Multispecies biofilm architecture determines bacterial exposure to phages

Abstract

Numerous ecological interactions among microbes - for example, competition for space and resources, or interaction among phages and their bacterial hosts - are likely to occur simultaneously in multispecies biofilm communities. While biofilms formed by just a single species occur, multispecies biofilms are thought to be more typical of microbial communities in the natural environment. Previous work has shown that multispecies biofilms can increase, decrease, or have no measurable impact on phage exposure of a host bacterium living alongside another species that the phages cannot target. The reasons causing this variability are not well understood, and how phage-host encounters change within multispecies biofilms remains mostly unexplored at the cellular spatial scale. Here, we explore how the microscale biofilm structure of a model 2-species biofilms impacts cell-cell and cell-phage interactions underlying larger population wide patterns. Our system consists of dual-culture biofilms of Escherichia coli and Vibrio cholerae under exposure to T7 phages or λ phages. In the absence of phages, the two species compete with each other for limited space and resources. As shown previously, sufficiently mature biofilms of E. coli can protect themselves from phage exposure via their curli matrix. Before this stage of biofilm structural maturity, E. coli is highly susceptible to phages, however we show that these bacteria can gain lasting protection against phage exposure if they have become embedded within highly packed groups of V. cholerae in co-culture. In this manner, E. coli cells that are otherwise susceptible to T7 and λ phages can survive phage exposure in the absence of de novo resistance evolution. While co-culture growth allows for earlier protection from phages conferred by V. cholerae cells, it comes at the cost of competing with V. cholerae and a disruption of normal curli-mediated protection for E. coli even in dual species biofilms grown over long time scales.

Breakdown of clonal cooperative architecture in multispecies biofilms and the spatial ecology of predation

“…In this case, almost all cells of both species were killed off by the predators (SI Figure S3C,D). In a parallel study illustrating the generality of these results, we show that E. coli embedded along the bottom layers of expanding V. cholerae biofilm clusters are also protected from exposure to the obligate lytic phage T7 and temperate phage l in a manner dependent on the high packing structure of V. cholerae biofilms (78).…”

“…In this case, almost all cells of both species were killed off by the predators (SI Figure S3C,D). In a parallel study, we show that all of these results are replicated if virulent lytic or temperate phages targeting E. coli are introduced to biofilms of E. coli and V. cholerae grown in co-culture (Winans et al, 2022, bioRxiv) 77 .…”

“…E. coli within-species cell packing, on the other hand, was not statistically different in ordered versus disordered cell groups (Figure 3F). On longer time scales, it should be noted, E. coli cell groups that have been enveloped within ordered V. cholerae colonies can also be driven to high within-species cell packing by the confinement imposed by V. cholerae biofilm architecture (78).…”

“…Following this first imaging step, B. bacteriovorus was introduced as described above; 48 h later, biofilms were imaged again. Biofilms were then dispersed by removing the tubing from the microfluidic devices before vigorously pipetting M9 media and air bubbles back and forth between the inlet and outlet ports of the biofilm chambers; this technique most effectively removes the majority of the E. coli population residing within highly packed V. cholerae microcolonies (78). The resulting cell suspension was vortexed before being serially diluted and plated for E. coli CFUs.…”

Sustainable Diversity of Phage-Bacteria Systems

“…The collective protection from phage attack in a colony is empowered further when the bacteria produce extracellular structures that constitute a large part of the biofilm [46]. For example, the collective protection by expression of curli polymer that can trap T7 phage enables matured enough E. coli biofilm to keep growing under phage attack [47,48]; more complex collective protection has been reported recently in a multispecies biofilm [49]. A simulation study confirmed that the mobility of phage is the key when attacking a biofilm [50].…”