Stabbed while Sleeping: Synthetic Retinoid Antibiotics Kill Bacterial Persister Cells.

Antibiotic-tolerant persister cells are difficult to eradicate by conventional classes of antibiotics. Kim and colleagues have discovered a new class of synthetic retinoid antibiotics that kill Staphylococcus aureus persisters by disrupting their cytoplasmic membrane.

Obg and Membrane Depolarization Are Part of a Microbial Bet-Hedging Strategy that Leads to Antibiotic Tolerance.

Within bacterial populations, a small fraction of persister cells is transiently capable of surviving exposure to lethal doses of antibiotics. As a bet-hedging strategy, persistence levels are determined both by stochastic induction and by environmental stimuli called responsive diversification. Little is known about the mechanisms that link the low frequency of persisters to environmental signals. Our results support a central role for the conserved GTPase Obg in determining persistence in Escherichia coli in response to nutrient starvation. Obg-mediated persistence requires the stringent response alarmone (p)ppGpp and proceeds through transcriptional control of the hokB-sokB type I toxin-antitoxin module. In individual cells, increased Obg levels induce HokB expression, which in turn results in a collapse of the membrane potential, leading to dormancy. Obg also controls persistence in Pseudomonas aeruginosa and thus constitutes a conserved regulator of antibiotic tolerance. Combined, our findings signify an important step toward unraveling shared genetic mechanisms underlying persistence.

Population Bottlenecks Strongly Affect the Evolutionary Dynamics of Antibiotic Persistence.

Bacterial persistence is a potential cause of antibiotic therapy failure. Antibiotic-tolerant persisters originate from phenotypic differentiation within a susceptible population, occurring with a frequency that can be altered by mutations. Recent studies have proven that persistence is a highly evolvable trait and, consequently, an important evolutionary strategy of bacterial populations to adapt to high-dose antibiotic therapy. Yet, the factors that govern the evolutionary dynamics of persistence are currently poorly understood. Theoretical studies predict far-reaching effects of bottlenecking on the evolutionary adaption of bacterial populations, but these effects have never been investigated in the context of persistence. Bottlenecking events are frequently encountered by infecting pathogens during host-to-host transmission and antibiotic treatment. In this study, we used a combination of experimental evolution and barcoded knockout libraries to examine how population bottlenecking affects the evolutionary dynamics of persistence. In accordance with existing hypotheses, small bottlenecks were found to restrict the adaptive potential of populations and result in more heterogeneous evolutionary outcomes. Evolutionary trajectories followed in small-bottlenecking regimes additionally suggest that the fitness landscape associated with persistence has a rugged topography, with distinct trajectories toward increased persistence that are accessible to evolving populations. Furthermore, sequencing data of evolved populations and knockout libraries after selection reveal various genes that are potentially involved in persistence, including previously known as well as novel targets. Together, our results do not only provide experimental evidence for evolutionary theories, but also contribute to a better understanding of the environmental and genetic factors that guide bacterial adaptation to antibiotic treatment.

Bacteria under antibiotic attack: Different strategies for evolutionary adaptation.

Bacteria are well known for their extremely high adaptability in stressful environments. The clinical relevance of this property is clearly illustrated by the ever-decreasing efficacy of antibiotic therapies. Frequent exposures to antibiotics favor bacterial strains that have acquired mechanisms to overcome drug inhibition and lethality. Many strains, including life-threatening pathogens, exhibit increased antibiotic resistance or tolerance, which considerably complicates clinical practice. Alarmingly, recent studies show that in addition to resistance, tolerance levels of bacterial populations are extremely flexible in an evolutionary context. Here, we summarize laboratory studies providing insight in the evolution of resistance and tolerance and shed light on how the treatment conditions could affect the direction of bacterial evolution under antibiotic stress.

Biochemical determinants of ObgE-mediated persistence.

