3D imaging-driven assembly of multi-species biofilms with antagonistic activity against undesirable bacteria

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 multi-strain 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.