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Understanding the biofilm microbiome and the biofilm-forming capacity of its dominant species is therefore essential, particularly for identifying species that coexist and enhance biofilm formation through interactions. This study investigated 248 bacterial isolates representing the dominant microbiota of biofilm samples collected from drinking water systems in broiler houses and pig nursery units. Biofilm formation was assessed in both single-species cultures and 341 multispecies combinations. Of the tested isolates, 80% formed biofilms in single culture; however, only 25% were classified as strong biofilm formers. In multispecies combinations with up to four species or isolates, antagonistic or competitive interactions were dominant, whereas biofilm mass enhancement was observed in only a limited number of combinations. These included opportunistic pathogens, with\n                      <jats:italic toggle=\"yes\">Citrobacter freundii<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">Staphylococcus nepalensis<\/jats:italic>\n                      identified as key species driving the interactions. Our research highlights the importance of specific microbial interactions in biofilm development and provides a potentially practice relevant four-species model to guide future research aimed at improving strategies for controlling biofilms in livestock drinking water systems.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>Ensuring the quality of drinking water on livestock production farms benefits animal production and health. Persistent biofilms on water line surfaces can lead to microbial contamination, resulting in lower-quality drinking water. This study examines bacteria commonly found in biofilms within livestock drinking water systems in pig nursery units and broiler houses. Most bacterial isolates can form biofilms in monoculture. Specific interactions among certain species primarily mediate bacterial interactions that enhance biofilm biomass in multispecies communities. Identifying these key interactions enables the development of a synthetic model to guide research on controlling biofilms in livestock drinking water systems.<\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.01084-26","type":"journal-article","created":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T13:00:16Z","timestamp":1789131616000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["Biofilm formation and multispecies interactions of bacteria recovered from drinking water systems in broiler houses and piglet nursery units"],"prefix":"10.1128","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-8134-034X","authenticated-orcid":true,"given":"Ulric","family":"Van Rossum","sequence":"first","affiliation":[{"name":"Technology and Food Unit, Flanders Research Institute for Agriculture, Fisheries and Food","place":["Merelbeke-Melle, 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