Obg is a versatile GTPase that plays a pivotal role in bacterial persistence. We previously showed that the Escherichia coli homolog ObgE exerts this activity through transcriptional activation of a toxin-antitoxin module and subsequent membrane depolarization. Here, we assessed the role of G-domain functionality in ObgE-mediated persistence. Through screening of a mutant library, we identified five obgE alleles (with substitutions G166V, D246G, S270I, N283I and I313N) that have lost their persistence function and no longer activate hokB expression. These alleles support viability of a strain otherwise deprived of ObgE, indicating that ObgE's persistence function can be uncoupled from its essential role. Based on the ObgE crystal structure, we designed two additional mutant proteins (T193A and D286Y), one of which (D286Y) no longer affects persistence. Using isothermal titration calorimetry, stopped-flow experiments and kinetics, we subsequently assessed nucleotide binding and GTPase activity in all mutants. With the exception of the S270I mutant that is possibly affected in protein-protein interactions, all mutants that have lost their persistence function display severely reduced binding to GDP or the alarmone ppGpp. However, we find no clear relation between persistence and GTP or pppGpp binding nor with GTP hydrolysis. Combined, our results signify an important step toward understanding biochemical determinants underlying persistence.

Synthetic reconstruction of extreme high hydrostatic pressure resistance in Escherichia coli.

Although high hydrostatic pressure (HHP) is an interesting parameter to be applied in bioprocessing, its potential is currently limited by the lack of bacterial chassis capable of surviving and maintaining homeostasis under pressure. While several efforts have been made to genetically engineer microorganisms able to grow at sublethal pressures, there is little information for designing backgrounds that survive more extreme pressures. In this investigation, we analyzed the genome of an extreme HHP-resistant mutant of E. coli MG1655 (designated as DVL1), from which we identified four mutations (in the cra, cyaA, aceA and rpoD loci) causally linked to increased HHP resistance. Analysing the functional effect of these mutations we found that the coupled effect of downregulation of cAMP/CRP, Cra and the glyoxylate shunt activity, together with the upregulation of RpoH and RpoS activity, could mechanistically explain the increased HHP resistance of the mutant. Using combinations of three mutations, we could synthetically engineer E. coli strains able to comfortably survive pressures of 600-800 MPa, which could serve as genetic backgrounds for HHP-based biotechnological applications.

Frequency-based haplotype reconstruction from deep sequencing data of bacterial populations.

Clonal populations accumulate mutations over time, resulting in different haplotypes. Deep sequencing of such a population in principle provides information to reconstruct these haplotypes and the frequency at which the haplotypes occur. However, this reconstruction is technically not trivial, especially not in clonal systems with a relatively low mutation frequency. The low number of segregating sites in those systems adds ambiguity to the haplotype phasing and thus obviates the reconstruction of genome-wide haplotypes based on sequence overlap information.Therefore, we present EVORhA, a haplotype reconstruction method that complements phasing information in the non-empty read overlap with the frequency estimations of inferred local haplotypes. As was shown with simulated data, as soon as read lengths and/or mutation rates become restrictive for state-of-the-art methods, the use of this additional frequency information allows EVORhA to still reliably reconstruct genome-wide haplotypes. On real data, we show the applicability of the method in reconstructing the population composition of evolved bacterial populations and in decomposing mixed bacterial infections from clinical samples.

Molecular mechanisms and clinical implications of bacterial persistence.

Any bacterial population harbors a small number of phenotypic variants that survive exposure to high concentrations of antibiotic. Importantly, these so-called 'persister cells' compromise successful antibiotic therapy of bacterial infections and are thought to contribute to the development of antibiotic resistance. Intriguingly, drug-tolerant persisters have also been identified as a factor underlying failure of chemotherapy in tumor cell populations. Recent studies have begun to unravel the complex molecular mechanisms underlying persister formation and revolve around stress responses and toxin-antitoxin modules. Additionally, in vitro evolution experiments are revealing insights into the evolutionary and adaptive aspects of this phenotype. Furthermore, ever-improving experimental techniques are stimulating efforts to investigate persisters in their natural, infection-associated, in vivo environment. This review summarizes recent insights into the molecular mechanisms of persister formation, explains how persisters complicate antibiotic treatment of infections, and outlines emerging strategies to combat these tolerant cells.

In Vitro Emergence of High Persistence upon Periodic Aminoglycoside Challenge in the ESKAPE Pathogens.

