Definitions and Guidelines for Research on Antibiotic Persistence

Corrected: Publisher Correction CONSENSUS STATEMENT Definitions and guidelines for research on antibiotic persistence Nathalie Q. Balaban 1*, Sophie Helaine 2, Kim Lewis3, Martin Ackermann4,5, Bree Aldridge6, Dan I. Andersson 7, Mark P. Brynildsen8, Dirk Bumann9, Andrew Camilli6, James J. Collins 10,11,12, Christoph Dehio9, Sarah Fortune 13, Jean- Marc Ghigo14, Wolf- Dietrich Hardt15, Alexander Harms9, Matthias Heinemann 16, Deborah T. Hung12, Urs Jenal9, Bruce R. Levin17, Jan Michiels18, Gisela Storz19, Man- Wah Tan20, Tanel Tenson21, Laurence Van Melderen22 and Annelies Zinkernagel23 Abstract | Increasing concerns about the rising rates of antibiotic therapy failure and advances in single-cellanalyseshaveinspiredasurgeofresearchintoantibioticpersistence.Bacterial persister cells represent a subpopulation of cells that can survive intensive antibiotic treatment withoutbeingresistant.Severalapproacheshaveemergedtodefineandmeasurepersistence,and it is now time to agree on the basic definition of persistence and its relation to the other mechanisms bywhichbacteriasurviveexposuretobactericidalantibiotictreatments,suchasantibiotic resistance,heteroresistanceortolerance.InthisConsensusStatement,weprovidedefinitionsof persistencephenomena,distinguishbetweentriggeredandspontaneouspersistenceandprovide aguidetomeasuringpersistence.Antibioticpersistenceisnotonlyaninterestingexampleof non-geneticsingle-cellheterogeneity,itmayalsohavearoleinthefailureofantibiotictreatments. Therefore,itisourhopethattheguidelinesoutlinedinthisarticlewillpavethewayforbetter characterizationofantibioticpersistenceandforunderstandingitsrelevancetoclinicaloutcomes. More than 70 years ago, Hobby1 and Bigger2 observed that 2018) in Ascona, Switzerland, which brought together antibiotics that are considered bactericidal and kill bacte- 121 investigators involved in antibiotic persistence ria in fact fail to sterilize cultures. Bigger realized that the research from 21 countries, a discussion panel laid the small number of bacteria that manage to survive inten- main themes for a Consensus Statement on the definition sive antibiotic treatments are a distinct subpopulation and detection procedure of antibiotic persistence detailed of bacteria that he named ‘persisters’. below. In light of the potential role that antibiotic per- Fuelled in part by increasing concerns about anti- sistence can have in antibiotic treatment regimens, it is biotic resistance but also by technological advances in our hope that clarification and standardization of experi- single- cell analyses, the past 15 years have witnessed a mental procedures will facilitate the translation of basic great deal of research on antibiotic persistence by inves- science research into practical guidelines. tigators with different backgrounds and perspectives. As the number of scientists that tackle the puzzles and Defining the persistence phenomena challenges of antibiotic persistence from many differ- We adopt here a phenomenological definition of anti- ent angles has profoundly increased, it is now time to biotic persistence that is based on a small set of obser- agree on the basic definition of persistence and its dis- vations that can be made from experiments performed tinction from the other mechanisms by which bacteria in vitro and that does not assume a specific mechanism. survive exposure to bactericidal antibiotic treatments3. We focus on the differences and similarities between Several approaches have independently emerged to antibiotic persistence and other processes enabling bac- define and measure persistence. Research groups fol- teria to survive exposure to antibiotic treatments that lowing seemingly similar procedures may reach differ- could kill them, such as resistance, tolerance and hetero- ent results, and careful examination of the experimental resistance. We identify different types of persistence *e- mail: nathalie.balaban@ procedures often reveals that results of different groups that should be measured differently to obtain meaning- mail.huji.ac.il cannot be compared. During the European Molecular ful results; therefore, the definition of these types goes https://doi.org/10.1038/ Biology Organization (EMBO) Workshop ‘Bacterial beyond semantics. For the more mathematically oriented s41579-019-0196-3 Persistence and Antimicrobial Therapy’ (10–14 June readers, we provide a mathematical definition of the NATURE REVIEWS | MICROBIOLOGY VOLUME 17 | JULY 2019 | 441 CONSENSUS STATEMENT various terms based on a widely used phenomenological Persistence versus resistance. ‘Resistance’ is the ability model for survival under drug exposure in BOx 1. of bacteria to replicate and not just