# Microbial Interactions Shape Spatial Organisation and Transcriptional Responses in a Model Mixed-Species Biofilm

## TL;DR

This study shows how microbial interactions in a three-species biofilm affect their spatial organization and gene activity, with implications for controlling biofilms in industrial settings.

## Contribution

The study reveals how specific microbial interactions shape both spatial structure and transcriptional responses in a model mixed-species biofilm.

## Key findings

- S. rhizophila dominates the biofilm and shows significant gene expression changes when co-cultured with M. lacticum.
- M. lacticum, though low in abundance, acts as the initial coloniser and influences spatial organisation.
- Co-culture with B. licheniformis induces minimal transcriptional changes in S. rhizophila, indicating a neutral interaction.

## Abstract

Dynamic social interactions within bacterial biofilms drive distinct spatial organisation and transcriptional responses. Here, we combine fluorescence in situ hybridisation (FISH), confocal laser scanning microscopy (CLSM), and RNA sequencing (RNA-Seq) to investigate a model three-species biofilm community derived from a dairy pasteuriser, comprising Stenotrophomonas rhizophila, Microbacterium lacticum, and Bacillus licheniformis. CLSM revealed species-specific biovolume dynamics and stratified 3D structures over 24 h, with S. rhizophila as the dominant species and M. lacticum exhibiting the lowest abundance yet playing an essential role as the initial coloniser. Spatial patterns reflected known pairwise interactions – commensalism, exploitation, and neutral interaction. Transcriptomic profiling of S. rhizophila revealed extensive gene expression changes in dual-species biofilms with M. lacticum, including upregulation of genes related to flagellar motility, nutrient acquisition, energy metabolism, and TonB-dependent transport. In contrast, co-culture with B. licheniformis induced minimal transcriptional changes in S. rhizophila, consistent with a neutral interaction among the two. Our findings demonstrate how interspecies interactions govern both spatial topology and functional specialisation in mixed-species biofilms which is of relevance to microbial ecology, industrial biofilm control, and the targeting of keystone biofilm species.

The online version contains supplementary material available at 10.1007/s00248-026-02701-w.