Single-cell scattering and auto-fluorescence-based fast antibiotic susceptibility testing for gram-negative and...

Area of Science:

• Microbiology
• Biophysics
• Analytical Chemistry

Background:

• Antimicrobial susceptibility testing (AST) is crucial for guiding antibiotic treatment.
• Current AST methods can be time-consuming, delaying effective patient care.
• Label-free techniques offer potential for faster, more efficient diagnostics.

Purpose of the Study:

• To develop a fast (<2 hours), label-free phenotypic antimicrobial susceptibility testing (AST) method.
• To assess the utility of single bacterial cell light scattering and auto-fluorescence for AST.
• To differentiate between susceptible and resistant bacterial phenotypes.

Main Methods:

• Label-free flow cytometry was employed to monitor bacterial auto-fluorescence (FAD, NADH) and light scattering.
• Single bacterial cells (Escherichia coli, Staphylococcus epidermidis) were incubated with antibiotics (aminoglycosides, beta-lactams).
• Dimensionality reduction techniques (PCA, one-class SVM) were used for data analysis and phenotype prediction.

Main Results:

• The label-free flow cytometry approach demonstrated feasibility for AST in under 2 hours.
• Single-cell auto-fluorescence provided added value for phenotyping compared to scattering alone.
• This method showed particular effectiveness for identifying resistant Staphylococcus strains (mecA+) treated with oxacillin.

Conclusions:

• Label-free flow cytometry is a viable method for rapid phenotypic AST.
• Combining light scattering and auto-fluorescence enhances the accuracy of bacterial susceptibility determination.
• This technique has significant potential for improving antibiotic stewardship and patient outcomes.