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Minimum Inhibitory Concentration Assays for Multidrug Resistant Gram‐Negative Bacterial Pathogens
Author(s) -
Edgar Jennifer M.,
Zhao Hang,
Brakke Sabrina A.,
Wise John G.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.04561
Subject(s) - antibiotics , microbiology and biotechnology , efflux , chloramphenicol , minimum inhibitory concentration , biology , antibiotic resistance , multiple drug resistance , bacteria , escherichia coli , neisseria gonorrhoeae , gene , biochemistry , genetics
Antibiotic resistance by bacterial pathogens is a persistent problem in medicine. One method of resistance is through RND efflux transporters, which can move antibiotics out of the cells and the periplasm, thus preventing cell death. Many of these transporters can move several very different antibiotics out of the cells, thus conferring multidrug resistance to the pathogens. This project focuses on one such RND transporter found in Neisseria gonorrhoeae known as mtrCDE. Our goal is to identify potential inhibitors of mtrCDE by observing their effects on the minimum inhibitory concentrations (MICs) of antibiotics. Ultimately, we aim to develop inhibitors of mtrCDE with the goal of restoring antibiotic sensitivity to antibiotic‐resistant N. gonorrhoeae . Potential inhibitors have been identified through computational means. In the system described here, the genes for mtrCDE have been transformed into an E. coli strain that has had the homologous E. coli RND transporter deleted for facilitating testing. We have attempted to optimize MIC assays to determine the minimum concentration of antibiotic required to inhibit the growth of cells expressing the mtrCDE genes. To date, the largest effects in these assays have been observed with the detergent Triton X‐100. In addition, the plasmid encoding mtrCDE included a gene conferring chloramphenicol resistance for selection. The media for initial growth of cells contained chloramphenicol to maintain this selection. Initial studies showed that inclusion of chloramphenicol with other antibiotics overwhelms even resistant bacteria. The effects of inclusion of dimethyl sulfoxide (DMSO), a commonly used carrier vehicle for experimental compounds, was tested in the assays since our compound libraries use this solvent. Experiments suggested that inclusion of low concentrations of DMSO does not affect the MIC values observed, but that the cells do reflect a dose response to varying concentrations of DMSO that needs to be carefully controlled. Continuing optimization of these assays with various antibiotics is being performed to determine MIC values, compare them with reported MIC values, and identify antibiotics that are effluxed by mtrCDE that have no published MIC values for N. gonorrhoeae or E. coli . We report here these and other results of our MIC assay optimization for multidrug resistant, gram‐negative bacterial pathogens. Support or Funding Information Department of Biological Sciences, the Center for Drug Discovery, Design and Delivery (CD4), the Center for Scientific Computation, Engaged Learning, and the Hamilton Scholars Program, Southern Methodist University, Dallas, TX