High-throughput quantification of population dynamics using luminescence

Bacterial population decline at antibiotic concentrations above the minimum inhibitory concentration (MIC) remains poorly characterized. This is because colony-forming units (CFU), the standard method to quantify inhibition, are slow, labor-intensive, and costly. Luminescence assays are widely used to quantify population dynamics at subinhibitory concentrations, yet their limitations and reliability at high concentrations remain underexplored. Here, we compared luminescence- and CFU-based rates in Escherichia coli across 20 antimicrobials. In our experiments, luminescence- and CFU-based rates did not differ significantly for half of them. For the other half, CFU-based decline rates were consistently higher. The estimates differed for two main reasons: First, because light intensity tracks biomass more closely than population size, luminescence declined more slowly than the population when bacteria filamented. Second, CFU-based estimates indicated a steeper decline when treatment reduced the number of colonies formed per plated bacterium. This can result from changes in clustering behavior, physiological changes that impair culturability, or antimicrobial carryover. Thus, the suitability of luminescence to quantify bacterial decline depends on the physiological effects of the antimicrobial and whether the quantity of interest is cell number or biomass. Within these limitations, luminescence can serve as an efficient, high-throughput alternative for quantifying bacterial dynamics at super-MIC concentrations.