Development of a high-throughput bioassay for screening of antibiotics in aquatic environmental samples

The goal of the present study was to select a Gram-positive (Gram+) and Gram-negative (Gram-) strain to measure antimicrobial activity in environmental samples, allowing high-throughput environmental screening. The sensitivity of eight pre-selected bacterial strains were tested to a training set of ten antibiotics, i.e. three Gram+ Bacillus subtilis strains with different read-outs, and five Gram- strains. The latter group consisted of a bioluminescent Allivibrio fischeri strain and four Escherichia coli strains, i.e. a wild type (WT) and three strains with a modified cell envelope to increase their sensitivity. The WT B. subtilis and an E. coli strain newly developed in this study, were most sensitive to the training set. This E. coli strain carries an open variant of an outer membrane protein combined with an inactivated multidrug efflux transport system. The assay conditions of these two strains were optimized and validated by exposure to a validation set of thirteen antibiotics with clinical and environmental relevance. The assay sensitivity ranged from the ng/mL to μg/mL range. The applicability of the assays for toxicological characterization of aquatic environmental samples was demonstrated for hospital effluent extract. A future application includes effect-directed analysis to identify yet unknown antibiotic contaminants or their transformation products.