Objective:

To identify bloodstream pathogens and measure their antimicrobial susceptibility within hours using a single-cell analysis platform.

Approach:

• Workflow Description: The system combined bacterial enrichment, fluorescence-based identification, and phenotypic antimicrobial susceptibility testing (AST).
• Sedimentation-Assisted Enrichment: The first stage, STREAM, involved mixing whole blood with culture broth and dextran to separate bacteria from red blood cells.
• Bacterial Identification: Molecularly barcoded sequential fluorescence in situ hybridization was used to identify individual bacteria by targeting bacterial 16S ribosomal RNA.
• Susceptibility Testing: Bacteria were immobilized in agarose gel and exposed to antimicrobial concentrations, with time-lapse imaging used to measure changes.

Key Findings:

• Results were produced in 6.75 to 17 hours for whole blood spiked with clinical bacterial isolates.
• 96% concordance with clinical laboratory identification in 104 positive blood culture samples.
• 98% essential agreement and 94% categorical agreement with clinical laboratory results for susceptibility testing.
• 4% very major error rate for resistant isolates misclassified as susceptible.

Interpretation:

The workflow demonstrated rapid identification and susceptibility testing of bloodstream pathogens, but further validation in clinical settings is needed.

Limitations:

• Testing was conducted on spiked samples rather than actual patient blood.
• Potential effects of prior antimicrobial exposure on bacterial recovery.
• Faster-growing organisms may obscure additional pathogens in polymicrobial infections.
• Intracellular, nonculturable, anaerobic, or fastidious organisms may require different processing conditions.
• The multistep workflow requires specialized imaging and further automation.

Conclusion:

The study presents a promising method for rapid identification and susceptibility testing of bloodstream pathogens.

Sources:

• Science Advances