High-Throughput Quantification of Population Dynamics using Luminescence

The dynamics of bacterial population decline at antibiotic concentrations above the minimum inhibitory concentration (MIC), remain poorly characterized. This is because measuring colony-forming units (CFU), the standard assay to quantify inhibition, is too slow, labour-intensive, and costly, and can be unreliable at high drug concentrations. To offer an alternative for high antimicrobial concentrations, we measured the change in light intensity from bioluminescent Escherichia coli over time and compared it with the change in CFU counts as a proxy for population size. Currently, luminescence assays are commonly used below the MIC, but have not yet been evaluated extensively in the super-MIC range. For 12 of the 21 antimicrobials tested, luminescence- and CFU-based rates did not differ significantly. To investigate the source of the discrepancies observed for the remaining 9 antibiotics, we introduced a size-structured population model with a continuum of cell sizes to simulate the impact of filamentation on the light signal, and complemented the findings with microscopy imaging. Discrepancies between the two rates fell into two classes. First, because light intensity tracks biomass more closely than population size, luminescence can indicate a shallower population decline when bacteria filament. Second, CFU-based estimates can indicate a steeper population decline when antimicrobial treatment reduces the number of colonies formed per viable bacterium. This effect can result from changes in clustering behaviour, physiological changes that impair culturability, or antimicrobial carry-over. With these caveats addressed, bioluminescence offers an efficient, high-throughput alternative for quantifying bacterial dynamics at super-MIC concentrations, although its suitability depends on the chosen metric (biomass vs. cell number) and on whether the antimicrobial induces filamentation or affects culturability.