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

With the spread of multidrug-resistant bacteria, there has been an increasing focus on molecular classes that have not yet yielded an antibiotic. A key capability for assessing and prescribing new antibacterial treatments is to compare the effects antibacterial agents have on bacterial growth at a phenotypic, single-cell level. Here, we combined time-lapse microscopy with microfluidics to investigate the concentration-dependent killing kinetics of stationary-phase Escherichia coli cells. We used antibacterial agents from three different molecular classes, β-lactams and fluoroquinolones, with the known antibiotics ampicillin and ciprofloxacin, respectively, and a new experimental class, protein Ψ-capsids. We found that bacterial cells elongated when treated with ampicillin and ciprofloxacin used at their minimum inhibitory concentration (MIC). This was in contrast to Ψ-capsids, which arrested bacterial elongation within the first two hours of treatment. At concentrations exceeding the MIC, all the antibacterial agents tested arrested bacterial growth within the first 2 h of treatment. Further, our single-cell experiments revealed differences in the modes of action of three different agents. At the MIC, ampicillin and ciprofloxacin caused the lysis of bacterial cells, whereas at higher concentrations, the mode of action shifted toward membrane disruption. The Ψ-capsids killed cells by disrupting their membranes at all concentrations tested. Finally, at increasing concentrations, ampicillin and Ψ-capsids reduced the fraction of the population that survived treatment in a viable but nonculturable state, whereas ciprofloxacin increased this fraction. This study introduces an effective capability to differentiate the killing kinetics of antibacterial agents from different molecular classes and offers a high content analysis of antibacterial mechanisms at the single-cell level.