ABSTRACTClinicians are increasingly confronted with the limitations of antibiotics to clear bacterial infections in patients. It has long been assumed that only antibiotic resistance plays a pivotal role in this phenomenon. Indeed, the worldwide emergence of antibiotic resistance is considered one of the major health threats of the 21st century. However, the presence of persister cells also has a significant influence on treatment outcomes. These antibiotic-tolerant cells are present in every bacterial population and are the result of the phenotypic switching of normal, antibiotic-sensitive cells. Persister cells complicate current antibiotic therapies and contribute to the development of resistance. In the past, extensive research has been performed to investigate persistence in laboratory settings; however, antibiotic tolerance under conditions that mimic the clinical setting remain poorly understood. In this study, we optimized a mouse model for lung infections with the opportunistic pathogenPseudomonas aeruginosa. In this model, mice are intratracheally infected withP. aeruginosaembedded in seaweed alginate beads and subsequently treated with tobramycin via nasal droplets. A diverse panel of 18P. aeruginosastrains originating from environmental, human, and animal clinical sources was selected to assess survival in the animal model. Survival levels were positively correlated with the survival levels determined via time-kill assays, a common method to study persistence in the laboratory. We showed that survival levels are comparable and thus that the classical persister assays are indicative of antibiotic tolerance in a clinical setting. The optimized animal model also enables us to test potential antipersister therapies and study persistence in relevant settings.IMPORTANCEThe importance of targeting persister cells in antibiotic therapies is becoming more evident, as these antibiotic-tolerant cells underlie relapsing infections and resistance development. Here, we studied persistence in a clinically relevant pathogen,Pseudomonas aeruginosa. It is one of the six ESKAPE pathogens (Enterococcus faecium,Staphylococcus aureus,Klebsiella pneumoniae,Acinetobacter baumannii,P. aeruginosa, andEnterobacterspp.), which are considered major health threats.P. aeruginosais mostly known to cause chronic lung infections in cystic fibrosis patients. We mimicked these lung infections in a mouse model to study persistence under more clinical conditions. It was shown that the survival levels of naturalP. aeruginosaisolates in this model are positively correlated with the survival levels measured in classical persistence assaysin vitro. These results not only validate the use of our current techniques to study persistence but also open opportunities to study new persistence mechanisms or evaluate new antipersister strategiesin vivo.