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Despite the mounting clinical evidence supporting the importance of bacterial heterogeneity in acute infections, current antimicrobial susceptibility tests (ASTs) are typically insensitive to cell\u2010to\u2010cell differences as they only measure population\u2010wide averages. Herein, the use of single\u2010cell motility to address this issue is demonstrated. It is shown for the first time that antibiotic susceptibility detected as a change in single\u2010cell 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\u2010throughput, single\u2010cell motility assay. The method allows for the detection of heteroresistance in <jats:italic>Escherichia coli<\/jats:italic> and <jats:italic>Salmonella typhimurium<\/jats:italic> in 2\u2009h or less and quantifies the MIC of an antibiotic in 1.5\u2009h. The findings emphasize the need for characterizing bacterial heterogeneity, and they highlight the importance of single\u2010cell bacterial motility in assessing both antibiotic susceptibility and population\u2010wide heteroresistance.<\/jats:p><\/jats:sec>","DOI":"10.1002\/smsc.202100123","type":"journal-article","created":{"date-parts":[[2022,3,24]],"date-time":"2022-03-24T22:43:33Z","timestamp":1648161813000},"source":"Crossref","is-referenced-by-count":8,"title":["Single\u2010Cell Motility Rapidly Quantifying Heteroresistance in Populations of <i>Escherichia coli<\/i> and <i>Salmonella typhimurium<\/i>"],"prefix":"10.1002","volume":"2","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8030-7699","authenticated-orcid":false,"given":"Giampaolo","family":"Pitruzzello","sequence":"first","affiliation":[{"name":"Department of Physics University of York  York YO10 5DD 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