Image-based dynamic phenotyping reveals genetic determinants of filamentation-mediated beta-lactam tolerance

Antibiotic tolerance characterized by slow killing of bacteria in response to a drug can lead to treatment failure and promote the emergence of resistance. beta-lactam antibiotics inhibit cell wall growth in bacteria and many of them cause filamentation followed by cell lysis. Hence delayed cell lysis can lead to beta-lactam tolerance. Systematic discovery of genetic factors that affect beta-lactam killing kinetics has not been performed before due to challenges in high-throughput, dynamic analysis of viability of filamented cells during bactericidal action. We implemented a high-throughput time-resolved microscopy approach in a gene deletion library of Escherichia coli to monitor the response of mutants to the beta-lactam cephalexin. Changes in frequency of lysed and intact cells due to the antibiotic action uncovered several strains with atypical lysis kinetics. Filamentation confers tolerance because antibiotic removal before lysis leads to recovery through numerous concurrent divisions of filamented cells. Filamentation-mediated tolerance was not associated with resistance, and therefore this phenotype is not discernible through most antibiotic susceptibility methods. We find that deletion of Tol-Pal proteins TolQ, TolR, or Pal but not TolA, TolB, or CpoB leads to rapid killing by beta-lactams. We also show that the timing of cell wall degradation determines the lysis and killing kinetics after beta-lactam treatment. Altogether, this study uncovers numerous genetic determinants of hitherto unappreciated filamentation-mediated beta-lactam tolerance and support the growing call for considering antibiotic tolerance in clinical evaluation of pathogens. More generally, the microscopy screening methodology described here can easily be adapted to study lysis in large numbers of strains

High-throughput time-resolved morphology screening in bacteria reveals phenotypic responses to antibiotics

Image-based high-throughput screening strategies for quantifying morphological phenotypes have proven widely successful. Here we describe a combined experimental and multivariate image analysis approach for systematic large-scale phenotyping of morphological dynamics in bacteria. Using off-the-shelf components and software, we established a workflow for high-throughput time-resolved microscopy. We then screened the single-gene deletion collection of Escherichia coli for antibiotic-induced morphological changes. Using single-cell quantitative descriptors and supervised classification methods, we measured how different cell morphologies developed over time for all strains in response to the β-lactam antibiotic cefsulodin. 191 strains exhibit significant variations under antibiotic treatment. Phenotypic clustering provided insights into processes that alter the antibiotic response. Mutants with stable bulges show delayed lysis, contributing to antibiotic tolerance. Lipopolysaccharides play a crucial role in bulge stability. This study demonstrates how multiparametric phenotyping by high-throughput time-resolved imaging and computer-aided cell classification can be used for comprehensively studying dynamic morphological transitions in bacteria.status: publishe

High-throughput time-resolved morphology screening in bacteria reveals phenotypic responses to antibiotics

Image-based high-throughput screening strategies for quantifying morphological phenotypes have proven widely successful. Here we describe a combined experimental and multivariate image analysis approach for systematic large-scale phenotyping of morphological dynamics in bacteria. Using off-the-shelf components and software, we established a workflow for high-throughput time-resolved microscopy. We then screened the single-gene deletion collection of Escherichia coli for antibiotic-induced morphological changes. Using single-cell quantitative descriptors and supervised classification methods, we measured how different cell morphologies developed over time for all strains in response to the β-lactam antibiotic cefsulodin. 191 strains exhibit significant variations under antibiotic treatment. Phenotypic clustering provided insights into processes that alter the antibiotic response. Mutants with stable bulges show delayed lysis, contributing to antibiotic tolerance. Lipopolysaccharides play a crucial role in bulge stability. This study demonstrates how multiparametric phenotyping by high-throughput time-resolved imaging and computer-aided cell classification can be used for comprehensively studying dynamic morphological transitions in bacteria.status: publishe

Image-Based Dynamic Phenotyping Reveals Genetic Determinants of Filamentation-Mediated β-Lactam Tolerance

