Adhesive interactions within microbial consortia can be differentiated at the single-cell level through expansion...

Area of Science:

• Microbial Ecology
• Microscopy Techniques
• Biofilm Research

Background:

• Microbe-microbe interactions are key to understanding microbial communities, but dense biofilms hinder single-cell analysis.
• Existing methods struggle to visualize individual cells and their interactions within complex, multi-species biofilms.

Purpose of the Study:

• To develop and apply a novel expansion microscopy technique for visualizing and analyzing single-cell interactions in dense microbial biofilms.
• To quantify the adhesive forces between bacterial species within model and synthetic biofilm communities.

Main Methods:

• Adaptation of expansion microscopy to achieve heterotropic expansion, selectively increasing space between cells but not within them.
• Application to mono-, dual-, and three-species biofilms including *Streptococcus*, *Fusobacterium nucleatum*, and *Schaalia-TM7*.
• Utilized adhesion mutants and quantitative proximity analysis to study specific interspecies interactions, such as *F. nucleatum* RadD and *Schaalia-TM7* epibiont associations.

Main Results:

• Heterotropic expansion microscopy successfully 'decrowded' dense biofilms, improving visualization of individual bacterial cells.
• The technique allowed for the quantitative analysis of microbe-microbe adhesive interactions at the single-cell level.
• Differential adhesion strengths and specific interspecies interactions were elucidated in model communities.

Conclusions:

• Heterotropic expansion microscopy is a powerful tool for resolving cellular architecture and interactions in crowded microbial communities.
• This method enhances the study of biofilm dynamics, interspecies adhesion, and the ecological roles of individual microbes.
• The findings provide new insights into the forces governing biofilm formation and stability.