Precise spatial structure impacts antimicrobial susceptibility of
 S. aureus
 in polymicrobial wound infections


 A hallmark of microbial ecology is that interactions between members of a community shape community function. This includes microbial communities in human infections, such as chronic wounds, where interactions can result in more severe diseases.
 Staphylococcus aureus
 is the most common organism isolated from human chronic wound infections and has been shown to have both cooperative and competitive interactions with
 Pseudomonas aeruginosa
 . Still, despite considerable study, most interactions between these microbes have been characterized using in vitro well-mixed systems, which do not recapitulate the infection environment. Here, we characterized interactions between
 S. aureus
 and
 P. aeruginosa
 in chronic murine wounds, focusing on the role that both macro- and micro-scale spatial structures play in disease. We discovered that
 S. aureus
 and
 P. aeruginosa
 coexist at high cell densities in murine wounds. High-resolution imaging revealed that these microbes establish a patchy distribution, only occupying 5 to 25% of the wound volume. Using a quantitative framework, we identified a precise spatial structure at both the macro (mm)- and micro (µm)-scales, which was largely mediated by
 P. aeruginosa
 production of the antimicrobial 2-heptyl-4-hydroxyquinoline N-oxide, while the antimicrobial pyocyanin had no impact. Finally, we discovered that this precise spatial structure enhances
 S. aureus
 tolerance to aminoglycoside antibiotics but not vancomycin. Our results provide mechanistic insights into the biogeography of
 S. aureus
 and
 P. aeruginosa
 coinfected wounds and implicate spatial structure as a key determinant of antimicrobial tolerance in wound infections.
