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Birth and Resuscitation of (p)ppGpp Induced Antibiotic Tolerant Persister Cells.

ABSTRACT: Transient antibiotic treatment typically eradicates most sensitive bacteria except a few survivors called persisters. The second messenger (p)ppGpp plays a key role in persister formation in Escherichia coli populations but the underlying mechanisms have remained elusive. In this study we induced (p)ppGpp synthesis by modulating tRNA charging and then directly observed the stochastic appearance, antibiotic tolerance, and resuscitation of persister cells using live microscopy. Different physiological parameters of persister cells as well as their regularly growing ancestors and sisters were continuously monitored using fluorescent reporters. Our results confirmed previous findings that high (p)ppGpp levels are critical for persister formation, but the phenomenon remained strikingly stochastic without any correlation between (p)ppGpp levels and antibiotic tolerance on the single-cell level. We could not confirm previous notions that persisters exhibit markedly low concentrations of intracellular ATP or were linked to post-transcriptional effects of (p)ppGpp through the activation of small genetic elements known as toxin-antitoxin (TA) modules. Instead, we suggest that persister cell formation under regular conditions is driven by the transcriptional response to increased (p)ppGpp levels.

SUBMITTER: Svenningsen MS

PROVIDER: S-EPMC6465370 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Birth and Resuscitation of (p)ppGpp Induced Antibiotic Tolerant Persister Cells.

Svenningsen Mikkel Skjoldan MS Veress Alexandra A Harms Alexander A Mitarai Namiko N Semsey Szabolcs S

Scientific reports 20190415 1

Transient antibiotic treatment typically eradicates most sensitive bacteria except a few survivors called persisters. The second messenger (p)ppGpp plays a key role in persister formation in Escherichia coli populations but the underlying mechanisms have remained elusive. In this study we induced (p)ppGpp synthesis by modulating tRNA charging and then directly observed the stochastic appearance, antibiotic tolerance, and resuscitation of persister cells using live microscopy. Different physiolog ...[more]

PMID: 30988388

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Project description:Bacterial persisters are phenotypic variants that survive antibiotic treatment in a dormant state and can be formed by multiple pathways. We recently proposed that the second messenger (p)ppGpp drives Escherichia coli persister formation through protease Lon and activation of toxin-antitoxin (TA) modules. This model found considerable support among researchers studying persisters but also generated controversy as part of recent debates in the field. In this study, we therefore used our previous work as a model to critically examine common experimental procedures to understand and overcome the inconsistencies often observed between results of different laboratories. Our results show that seemingly simple antibiotic killing assays are very sensitive to variations in culture conditions and bacterial growth phase. Additionally, we found that some assay conditions cause the killing of antibiotic-tolerant persisters via induction of cryptic prophages. Similarly, the inadvertent infection of mutant strains with bacteriophage ?80, a notorious laboratory contaminant, apparently caused several of the phenotypes that we reported in our previous studies. We therefore reconstructed all infected mutants and probed the validity of our model of persister formation in a refined assay setup that uses robust culture conditions and unravels the dynamics of persister cells through all bacterial growth stages. Our results confirm the importance of (p)ppGpp and Lon but no longer support a role of TA modules in E. coli persister formation under unstressed conditions. We anticipate that the results and approaches reported in our study will lay the ground for future work in the field.IMPORTANCE The recalcitrance of antibiotic-tolerant persister cells is thought to cause relapsing infections and antibiotic treatment failure in various clinical setups. Previous studies identified multiple genetic pathways involved in persister formation but also revealed reproducibility problems that sparked controversies about adequate tools to study persister cells. In this study, we unraveled how typical antibiotic killing assays often fail to capture the biology of persisters and instead give widely differing results based on poorly controlled experimental parameters and artifacts caused by cryptic as well as contaminant prophages. We therefore established a new, robust assay that enabled us to follow the dynamics of persister cells through all growth stages of bacterial cultures without distortions by bacteriophages. This system also favored adequate comparisons of mutant strains with aberrant growth phenotypes. We anticipate that our results will contribute to a robust, common basis for future studies on the formation and eradication of antibiotic-tolerant persisters.