survive in the presence of a drug (BOx 2). The most common measure of Persistent infection versus antibiotic persistence. First, the level of resistance is the minimum inhibitory con- we would like to distinguish ‘antibiotic persistence’ from centration (MIC), which is the lowest concentration of ‘persistent infection’4,5 (Fig. 1). The latter is generally used the antibiotic required to prevent the replication of the to describe infections in the host that are not cleared bacteria. A higher MIC corresponds with a higher level by the host immune system, whereas antibiotic persis- of resistance (Fig. 2a). Resistance is inherited and may tence describes a bacterial population that is refractory be acquired by horizontal gene transfer of resistance- to antibiotic treatments, whether in vitro or possibly in encoding genes (for example, encoding antibiotic the host. Persistent infections are typically multifactorial inactivating enzymes10 or efflux pumps11) or mutations and involve mechanisms evolved by different pathogens (for example, leading to modification of the antibi- to evade the immune system, such as antigenic mimicry otic target) that confer the resistance phenotype to the in Helicobacter pylori, antigenic variation in Neisseria bacterial population12. gonorrhoeae and inhibition of phagocytosis4 and imm- ‘Persistence’ is the ability of a subset of the popula- une evasion in Mycobacterium tuberculosis6. As antibiotic tion to survive exposure to a bactericidal drug concen- persistence specifically addresses the ability of bacteria tration (Fig. 2). Therefore, persistence is defined only to survive antibiotic treatments, it may be an additional for bactericidal antibiotics. Several features distinguish factor for the prolongation of persistent infections persistence from resistance. First, the hallmark of anti- despite antibiotic treatment, for example, in recurrent biotic persistence is the biphasic killing curve (Fig. 2c); urinary tract infections7,8. Moreover, the same mecha- that is, the observation that not all bacteria in a clonal nisms may be involved in both immune evasion and culture are killed at the same rate. Second, when per- antibiotic persistence, for example, biofilm formation9. sister cells regrow without antibiotics (see below), their Historically, the term ‘persistent infection’ was used progeny give rise to a population that is as susceptible before antibiotics were available to treat infections. to drugs as the parental population it was isolated To avoid ambiguity, we suggest using the term antibiotic from. Third, the level of persistence, namely, the size of persistence to distinguish between these pheno mena the persister subpopulation, will only weakly depend when first mentioned in a publication. The focus in this on the concentration of the drug as long as it is far above article is on antibiotic persistence, although for simplicity, the MIC. In addition, the survival advantage of persister tradition and brevity, below, we use the word persistence. bacteria is often observed for antibiotic treatments belonging to different classes of antibiotics, for example, 13 Author addresses β-lactams and fluoroquinolones . Fourth, in contrast to resistant cells, persister bacteria cannot replicate in the 1 Racah Institute of Physics, The Hebrew University, Jerusalem, Israel. presence of the drug any better than the non- persister 2 MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, UK. cells but are killed at a lower rate than the susceptible 3Department of Biology, Northeastern University, Boston, MA, USA. population from which they arose. This property also 4Institute of Biogeochemistry and Pollutant Dynamics, ETH Zurich, Zurich, Switzerland. 5Department of Environmental Microbiology, Eawag, Dubendorf, Switzerland. distinguishes persistence from heteroresistance, a phe- 6Department of Molecular Biology and Microbiology, Tufts University School of Medicine, nomenon in which a small subpopulation transiently Boston, MA, USA. displays a substantially (more than eightfold) higher 7Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, MIC14 (see BOx 1). Sweden. 8Department of Chemical and Biological Engineering, Princeton University, Princeton, Persistence and tolerance. ‘Tolerance’ and persistence NJ, USA. are similar phenomena of increased survival in the pres- 9 Focal Area Infection Biology, Biozentrum of the University of Basel, Basel, Switzerland. ence of an antibiotic without an increase in the MIC. In 10 Institute for Medical Engineering & Science, Department of Biological Engineering, and studies that focus on only a qualitative understanding of Synthetic Biology Center, Massachusetts Institute of Technology,

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