A double-layer plate method for rapid screening of cell wall-targeting antibiotic producers using a microbial whole-cell biosensor.

The escalating antimicrobial resistance crisis demands innovative strategies for antibiotic discovery. Conventional approaches for identifying antibiotic-producing microorganisms from environmental samples are often laborious and low-throughput, requiring prior isolation and purification of individual strains. Here, we developed a high-throughput screening platform integrating a microbial whole-cell biosensor into a double-layer plate assay, enabling rapid identification of bacteria producing cell wall-targeting antibiotics from environmental samples. The biosensor is based on the PghKR two-component system from the gram-negative bacterium IMPORTANCE: The rise of antimicrobial resistance calls for faster, more efficient ways to discover new antibiotics from environmental microbes. Traditional methods are slow because they require laborious, one-by-one isolation and purification of individual strains before any activity testing. To overcome this bottleneck, we developed a simple double-layer plate assay that directly identifies bacteria producing cell wall-targeting antibiotics while they grow. This "grow-and-detect" strategy bypasses traditional isolation steps, dramatically speeding up the initial discovery pipeline. Our platform enables large-scale, low-cost screening of environmental samples for antibiotic producers.