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Although antibiotics and synthetic antimicrobial agents can combat these pathogens, antibiotic resistance remains a major concern. Recent decades have seen growing interest in antibiotic alternatives. Juglone, a natural naphthoquinone compound from Juglandaceae plant, exhibits strong antimicrobial activity against\n                      <jats:italic>S. aureus<\/jats:italic>\n                      . However, its antimicrobial mechanism is not yet fully understood. This study investigated the antimicrobial mechanism of juglone from the perspectives of cell biology, cell morphology, and transcriptomics.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Juglone had potent antimicrobial effects against\n                      <jats:italic>E. coli<\/jats:italic>\n                      ,\n                      <jats:italic>S. aureus<\/jats:italic>\n                      , and\n                      <jats:italic>S. pullorum<\/jats:italic>\n                      . The minimum inhibitory concentration (MIC) of juglone against all three bacterial strains was 15.6\u00a0\u00b5g\/mL. Treatment with juglone decreased bacterial metabolic activity, reduced the intracellular DNA and RNA fluorescence intensity, resulted in the leakage of intracellular alkaline phosphatase (AKP) and ions, and caused a decline in ATP content and ATPase activity. Scanning electron microscopy (SEM) revealed significant membrane damage in each of the three bacterial species following juglone treatment. Transcriptomic sequencing and Gene Ontology (GO) enrichment analysis of\n                      <jats:italic>S. pullorum<\/jats:italic>\n                      revealed that juglone treatment resulted in a significant upregulation of GO terms related to translation, while those terms associated with transport, localization, and membrane functions were significantly downregulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the pathways related to oxidative phosphorylation and the citrate cycle were significantly upregulated, whereas those pathways related to ABC transporters and quorum sensing were significantly downregulated.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>\n                      These findings suggest that juglone compromises the permeability and integrity of the cell envelope in\n                      <jats:italic>E. coli<\/jats:italic>\n                      ,\n                      <jats:italic>S. aureus<\/jats:italic>\n                      , and\n                      <jats:italic>S. pullorum<\/jats:italic>\n                      , resulting in cytoplasmic leakage and metabolic impairment. Additionally, juglone alters the gene expression of transporters, interferes with the energy metabolism, protein synthesis and transport, quorum sensing, and biofilm formation of\n                      <jats:italic>S. pullorum<\/jats:italic>\n                      , thereby exerting antimicrobial effects.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s12866-025-04354-0","type":"journal-article","created":{"date-parts":[[2025,10,17]],"date-time":"2025-10-17T13:58:39Z","timestamp":1760709519000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["Antimicrobial activity and possible mechanisms of juglone against Escherichia coli, Staphylococcus aureus, and Salmonella pullorum"],"prefix":"10.1186","volume":"25","author":[{"given":"Lei","family":"Wang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mingxin","family":"Qiu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xuanyue","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mengjian","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luyu","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8818-4921","authenticated-orcid":false,"given":"Yong","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,10,17]]},"reference":[{"key":"4354_CR1","doi-asserted-by":"publisher","first-page":"1006676","DOI":"10.3389\/fvets.2022.1006676","volume":"9","author":"K Wang","year":"2022","unstructured":"Wang K, Cha J, Liu K, Deng J, Yang B, Xu H, Wang J, Zhang L, Gu X, Huang C, Qu W. 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