Metabolic dependency profiling guides staggered antibiotic administration to enhance killing of antibiotic-tolerant Acinetobacter baumannii

Background Acinetobacter baumannii infections are difficult to eradicate, and bacterial physiological state influences antibiotic killing. However, antibiotic-specific activity across nutrient-associated physiological states and quantitative comparison of these differences remain insufficiently understood. Methods Acinetobacter baumannii ATCC 19606 T was cultured across a nutrient gradient (0–100% LB) to generate nutrient-associated physiological states. Bacterial growth, CFU-normalized intracellular ATP levels, and killing by six antibiotics were quantified. ATP–survival relationships were assessed across standardized concentrations of 0.5×−64×MIC, and a Metabolic Dependency Index (MDI) was developed as a continuous empirical measure of ATP-associated variation in antibiotic killing. Meropenem (MEM) and amikacin (AMK), representing opposite ends of the observed MDI spectrum, were evaluated using simultaneous, MEM-first staggered, and AMK-first staggered administration. The MEM–AMK combination was also examined in five clinical isolates. Results Nutrient limitation reduced bacterial growth and CFU-normalized intracellular ATP levels and produced antibiotic- and concentration-dependent changes in killing. MDI values differed among antibiotics, with meropenem showing the highest and amikacin the lowest MDI. Simultaneous MEM–AMK treatment enhanced killing compared with the corresponding monotherapies under nutrient-limited conditions. MEM-first staggered administration further reduced bacterial counts relative to simultaneous treatment by 1.21 log 10 CFU (95% CI, 0.48–1.94; p = 0.019) and 0.86 log 10 CFU (95% CI, 0.47–1.26; p = 0.011) at two prespecified concentration combinations. Reverse-order AMK-first staggered treatment produced killing comparable to or greater than that achieved with MEM-first staggered treatment under several concentration combinations, indicating that the benefit of staggered exposure was not specific to a predefined MEM-first sequence. Similar differences between simultaneous and staggered MEM–AMK administration were observed across five clinical isolates, although the magnitude of these differences varied among isolates. Meropenem and amikacin MICs remained unchanged after antibiotic-free recovery of surviving populations, supporting a tolerance-associated phenotype rather than stable resistance. Conclusion Antibiotic killing in A. baumannii varies across nutrient-associated physiological states in an antibiotic- and concentration-dependent manner. The MDI provides a continuous framework for comparing these differences, while the MEM–AMK experiments show that combination activity can be further influenced by antibiotic administration pattern. These findings provide a basis for integrating bacterial physiological state into the evaluation and optimization of antibiotic combinations under nutrient-limited conditions.