(15a) A New High-Throughput Assay for Quantification of Antibiotic Penetration in Gram-Negative Bacterial Cells | AIChE

(15a) A New High-Throughput Assay for Quantification of Antibiotic Penetration in Gram-Negative Bacterial Cells


Gu, H. - Presenter, University of New Haven
Jiang, Z., Syracuse University
Ren, D., Syracuse Biomaterials Institute
A new high-throughput assay for quantification
of antibiotic penetration in Gram-negative bacterial cells

Huan Gu1,2, Zhaowei
Jiang1,2,and Dacheng Ren1,2,3,4*

of Biomedical and Chemical Engineering, 2Syracuse Biomaterials
Institute, 3Department of Civil and Environmental Engineering, 4Department
of Biology, Syracuse University, Syracuse, NY 13244, USA

resistance is a grand challenge with paramount societal impacts and requires
immediate action to discover new antimicrobials. However, the discovery of new
antibiotics has been at a standstill due to the lack of technologies to
quantify antibiotic penetration in bacteria, especially the Gram-negative
species. Here, we present a new assay that can rapidly quantify antimicrobial molecules
in the periplasm and cytoplasm of Gram-negative bacteria. The premise of this
method is separate lysis of outer and inner membranes, which allows the drug
concentration to be determined using a reporter strain. Following the
development of this assay, Pseudomonas
was selected as the model
microorganism for a proof-of-concept study because
of its well-documented resistance
to conventional antibiotics due to its double-layered membranes and narrow
pores. By evaluating the penetration of ciprofloxacin (CIP, 200 µg/mL) into P. aeruginosa PAO1 using this assay, we
found that its outer membrane is the major barrier to antibiotic penetration. In stationary phase P. aeruginosa PAO1 cells, CIP concentration in the periplasm is 300
times higher than that in the cytoplasm (107 vs. 0.3 µg/mL), where DNA gyrase,
the target of CIP, locates. Penetration of CIP into
the cytoplasm of stationary phase P.
cells was significantly improved by mutating the gene mexB encoding an efflux pump protein in
the inner membrane of P. aeruginosa.
This new assay may have broad applications in antibiotic discovery and
mechanistic study of antibiotic resistance.