Quantitative imaging of efflux pumps in planktonic and biofilm-associated bacteria through single-molecule localization microscopy

When bacteria switch from a planktonic to a biofilm-associated lifestyle, a wide remodeling of gene expression takes place. In particular, efflux pump genes such as the acrAB operon are known to be upregulated under biofilm growth when compared to planktonic bacteria, and this phenomenon plays an important role in determining an increased resistance of biofilms to antimicrobial treatments. Although efflux pump expression in biofilms has been previously investigated through indirect methods, direct observation and quantification of efflux pumps at the single-cell level has never been reported to our knowledge. Here, we implement a method involving CRISPR/Cas9-assisted genome editing coupled to single-molecule localization microscopy to obtain quantitative data on the expression and distribution of the AcrB efflux pump in both planktonic and biofilm-associated Escherichia coli cells.