Comprehensive validation of bactericidal efficacy of umbelliferone against multidrug-resistant pathogens through multifaceted antimicrobial assays.

Natural plants products and their derivatives have emerged as a promising candidate to address antibiotic resistance in pathogenic bacteria due to their diverse chemical structures and potent antimicrobial activity. One such coumarin-based derivative known as umbelliferone (7-hydroxycoumarin) is particularly known for its high antibacterial and antibiofilm activities. This compound is commonly found in both Rutaceae and Apiaceae (Umbelliferae) plant families. The present study focuses on investigating antibacterial and antibiofilm potential of umbelliferone and elucidating its mechanism of action against Bacillus cereus, Bacillus subtilis, and Klebsiella pneumoniae. The bactericidal efficacy of umbelliferone was determined using microtiter broth dilution, agar well diffusion and time-kill curve assays. The results showed complete inhibition at minimum inhibitory concentration (MIC) of 0.31 mg mL− 1 (1.92 µM) for B. cereus and 0.62 mg mL− 1 (3.85 µM) for both K. pneumoniae and B. subtilis. Notably, agar diffusion assays revealed that umbelliferone was most effective against K. pneumoniae as compared to other tested strains. Further assays evaluating antibiofilm and exopolysaccharide inhibition demonstrated that the umbelliferone could significantly suppressed the growth of tested strains and inhibited biofilm formation in concentration-dependent manner. The most notable effects were observed at 1MIC, where umbelliferone achieved maximum biofilm inhibition (85.86 ± 2.10%) and reduced exopolysaccharide production to 10.07 ± 1.58 µg mL⁻¹ against B. cereus and B. subtilis, respectively. Additionally, umbelliferone triggered a time-dependent release of intracellular proteins, and propidium iodide staining under fluorescence microscopy indicated membrane disruption. These findings were corroborated by scanning electron microscopy (SEM), which revealed deformation of the bacterial cell membrane and leakage of intracellular contents. Overall, the findings suggest that umbelliferone is a promising candidate for managing drug-resistant infections, primarily acting through bacterial cell membranes, a mechanism that indicates a low potential for resistance development, making it an ideal antibacterial agent or adjuvant to enhance the efficacy of existing antibiotics.