Microfluidic single-cell phenotyping of the activity of peptide-based antimicrobials

Abstract

Antibiotic resistance is a major challenge for modern medicine, and there is a dire need to refresh the antibiotic development pipeline to treat infections that are resistant to currently available drugs. Peptide-based antimicrobials represent a promising source of novel anti-infectives, but their development is severely impeded due to the lack of suitable techniques to accurately quantify their antimicrobial efficacy. A major problem involves the heterogeneity of cellular phenotypes in response to these peptides, even within a clonal population of bacteria. There is thus a need to develop single-cell resolution assays to quantify drug efficacy for these novel therapeutics. We present here a detailed microfluidics-microscopy protocol for testing the efficacy of peptide-based antimicrobials on hundreds to thousands of individual bacteria in well-defined microenvironments. This enables the study of cell-to-cell differences in drug response within a clonal population. It is a highly versatile tool, which can be used to quantify drug efficacy, including the number of individual survivors at defined drug doses; it even enables the potential exploration of the molecular mechanisms of action of the drug, which are often unknown in the early stages of drug development. We present here protocols for working with Escherichia coli, but organisms of different geometric shapes and sizes may also be tested with suitable modifications of the microfluidic device.