Antibiotic Tolerance and Persistence Studied Throughout Bacterial Growth Phases.

Antibiotic tolerance and persistence allow bacteria to survive lethal doses of antibiotic drugs in the absence of genetic resistance. Despite the urgent need to address these phenomena as a cause of clinical antibiotic treatment failure, studies on antibiotic tolerance and persistence are notorious for contradictory and inconsistent findings. Many of these problems are likely caused by differences in the methodology used to study antibiotic tolerance and persistence in the laboratory. Standardized experimental procedures would therefore greatly promote research in this field by facilitating the integrated analysis of results obtained by different research groups. Here, we present a robust and adaptable methodology to study antibiotic tolerance/persistence in broth cultures of Escherichia coli and Pseudomonas aeruginosa . The hallmark of this methodology is that the formation and disappearance of antibiotic-tolerant cells is recorded throughout all bacterial growth phases from lag after inoculation over exponential growth into early and then late stationary phase. In addition, all relevant experimental conditions are rigorously controlled to obtain highly reproducible results. We anticipate that this methodology will promote research on antibiotic tolerance and persistence by enabling a deeper view at the growth-dependent dynamics of this phenomenon and by contributing to the standardization or at least comparability of experimental procedures used in the field.

PasT of Escherichia coli sustains antibiotic tolerance and aerobic respiration as a bacterial homolog of mitochondrial Coq10.

Antibiotic-tolerant persisters are often implicated in treatment failure of chronic and relapsing bacterial infections, but the underlying molecular mechanisms have remained elusive. Controversies revolve around the relative contribution of specific genetic switches called toxin-antitoxin (TA) modules and global modulation of cellular core functions such as slow growth. Previous studies on uropathogenic Escherichia coli observed impaired persister formation for mutants lacking the pasTI locus that had been proposed to encode a TA module. Here, we show that pasTI is not a TA module and that the supposed toxin PasT is instead the bacterial homolog of mitochondrial protein Coq10 that enables the functionality of the respiratory electron carrier ubiquinone as a "lipid chaperone." Consistently, pasTI mutants show pleiotropic phenotypes linked to defective electron transport such as decreased membrane potential and increased sensitivity to oxidative stress. We link impaired persister formation of pasTI mutants to a global distortion of cellular stress responses due to defective respiration. Remarkably, the ectopic expression of human coq10 largely complements the respiratory defects and decreased persister levels of pasTI mutants. Our work suggests that PasT/Coq10 has a central role in respiratory electron transport that is conserved from bacteria to humans and sustains bacterial tolerance to antibiotics.

Type II and type IV toxin-antitoxin systems show different evolutionary patterns in the global Klebsiella pneumoniae population.

The Klebsiella pneumoniae species complex includes important opportunistic pathogens which have become public health priorities linked to major hospital outbreaks and the recent emergence of multidrug-resistant hypervirulent strains. Bacterial virulence and the spread of multidrug resistance have previously been linked to toxin-antitoxin (TA) systems. TA systems encode a toxin that disrupts essential cellular processes, and a cognate antitoxin which counteracts this activity. Whilst associated with the maintenance of plasmids, they also act in bacterial immunity and antibiotic tolerance. However, the evolutionary dynamics and distribution of TA systems in clinical pathogens are not well understood. Here, we present a comprehensive survey and description of the diversity of TA systems in 259 clinically relevant genomes of K. pneumoniae. We show that TA systems are highly prevalent with a median of 20 loci per strain. Importantly, these toxins differ substantially in their distribution patterns and in their range of cognate antitoxins. Classification along these properties suggests different roles of TA systems and highlights the association and co-evolution of toxins and antitoxins.

SLING: a tool to search for linked genes in bacterial datasets.

Gene arrays and operons that encode functionally linked proteins form the most basic unit of transcriptional regulation in bacteria. Rules that govern the order and orientation of genes in these systems have been defined; however, these were based on a small set of genomes that may not be representative. The growing availability of large genomic datasets presents an opportunity to test these rules, to define the full range and diversity of these systems, and to understand their evolution. Here we present SLING, a tool to Search for LINked Genes by searching for a single functionally essential gene, along with its neighbours in a rule-defined proximity (https://github.com/ghoresh11/sling/wiki). Examining this subset of genes enables us to understand the basic diversity of these genetic systems in large datasets. We demonstrate the utility of SLING on a clinical collection of enteropathogenic Escherichia coli for two relevant operons: toxin antitoxin (TA) systems and RND efflux pumps. By examining the diversity of these systems, we gain insight on distinct classes of operons which present variable levels of prevalence and ability to be lost or gained. The importance of this analysis is not limited to TA systems and RND pumps, and can be expanded to understand the diversity of many other relevant gene arrays.

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

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 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.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.