Single Cell Killing Kinetics Differentiate Phenotypic Bacterial Responses to Different Antibacterial Classes

Area of Science:

• Microbiology and Molecular Biology
• Antimicrobial Resistance
• Drug Discovery and Development

Background:

• The rise of multidrug-resistant bacteria necessitates the exploration of novel antibiotic classes.
• Understanding antibacterial agent effects at a single-cell level is crucial for assessing new treatments.
• Phenotypic variations in bacteria can impact antibiotic efficacy, highlighting the need for detailed mechanistic studies.

Purpose of the Study:

• To investigate the concentration-dependent killing kinetics of stationary-phase Escherichia coli using different antibacterial agents.
• To compare the single-cell responses to known antibiotics (ampicillin, ciprofloxacin) and a novel class (protein Ψ-capsids).
• To differentiate the modes of action and survival dynamics of these agents at the single-cell level.

Main Methods:

• Combined time-lapse microscopy with microfluidics for high-resolution, single-cell analysis.
• Investigated stationary-phase Escherichia coli treated with ampicillin, ciprofloxacin, and protein Ψ-capsids at various concentrations.
• Quantified bacterial elongation, growth arrest, cell lysis, membrane disruption, and viable but nonculturable states.

Main Results:

• Protein Ψ-capsids arrested bacterial elongation within 2 hours, contrasting with ampicillin and ciprofloxacin which caused elongation at minimum inhibitory concentration (MIC).
• All agents arrested growth within 2 hours at concentrations above MIC.
• Ampicillin and ciprofloxacin induced lysis at MIC, shifting to membrane disruption at higher concentrations; Ψ-capsids caused membrane disruption at all tested concentrations.

Conclusions:

• The study establishes a microfluidics-based method to differentiate antibacterial killing kinetics and mechanisms at the single-cell level.
• Protein Ψ-capsids exhibit a distinct mode of action, arresting elongation unlike traditional antibiotics.
• Findings provide a foundation for comparative design of new antibacterial therapies and clinical susceptibility testing.