{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T13:16:11Z","timestamp":1785417371328,"version":"3.56.0"},"reference-count":41,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,4,30]],"date-time":"2026-04-30T00:00:00Z","timestamp":1777507200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32230106"],"award-info":[{"award-number":["32230106"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"award":["32230106"],"award-info":[{"award-number":["32230106"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Pinduoduo-China Agricultural University Research Fund","award":["PC2024B02001"],"award-info":[{"award-number":["PC2024B02001"]}]},{"DOI":"10.13039\/501100002365","name":"Basic Research Funding of China Agricultural University","doi-asserted-by":"publisher","award":["2025TC024"],"award-info":[{"award-number":["2025TC024"]}],"id":[{"id":"10.13039\/501100002365","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Antibiotics"],"abstract":"<jats:p>Background: Phospholipids are essential components of bacterial membranes and play central roles in membrane integrity and adaptation to antibiotic stress. However, confident annotation of phospholipid molecular species remains challenging due to the complexity of the lipidome and the limited structural constraints in conventional lipidomics workflows. Methods: Here, we present a bacterial phospholipidomic framework that integrates orthogonal structural evidence to achieve high-confidence and traceable annotation. Thin-layer chromatography (TLC) provides phospholipid headgroup assignment, gas chromatography\u2013mass spectrometry (GC\u2013MS) defines the acyl-chain pool, and Patern\u00f2\u2013B\u00fcchi derivatization enables C=C localization, collectively restricting the structural search space prior to liquid chromatography\u2013tandem mass spectrometry (LC\u2013MS\/MS) analysis. A rule-based ion prediction library further standardizes diagnostic ion assignment and reduces annotation ambiguity. Results: Applying this platform, we found Escherichia coli in the stationary phase remodeled the membrane phospholipids, with cardiolipin (CL) increasing from ~5% to ~10% and cyclopropane-containing phospholipid species rising to ~75%. Similar remodeling patterns are observed under diverse antibiotic exposures at sub-inhibitory concentrations, consistent with convergence toward a tolerance-associated membrane state. Extension of the framework to Enterococcus faecium supports proof-of-concept application in an additional Gram-positive model, with vancomycin-resistant strains exhibiting pronounced phosphatidylglycerol (PG) enrichment and reduced CL. Conclusions: Our work provides a scalable and reproducible strategy for bacterial phospholipid annotation, enabling molecular-species-resolved investigation of membrane adaptation and offering a framework for future exploration of lipid homeostasis pathways as potential antimicrobial targets.<\/jats:p>","DOI":"10.3390\/antibiotics15050459","type":"journal-article","created":{"date-parts":[[2026,5,1]],"date-time":"2026-05-01T06:50:01Z","timestamp":1777618201000},"page":"459","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Rule-Based Ion Prediction with Orthogonal Constraints Reveals Bacterial Phospholipid Remodeling Signatures"],"prefix":"10.3390","volume":"15","author":[{"given":"Wanying","family":"Hu","sequence":"first","affiliation":[{"name":"State Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenhan","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meirong","family":"Song","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4352-5344","authenticated-orcid":false,"given":"Jianfei","family":"Zhu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8242-3952","authenticated-orcid":false,"given":"Kui","family":"Zhu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China"},{"name":"Engineering Research Center of Animal Innovative Drugs and Safety Evaluation, Ministry of Education, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China"},{"name":"Technology Innovation Center for Food Safety Surveillance and Detection (Hainan), Sanya Institute of China Agricultural University, Sanya 572025, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1016\/j.cbpa.2019.05.025","article-title":"The role of bacterial lipid diversity and membrane properties in modulating antimicrobial peptide activity and drug resistance","volume":"52","author":"Lee","year":"2019","journal-title":"Curr. 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