Combinatorial action of regulatory systems generates colistin heteroresistance

The mechanism by which phenotypic heterogeneity is generated within a bacterial population and leads to colistin heteroresistance has been unclear. Heteroresistance is a form of antibiotic resistance in which a minor subpopulation of resistant cells coexists with a majority susceptible population. Colistin heteroresistance is common among Enterobacter isolates, threatens its utility as a therapeutic, and has become a model to study the fundamental bases of heteroresistance. Here, we identify the sigma factor σ^E as the source of heterogeneity in colistin resistance. Single-cell tracking revealed that σ^E is active in 1% of the population, and only those cells survive colistin exposure. However, σ^E expression and heterogeneity were insufficient for survival, as a mutant lacking the PhoPQ system controlling lipid A modifications necessary for colistin resistance retained heterogeneity but lost resistance. These findings lead to a paradigm in heteroresistance; the combinatorial action of regulatory systems, encompassing a heterogeneity generator and a distinct resistance generator, gives rise to colistin heteroresistance.