Antibiotic tolerance characterized by slow killing of bacteria in response to a drug can lead to treatment failure and promote the emergence of resistance. β-lactam antibiotics inhibit cell wall growth in bacteria and many of them cause filamentation followed by cell lysis. Hence delayed cell lysis can lead to β-lactam tolerance. Systematic discovery of genetic factors that affect β-lactam killing kinetics has not been performed before due to challenges in high-throughput, dynamic analysis of viability of filamented cells during bactericidal action. We implemented a high-throughput time-resolved microscopy approach in a gene deletion library of Escherichia coli to monitor the response of mutants to the β-lactam cephalexin. Changes in frequency of lysed and intact cells due to the antibiotic action uncovered several strains with atypical lysis kinetics. Filamentation confers tolerance because antibiotic removal before lysis leads to recovery through numerous concurrent divisions of filamented cells. Filamentation-mediated tolerance was not associated with resistance, and therefore this phenotype is not discernible through most antibiotic susceptibility methods. We find that deletion of Tol-Pal proteins TolQ, TolR, or Pal but not TolA, TolB, or CpoB leads to rapid killing by β-lactams. We also show that the timing of cell wall degradation determines the lysis and killing kinetics after β-lactam treatment. Altogether, this study uncovers numerous genetic determinants of hitherto unappreciated filamentation-mediated β-lactam tolerance and support the growing call for considering antibiotic tolerance in clinical evaluation of pathogens. More generally, the microscopy screening methodology described here can easily be adapted to study lysis in large numbers of strains.status: Published onlin

Enrichment of persisters enabled by a beta-lactam-induced filamentation method reveals their stochastic single-cell awakening

When exposed to lethal doses of antibiotics, bacterial populations are most often not completely eradicated. A small number of phenotypic variants, defined as 'persisters', are refractory to antibiotics and survive treatment. Despite their involvement in relapsing infections, processes determining phenotypic switches from and to the persister state largely remain elusive. This is mainly due to the low frequency of persisters and the lack of reliable persistence markers, both hampering studies of persistence at the single-cell level. Here we present a highly effective persister enrichment method involving cephalexin, an antibiotic that induces extensive filamentation of susceptible cells. We used our enrichment method to monitor outgrowth of Escherichia coli persisters at the single-cell level, thereby conclusively demonstrating that persister awakening is a stochastic phenomenon. We anticipate that our approach can have far-reaching consequences in the persistence field, by allowing single-cell studies at a much higher throughput than previously reported.status: publishe

Enrichment of persisters enabled by a ß-lactam-induced filamentation method reveals their stochastic single-cell awakening

International audienceAbstract When exposed to lethal doses of antibiotics, bacterial populations are most often not completely eradicated. A small number of phenotypic variants, defined as ‘persisters’, are refractory to antibiotics and survive treatment. Despite their involvement in relapsing infections, processes determining phenotypic switches from and to the persister state largely remain elusive. This is mainly due to the low frequency of persisters and the lack of reliable persistence markers, both hampering studies of persistence at the single-cell level. Here we present a highly effective persister enrichment method involving cephalexin, an antibiotic that induces extensive filamentation of susceptible cells. We used our enrichment method to monitor outgrowth of Escherichia coli persisters at the single-cell level, thereby conclusively demonstrating that persister awakening is a stochastic phenomenon. We anticipate that our approach can have far-reaching consequences in the persistence field, by allowing single-cell studies at a much higher throughput than previously reported

Model-Driven Controlled Alteration of Nanopillar Cap Architecture Reveals its Effects on Bactericidal Activity

Nanostructured surfaces can be engineered to kill bacteria in a contact-dependent manner. The study of bacterial interactions with a nanoscale topology is thus crucial to developing antibacterial surfaces. Here, a systematic study of the effects of nanoscale topology on bactericidal activity is presented. We describe the antibacterial properties of highly ordered and uniformly arrayed cotton swab-shaped (or mushroom-shaped) nanopillars. These nanostructured surfaces show bactericidal activity against Staphylococcus aureus and Pseudomonas aeruginosa. A biophysical model of the cell envelope in contact with the surface, developed ab initio from the infinitesimal strain theory, suggests that bacterial adhesion and subsequent lysis are highly influenced by the bending rigidity of the cell envelope and the surface topography formed by the nanopillars. We used the biophysical model to analyse the influence of the nanopillar cap geometry on the bactericidal activity and made several geometrical alterations of the nanostructured surface. Measurement of the bactericidal activities of these surfaces confirms model predictions, highlights the non-trivial role of cell envelope bending rigidity, and sheds light on the effects of nanopillar cap architecture on the interactions with the bacterial envelope. More importantly, our results show that the surface nanotopology can be rationally designed to enhance the bactericidal efficiency