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There is a rich history of work studying the relative advantages and disadvantages of these two phage life history strategies, but little of this work has addressed the spatial constraints common to biofilm environments. We developed a live imaging system to track lytic infections, lysogenic infections, and uninfected cells at single-cell resolution within three-dimensional\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    biofilms. We find that biofilm structure substantially impacts the ecological success of different phage infection strategies. Temperate phages have the unique capacity to release phages from lysogens that have undergone lytic induction from within the interior of mature biofilms. When this occurs in biofilm contexts that do not limit phage diffusion, lytic infections expand rapidly, but lysogenic infections are favored as phage mobility declines in densely packed biofilm architectures. In matrix-replete biofilms that do limit phage mobility, lytic phage infection is more limited, favoring lysogenic growth. Direct competition assays between lysogenized host bacteria and obligately lytic phages\u2014with or without the ability to superinfect lysogens\u2014revealed that spatial structure and superinfection potential together greatly impact phage competition outcomes during co-invasion into pre-existing, phage-susceptible biofilm populations. Highly packed, phage diffusion-impeding biofilms disproportionately favored temperate phages in the lysogenic cycle over obligate lytic phages, highlighting how biofilm architecture can constrain lytic phage infection and promote vertical phage genome transmission strategies.\n                  <\/jats:p>","DOI":"10.1371\/journal.pbio.3003737","type":"journal-article","created":{"date-parts":[[2026,3,31]],"date-time":"2026-03-31T17:33:28Z","timestamp":1774978408000},"page":"e3003737","update-policy":"https:\/\/doi.org\/10.1371\/journal.pbio.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Biofilm spatial structure and superinfection immunity modulate inter-phage competition"],"prefix":"10.1371","volume":"24","author":[{"given":"James B.","family":"Winans","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1751-4895","authenticated-orcid":true,"given":"Carey D.","family":"Nadell","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2026,3,31]]},"reference":[{"key":"pbio.3003737.ref001","doi-asserted-by":"crossref","unstructured":"Fillol-Salom A, et al. 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