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ABSTRACTMicrobes have developed resistance to nearly every antibiotic, yet the steps leading to drug resistance remain unclear. Here we report a multistage process by which Pseudomonas aeruginosaacquires drug resistance following exposure to ciprofloxacin at levels ranging from 0.5× to 8× the initial MIC. In stage I, susceptible cells are killed en masseby the exposure. In stage II, a small, slow to nongrowing population survives antibiotic exposure that does not exhibit significantly increased resistance according to the MIC measure. In stage III, exhibited at 0.5× to 4× the MIC, a growing population emerges to reconstitute the population, and these cells display heritable increases in drug resistance of up to 50 times the original level. We studied the stage III cells by proteomic methods to uncover differences in the regulatory pathways that are involved in this phenotype, revealing upregulation of phosphorylation on two proteins, succinate-semialdehyde dehydrogenase (SSADH) and methylmalonate-semialdehyde dehydrogenase (MMSADH), and also revealing upregulation of a highly conserved protein of unknown function. Transposon disruption in the encoding genes for each of these targets substantially dampened the ability of cells to develop the stage III phenotype. Considering these results in combination with computational models of resistance and genomic sequencing results, we postulate that stage III heritable resistance develops from a combination of both genomic mutations and modulation of one or more preexisting cellular pathways.