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

Type II toxin–antitoxin (TA) systems are generally composed of two genes organized in an operon, encoding a labile antitoxin and a stable toxin. They were first discovered on plasmids where they contribute to plasmid stability by a phenomenon denoted as ‘addiction’, and subsequently in bacterial chromosomes. To discover novel families of antitoxins and toxins, we developed a bioinformatics approach based on the ‘guilt by association’ principle. Extensive experimental validation in Escherichia coli of predicted antitoxins and toxins increased significantly the number of validated systems and defined novel toxin and antitoxin families. Our data suggest that toxin families as well as antitoxin families originate from distinct ancestors that were assembled multiple times during evolution. Toxin and antitoxin families found on plasmids tend to be promiscuous and widespread, indicating that TA systems move through horizontal gene transfer. We propose that due to their addictive properties, TA systems are likely to be maintained in chromosomes even though they do not necessarily confer an advantage to their bacterial hosts. Therefore, addiction might play a major role in the evolutionary success of TA systems both on mobile genetic elements and in bacterial chromosomes.

Bacterial toxin–antitoxin (TA) systems are diverse and widespread in the prokaryotic kingdom. They are composed of closely linked genes encoding a stable toxin that can harm the host cell and its cognate labile antitoxin, which protects the host from the toxin's deleterious effect. TA systems are thought to invade bacterial genomes through horizontal gene transfer. Some TA systems might behave as selfish elements and favour their own maintenance at the expense of their host. As a consequence, they may contribute to the maintenance of plasmids or genomic islands, such as super-integrons, by post-segregational killing of the cell that loses these genes and so suffers the stable toxin's destructive effect. The function of the chromosomally encoded TA systems is less clear and still open to debate. This Review discusses current hypotheses regarding the biological roles of these evolutionarily successful small operons. We consider the various selective forces that could drive the maintenance of TA systems in bacterial genomes.

Streptococcus pyogenes ranks among the main causes of mortality from bacterial infections worldwide. Currently there is no vaccine to prevent diseases such as rheumatic heart disease and invasive streptococcal infection. The streptococcal M protein that is used as the substrate for epidemiological typing is both a virulence factor and a vaccine antigen. Over 220 variants of this protein have been described, making comparisons between proteins difficult, and hindering M protein-based vaccine development. A functional classification based on 48 emm-clusters containing closely related M proteins that share binding and structural properties is proposed. The need for a paradigm shift from type-specific immunity against S. pyogenes to emm-cluster based immunity for this bacterium should be further investigated. Implementation of this emm-cluster-based system as a standard typing scheme for S. pyogenes will facilitate the design of future studies of M protein function, streptococcal virulence, epidemiological surveillance, and vaccine development.

Toxin-antitoxin systems are widespread in bacterial genomes. They are usually composed of two elements: a toxin that inhibits an essential cellular process and an antitoxin that counteracts its cognate toxin. In the past decade, a number of new toxin-antitoxin systems have been described, bringing new growth inhibition mechanisms to light as well as novel modes of antitoxicity. However, recent advances in the field profoundly questioned the role of these systems in bacterial physiology, stress response and antimicrobial persistence. This shifted the paradigm of the functions of toxin-antitoxin systems to roles related to interactions between hosts and their mobile genetic elements, such as viral defence or plasmid stability. In this Review, we summarize the recent progress in understanding the biology and evolution of these small genetic elements, and discuss how genomic conflicts could shape the diversification of toxin-antitoxin systems.

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Type II toxin-antitoxin (TA) systems are small genetic elements composed of a toxic protein and its cognate antitoxin protein, the latter counteracting the toxicity of the former. While TA systems were initially discovered on plasmids, functioning as addiction modules through a phenomenon called postsegregational killing, they were later shown to be massively present in bacterial chromosomes, often in association with mobile genetic elements. Extensive research has been conducted in recent decades to better understand the physiological roles of these chromosomally encoded modules and to characterize the conditions leading to their activation. The diversity of their proposed roles, ranging from genomic stabilization and abortive phage infection to stress modulation and antibiotic persistence, in conjunction with the poor understanding of TA system regulation, resulted in the generation of simplistic models, often refuted by contradictory results. This review provides an epistemological and critical retrospective on TA modules and highlights fundamental questions concerning their roles and regulations that still remain unanswered. FIG 1 Type II TA systems, postsegregational killing and distribution. (A) Nonviable segregant or postsegregational killing model. TA genes, as well as proteins, are represented in red (toxins) and green (antitoxins).Rectangles denote TA genes encoded on a plasmid, and round shapes denote TA proteins produced from these genes. A TA-encoding plasmid can be lost during division in a way that one of the daughter cells does not inherit a plasmid copy. In these cells, TA proteins cannot be replenished due to the absence of TA genes. Since the antitoxin is degraded while its cognate toxin is stable, the free toxin concentration will increase, exert its activity, and, in time, induce cell death, therefore killing plasmid-free segregants. (B) Distribution of type II TA systems in various E. coli reference strains generated by TAfinder (23). Asterisks indicate systems that were not validated experimentally. Parentheses include name of the prophage a TA is encoded on when applicable. The strains are MG1655 (NCBI U00096.3), a common lab strain from phylogroup A; W (CP002967.1), a soil isolate from phylogroup B1; EDL933 (AE005174.2), an enterohemorrhagic pathogen from phylogroup E; and UTI89 (CP000243.1), a uropathogen from phylogroup B2. No TA systems are conserved within these four distantly related E. coli strains.

Genes for toxin-antitoxin (TA) complexes are widespread in prokaryote genomes, and species frequently possess tens of plasmid and chromosomal TA loci. The complexes are categorized into three types based on genetic organization and mode of action. The toxins universally are proteins directed against specific intracellular targets, whereas the antitoxins are either proteins or small RNAs that neutralize the toxin or inhibit toxin synthesis. Within the three types of complex, there has been extensive evolutionary shuffling of toxin and antitoxin genes leading to considerable diversity in TA combinations. The intracellular targets of the protein toxins similarly are varied. Numerous toxins, many of which are sequence-specific endoribonucleases, dampen protein synthesis levels in response to a range of stress and nutritional stimuli. Key resources are conserved as a result ensuring the survival of individual cells and therefore the bacterial population. The toxin effects generally are transient and reversible permitting a set of dynamic, tunable responses that reflect environmental conditions. Moreover, by harboring multiple toxins that intercede in protein synthesis in response to different physiological cues, bacteria potentially sense an assortment of metabolic perturbations that are channeled through different TA complexes. Other toxins interfere with the action of topoisomersases, cell wall assembly, or cytoskeletal structures. TAs also play important roles in bacterial persistence, biofilm formation and multidrug tolerance, and have considerable potential both as new components of the genetic toolbox and as targets for novel antibacterial drugs.