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

Frontiers in Microbiology

Sabine Franke¹  Johan Hofkens²  Rafael Camacho²  Bram Weytjens⁴  Kathleen Marchal⁴  Maarten Fauvart⁵  Taiyeb Zahir⁶  Dorien Wilmaerts⁶  Jan Michiels⁶ 

[1] Centre of Microbial and Plant Genetics, KU Leuven, Leuven, Belgium;Department of Chemistry, KU Leuven – University of Leuven, Leuven, Belgium;Department of Information Technology, IDLab Group, Ghent University, Ghent, Belgium;Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium;Interuniversity Microelectronics Centre (IMEC), Leuven, Belgium;VIB-KU Leuven Center of Microbiology, Leuven, Belgium;

关键词: β-lactam;    antibiotic tolerance;    filamentation;    high-throughput microscopy;    bacteriolysis;   

DOI  :  10.3389/fmicb.2020.00374

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown