Title : Marine cyanobacterial metabolites as dual inhibitors of bacterial efflux pumps and biofilm formation

Antimicrobial resistance (AMR) has become a major global health challenge, highlighting the urgent need for alternative therapeutic strategies. Marine cyanobacteria are an underexplored source of structurally diverse natural products with significant potential for combating bacterial resistance mechanisms.This study investigated the efflux pump inhibitory and antibiofilm activities of two cyanobacterial metabolites, γ Lactone 2 isolated from Oscillatoria sp. and Nhatrangin A from Halomicronema sp. Their activities were evaluated against Enterotoxigenic Escherichia coli (ETEC), Enteropathogenic E. coli (EPEC), and methicillin-resistant Staphylococcus aureus (MRSA; Mu50 and F-182). Efflux pump inhibition targeting AcrB (Gram-negative) and NorA (Grampositive) transporters was assessed using ethidium bromide (EtBr) accumulation assays, while antibiofilm activity was determined by crystal violet staining. Molecular docking and molecular dynamics simulations were performed to investigate ligand-protein interactions.Both metabolites significantly increased intracellular EtBr accumulation, indicating effective inhibition of bacterial efflux pumps. Nhatrangin A and γ Lactone 2 exhibited efflux inhibition activity with minimum inhibitory concentrations (MICs) of 10 µg/mL and 16 µg/mL, respectively. In addition, both compounds significantly inhibited biofilm formation, demonstrating a dual mechanism for attenuating bacterial defence systems. Computational analyses further supported stable binding of both metabolites to AcrB and NorA efflux transporters, providing mechanistic insights into their inhibitory activity.These findings identify γ Lactone 2 and Nhatrangin A as promising marine cyanobacterial metabolites capable of simultaneously targeting bacterial efflux pumps and biofilm formation. Their dual mode of action highlights their potential as lead molecules for the development of novel natural product-based therapeutics to combat antimicrobial resistance.

Nousi Parvin is a Senior Research Fellow and Ph.D. candidate in the Department of Botany at Visva Bharati University, India. Her research focuses on marine cyanobacterial natural products, antimicrobial resistance, quorum sensing inhibition, efflux pump inhibition, and antibiofilm therapeutics. She has worked on a SERB-funded project exploring cyanobacterial metabolites as novel anti-infective agents. Her expertise includes microbiology, natural product chemistry, LC-MS/MS-based metabolomics, molecular docking, and molecular dynamics simulations. Her current research aims to identify bioactive cyanobacterial metabolites as lead compounds for developing innovative strategies to combat multidrug-resistant bacterial infections.