A Rapid Single-Cell Antimicrobial Susceptibility Testing Workflow for Bloodstream Infections

Area of Science:

• Microbiology
• Biotechnology
• Medical Diagnostics

Background:

• Bloodstream infections cause significant global morbidity and mortality.
• Rapid antibiotic treatment is crucial but hindered by blood's complexity and slow culture methods.
• Current diagnostics face challenges from blood matrix effects and lengthy procedures.

Purpose of the Study:

• To develop a culture-free workflow for rapid bacterial pathogen isolation and enrichment from whole blood.
• To enable single-cell antimicrobial susceptibility testing (AST) for bloodstream infections.
• To significantly reduce the time to actionable diagnostic results.

Main Methods:

• Implemented a dextran sedimentation step for rapid red blood cell depletion (4 orders of magnitude in 20-30 min).
• Utilized a centrifugation-based enrichment step for sepsis-relevant bacterial concentrations.
• Applied a microfluidic single-cell trapping device for pathogen analysis.
• Validated workflow compatibility with antibiotic-resistant bacteria and impact on minimum inhibitory concentrations.

Main Results:

• Achieved a 4-log reduction in blood cells within 30 minutes while preserving bacterial viability.
• Demonstrated successful enrichment of bacteria at clinically relevant concentrations.
• Confirmed workflow compatibility with common antibiotic-resistant strains without affecting minimum inhibitory concentrations.
• Successfully performed single-cell AST, yielding categorical results in under two hours.

Conclusions:

• The developed culture-free workflow enables rapid, single-cell AST from whole blood.
• This method significantly shortens the time to results for bloodstream infections, potentially improving treatment efficacy.
• The workflow offers a promising advancement in diagnosing and managing bacterial infections.