Multiparameter flow cytometry of bacteria: implications for diagnostics and therapeutics

Area of Science:

• Microbiology
• Flow Cytometry
• Bacterial Physiology

Background:

• Traditional flow cytometry for bacterial antibiotic susceptibility measures single parameters like membrane potential or permeability.
• Simultaneous staining with membrane potential and permeability dyes reveals unexpected complexity in bacterial responses.

Purpose of the Study:

• To investigate the complexity of bacterial responses using multiparameter flow cytometry.
• To evaluate the potential of flow cytometry for developing novel antibiotic susceptibility assays and therapeutic approaches.

Main Methods:

• Three bacterial species were stained with hexamethylindocarbocyanine [DiIC1(3)] (membrane potential) and TO-PRO-3 (permeability).
• Analysis was performed using a dual-laser flow cytometer, with and without a proton ionophore.
• Bacterial cultures were examined under suboptimal growth conditions and following antibiotic exposure.

Main Results:

• Bacterial cells under suboptimal conditions showed TO-PRO-3 uptake while maintaining membrane potential.
• Some events indicated both high membrane potential and high permeability, potentially representing cell clumps.
• Metabolic variations were observed between species and within organisms under stress.

Conclusions:

• Variations in bacterial metabolic patterns complicate the development of standard flow cytometric antibiotic susceptibility tests.
• Transient permeabilization of resistant bacteria by sublethal antibiotic doses may enable treatment with toxic molecules.
• Multiparameter flow cytometry is a valuable tool for exploring these complex interactions and developing new therapies.