A motile doublet form of Salmonella Typhimurium diversifies target search behavior at the epithelial surface

The behaviors of infectious bacteria are commonly studied in bulk. This is effective to define the general properties of a given isolate, but insufficient to resolve subpopulations and unique single‐microbe behaviors within the bacterial pool. We here employ microscopy to study single‐bacterium characteristics among Salmonella enterica serovar Typhimurium (S.Tm), as they prepare for and launch invasion of epithelial host cells. We find that during the bacterial growth cycle, S.Tm populations switch gradually from fast planktonic growth to a host cell‐invasive phenotype, characterized by flagellar motility and expression of the Type‐three‐secretion‐system‐1. The indistinct nature of this shift leads to the establishment of a transient subpopulation of S.Tm “doublets”—waist‐bearing bacteria anticipating cell division—which simultaneously express host cell invasion machinery. In epithelial cell culture infections, these S.Tm doublets outperform their “singlet” brethren and represent a hyperinvasive subpopulation. Atop both glass and enteroid‐derived monolayers, doublets swim along markedly straighter trajectories than singlets, thereby diversifying search patterns and improving the surface exploration capacity of the total bacterial population. The straighter swimming, combined with an enhanced cell‐adhesion propensity, suffices to account for the hyperinvasive doublet phenotype. This work highlights bacterial cell length heterogeneity as a key determinant of target search patterns atop epithelia.