# Droplet Microfluidics for High-Throughput Analysis of Antibiotic Susceptibility in Bacterial Cells and Populations

## TL;DR

Droplet microfluidics enables high-throughput analysis of antibiotic resistance in bacteria, offering insights into resistance distribution and aiding in rapid diagnostics.

## Contribution

A novel detection system using light scattering for high-throughput droplet-based antibiotic susceptibility testing is developed.

## Key findings

- Droplet techniques reveal resistance level distributions in clonally identical bacterial populations.
- A light scattering detection system enables rapid, label-free analysis of bacterial resistance in thousands of droplets per second.

## Abstract

Antibiotic-resistant bacteria are an increasing concern both in
everyday life and specialized environments such as healthcare. As
the rate of antibiotic-resistant infections rises, so do complications
to health and the risk of disability and death. Urgent action is required
regarding the discovery of new antibiotics and rapid diagnosis of
the resistance profile of an infectious pathogen as well as a better
understanding of population and single-cell distribution of the resistance
level. High-throughput screening is the major affordance of droplet
microfluidics. Droplet screens can be exploited both to look for combinations
of drugs that could stop an infection of multidrug-resistant bacteria
and to search for the source of resistance via directed-evolution
experiments or the analysis of various responses to a drug by genetically
identical bacteria. In droplet techniques that have been used in this
way for over a decade, aqueous droplets containing antibiotics and
bacteria are manipulated both within and outside of the microfluidic
devices. The diagnostics problem was approached by producing a series
of microfluidic systems with integrated dilution modules for automated
preparation of antibiotic concentration gradients, achieving the speed
that allowed for high-throughput combinatorial assays. We developed
a method for automated emulsification of a series of samples that
facilitated measuring the resistance levels of thousands of individual
cells encapsulated in droplets and quantifying the inoculum effect,
the dependence of resistance level on bacterial cell count. Screening
of single cells encapsulated in droplets with varying antibiotic contents
has revealed a distribution of resistance levels within populations
of clonally identical cells. To be able to screen bacteria from clinical
samples, a study of fluorescent dyes in droplets determined that a
derivative of a popular viability marker is more suitable for droplet
assays. We have developed a detection system that analyzes the growth
or death state of bacteria with antibiotics for thousands of droplets
per second by measuring the scattering of light hitting the droplets
without labeling the cells or droplets. The droplet-based microchemostats
enabled long-term evolution of resistance experiments, which will
be integrated with high-throughput single-cell assays to better understand
the mechanism of resistance acquisition and loss. These techniques
underlie automated combinatorial screens of antibiotic resistance
in single cells from clinical samples. We hope that this Account will
inspire new droplet-based research on the antibiotic susceptibility
of bacteria.