A Simple Label-Free Method Reveals Bacterial Growth Dynamics and Antibiotic Action in Real-Time

Abstract

Background: Understanding the response of bacteria to environmental stress is hampered by the relative insensitivity of methods to detect growth. This means studies of antibiotic resistance and other physiological methods often exceed 24 hours. Methods: We developed a scattered light and detection system (SLIC) to address this challenge, establishing the LoD (LoD), and the time to growth detection of small inocula. We compared the light-scattering of bacteria grown in different nutrient conditions and the growth dynamics of two closely related organisms. Scattering data was modelled using Gompertz and Broken Stick equations. Bacteria were also exposed to meropenem, gentamicin, and cefoxitin at various concentrations and light-scattering was captured in real-time. Findings: We established the LoD for SLIC to be between 10-100 cfu mL-1 in a volume of 1-2mL. Quantitative measurements of the different nutrient effects on bacteria were obtained in less than four hours and it was possible to rapidly distinguish differences in growth dynamics of Klebsiella pneumoniae strains with and without the BlaKPC gene. There was a dose-dependent difference in the speed of action of each antibiotic tested at supra-MIC concentrations. The lethal effect of gentamicin, lytic effect of meropenem, and the slow bactericidal effect of cefoxitin were demonstrated in real-time. Significantly, strains that were sensitive to antibiotics could be identified in seconds. Interpretation: This research demonstrates the critical importance of improving the sensitivity of bacterial detection. The ability to rapidly assess susceptibility and to capture a wealth of data on bacterial growth dynamics is presented. The rapid bactericidal effect at supra-MIC concentrations is an important finding that needs to be incorporated into pharmacokinetic and pharmacodynamic models. Importantly, enhanced sensitivity of bacterial detection opens the possibility of susceptibility results being reportable clinically in a few minutes. Funding Information: Scottish Enterprise, NESTA Longitude Prize, and the University of St Andrews Declaration of Interests: None to declare.