More than 70 years ago, Hobby 1 and Bigger 2 observed that antibiotics that are considered bactericidal and kill bacteria in fact fail to sterilize cultures. Bigger realized that the small number of bacteria that manage to survive intensive antibiotic treatments are a distinct subpopulation of bacteria that he named 'persisters'. Fuelled in part by increasing concerns about antibiotic resistance but also by technological advances in single-cell analyses, the past 15 years have witnessed a great deal of research on antibiotic persistence by investigators with different backgrounds and perspectives. As the number of scientists that tackle the puzzles and challenges of antibiotic persistence from many different angles has profoundly increased, it is now time to agree on the basic definition of persistence and its distinction from the other mechanisms by which bacteria survive exposure to bactericidal antibiotic treatments 3. Several approaches have independently emerged to define and measure persistence. Research groups following seemingly similar procedures may reach different results, and careful examination of the experimental procedures often reveals that results of different groups cannot be compared. During the European Molecular Biology Organization (EMBO) Workshop 'Bacterial Persistence and Antimicrobial Therapy' (10-14 June 2018) in Ascona, Switzerland, which brought together 121 investigators involved in antibiotic persistence research from 21 countries, a discussion panel laid the main themes for a Consensus Statement on the definition and detection procedure of antibiotic persistence detailed below. In light of the potential role that antibiotic persistence can have in antibiotic treatment regimens, it is our hope that clarification and standardization of experimental procedures will facilitate the translation of basic science research into practical guidelines. Defining the persistence phenomena We adopt here a phenomenological definition of antibiotic persistence that is based on a small set of observations that can be made from experiments performed in vitro and that does not assume a specific mechanism. We focus on the differences and similarities between antibiotic persistence and other processes enabling bacteria to survive exposure to antibiotic treatments that could kill them, such as resistance, tolerance and heteroresistance. We identify different types of persistence that should be measured differently to obtain meaningful results; therefore, the definition of these types goes beyond semantics. For the more mathematically oriented readers, we provide a mathematical definition of the

Many bacteria can infect and persist inside their hosts for long periods of time. This can be due to immunosuppression of the host, immune evasion by the pathogen and/or ineffective killing by antibiotics. Bacteria can survive antibiotic treatment if they are resistant or tolerant to a drug. Persisters are a subpopulation of transiently antibiotic-tolerant bacterial cells that are often slow-growing or growth-arrested, and are able to resume growth after a lethal stress. The formation of persister cells establishes phenotypic heterogeneity within a bacterial population and has been hypothesized to be important for increasing the chances of successfully adapting to environmental change. The presence of persister cells can result in the recalcitrance and relapse of persistent bacterial infections, and it has been linked to an increase in the risk of the emergence of antibiotic resistance during treatment. If the mechanisms of the formation and regrowth of these antibiotic-tolerant cells were better understood, it could lead to the development of new approaches for the eradication of persistent bacterial infections. In this Review, we discuss recent developments in our understanding of bacterial persisters and their potential implications for the treatment of persistent infections.

Many bacterial pathogens cause persistent infections despite repeated antibiotic exposure. Bacterial persisters are antibiotic-tolerant cells, but little is known about their growth status and the signals and pathways leading to their formation in infected tissues. We used fluorescent single-cell analysis to identify Salmonella persisters during infection. These were part of a nonreplicating population formed immediately after uptake by macrophages and were induced by vacuolar acidification and nutritional deprivation, conditions that also induce Salmonella virulence gene expression. The majority of 14 toxin-antitoxin modules contributed to intracellular persister formation. Some persisters resumed intracellular growth after phagocytosis by naïve macrophages. Thus, the vacuolar environment induces phenotypic heterogeneity, leading to either bacterial replication or the formation of nonreplicating persisters that could provide a reservoir for relapsing infection.

Several important pathogens cause disease by surviving and replicating within host cells. Bacterial proliferation is the product of both replication and killing undergone by the population. However, these processes are difficult to distinguish, and are usually assessed together by determination of net bacterial load. In addition, measurement of net load does not reveal heterogeneity within pathogen populations. This is particularly important in persistent infections in which slow or nongrowing bacteria are thought to have a major impact. Here we report the development of a reporter system based on fluorescence dilution that enables direct quantification of the replication dynamics of Salmonella enterica serovar Typhimurium (S. Typhimurium) in murine macrophages at both the population and single-cell level. We used this technique to demonstrate that a major S. Typhimurium virulence determinant, the Salmonella pathogenicity island 2 type III secretion system, is required for bacterial replication but does not have a major influence on resistance to killing. Furthermore, we found that, upon entry into macrophages, many bacteria do not replicate, but appear to enter a dormant-like state. These could represent an important reservoir of persistent bacteria. The approach could be extended to other pathogens to study the contribution of virulence and host resistance factors to replication and killing, and to identify and characterize nonreplicating bacteria associated with chronic or latent infections.