Bacteria under antibiotic selective pressure that acquire resistance via plasmid copy-number amplification accumulate a disproportionately high mutational load in plasmid-encoded housekeeping functions as a consequence of error-prone replication at high copy number. A two-stage evolutionary trap therapy — stage 1 agent forces resistance via predictable plasmid amplification, stage 2 agent is selectively toxic to high-copy-number cells via amplification-driven toxin-antitoxin imbalance — creates an escape-proof treatment strategy because the only resistance route to stage 2 (reducing copy number) restores susceptibility to stage 1. This is formalizable as a directed acyclic graph over resistance states with no sink node outside the susceptible state, provable by exhaustive enumeration of known plasmid resistance mechanisms in the CARD and ResFinder databases.

Two linked claims: (H1) Bacteria that evolve antibiotic resistance predominantly via plasmid copy-number (CN) amplification (rather than point mutations or horizontal acquisition of novel genes) accumulate a statistically elevated mutation rate/load specifically in plasmid-encoded housekeeping genes (replication initiators, partitioning genes, toxin-antitoxin modules, core metabolic genes carried on the plasmid), relative to (a) chromosomal genes in the same cells and (b) plasmid genes in low-copy control lineages, due to error-prone replication and reduced per-copy proofreading fidelity at high copy number. (H2) A sequential two-drug regimen exploiting this — Stage 1 selecting for CN amplification, Stage 2 selectively toxic to high-CN cells via TA-module stoichiometric imbalance — constitutes an 'evolutionary trap' in which the only accessible resistance path to Stage 2 (CN reduction) reverts susceptibility to Stage 1, formalizable as a DAG over resistance states with the susceptible state as the unique long-run absorbing/recurrent state under alternating selection.