Real‐Time Imaging of the Mechanobactericidal Action of Colloidal Nanomaterials and Nanostructured Topographies

Abstract

Mechanobactericidal nanomaterials, such as low‐dimensional nanoparticles in suspension or high‐aspect‐ratio nanofabricated topographies, rely on their mechanical or physical interactions with bacteria and are promising antimicrobial strategies that overcome bacterial resistance to classical antibiotics. However, the underlying killing mechanisms are poorly understood, given the challenges associated with the real‐time characterization of the mechanical interaction in a biologically relevant environment. Indeed, different death mechanisms have been proposed depending on the magnitude of the interaction forces. Herein, the real‐time and single‐cell response of bacteria to weak mechanical interactions with nanostructured topographies and “nanodarts,” exemplified by flowing single‐walled carbon nanotubes, is investigated. To that end, an advanced reporting strategy is used to follow sublethal physiological effects on bacteria upon contact with these materials. With this method, it is estimated that the contact time at which the initial stages of bacterial death occur is in the order of a few tens of minutes. This information contributes to a full understanding of the complex mechanisms of mechanically induced bacterial death.

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

“…During that irradiation period, 4 min movies were acquired at the start and before the end of irradiation. After the 660 nm LED was turned off, bacteria were left in the dark for 6 min and a final movie was acquired postirradiation, since we have previously shown that it can take up to 10–20 min for the activation of stress responses or metabolic changes that lead to an observable change in the oscillation period Figure shows representative behavior for a bacterium in an irreversibly loaded hydrogel during the imaging steps described above.…”

“…After the 660 nm LED was turned off, bacteria were left in the dark for 6 min and a final movie was acquired postirradiation, since we have previously shown that it can take up to 10−20 min for the activation of stress responses or metabolic changes that lead to an observable change in the oscillation period. 25 Figure 4 shows representative behavior for a bacterium in an irreversibly loaded hydrogel during the imaging steps described above. It can be clearly seen that the oscillation period increases immediately after MB irradiation and becomes even slower as the experiment progresses.…”

“…Under normal growth conditions, MinD is associated with the membrane and undergoes pole-to-pole oscillations . The period of these oscillations is affected by environmental stress factors such as (sublethal) concentrations of antibiotics or mechanical stress ,− and likely reflects changes in membrane potential . The use of Min oscillations as a reporter has also been explored in the context of aPDT, showing that the oscillation pattern is sensitive to photosensitizer concentration, and can report on mild photodynamic effects that are overlooked by traditional methods .…”

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

“…A subsequent study has investigated how Min oscillations in E. coli respond to weak mechanical perturbations in a more realistic scenario of interactions between bacteria and mechano-bactericidal nanomaterials [ 48 ]. To that end, nanostructured topographies and ‘nanodarts’, exemplified by flowing SWCNTs, were used to inflict a physiological response on bacteria.…”

“…These advantages are similar to those provided by fluorescence lifetime imaging from the quantitative point of view, namely an independence of heterogeneities in the sample and imaging system, as well as in the fluorophore concentration [ 65 ]. This is particularly interesting for experiments in microfluidic systems or on non-uniform surfaces such as nanofabricated topographies mentioned above [ 48 ].…”