Exploring Bacterial Biofilm Responses Through 3D Mass Spectrometry Imaging and Microfluidic Simulation of Treatment Conditions

Bacterial biofilms, found in diverse environments including the human body, pose
significant challenges due to their complex growth dynamics and regulatory systems.
These microbial colonies are often responsible for persistent infections in lung
infections, chronic wounds, and implanted medical devices. There is an urgent need for
new strategies and therapeutic agents to effectively eradicate biofilm infections. While
existing research has focused on biofilm resistance mechanisms, less understanding has
been acquired to their complex structure, environmental heterogeneity, and adaptive
responses, all of which contribute to biofilms' resilience and inform potential treatment
strategies. Understanding these facets is crucial for developing effective elimination
methods.
Additionally, the incubation model significantly impacts biofilm studies. Most research
on biofilm responses to external stimuli has utilized flowcell models, simulating highshear environments. However, biofilms also thrive in low-shear conditions, such as those
grown on the gas-tissue interfaces in lung and diabetic foot. Without the stress and
draining effect from the shear flow, these static biofilms have distinct characters, while
metabolic and signaling molecular distribution could be better preserved for analysis.
Furthermore, antibiotic testing typically involves fixed concentrations, which does not
reflect clinical scenarios where fluctuating antibiotic concentrations diffuse into infected
tissues and biofilm, exposing some bacteria in the biofilms to lower concentrations that
complicate eradication efforts. Similarly, some dispersal agents were reported to enhance
treatment efficacy in a flowcell model, but it remains unclear whether the conclusion is
valid for the significantly different static biofilms. A better model is demanded for
studies on this topic.
To address these challenges, we propose developing a microfluidic device that simulates
realistic biofilm treatment under localized stimulation. This platform will create
controllable drug gradients, allowing us to study biofilm responses in heterogeneous
environments without shear flow. Also, we will employ a novel moisture-assisted cryosection (MACS) technique we recently developed for high-resolution 3D mass
spectrometry imaging (MSI) of bacterial colonies. This technique provides detailed
visualization of the spatial distribution of molecules within biofilms, revealing their
multi-layered molecular organization. Using this method, we will compare the signaling
molecules in biofilms grown on this platform with those cultured conventionally,
validating the visualization of quorum sensing (QS) regulation processes. We will also
investigate the fundamental questions with dispersal agents under static biofilm
condition, and investigate the potential of using adjuvants to enhance the effectiveness
of antibiotic treatment. This work aims to provide an upgraded framework for
understanding QS regulation and dispersion, and developing future treatment strategies.