High-Throughput Screening for Antimicrobial Compounds Using a 96-Well Format Bacterial Motility Absorbance Assay

Abstract

There is a pressing need to develop new antimicrobial drugs because of the increasing resistance of pathogenic bacteria to existing antibiotics. The preliminary development and validation of a novel methodology for the high-throughput screening of antimicrobial compounds and inhibitors of bacterial motility is described. This method uses a bacterial motility swarming agar assay, combined with the use of offset inoculation of the wells in a standard, clear, 96-well plate, to enable rapid screening of compounds for potential antibiotic and antimotility properties with a standard absorbance microplate reader. Thus, the methodology should be compatible with 96-well laboratory automation technology used in drug discovery and chemical biology studies. To validate the screening method, the Genesis Plus structurally diverse library of 960 biologically active compounds was screened against a motile strain of the gram-negative bacterial pathogen Salmonella typhimurium. The average Z′ value for the positive and negative motility controls on all 12 compound plates was 0.67 ± 0.14, and the signal-to-baseline ratio calculated from the positive and negative controls was 5.9 ± 1.1. A collection of 70 compounds with well-known antimicrobial properties was successfully identified using this assay. (Journal of Biomolecular Screening 2007:849-854)

A High-Throughput Screening Assay for Inhibitors of Bacterial Motility Identifies a Novel Inhibitor of the Na+-Driven Flagellar Motor and Virulence Gene Expression in Vibrio cholerae

“…Thus, an HTS assay for inhibitors of bacterial motility could be instrumental for the discovery of novel anti-infective drugs. Here we describe the development of an HTS assay for inhibitors of bacterial motility based on a previous method using a soft agar motility assay in 96-well microtiter plates (38). To miniaturize the assay, we first confirmed that in contrast to the case for motile V. cholerae strain C7258, inoculation of the isogenic nonmotile strain C7258 Mot Ϫ in 384-well plates did not interfere with the OD 615 reading even after 24 h of incubation (data not shown).…”

“…We have developed and validated an HTS assay for smallmolecule inhibitors of bacterial motility based on a previous assay for S. Typhimurium antibacterial compounds that used a 96-well off-center inoculation method (38). The two major shortcomings of the former method were its low throughput and the inability to distinguish between compounds that inhibit motility and those that affect viability.…”

“…The HTS assay described in this study was based on a recently described 96-well assay for Salmonella enterica serovar Typhimurium antimicrobial compounds that uses motility as a readout (38). This assay is based on (i) off-center inoculation of a bacterial culture into wells containing soft agar, (ii) an incubation period allowing motile bacteria to spread throughout the well, and (iii) a diagonal off-center reading of absorbance (optical density at 615 nm [OD 615 ]) to estimate the extent to which motile bacteria spread across the well.…”

Genomic sequencing-based mutational enrichment analysis identifies motility genes in a genetically intractable gut microbe

“…After overnight incubation, bacteria at the initial site of inoculation were collected and reinoculated in the center of another 0.3% agar plate to enrich for nonswarming mutants (45). After three rounds of enrichment, individual bacterial colonies were tested in a 96-well plating assay (46) to identify mutants that failed to swarm in lowpercentage agar. Fig.…”

Acoustic Droplet Ejection Applications for High-Throughput Screening of Infectious Agents

“…As early as 2007, the manual assay that had historically been done in 100 mm dishes or low-density multiwell plates, such as 6-or 24-well plates, and inoculated manually was adapted to the 96-well format and a small library of 960 compounds was run as proof of principle. 23 In 2010, bacteriologists at Southern Research proposed screening up to 200,000 compounds in a motility assay using Vibrio cholera. While it could be done in the 96-well format, the cost and time required could both be reduced if the assay could be further miniaturized to the 384-well format.…”