods. This study presents a new way to study bacterial colonies and interactions between bacteria and their viruses, bacteriophages (phages), in situ on agar plates using helium ion microscopy (HIM). In biological imaging, HIM has advantages over traditional scanning electron microscopy with its sub-nanometer resolution, increased surface sensitivity, and the possibility to image nonconductive samples. Furthermore, by controlling the He beam dose or by using heavier Ne ions, the HIM instrument provides the possibility to mill out material in the samples, allowing for subsurface imaging and in situ sectioning. Here, the first HIM-images of bacterial colonies and phage-bacterium interactions are presented at different stages of the infection as they occur on an agar culture. The feasibility of neon and helium milling is also demonstrated to reveal the subsurface structures of bacterial colonies on agar substrate, and in some cases also structure inside individual bacteria after cross-sectioning. The study concludes that HIM offers great opportunities to advance the studies of microbial imaging, in particular in the area of interaction of viruses with cells.