In Situ Single-Cell Bacterial Imaging Provides Mechanistic Insight into the Photodynamic Action of Photosensitizer-Loaded Hydrogels.

Hydrogels, three-dimensional hydrophilic polymeric networks with high water retaining capacity, have gained prominence in wound management and drug delivery due to their tunability, softness, permeability, and biocompatibility. Electron-beam polymerized poly(ethylene glycol) diacrylate (PEGDA) hydrogels are particularly useful for phototherapies such as antimicrobial photodynamic therapy (aPDT) due to their excellent optical properties. This work takes advantage of the transparency of PEGDA hydrogels to investigate bacterial responses to aPDT at the single-cell level, in real-time and in situ. The photosensitizer methylene blue (MB) was loaded in PEGDA hydrogels by using two methods: reversible loading and irreversible immobilization within the 3D polymer network. MB release kinetics and singlet oxygen generation were studied, revealing the distinct behaviors of both hydrogels. Real-time imaging of Escherichia coli was conducted during aPDT in both hydrogel types, using the Min protein system to report changes in bacterial physiology. Min oscillation patterns provided mechanistic insights into bacterial photoinactivation, revealing a dependence on the hydrogel preparation method. This difference was attributed to the mobility of MB within the hydrogel, affecting its direct interaction with bacterial membranes. These findings shed light on the complex interplay between hydrogel properties and the bacterial response during aPDT, offering valuable insights for the development of antibacterial wound dressing materials. The study demonstrates the capability of real-time, single-cell fluorescence microscopy to unravel dynamic bacterial behaviors in the intricate environment of hydrogel surfaces during aPDT.

Min oscillations in bacteria as real-time reporter of environmental challenges at the single-cell level.

Min oscillations are a fascinating mechanism used by Escherichia coli to find their middle. Beyond their biological role, they provide a convenient and relatively unexplored method to monitor the effect of sublethal environmental challenges on bacterial physiology in real-time and at the single-cell level. In this review, we discuss the original papers that put forward the idea of using Min oscillations as a reporting tool to monitor the effect of extracellular cationic compounds, including antibiotics. More recent work from our laboratory explores this tool to follow bacterial response to other challenges such as weak mechanical interactions with nanomaterials or photodynamic treatment. We discuss the physiological meaning of the changes in Min oscillation period, likely related to membrane potential dynamics, as well as the benefits and limitations of using oscillations as a reporter in fluorescence microscopy. Overall, Min oscillations are a useful addition to the fluorescence microscopy toolbox in order to visualize stress responses in E. coli, and have the potential to provide full mechanistic understanding of the events that lead to bacterial cell death in different contexts.

Min Oscillations as Real-time Reporter of Sublethal Effects in Photodynamic Treatment of Bacteria.

The Min protein system is a cell division regulator in Escherichia coli. Under normal growth conditions, MinD is associated with the membrane and undergoes pole-to-pole oscillations. The period of these oscillations has been previously proposed as a reporter for the bacterial physiological state at the single-cell level and has been used to monitor the response to sublethal challenges from antibiotics, temperature, or mechanical fatigue. Using real-time single-cell fluorescence imaging, we explore here the effect of photodynamic treatment on MinD oscillations. Irradiation of bacteria in the presence of the photosensitizer methylene blue disrupts the MinD oscillation pattern depending on its concentration. In contrast to antibiotics, which slow down the oscillation, photodynamic treatment results in an abrupt interruption, reflecting divergent physiological mechanisms leading to bacterial death. We show that MinD oscillations are sensitive to mild photodynamic effects that are overlooked by traditional methods, expanding the toolbox for mechanistic studies in antimicrobial photodynamic therapy.