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Treatment failure and recurrent infections can occur even when patients are treated with antibiotics to which bacteria are susceptible. These treatment failures could be due to a sub-population of bacteria persisting through the treatment. In this study, we tested the hypothesis that such bacterial persisters manifest in clinical samples as small colony variants (SCVs). We worked with the bacterial pathogen Staphylococcus aureus, where SCVs are frequently observed in clinics. The small size of SCV-colonies is often considered to be the result of mutations that reduce the bacterial growth rate. Alternatively, it is possible that the small colony size could result from a long lag time of bacteria in plated samples. We used automated plate imaging and single-cell microscopy to precisely quantify growth kinetics of bacteria sampled from patient and mice abscesses, and from low-pH in vitro cultures mimicking the host. Under these conditions, the small colony size was the consequence of a long lag time and was associated with tolerance towards antibiotics. We found that bacteria with a long lag time emerged de novo during the growth phase in the murine abscess as well as in the low-pH in vitro cultures, and that their proportion increased during stationary phase. Antibiotic exposure further increased the proportion of bacteria with a long lag time. Thus, the persisters found in low-pH host compartments could be a cause of treatment failure, and antibiotic treatment potentially aggravates the problem. These insights call for antibiotic treatment strategies that explicitly address the problem of persister formation.Significance StatementBacterial pathogens often contain subpopulations that are multidrug tolerant and metabolically inactive. These subpopulations are known as persisters and are hypothesised to be linked to treatment failure and recurrent infections even in bacteria that are not genetically resistant to antibiotics. Identifying persisters in clinical samples remains a challenge due to their metabolicaly active state. Here, we used time-lapse plate imaging and single cell microscopy to link the presence of persisters to the nonstable small colony variant (SCV) phenotype that is observed in some Staphylococcus aureus infections. These findings may help to identify treatment regimens that prevent or address the emergence of persistence and thus to reduce relapses.