The Quantification of Drug Accumulation within Gram-Negative Bacteria

TL;DR

This paper introduces a new method to measure how drugs accumulate inside Gram-negative bacteria, which is important for drug discovery and understanding bacterial resistance.

Contribution

A novel LC-MS-based platform is developed to quantify drug accumulation and metabolism in Gram-negative bacteria without drug labeling.

Findings

The method was validated with E. coli, A. baumannii, K. pneumoniae, and P. aeruginosa in single-compound and high-throughput formats.

Drug accumulation correlates with minimum inhibitory concentration (MIC) in wild-type and efflux-deficient bacterial strains.

High-throughput screening using this platform is feasible and adaptable for translational and fundamental studies.

Abstract

Intrabacterial drug
accumulation, mediated by the bacterial permeability
barrier, efflux, and intrabacterial drug metabolism, is of general
significance to the interaction between small molecules and bacteria.
For example, the ability of a small molecule to accumulate within
a bacterium influences its ability to serve as a chemical probe of
an intracellular protein target and/or its efficacy as an antibacterial
drug discovery entity. A general method to quantitatively interrogate
both intrabacterial drug accumulation and metabolism (IBDM) is presented
for Gram-negative bacteria and exemplified with Escherichia
coli, Acinetobacter baumanni
i, Klebsiella pneumoniae, and Pseudomonas aeruginosa in both
single-compound and high-throughput formats. The liquid chromatography–mass
spectrometry-based platform does not depend on drug labeling, and
its utility is highlighted through the exploration of the relationship
between drug accumulation and drug minimum inhibitory concentration
(MIC) for both wild-type and efflux-deficient strains of E. coli and K. pneumoniae clinical and laboratory strains of varying degrees of drug resistance.
Furthermore, an investigation of drug synergy implicates the selective
enhancement of the accumulation of one drug by its partner therapy.
Finally, a high-throughput format is validated and deployed, which
provides a readily adaptable approach to screening assays. We anticipate
the further applications of this platform to both the translational
and the fundamental studies of the interactions of small molecules
with bacteria.

Figures