# 3D imaging-driven assembly of multispecies biofilms with antagonistic activity against undesirable bacteria

## TL;DR

This paper shows how to build multispecies biofilms that block harmful bacteria using 3D imaging and mathematical models.

## Contribution

A new bottom-up approach combining 3D imaging and high-throughput analysis to assemble multispecies biofilms with controlled antagonistic activity.

## Key findings

- Competitive strains can exclude both harmful and beneficial microbes, emphasizing compatibility control.
- SynComs of Bacillus velezensis and Pediococcus spp. improved pathogen exclusion compared to single strains.
- Pathogen exclusion was driven by nutritional competition and interference dynamics in biofilms.

## Abstract

Engineered synthetic microbial communities (SynComs) forming biofilms with antagonistic activity offer a promising strategy in biotechnology to prevent harmful bacterial settlement and reduce reliance on chemical antimicrobials. However, strain selection criteria and antagonistic mechanisms remain unclear. This study presents a bottom-up approach integrating 3D fluorescence imaging with high-throughput analysis of multistrain biofilms. Our findings reveal that competitive strains against undesirable bacteria may also exclude desirable community members, highlighting the need for compatibility control in SynCom assembly. SynComs composed of Bacillus velezensis and Pediococcus spp. enhanced pathogen exclusion compared to single strains. Temporal analysis of biofilm interactions, supported by mathematical models, showed that pathogen exclusion was primarily driven by nutritional competition (Jameson effect) with additional specific interference dynamics (prey–predator Lotka-Volterra model). Furthermore, pre-established SynComs significantly increased pathogen inhibition, indicating a distinct biofilm-associated exclusion effect. These insights provide a framework for SynCom assembly and refine our understanding of interaction dynamics driving antagonistic biofilm applications.