A comprehensive analytical framework integrating liquid chromatography-tandem mass spectrometry metabolomics with chemometrics for metabolite profiling of lettuce varieties and discovery of antibacterial agents

correlation between the elucidated profiles and their antivirulence effects. A total of 195 metabolites were annotated

using LC-QTOF-MS/MS metabolomics assisted by molecular networking and integrated with chemometrics.

Red varieties (red longifolia and lolla rosa) demonstrated higher chlorogenic and chicoric acids

suggesting their antioxidant properties. In parallel, amino acids and disaccharides were enriched in romaine

longifolia rationalizing its palatable taste and nutritional potential, while crispa, capitata, and lolla bionda presented

a high β-carboline alkaloid content. The antibacterial and antihemolytic potential of all varieties against

methicillin-sensitive and methicillin-resistant Staphylococcus aureus was assessed and validated by prominent

downregulation of α-hemolysin transcriptional levels in both strains. Moreover, correlation analysis revealed

sesquiterpenes, β-carboline alkaloids, amino acids, and oxy-fatty acids as the main bioactives. Results emphasize

lettuce significance as a functional food and nutraceutical source, and highlight varieties naturally rich in antibacterial agents to adapt breeding programs.