Health care-associated infections present a major threat to modern medical care. Six worrisome nosocomial pathogens-Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.-are collectively referred to as the "ESKAPE bugs." They are notorious for extensive multidrug resistance, yet persistence, or the phenotypic tolerance displayed by a variant subpopulation, remains underappreciated in these pathogens. Importantly, persistence can prevent eradication of antibiotic-sensitive bacterial populations and is thought to act as a catalyst for the development of genetic resistance. Concentration- and time-dependent aminoglycoside killing experiments were used to investigate persistence in the ESKAPE pathogens. Additionally, a recently developed method for the experimental evolution of persistence was employed to investigate adaptation to high-dose, extended-interval aminoglycoside therapy in vitro We show that ESKAPE pathogens exhibit biphasic killing kinetics, indicative of persister formation. In vitro cycling between aminoglycoside killing and persister cell regrowth, evocative of clinical high-dose extended-interval therapy, caused a 37- to 213-fold increase in persistence without the emergence of resistance. Increased persistence also manifested in biofilms and provided cross-tolerance to different clinically important antibiotics. Together, our results highlight a possible drawback of intermittent, high-dose antibiotic therapy and suggest that clinical diagnostics might benefit from taking into account persistence.

Fitness trade-offs explain low levels of persister cells in the opportunistic pathogen Pseudomonas aeruginosa.

Microbial populations often contain a fraction of slow-growing persister cells that withstand antibiotics and other stress factors. Current theoretical models predict that persistence levels should reflect a stable state in which the survival advantage of persisters under adverse conditions is balanced with the direct growth cost impaired under favourable growth conditions, caused by the nonreplication of persister cells. Based on this direct growth cost alone, however, it remains challenging to explain the observed low levels of persistence (<<1%) seen in the populations of many species. Here, we present data from the opportunistic human pathogen Pseudomonas aeruginosa that can explain this discrepancy by revealing various previously unknown costs of persistence. In particular, we show that in the absence of antibiotic stress, increased persistence is traded off against a lengthened lag phase as well as a reduced survival ability during stationary phase. We argue that these pleiotropic costs contribute to the very low proportions of persister cells observed among natural P. aeruginosa isolates (3 × 10(-8) -3 × 10(-4)) and that they can explain why strains with higher proportions of persister cells lose out very quickly in competition assays under favourable growth conditions, despite a negligible difference in maximal growth rate. We discuss how incorporating these trade-offs could lead to models that can better explain the evolution of persistence in nature and facilitate the rational design of alternative therapeutic strategies for treating infectious diseases.

A study of SeqA subcellular localization in Escherichia coli using photo-activated localization microscopy.

Escherichia coli (E. coli) cells replicate their genome once per cell cycle to pass on genetic information to the daughter cells. The SeqA protein binds the origin of replication, oriC, after DNA replication initiation and sequesters it from new initiations in order to prevent overinitiation. Conventional fluorescence microscopy studies of SeqA localization in bacterial cells have shown that the protein is localized to discrete foci. In this study we have used photo-activated localization microscopy (PALM) to determine the localization of SeqA molecules, tagged with fluorescent proteins, with a localization precision of 20-30 nm with the aim to visualize the SeqA subcellular structures in more detail than previously possible. SeqA-PAmCherry was imaged in wild type E. coli, expressed from plasmid or genetically engineered into the bacterial genome, replacing the native seqA gene. Unsynchronized cells as well as cells with a synchronized cell cycle were imaged at various time points, in order to investigate the evolution of SeqA localization during the cell cycle. We found that SeqA indeed localized into discrete foci but these were not the only subcellular localizations of the protein. A significant amount of SeqA-PAmCherry molecules was localized outside the foci and in a fraction of cells we saw patterns indicating localization at the membrane. Using quantitative PALM, we counted protein copy numbers per cell, protein copy numbers per focus, the numbers of foci per cell and the sizes of the SeqA clusters. The data showed broad cell-to-cell variation and we did not observe a correlation between SeqA-PAmCherry protein numbers and the cell cycle under the experimental conditions of this study. The numbers of SeqA-PAmCherry molecules per focus as well as the foci sizes also showed broad distributions indicating that the foci are likely not characterized by a fixed number of molecules. We also imaged an E. coli strain devoid of the dam methylase (Δdam) and observed that SeqA-PAmCherry no longer formed foci, and was dispersed throughout the cell and localized to the plasma membrane more readily. We discuss our results in the context of the limitations of the technique.

Antibiotic dose and nutrient availability differentially drive the evolution of antibiotic resistance and persistence.

