Study of β-lactam induced bacteriolysis using high-throughput time-resolved microscopy and mechanical bacteriolysis by nanostructured surfaces

Bacteriolysis is one of the most widely used modes of killing bacteria. β-lactam antibiotics, antimicrobial peptides, glycopeptides, phage therapy and nanostructured antibacterial surfaces kill bacteria principally by inducing lysis. A deeper understanding of the process of bacteriolysis can inspire new ways to prevent bacterial colonization and increase antibiotic efficacy. 

In the first part of this work, we develop a high-throughput microscopy-based methodology to monitor morphological changes in bacteria due to environmental perturbation. Using off-the-shelf microscopy hardware and software utilities we implement a protocol for imaging bacteria in 96-well plates. To demonstrate the capabilities of the method, we monitored morphological changes and lysis of around 4000 Escherichia coli mutants in response to the β-lactam antibiotic cefsulodin. We also established a novel image analysis pipeline for automated classification of cells based on their shape and intensity features. Based on changes in frequencies of cell morphotypes we identified mutants that displayed atypical morphological dynamics. The aberrant phenotypes were further clustered to reveal the distinct morphological responses of mutants to cefsulodin. Stable bulge formation in certain mutants promotes antibiotic tolerance, as bulging cells are capable of reverting back to normal growth after the antibiotic is removed. This methodology is highly versatile and can be applied to find genetic modulators of bacterial morphological responses to different kinds of perturbations like antibiotics.    

In the second part of this thesis, we applied the imaging methodology developed in the first part to measure lysis kinetics of around 4000 E. coli mutants in response to the β-lactam antibiotic cephalexin. We found that the period of filamentation before lysis differs widely among the mutants and that lysis kinetics correlate with survival. Delay in lysis confers antibiotic tolerance because when the antibiotic is removed, the filamented cells can successfully form multiple septations simultaneously and divide into multiple progenies. We found that deletion of tol-pal genes tolQ, tolR, ybgC and pal results in rapid lysis without filamentation upon treatment with β-lactams. These results emphasize the importance of considering antibiotic tolerance during antibiotic therapy.    

In the final part, we describe the antibacterial activity of cotton swab shaped nanostructures. These nanostructures kill bacteria in a physical contact-dependent manner. A biophysical model was developed from infinitesimal strain theory to investigate the effects of changes in surface topography on the bactericidal activity. We made several controlled geometrical alterations of the cotton swab shaped nanostructures. Measurement of bactericidal activities of these nanostructured surfaces confirmed model predictions and highlighted the non-trivial role of cell envelope bending rigidity in the process of bacteriolysis by nanostructures.   

In conclusion, the high-throughput time-resolved morphology screening methodology presented in this work provides an easy-to-implement protocol for rapid imaging of large number of bacterial strains and analyzing single-cell morphological changes in response to any perturbant. Additionally, results presented in this thesis yield novel insights that will potentially contribute to the development of better prevention and treatment strategies needed to successfully combat bacterial infections.