Single‐Cell Motility Rapidly Quantifying Heteroresistance in Populations of Escherichia coli and Salmonella typhimurium

Heterogeneous bacterial populations can display increased resistance to external threats, such as exposure to antibiotics. Despite the mounting clinical evidence supporting the importance of bacterial heterogeneity in acute infections, current antimicrobial susceptibility tests (ASTs) are typically insensitive to cell‐to‐cell differences as they only measure population‐wide averages. Herein, the use of single‐cell motility to address this issue is demonstrated. It is shown for the first time that antibiotic susceptibility detected as a change in single‐cell motility is an excellent proxy for polyclonal and monoclonal heteroresistance. It is also demonstrated that motility and growth are both inhibited by an antibiotic with strikingly similar patterns, thus enabling the quantification of minimum inhibitory concentration (MIC) using a high‐throughput, single‐cell motility assay. The method allows for the detection of heteroresistance in Escherichia coli and Salmonella typhimurium in 2 h or less and quantifies the MIC of an antibiotic in 1.5 h. The findings emphasize the need for characterizing bacterial heterogeneity, and they highlight the importance of single‐cell bacterial motility in assessing both antibiotic susceptibility and population‐wide heteroresistance.