Effective treatment of bacterial infections proves increasingly challenging due to the emergence of bacterial variants that endure antibiotic exposure. Antibiotic resistance and persistence have been identified as two major bacterial survival mechanisms, and several studies have shown a rapid and strong selection of resistance or persistence mutants under repeated drug treatment. Yet, little is known about the impact of the environmental conditions on resistance and persistence evolution and the potential interplay between both phenotypes. Based on the distinct growth and survival characteristics of resistance and persistence mutants, we hypothesized that the antibiotic dose and availability of nutrients during treatment might play a key role in the evolutionary adaptation to antibiotic stress. To test this hypothesis, we combined high-throughput experimental evolution with a mathematical model of bacterial evolution under intermittent antibiotic exposure. We show that high nutrient levels during antibiotic treatment promote selection of high-level resistance, but that resistance mainly emerges independently of persistence when the antibiotic concentration is sufficiently low. At higher doses, resistance evolution is facilitated by the preceding or concurrent selection of persistence mutants, which ensures survival of populations in harsh conditions. Collectively, our experimental data and mathematical model elucidate the evolutionary routes toward increased bacterial survival under different antibiotic treatment schedules, which is key to designing effective antibiotic therapies.

The number of fatalities drives disaster aid: increasing sensitivity to people in need.

In the studies reported here, an analysis of financial donations in response to natural disasters showed that the amount of money allocated for humanitarian aid depends on the number of fatalities but not on the number of survivors who are affected by the disaster (i.e., the actual beneficiaries of the aid). On the basis of the experimental evidence, we discuss the underlying cause and provide guidelines to increase sensitivity to people in need.

Testosterone inhibits trust but promotes reciprocity.

The steroid hormone testosterone has been associated with behavior intended to obtain or maintain high social status. Although such behavior is typically characterized as aggressive and competitive, it is clear that high social status is achieved and maintained not only through antisocial behavior but also through prosocial behavior. In the present experiment, we investigated the impact of testosterone administration on trust and reciprocity using a double-blind randomized control design. We found that a single dose of 0.5 mg of testosterone decreased trust but increased generosity when repaying trust. These findings suggest that testosterone may mediate different types of status-seeking behavior. It may increase competitive, potentially aggressive, and antisocial behavior when social challenges and threats (i.e., abuse of trust and betrayal) need to be considered; however, it may promote prosocial behavior in the absence of these threats, when high status and good reputation may be best served by prosocial behavior.

In Vitro Persistence Level Reflects In Vivo Antibiotic Survival of Natural Pseudomonas aeruginosa Isolates in a Murine Lung Infection Model.

Clinicians 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 pathogen Pseudomonas aeruginosa. In this model, mice are intratracheally infected with P. aeruginosa embedded in seaweed alginate beads and subsequently treated with tobramycin via nasal droplets. A diverse panel of 18 P. aeruginosa strains 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. IMPORTANCE The 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, and Enterobacter spp.), which are considered major health threats. P. aeruginosa is 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 natural P. aeruginosa isolates in this model are positively correlated with the survival levels measured in classical persistence assays in 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 strategies in vivo.

Bugs on Drugs: A Drosophila melanogaster Gut Model to Study In Vivo Antibiotic Tolerance of E. coli.

With an antibiotic crisis upon us, we need to boost antibiotic development and improve antibiotics' efficacy. Crucial is knowing how to efficiently kill bacteria, especially in more complex in vivo conditions. Indeed, many bacteria harbor antibiotic-tolerant persisters, variants that survive exposure to our most potent antibiotics and catalyze resistance development. However, persistence is often only studied in vitro as we lack flexible in vivo models. Here, I explored the potential of using Drosophila melanogaster as a model for antimicrobial research, combining methods in Drosophila with microbiology techniques: assessing fly development and feeding, generating germ-free or bacteria-associated Drosophila and in situ microscopy. Adult flies tolerate antibiotics at high doses, although germ-free larvae show impaired development. Orally presented E. coli associates with Drosophila and mostly resides in the crop. E. coli shows an overall high antibiotic tolerance in vivo potentially resulting from heterogeneity in growth rates. The hipA7 high-persistence mutant displays an increased antibiotic survival while the expected low persistence of ΔrelAΔspoT and ΔrpoS mutants cannot be confirmed in vivo. In conclusion, a Drosophila model for in vivo antibiotic tolerance research shows high potential and offers a flexible system to test findings from in vitro assays in a broader, more complex condition.

