Prophages and Growth Dynamics Confound Experimental Results with Antibiotic-Tolerant Persister Cells.

Joerg Vogel,Alexander Harms,Szabolcs Semsey,Michael A Sørensen,Kenn Gerdes,Cinzia Fino

doi:10.1128/mbio.01964-17

Abstract

ABSTRACTBacterial 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 ϕ80, 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.

Highlights

Bacterial persisters are phenotypic variants that survive antibiotic treatment in a dormant state and can be formed by multiple pathways
The formation of persister cells is typically measured by determining the fraction of antibiotic-tolerant cells in bacterial cultures that are considered to be exponentially growing some hours after inoculation from dense overnight cultures (Fig. 1A)
Despite the apparent simplicity of this experimental setup, persister assays are known to be sensitive to even minor variations of the experimental conditions and have often given inconsistent results in different laboratories [5, 16, 20, 21]. We suspected that this simple assay setup may be inadequate to represent the dynamic nature of bacterial persistence and could give results that are strongly affected by biological or technical parameters that are usually not controlled in persistence assays

Summary

Introduction

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 and generated controversy as part of recent debates in the field. A large body of studies from several laboratories has uncovered genetic pathways that control and execute the formation of persister cells of Escherichia coli K-12 as a phenotypic conversion into dormancy [1, 2] These mechanisms include a drop of cellular ATP levels, the modulation of nucleoidassociated proteins, changes in metabolic fluxes, high expression of drug efflux pumps, or the activation of different sets of toxin-antitoxin (TA) modules [3,4,5,6,7,8,9,10]. We could confirm a role of (p)ppGpp, polyphosphate, and Lon in bacterial persister formation and/or survival but did not find strong evidence for the involvement of TA modules or the connection of these components in a single pathway of persister formation under unstressed conditions

Methods

Results

Conclusion