Droplet microfluidics strategies for overcoming bottlenecks in antibiotics discovery.

Antimicrobial resistance (AMR) presents a mounting global health crisis. Traditional antibiotic discovery methods are hindered by low throughput, frequent rediscovery of known compounds, and limited access to unculturable microbes and silent biosynthetic gene clusters (BGCs). Droplet microfluidics offers a transformative platform to overcome these barriers by enabling massively parallel, compartmentalized analysis at the single-cell level. In this perspective, we discuss how droplet microfluidics is reshaping the antibiotic discovery landscape by addressing long-standing limitations in cultivation, screening, and compound identification. We highlight recent advances in: (1) accessing microbial dark matter through high-throughput single-cell technologies, (2) activating cryptic BGCs and dissecting microbial interactions using microfluidic coculture platforms, (3) accelerating chemical dereplication and discovery of novel metabolites through integration with mass spectrometry-based tools, and (4) uncovering resistance and persistence mechanisms by linking antibiotic resistance genes to individual microbial hosts and single cell transcriptomes. Together, these innovations position droplet microfluidics as a powerful engine to accelerate antibiotic discovery and combat the global threat of AMR.