Antibiotic Tolerance Indicative of Persistence Is Pervasive among Clinical Streptococcus pneumoniae Isolates and Shows Strong Condition Dependence.

Streptococcus pneumoniae is an important human pathogen, being one of the most common causes of community-acquired pneumonia and otitis media. Antibiotic resistance in S. pneumoniae is an emerging problem, as it depletes our arsenal of effective drugs. In addition, persistence also contributes to the antibiotic crisis in many other pathogens, yet for S. pneumoniae, little is known about antibiotic-tolerant persisters and robust experimental means are lacking. Persister cells are phenotypic variants that exist as a subpopulation within a clonal culture. Being tolerant to lethal antibiotics, they underly the chronic nature of a variety of infections and even help in acquiring genetic resistance. In this study, we set out to identify and characterize persistence in S. pneumoniae. Specifically, we followed different strategies to overcome the self-limiting nature of S. pneumoniae as a confounding factor in the prolonged monitoring of antibiotic survival needed to study persistence. Under optimized conditions, we identified genuine persisters in various growth phases and for four relevant antibiotics through biphasic survival dynamics and heritability assays. Finally, we detected a high variety in antibiotic survival levels across a diverse collection of S. pneumoniae clinical isolates, which assumes that a high natural diversity in persistence is widely present in S. pneumoniae. Collectively, this proof of concept significantly progresses the understanding of the importance of antibiotic persistence in S. pneumoniae infections, which will set the stage for characterizing its relevance to clinical outcomes and advocates for increased attention to the phenotype in both fundamental and clinical research. IMPORTANCE S. pneumoniae is considered a serious threat by the Centers for Disease Control and Prevention because of rising antibiotic resistance. In addition to resistance, bacteria can also survive lethal antibiotic treatment by developing antibiotic tolerance, more specifically, antibiotic tolerance through persistence. This phenotypic variation seems omnipresent among bacterial life, is linked to therapy failure, and acts as a catalyst for resistance development. This study gives the first proof of the presence of persister cells in S. pneumoniae and shows a high variety in persistence levels among diverse strains, suggesting that persistence is a general trait in S. pneumoniae cultures. Our work advocates for higher interest for persistence in S. pneumoniae as a contributing factor for therapy failure and resistance development.

Mutations in respiratory complex I promote antibiotic persistence through alterations in intracellular acidity and protein synthesis.

Antibiotic persistence describes the presence of phenotypic variants within an isogenic bacterial population that are transiently tolerant to antibiotic treatment. Perturbations of metabolic homeostasis can promote antibiotic persistence, but the precise mechanisms are not well understood. Here, we use laboratory evolution, population-wide sequencing and biochemical characterizations to identify mutations in respiratory complex I and discover how they promote persistence in Escherichia coli. We show that persistence-inducing perturbations of metabolic homeostasis are associated with cytoplasmic acidification. Such cytoplasmic acidification is further strengthened by compromised proton pumping in the complex I mutants. While RpoS regulon activation induces persistence in the wild type, the aggravated cytoplasmic acidification in the complex I mutants leads to increased persistence via global shutdown of protein synthesis. Thus, we propose that cytoplasmic acidification, amplified by a compromised complex I, can act as a signaling hub for perturbed metabolic homeostasis in antibiotic persisters.

Enrichment of Persister Cells Through Β-Lactam-Induced Filamentation and Size Separation.

Analyzing persisters at the single-cell level is crucial to properly define their phenotypic traits. However, single-cell analyses are challenging due to the rare and temporary nature of persister cells, thus requiring their rapid and efficient enrichment in a culture. Existing methods to isolate persisters from a bacterial population show important shortcomings, including contamination with susceptible cells and/or cell debris, which complicate subsequent microscopic analyses. We here describe a protocol to enrich persisters in a culture using ß-lactam-induced filamentation followed by size separation. This protocol minimizes the amount of cell debris in the final sample, facilitating single-cell studies of persister cells.

Author Correction: Canonical germinant receptor is dispensable for spore germination in Clostridium botulinum group II strain NCTC 11219.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.