A Live Bacterial Screening Assay for Membrane-Active Antimicrobial Compounds Using Imaging Fluorescence Correlation Spectroscopy.

There is a growing need in the personal hygiene industry to develop a new generation of effective antimicrobial actives, to be used as functional antibacterial ingredients and preservatives. Antimicrobials that attack bacterial membranes are an attractive target due to the relatively conserved structure compositions of the bacterial membrane, which bacteria cannot easily change without influences on the function of membrane-embedded proteins. However, current screening is slow and there is a demand for rapid screening methodologies to overcome the time-consuming nature of existing screening tools. Imaging total internal reflection-fluorescence correlation spectroscopy (ITIR-FCS) is a powerful technique that can measure membrane dynamics and identify changes with high accuracy and precision. We therefore combine ITIR-FCS with a segmentation algorithm to automatically identify bacterial cells to screen the effect of antimicrobial compounds on the dynamics of bacterial membranes as a function of antimicrobial concentration and incubation time. This allows to assess membrane activity within less than 30 min and generates dose-response curves within a span of 2 h. The technique detects antimicrobial activity at lower concentrations and an order of magnitude faster than commonly used susceptibility testing assays.