Combinatorial action of regulatory systems generates colistin heteroresistance

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 cloacae clinical isolates, threatens its utility as a last-line therapeutic, and has become a model with which to understand the fundamental bases of heteroresistance. Despite numerous insights, the mechanism by which phenotypic heterogeneity is generated within the population and leads to colistin heteroresistance has been unclear. Here, using a transposon-based mutagenesis screen, we identify the sigma factor σ^Eas the source of heterogeneity in population-wide colistin resistance levels. Single-cell tracking experiments revealed that σ^Eis active in only one percent of the population at baseline, and only those cells with active σ^E survive colistin exposure. However, σ^E expression and population heterogeneity are insufficient for survival, as a mutant lacking the PhoPQ two-component system controlling lipid A modifications necessary for colistin resistance retains heterogeneity but loses colistin resistance. These findings lead to a new paradigm in heteroresistance, where the combinatorial action of multiple regulatory systems, encompassing a heterogeneity generator and a distinct resistance generator, are required to give rise to colistin heteroresistance.