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.

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.

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.

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.

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