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

Abstract

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.

Antibiotics modulate attractive interactions in bacterial colonies affecting survivability under combined treatment

“…Inhibition of cell division is observed in response to various stresses [38] and could explain why cells grow larger. An increase in cell volume has been implicated with increased antibiotic tolerance enabling cells to extend their survival time during antibiotic treatment [39]. We conclude that spherically shaped gonococci increase their cell volume in response to treatment with different classes of antibiotics.…”

Understanding Beta-Lactam-Induced Lysis at the Single-Cell Level

“…Recent studies have characterized the dynamics of lysis in E. coli cells treated with β-lactams (Yao et al, 2012 ; Wong and Amir, 2019 ; Zahir et al, 2019 , 2020 ). In previous work, Yao et al studied the dynamics of lysis in single cells treated with cephalexin and ampicillin using high-resolution microscopy (Yao et al, 2012 ); here, we build on this study to explore additional perturbations involving turgor pressure, mechanosensitive channels (MSCs), and cell shape changes.…”

A model-based approach to characterize enzyme-mediated response to antibiotic treatments: going beyond the SIR classification

“…Modelling cell length and filamentation is needed in order to explain tolerance to β-lactam treatments 9 . However, simply considering that all the cells have the same length, which evolves with time because of filamentation, cannot explain the complex dynamics of cell lysis.…”

“…Many β-lactams cause filamentation, but some can also cause the apparition of spheroplasts, ovoid or bulging cells [6][7][8] . These processes delay lysis and hence are a form of tolerance 9 . Research has also been done on the response of a cell population to β-lactam treatments, highlighting the role of enzyme-mediated resilience (also called collective antibiotic tolerance (CAT)) either through OD measurements or cell counting [10][11][12] .…”