New techniques in antibiotic discovery and resistance: Raman spectroscopy

Area of Science:

• Biochemistry
• Microbiology
• Spectroscopy

Background:

• Antibiotic discovery faces challenges in quantifying intracellular drug concentrations and interactions.
• Understanding drug-target interactions is crucial for developing effective antibiotics and combating resistance.

Purpose of the Study:

• To present a Raman microscopy protocol for measuring intracellular drug populations and interactions.
• To investigate drug-target engagement within bacterial cells using a label-free spectroscopic approach.

Main Methods:

• Developed a protocol involving soaking live bacterial cells with drug candidates, followed by freezing and freeze-drying.
• Utilized non-resonant Raman microscopy to obtain quantitative data on intracellular drug molecules and their chemical reactions.
• Supported spectroscopic interpretation with in vitro Raman experiments in aqueous solution and single crystals.

Main Results:

• The protocol successfully measured intracellular drug molecule populations and revealed details of drug-target interactions.
• Demonstrated the ability to track cell-compound uptake kinetics on a minute timescale.
• Applied the method to study inhibitors of β-lactamase and dihydrofolate reductase in various bacterial cell lines.

Conclusions:

• Raman microscopy offers a powerful, label-free tool for antibiotic discovery and understanding resistance mechanisms.
• The developed protocol provides quantitative insights into in-cell drug chemistry and kinetics.
• Correlating in-cell data with in vitro studies enhances understanding of drug-target interactions.