Development and Optimization of a Higher-Throughput Bacterial Compound Accumulation Assay.

Area of Science:

• Microbiology
• Pharmacology
• Analytical Chemistry

Background:

• Gram-negative bacteria possess a challenging permeability barrier and efflux systems that hinder antibiotic drug discovery.
• Existing assays for compound penetration and efflux are often resource-intensive, small-scale, and lack efficiency.
• Developing effective antibiotics is crucial to combatting bacterial infections.

Purpose of the Study:

• To present BacPK, a novel label-free assay for measuring total cellular compound accumulation in Escherichia coli.
• To provide a high-throughput method for assessing antibiotic penetration and efflux pump activity.
• To facilitate antibiotic drug discovery by enabling efficient analysis of compound accumulation.

Main Methods:

• Utilized a solid phase extraction-mass spectrometry (SPE-MS)-based assay named BacPK.
• Employed a 96-well format allowing for rapid analysis (9 s/sample) of compound accumulation.
• Validated the assay using tetracycline analogs and diverse antibacterial compounds in isogenic bacterial strains.

Main Results:

• BacPK accurately measured differences in cellular accumulation for tetracycline and its analogs.
• The assay distinguished the impact of efflux pumps (TetA) versus other resistance mechanisms (TetM) on tetracycline accumulation.
• BacPK quantified accumulation differences in strains with altered permeability or efflux, correlating with observed susceptibility.

Conclusions:

• BacPK is an efficient, high-throughput assay for measuring bacterial compound accumulation.
• The assay aids in understanding antibiotic-drug interactions with bacterial permeability and efflux mechanisms.
• BacPK is a valuable tool for advancing antibiotic drug discovery against Gram-negative pathogens.