A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

Abstract

The envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measure Escherichia coli envelope permeability to a ␤-galactosidase chromogenic substrate. The signal produced by cytoplasmic ␤-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds and E. coli gene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8-to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinct E. coli strains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 M of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R > 0.5 g/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

New N -phenylpyrrolamide DNA gyrase B inhibitors: Optimization of efficacy and antibacterial activity

“…We therefore sought to explore systematically the limiting factors for lack of anti-Gram-negative activity of compound 11a. To investigate possible difficulties with its penetration into the bacterial cell, we tested compound 11a on a diverse panel of mutant E. coli strains carrying loss-of-function mutations in either dapF, mrcB or surA genes that disrupt the bacterial cell wall integrity [27]. As a parallel approach, to test the susceptibility of compounds to efflux, we treated the parental strains and their corresponding mutants with an efflux pump inhibitor phenylalanine-arginine β-naphthylamide (PAβN), which is in fact not a typical inhibitor but a substrate for efflux pumps that is preferentially effluxed.…”

Repurposing Azithromycin and Rifampicin Against Gram-Negative Pathogens by Combination With Peptidomimetics

“…Minimal inhibitory concentrations (MICs) of peptidomimetics, antibiotics and their combinations were determined by broth microdilution according to the Clinical Laboratory Standards Institute performance standards (Clinical and Laboratory Standards Institute, 2013). Based on previous studies indicating that among a range of tested antibiotics belonging to several classes azithromycin and rifampicin displayed a high propensity to synergize with peptides, and hence these antibiotics were chosen for the initial screening of peptidomimetics to identify possible antibiotic adjuvants (Jammal et al, 2015; Baker et al, 2016, 2018; Corbett et al, 2017; Lyu et al, 2017; Wu et al, 2017; Domalaon et al, 2018a,b; Yang et al, 2018). Here, a sub-MIC concentration of 0.5 μM peptidomimetic was tested in combination with 0.5 μg/mL rifampicin or 1 μg/mL azithromycin in 96-well plates, followed by inoculum addition according to CLSI guidelines.…”

Genome Analysis of Lactobacillus plantarum Isolated From Some Indian Fermented Foods for Bacteriocin Production and Probiotic Marker Genes

“…Most of the experiments on the mode of action is being done by conventional microbiological methods involving screening of antimicrobial compound by agar well diffusion assay, which are tedious and unwieldy (Balouiri et al, 2016). Nowadays, the application of high-throughput screening (HTS)with targeted cell-based assays, that carry reporters such as β-galactosidase or luciferase genes (Shobharani and Halami, 2016) for microbial compounds, is getting more popular and reliable (Baker et al, 2016).…”