Unstable Heteroresistance as a Stepping Stone to Stable Resistance via Compensatory Mutations

Antibiotic heteroresistance, a trait in which a drug-susceptible population harbors a rare resistant subpopulation, has been presented as an unstable intermediate state on the way to classic stable antibiotic resistance. However, the timescales and pathways under which this could occur has not been examined. Extending a recombination-based model of copy- heteroresistance to include compensatory mutations, with rates and costs from experimental estimates, we simulated populations across thousands of generations of growth with β-lactam exposure. In the first days a founder population survives by one of two paths, either amplifying the resistance gene to a high copy-number or gaining compensatory mutations and staying at low copy-number. However, both paths converge over weeks to months on the same low-copy compensated state, which would be indistinguishable from classical resistance. Importantly, the ending state would provide no information as to the route taken: an isolate sequenced afterward carries no sign of the intermediate amplified state. With the parameters estimated and across a wide range of parameters, we find that amplification-mediated heteroresistance can be a transient stepping stone across lethal selection that leaves no sequence trace in surviving isolates.