Automated time-resolved analysis of bacteria–substrate interactions using functionalized microparticles and flow cytometry

Surface biofouling poses an increasing problem in industrial and health care applications, driving research for surface coatings to prevent anti-microbial colonization and characterization of the efficacy of the same. The diversity and increasing sophistication of such coatings, which postulate different types of anti-microbial action on planktonic and surface adhering bacteria, challenge the suitability of current approaches to evaluate and compare the different approaches as well as the speed and accuracy at which screening can be made. We describe and provide proof of principle for a method to use microparticles functionalized with molecular coatings through self-assembly together with flow cytometry readout to evaluate Escherichia coli bacteria surface adhesion and killing efficiency. Advantages of the method are the automation of the method that allows recording an immense number of interactions and the possibility to simultaneously record effects on both surface adhering and planktonic bacteria. We demonstrate and discuss design criteria to obtain this information on two coatings, poly(L-lysine)-graft-C3H6N+(CH3)2C12H25 (PLL-g-QAC) and poly(L-lysine)-graft-poly(ethylene glycol)-C3H6N+(CH3)2C12H25 (PLL-g-PEG-QAC), which exemplify two different approaches to creating anti-microbial interfaces. Despite an apparent higher killing efficiency of the PLL-g-QAC during brief exposures, the rapid fouling of that surface quickly reduces its efficiency, whereas the PLL-g-PEG-QAC coating showed greater promise in reducing the growth and interfacial colonization of bacteria over longer time scales.