# Probing Antibiotic Inhibition in Small Bacterial Populations With Combinatorial Droplet Microfluidics

## TL;DR

This study uses microfluidics to explore how small bacterial populations respond to low antibiotic doses, revealing variability not captured by traditional bulk tests.

## Contribution

A combinatorial droplet microfluidics platform is introduced to study antibiotic effects on small bacterial populations at sub-inhibitory concentrations.

## Key findings

- Tetracycline causes nonmonotonic growth in small bacterial populations at low concentrations.
- Streptomycin leads to bistable outcomes, with some populations growing and others dying.
- Ampicillin induces stochastic filamentation in small bacterial populations.

## Abstract

Bacterial infections often involve small, local populations of bacteria, yet antibiotic treatment decisions are generally based on bulk population susceptibility assays. Stochastic variability among local small populations can influence susceptibility, limiting the predictive capability of bulk assays. Therefore there is a need to better understand antibiotic response in small populations. Droplet‐based microfluidics enables the high‐throughput production of tens of thousands of picolitre droplets, in which small populations of bacteria (e.g., 8 cells) can be encapsulated and their responses to different environmental conditions tracked. Here, we use a combinatorial droplet‐generation platform, combined with microscopy and image analysis, to interrogate the responses of small populations of Escherichia coli to different bulk‐determined sub‐inhibitory concentrations of the antibiotics tetracycline, streptomycin, and ampicillin within a single experiment. We observe qualitatively distinct small‐population responses for these antibiotics. For the bacteriostatic ribosome‐targeting antibiotic tetracycline, growth varies nonmonotonically at low antibiotic concentrations. For the bactericidal ribosome‐targeting antibiotic streptomycin, we observe apparent bistability, some replicate populations growing while others die. For the bactericidal cell‐wall targeting antibiotic ampicillin, we observe stochastic bacterial filamentation. Our study shows how distinct phenomena impacting antibiotic susceptibility may emerge in small bacterial populations, laying a foundation for deeper studies into potential treatment implications.

Small local populations of bacteria are often involved in bacterial infections; however, bulk susceptibility assays guide antibiotic treatment decisions. Furthermore, during the early and late stages of treatment, these small bacterial populations are exposed to sub‐inhibitory concentrations of antibiotics. Here, we utilized a combinatorial microfluidic droplet‐generation platform to evaluate the effects of bulk‐determined sub‐inhibitory antibiotic concentrations on small bacterial populations.© 2026 WILEY‐VCH GmbH