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Invisible aligners, although removable and easier to clean, are frequently exposed to external environments, which may allow contamination by non-oral microorganisms. This study aimed to evaluate the association and viability of oral and non-oral microorganisms in mono- and multispecies biofilm models formed on orthodontic appliances. Methods: Stimulated human saliva was used to form the acquired pellicle on specimens of invisible aligners, conventional brackets, and self-ligating brackets. The samples were exposed to\n                    <jats:italic>Streptococcus mutans<\/jats:italic>\n                    ,\n                    <jats:italic>Candida albicans<\/jats:italic>\n                    ,\n                    <jats:italic>Staphylococcus aureus<\/jats:italic>\n                    , and\n                    <jats:italic>Pseudomonas aeruginosa<\/jats:italic>\n                    , and incubated for 48\u00a0h under mono- and multispecies conditions. After ultrasonic detachment, viable cells were quantified by CFU\/mL enumeration, and extracellular polymeric substance (EPS) content was determined. Statistical analysis was performed using one-way ANOVA followed by Tukey\u2019s post hoc test for monospecies biofilms, and two-way ANOVA followed by Bonferroni\u2019s post hoc test for multispecies biofilms (\n                    <jats:italic>p<\/jats:italic>\n                    \u2009&lt;\u20090.05). Higher CFU\/mL values were observed for\n                    <jats:italic>S. aureus<\/jats:italic>\n                    and\n                    <jats:italic>P. aeruginosa<\/jats:italic>\n                    compared to\n                    <jats:italic>C. albicans<\/jats:italic>\n                    and\n                    <jats:italic>S. mutans<\/jats:italic>\n                    (\n                    <jats:italic>p<\/jats:italic>\n                    \u2009&lt;\u20090.05). In multispecies biofilms,\n                    <jats:italic>C. albicans<\/jats:italic>\n                    showed higher values than\n                    <jats:italic>S. mutans<\/jats:italic>\n                    (\n                    <jats:italic>p<\/jats:italic>\n                    \u2009&lt;\u20090.05). CFU\/mL increased over time, with no differences among orthodontic devices (\n                    <jats:italic>p<\/jats:italic>\n                    \u2009&gt;\u20090.05). EPS content was higher in conventional brackets, intermediate in self-ligating brackets, and lower in aligners (\n                    <jats:italic>p<\/jats:italic>\n                    \u2009&lt;\u20090.05). Oral and non-oral microorganisms remained viable and were recovered from biofilm models formed on orthodontic appliances. These findings suggest that such devices may act as substrates for the persistence of non-oral microorganisms under biofilm-forming conditions, highlighting the importance of appropriate hygiene and storage practices.\n                  <\/jats:p>","DOI":"10.1007\/s10266-026-01423-2","type":"journal-article","created":{"date-parts":[[2026,6,2]],"date-time":"2026-06-02T21:01:08Z","timestamp":1780434068000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Association of oral and non-oral microorganisms in a multispecies biofilm model formed on orthodontic appliances: an in vitro study"],"prefix":"10.1007","author":[{"given":"Greziele Barranco","family":"Passamani","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lucas","family":"de Oliveira Pereira","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thayn\u00e1 Alves","family":"Ferreira","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Priscila Vieira","family":"da Silva","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luana","family":"Backes","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joana","family":"de Freitas Santos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jana\u00edna Orlandi","family":"Sardi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tahyna Duda Deps","family":"de Almeida","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Let\u00edcia Targino","family":"Campos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexandre Meireles","family":"Borba","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9724-0147","authenticated-orcid":false,"given":"Diego","family":"Rom\u00e1rio-Silva","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,6,2]]},"reference":[{"key":"1423_CR1","doi-asserted-by":"publisher","first-page":"1773","DOI":"10.3390\/medicina59101773","volume":"59","author":"CH Kau","year":"2023","unstructured":"Kau CH, Soh J, Christou T, Mangal A. 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