Biofilm compactness and spatial community composition determine bacterial phage-susceptibility

Bacteriophages (phages) are viruses that infect bacteria, often posing a threat to their survival. In recent years, research has shifted from studying bacteria-phage interactions in homogeneous, well-mixed environments to investigating more complex heterogeneous, structured communities, thereby more accurately reflecting bacterial life in natural and disease-associated settings. So far, these studies predominantly tested interactions of phages and single-species biofilms, despite the dominance of highly diverse biofilm communities in nature. Here, we investigated how different lytic phages spread in and infect a synthetic community composed of Escherichia coli, Kluyvera cryocrescens, and Vibrio anguillarum. We found that E. coli was protected from eradication by the T7-coliphage when embedded in a multispecies biofilm and covered by the two other species, where the V. anguillarum biofilm matrix was essential for protection of E. coli. The protective nature of the V. anguillarum matrix also ensured survival of K. cryocrescens exposed to the lytic kluyveraphage Lyn. V. anguillarum mutants lacking the matrix synthesis genes vpsMNOP or rbmC failed to protect E. coli despite forming biofilms. Labelling of phages indicated that these were not trapped in the matrix of the wildtype. Instead, advanced image analysis revealed that the mutants displayed biofilms with lower cell density, resulting in the E. coli clusters being less tightly surrounded by V. anguillarum cells, which opened gaps for phage entry. These findings highlight that community context and matrix composition play a major role in phage-bacteria dynamics.