BACKGROUND: Metabolite production is essential for the proliferation and environmental adaptation of all living organisms. In pathogenic bacteria, metabolite exchange during host infection can regulate their physiology and virulence. However, there is still much unknown about which specific metabolic pathways in pathogenic bacteria respond to changes in the environment during infections. This study examines how pathogenic bacterium Acinetobacter baumannii uses particular metabolic pathways to regulate its ability to antibiotic persistence and pathogenesis. RESULTS: To determine specific metabolic pathways in pathogenic antibiotic resistance bacteria, metabolite profiles of bacteria were constructed using ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry and multivariate statistical analysis. A. baumannii generates amino acid derivative metabolites, which are precursors for fatty acid production. Comparative genomic analysis identified specific genes regulating the production of these metabolites and fatty acids in A. baumannii. Inactivation of genes involved in glutamate metabolism, gdhA, aspB, murI1, and racD, impairs antibiotic persistence, while inactivation of the hisC gene, encoding histidinol - phosphate aminotransferase enzyme in histidine metabolic pathway, increases bacterial survival inside macrophages during infections. CONCLUSIONS: This study reports that A. baumannii regulates antibiotic persistence and pathogenesis through glutamate and histidine metabolic pathways, respectively. These findings suggest that specific metabolic pathways regulate bacterial pathogenesis and antibiotic persistence during infections, providing potential therapeutic targets for pathogenic bacteria.