Label-free single-cell antimicrobial susceptibility testing in droplets with concentration gradient generation

Area of Science:

• Microfluidics
• Microbial genetics
• Antibiotic resistance

Background:

• Bacterial heterogeneity leads to antibiotic-resistant subpopulations, often undetected early in treatment.
• Current single-cell antibiotic susceptibility testing (AST) methods are inefficient for broad antibiotic and concentration screening.
• Rapid diagnostics are crucial for effective treatment and antibiotic stewardship.

Purpose of the Study:

• To develop a droplet-based microfluidic platform for rapid, high-throughput single-cell AST.
• To demonstrate the platform's ability to determine minimum inhibitory concentrations (MICs) quickly.
• To investigate phenotypic heterogeneity in antibiotic resistance within isogenic bacterial populations.

Main Methods:

• A droplet-based microfluidic system was engineered for bacteria and antibiotic mixing, cell encapsulation, and incubation.
• The platform facilitated rapid determination of MICs for various antibiotics against *E. coli* and *S. aureus*.
• Single-cell analysis within droplets was performed to assess phenotypic resistance levels.

Main Results:

• The microfluidic platform achieved AST results in 3 hours, significantly faster than conventional methods (1-2 days).
• MICs were accurately determined for clinical isolates, including MRSA and MDRSA, compared to CLSI standards.
• Phenotypic heterogeneity in antibiotic resistance was observed among genetically identical bacterial cells.

Conclusions:

• The developed microfluidic platform offers a rapid and efficient solution for single-cell AST.
• This technology can identify previously undetected resistance mechanisms and inform treatment strategies.
• The findings support improved antibiotic stewardship by revealing population-level resistance dynamics.