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ABSTRACT Chronic polymicrobial infections involvingPseudomonas aeruginosaandStaphylococcus aureusare prevalent, difficult to eradicate, and associated with poor health outcomes. Therefore, understanding interactions between these pathogens is important to inform improved treatment development. We previously demonstrated thatP. aeruginosais attracted toS. aureususing type IV pili (TFP)-mediated chemotaxis, but the impact of attraction onS. aureusgrowth and physiology remained unknown. Using live single-cell confocal imaging to visualize microcolony structure, spatial organization, and survival ofS. aureusduring coculture, we found that interspecies chemotaxis providesP. aeruginosaa competitive advantage by promoting invasion into and disruption ofS. aureusmicrocolonies. This behavior rendersS. aureussusceptible toP. aeruginosaantimicrobials. Conversely, in the absence of TFP motility,P. aeruginosacells exhibit reduced invasion ofS. aureuscolonies. Instead,P. aeruginosabuilds a cellular barrier adjacent toS. aureusand secretes diffusible, bacteriostatic antimicrobials like 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) into theS. aureuscolonies. Reduced invasion leads to the formation of denser and thickerS. aureuscolonies with increased HQNO-mediated lactic acid fermentation, a physiological change that could complicate treatment strategies. Finally, we show thatP. aeruginosamotility modifications of spatial structure enhance competition againstS. aureus. Overall, these studies expand our understanding of howP. aeruginosaTFP-mediated interspecies chemotaxis facilitates polymicrobial interactions, highlighting the importance of spatial positioning in mixed-species communities. IMPORTANCEThe polymicrobial nature of many chronic infections makes their eradication challenging. Particularly, coisolation ofPseudomonas aeruginosaandStaphylococcus aureusfrom airways of people with cystic fibrosis and chronic wound infections is common and associated with severe clinical outcomes. The complex interplay between these pathogens is not fully understood, highlighting the need for continued research to improve management of chronic infections. Our study unveils thatP. aeruginosais attracted toS. aureus, invades into neighboring colonies, and secretes anti-staphylococcal factors into the interior of the colony. Upon inhibition ofP. aeruginosamotility and thus invasion,S. aureuscolony architecture changes dramatically, wherebyS. aureusis protected fromP. aeruginosaantagonism and responds through physiological alterations that may further hamper treatment. These studies reinforce accumulating evidence that spatial structuring can dictate community resilience and reveal that motility and chemotaxis are critical drivers of interspecies competition. 

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 underlying 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 study how the cellular scale architecture of model 2-species biofilms impacts cell–cell and cell–phage interactions controlling larger scale population and community dynamics. Our system consists of dual culture biofilms ofEscherichia coliandVibrio choleraeunder exposure to T7 phages, which we study using microfluidic culture, high-resolution confocal microscopy imaging, and detailed image analysis. As shown previously, sufficiently mature biofilms ofE.colican protect themselves from phage exposure via their curli matrix. Before this stage of biofilm structural maturity,E.coliis highly susceptible to phages; however, we show that these bacteria can gain lasting protection against phage exposure if they have become embedded in the bottom layers of highly packed groups ofV.choleraein co-culture. This protection, in turn, is dependent on the cell packing architecture controlled byV.choleraebiofilm matrix secretion. In this manner,E.colicells that are otherwise susceptible to phage-mediated killing can survive phage exposure in the absence of de novo resistance evolution. While co-culture biofilm formation withV.choleraecan confer phage protection toE.coli, it comes at the cost of competing withV.choleraeand a disruption of normal curli-mediated protection forE.colieven in dual species biofilms grown over long time scales. This work highlights the critical importance of studying multispecies biofilm architecture and its influence on the community dynamics of bacteria and phages. 

Biofilm formation, including adherence to surfaces and secretion of extracellular matrix, is common in the microbial world, but we often do not know how interaction at the cellular spatial scale translates to higher-order biofilm community ecology. Here we explore an especially understudied element of biofilm ecology, namely predation by the bacteriumBdellovibrio bacteriovorus. This predator can kill and consume many different Gram-negative bacteria, includingVibrio choleraeandEscherichia coli.V. choleraecan protect itself from predation within densely packed biofilm structures that it creates, whereasE. colibiofilms are highly susceptible toB. bacteriovorus. We explore how predator–prey dynamics change whenV. choleraeandE. coliare growing in biofilms together. We find that in dual-species prey biofilms,E. colisurvival underB. bacteriovoruspredation increases, whereasV. choleraesurvival decreases.E. colibenefits from predator protection when it becomes embedded within expanding groups of highly packedV. cholerae. But we also find that the ordered, highly packed, and clonal biofilm structure ofV. choleraecan be disrupted ifV. choleraecells are directly adjacent toE. colicells at the start of biofilm growth. When this occurs, the two species become intermixed, and the resulting disordered cell groups do not block predator entry. Because biofilm cell group structure depends on initial cell distributions at the start of prey biofilm growth, the surface colonization dynamics have a dramatic impact on the eventual multispecies biofilm architecture, which in turn determines to what extent both species survive exposure toB. bacteriovorus.