Droplet microfluidics-based detection of very rare antibiotic-resistant subpopulation in clinical isolates of Escherichia coli from bloodstream infections

Population heterogeneity in bacterial phenotypes, such as antibiotic resistance, is increasingly recognized as a medical concern. Heteroresistance (HR) occurs when a predominantly susceptible bacterial population harbors a rare resistant subpopulation. During antibiotic exposure, these resistant bacteria can be selected and lead to treatment failure. Standard antibiotic susceptibility testing (AST) methods often fail to reliably detect these subpopulations due to their low frequency, highlighting the need for new diagnostic approaches. Here, we present a droplet microfluidics method where bacteria are encapsulated in droplets containing growth medium and antibiotics. The growth of rare resistant cells is detected by observing droplet shrinkage under microscopy. We validated this method for three clinically important antibiotics in Escherichia coli isolates obtained from bloodstream infections and showed that it can detect resistant subpopulations as infrequent as 10 ⁻⁶ using only 200 to 300 droplets. Additionally, we designed a multiplex microfluidic chip to increase the throughput of the assay.