The combination of amoxicillin (AMX) and clavulanic acid (CA) remains one of the most widely prescribed antibiotic therapies. However, its clinical efficacy is increasingly compromised by the emergence of bacterial resistance and persistence mechanisms, among both Gram-negative and Gram-positive pathogens.

To address this limitation, we developed a novel formulation combining AMX and CA with 1,8-cineole (CN), a natural compound well recognized in the pharmacopoeia for its broad pharmacological activities and favorable safety profile.

The obtained in vitro, in vivo and clinical trials demonstrate that this triple combination significantly enhances the antibacterial activity of AMX/CA against susceptible strains. Importantly, it also restores susceptibility in resistant and persistent bacterial populations.

In parallel, in silico investigations were conducted to elucidate the underlying mechanisms of action. Key bacterial targets were explored, including penicillin-binding proteins (PBPs), as well as molecular targets associated with bacterial persistence. These computational analyses suggest that AMX, CA, and CN can form multiple molecular complexes, including three binary combinations (AMX–CA, AMX–CN, and CA–CN) as well as a ternary complex (AMX–CA–CN). These entities may act simultaneously on bacterial targets and significantly reduce the probability of resistance emergence and bacterial survival in a persistent state.