Single-cell patterning and characterisation of antibiotic persistent bacteria using bio-sCAPA.

Area of Science:

• Microbiology
• Cell Biology
• Bacterial Physiology

Background:

• Studying rare bacterial subpopulations like antibiotic-tolerant persister cells is crucial for understanding chronic infections.
• Existing single-cell characterization methods lack scalability for analyzing rare phenotypes.
• Persister cells survive antibiotics without genetic resistance, posing a significant clinical challenge.

Purpose of the Study:

• To introduce bio-sCAPA, a novel technique for high-throughput, single-cell analysis of bacterial populations.
• To investigate the phenotypic characteristics of antibiotic-tolerant *Staphylococcus aureus* persister cells.
• To determine the basis of antibiotic tolerance in *S. aureus* at the single-cell level.

Main Methods:

• Development and application of bio-sCAPA for precise geometric patterning of bacterial cells (>10^5 cells per template).
• Parallel processing of multiple templates to monitor rare phenotypes across diverse nutrient environments.
• Single-cell phenotypic analysis of *Staphylococcus aureus* exposed to flucloxacillin and rifampicin.

Main Results:

• bio-sCAPA enables scalable, single-cell monitoring of bacterial populations under various conditions.
• Antibiotic-tolerant *Staphylococcus aureus* persister cells were analyzed using bio-sCAPA.
• Persister cells showed no significant growth rate heterogeneity; tolerance was linked to prolonged lag times.

Conclusions:

• bio-sCAPA is a powerful tool for dissecting bacterial population heterogeneity and rare phenotypes.
• Antibiotic tolerance in *S. aureus* is primarily mediated by extended lag phases rather than altered growth rates.
• This finding advances our understanding of persister cell biology and potential therapeutic strategies.