Deciphering Common Genetic Pathways to Antibiotic Resistance in Using a MEGA-Plate Evolution System.

Antimicrobial resistance (AMR) poses a significant global health threat, necessitating a deeper understanding of bacterial adaptation mechanisms. This study investigates the genotypic and phenotypic evolutionary trajectories of under meropenem and gentamicin selection, and it benchmarks these findings against florfenicol-evolved strains. Utilizing a downsized, three-layer acrylic modified "Microbial Evolution and Growth Arena (MEGA-plate) system"-scaled to 40 × 50 cm for sterile handling and uniform 37 °C incubation-we tracked adaptation over 9-13 days, enabling real-time visualization of movement across antibiotic gradients. Meropenem exposure elicited pronounced genetic heterogeneity and morphological remodeling (filamentous and circular forms), characteristic of SOS-mediated division arrest and DNA-damage response. In contrast, gentamicin exposure produced a uniform resistance gene profile and minimal shape changes, suggesting reliance on conserved defenses without major morphological adaptation. Comprehensive genomic analysis revealed a core resistome of 22 chromosomal loci shared across all three antibiotics, highlighting potential cross-resistance and the central roles of , , and in coordinating adaptive responses. Gene ontology enrichment underscored the positive regulation of gene expression and intracellular signaling as key themes in resistance evolution. Our findings illustrate the multifaceted strategies employs-combining metabolic flexibility with sophisticated regulatory networks-to withstand diverse antibiotic pressures. This study underscores the utility of the MEGA-plate system in dissecting spatiotemporal AMR dynamics in a controlled yet ecologically relevant context. The divergent responses to meropenem and gentamicin highlight the complexity of resistance development and reinforce the need for integrated, One Health strategies. Targeting shared regulatory hubs may open new avenues for antimicrobial intervention and help preserve the efficacy of existing drugs.