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

Abstract

ABSTRACT 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 Shewanella oneidensis MR-1. Upon exposure to cell wall-targeting antibiotics, PghKR activates the promoter of blaA, driving expression of the luxCDABE reporter and generating luminescence. A highly sensitive biosensor was engineered through the synergistic deletion of blaA and ampG, which greatly improved its responsiveness. The method was then applied to screen soil samples. From the primary screen, 103 colonies producing distinct luminescent signals were identified. Of these, 36 isolates consistently activated the biosensor in a confirmation assay, and 5 exhibited antibacterial activity against a multidrug-resistant indicator strain. This integrated approach combines microbial separation with immediate biosensor-based detection, thereby accelerating the discovery of novel antibiotic producers from complex environmental communities. 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. 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.