Application of green fluorescent protein to measure antimicrobial efficacy and the kinetics of cell death against Escherichia coli

Abstract

Industrial antimicrobials have been extensively used to control unwanted microbial growth by incorporation into a variety of products such as plastics and paints, reducing biodeterioration and biofouling and extending the lifespan of the product. Industrial antimicrobials generally have broad sites of action affecting core cellular functions such as central metabolism, enzyme function, cell wall or DNA synthesis and can either be biocidal or biostatic. In addition, susceptibility can be affected by the metabolic state of the microbe, with metabolically inactive cells generally more resistant than metabolically active cells. Previously it was demonstrated that cytosolically expressed green fluorescent protein could be used as a real-time viability indicator in the yeast Aureobasidium pullulans based on the pH dependent fluorescence of GFP and the collapse of the proton gradient across the cell membrane during cell death. In this study we report on the development and validation of an equivalent GFP fluorescence viability assay in Escherichia coli and used this assay to study the effect of five antimicrobials commonly used in plastics; 4,5-dichloro-2-octyl-isothiazol-3-one (DCOIT), sodium pyrithione, 1,2-benzisothiazol-3-one (BIT), 2-octyl-isothiazol-3-one (OIT) and n-butyl-1,2-benzisothiazol-3-one (BBIT). The results demonstrate broad differences amongst the antimicrobials in both relative efficacy, rate of effect and for some antimicrobials, marked differences in sensitivity toward growing and non-growing cells.