Project description:Persister cells are metabolically quiescent multi-drug tolerant fraction of a genetically sensitive bacterial population and are thought to be responsible for relapse of many persistent infections. Persisters can be formed naturally in the stationary phase culture, and also can be induced by bacteriostatic antibiotics. However, the molecular basis of bacteriostatic antibiotic induced persister formation is unknown. Here, we established a bacteriostatic antibiotic induced persister model and screened the Escherichia coli single gene deletion mutant library for mutants with defect in rifampin or tetracycline induced persistence to ofloxacin. Thirsty-seven and nine genes were found with defects in rifampin- and tetracycline-induced persister formation, respectively. Six mutants were found to overlap in both rifampin and tetracycline induced persister screens: recA, recC, ruvA, uvrD, fis, and acrB. Interestingly, four of these mutants (recA, recC, ruvA, and uvrD) mapped to DNA repair pathway, one mutant mapped to global transcriptional regulator (fis) and one to efflux (acrB). The stationary phase culture of the identified mutants and parent strain BW25113 were subjected to different antibiotics including ofloxacin, ampicillin, gentamicin, and stress conditions including starvation and acid pH 4.0. All the six mutants showed less tolerance to ofloxacin, but only some of them were more sensitive to other specific stress conditions. Complementation of five of the six common mutants restored the persister level to that of the parent strain in both stationary phase and static antibiotic-induced conditions. In addition to the DNA repair pathways shared by both rifampin and tetracycline induced persisters, genes involved in rifampin-induced persisters map also to transporters, LPS biosynthesis, flagella biosynthesis, metabolism (folate and energy), and translation, etc. These findings suggest that persisters generated by different ways may share common mechanisms of survival, and also shed new insight into the molecular basis of static antibiotic induced antagonism of cidal antibiotics.

Project description:BackgroundPersisters and viable but non-culturable (VBNC) cells are two phenotypic variants known to be highly tolerant to antibiotics. Although both cell types are stained as live and often appear as nongrowing during antibiotic treatment, the only distinguishing feature is the ability of persisters to recolonize in standard culture media in the absence of antibiotics. Despite considerable progress in the characterization of persister formation mechanisms, their resuscitation mechanisms remain unclear due to technical limitations in detecting and isolating these cell types in culture environments that are highly heterogeneous.ResultsIn this study, we used a methodology integrating flow cytometry, fluorescent protein expression systems and ampicillin-mediated cell lysing technique to monitor persister resuscitation at the single-cell level. With this method, we were able to investigate the effects of various culture conditions (e.g., antibiotic treatment time, the length of the stationary phase in overnight pre-cultures, or pretreatment of cells with a metabolic inhibitor) on persister resuscitation. Although we observed long-term pre-cultures have many more VBNC cells compared to short-term pre-cultures, only a small fraction of non-lysed cells was able to resuscitate in all conditions tested. Regardless of pre-culturing and ampicillin treatment times, these persister cells started to resuscitate within 1 hour, after they were transferred to fresh liquid media, with the same doubling time that normal cells have. Our analysis further showed that ampicillin was not able to lyse the cells in the presence of arsenate, a metabolic inhibitor commonly used to increase bacterial persistence. However, the removal of arsenate during antibiotic treatment resulted in cell lysis and a reduction in persister levels despite the significant decrease in ATP levels in the cells.ConclusionsThe strategy presented in this study helps us monitor persister resuscitation at the single-cell level, and simultaneously quantify persister, VBNC and dead cell subpopulations in ampicillin-treated cultures. Our results indicate that the characterization of persister resuscitation with flow cytometry will enhance the current molecular-level understanding of persistence and its evolution.

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Birth and Resuscitation of (p)ppGpp Induced Antibiotic Tolerant Persister Cells.

Publications

Birth and Resuscitation of (p)ppGpp Induced Antibiotic Tolerant Persister Cells.

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