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Additionally, they may be linked to unsuccessful therapies, which raises death rates, healthcare expenses, and the need for additional hospitalization. Therefore, to protect the environment and improve human health, there is a need for the creative synthesis of novel antibacterial materials. <jats:italic>Proteus mirabilis<\/jats:italic> strain PQ350419 was isolated, identified, and utilized as an efficient bio-nano-factory for biosynthesizing selenium nanoparticles (Se NPs) and optimizing procedures. This study showcases a simple and cost-effective approach for green-synthesizing a selenium\/chitosan\/ampicillin nanocomposite (Se\/CS\/AMP) as a novel antibacterial and antibiofilm agent. Several analyses, such as transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier-transform infrared (FTIR) spectroscopy, zeta analysis, and ultraviolet-visible (UV-Vis) spectroscopy, were utilized to confirm and characterize the production of Se NPs and Se\/CS\/AMP. The absorption peaks for Se NPs and Se\/CS\/AMP were identified to be between 350 and 360\u00a0nm. The XRD data revealed the crystalline composition of the Se NPs loaded with CS and AMP. The FTIR spectra confirmed the presence of proteins that act as supporting and binding agents during synthesis. The stability of the prepared nanomaterials is improved by a strong negative surface charge of \u2212\u200924.27 mV for Se NPs and \u2212\u200923.92 mV for Se\/CS\/AMP. The particle sizes of Se NPs and Se\/CS\/AMP are shown by TEM to be in the ranges of 88\u201398\u00a0nm and 86\u2013129\u00a0nm, respectively. Se NPs, either alone or in combination with chitosan (CS) and ampicillin (AMP), exhibited strong antibacterial activity against methicillin-resistant <jats:italic>Staphylococcus aureus<\/jats:italic> ATCC 43,300, <jats:italic>Bacillus cereus<\/jats:italic> ATCC 14,579, <jats:italic>Klebsiella pneumoniae<\/jats:italic> ATCC 11,296, and <jats:italic>P. mirabilis<\/jats:italic> PQ350419 in a dose-dependent manner. Compared to Se NPs and the common antibiotic AMP, the Se\/CS\/AMP combination demonstrated superior antibacterial activity. In comparison to Se NPs (40, 70, 110, and 150\u00a0\u00b5g\/ml, respectively), the nanocomposite produced MIC values of 30, 40, 60, and 100\u00a0\u00b5g\/ml against <jats:italic>B. cereus<\/jats:italic>, <jats:italic>S. aureus<\/jats:italic>, <jats:italic>K. pneumoniae<\/jats:italic>, and <jats:italic>P. mirabilis<\/jats:italic>. When compared to untreated cells, treated cells exhibited significant morphological changes and deformities, such as cell wall distortion, the separation of the cell wall from the plasma membrane, the formation of vacuoles, and complete cell lysis, according to TEM ultrastructure studies of bacteria treated with nanocomposite. Se\/CS\/AMP at 100\u00a0\u00b5g\/ml was sufficient to prevent biofilm formation by up to 50% in <jats:italic>S. aureus<\/jats:italic>, <jats:italic>K. pneumoniae<\/jats:italic>, and <jats:italic>P. mirabilis<\/jats:italic>. The cell viability of the Vero cell line was significantly reduced (<jats:italic>p<\/jats:italic>\u02c20.05) in the cytotoxicity test of Se NPs alone at a concentration of 40.95\u2009\u00b1\u20092.34\u00a0\u00b5g\/ml, and in its nanocomposite at a concentration of 199.09\u2009\u00b1\u20092.61\u00a0\u00b5g\/ml. This indicates the nanocomposite\u2019s safety by showing its minimal harmful impact on the Vero cell line.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusion<\/jats:title>\n            <jats:p>Se\/CS\/AMP has revealed an antibacterial and antibiofilm agent that could be useful in various industrial, medicinal, and environmental applications. This study introduces a work that presents an alternative, safe, promising, and efficient nanocomposite for treating harmful bacteria in humans and animals. This treatment is based on the synergistic effectiveness of Se NPs, CS, and AMP.<\/jats:p>\n          <\/jats:sec>","DOI":"10.1186\/s12934-025-02783-0","type":"journal-article","created":{"date-parts":[[2025,7,5]],"date-time":"2025-07-05T05:16:17Z","timestamp":1751692577000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":18,"title":["Biological activities of optimized biosynthesized selenium nanoparticles using Proteus mirabilis PQ350419 alone or combined with chitosan and ampicillin against common multidrug-resistant bacteria"],"prefix":"10.1186","volume":"24","author":[{"given":"Laila A.","family":"Elshikiby","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zakaria A.M.","family":"Baka","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2694-3720","authenticated-orcid":false,"given":"Mohamed M.","family":"El-Zahed","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,7,5]]},"reference":[{"key":"2783_CR1","doi-asserted-by":"publisher","first-page":"232","DOI":"10.14745\/ccdr.v42i11a03","volume":"42","author":"K Amaratunga","year":"2016","unstructured":"Amaratunga K, Tarasuk J, Tsegaye L, Archibald CP, Group ARAMRST. 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Pharmaceuticals. 2024;17:915.","journal-title":"Pharmaceuticals"}],"container-title":["Microbial Cell Factories"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s12934-025-02783-0.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/article\/10.1186\/s12934-025-02783-0\/fulltext.html","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s12934-025-02783-0.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,7,5]],"date-time":"2025-07-05T06:02:25Z","timestamp":1751695345000},"score":1,"resource":{"primary":{"URL":"https:\/\/microbialcellfactories.biomedcentral.com\/articles\/10.1186\/s12934-025-02783-0"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,7,5]]},"references-count":111,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["2783"],"URL":"https:\/\/doi.org\/10.1186\/s12934-025-02783-0","relation":{},"ISSN":["1475-2859"],"issn-type":[{"value":"1475-2859","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,7,5]]},"assertion":[{"value":"27 March 2025","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"24 June 2025","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"5 July 2025","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"The study was carried out in accordance with the guidelines and regulations of the scientific research ethics committee, Faculty of Science, Damietta University, Egypt. Written informed consent for enrolment, screening, and specimen collection was obtained from each patient or their parents or guardians. The data of the specimens were not exposed. The cytotoxicity experiments were conducted in compliance with the IACUC (The Institutional Animal Care and Use Committee) statement for using animals in research and teaching by the local ethical committee of AUHA (Al-Azhar University Housing Animals).","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"The authors declare no competing interests.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"159"}}