Synthesis of methylated rhamnolipid derivatives and their synergistic antibacterial effect alongside the photosensitizer toluidine blue O (TBO)

Bacterial resistance poses a growing challenge that requires innovative antibacterial solutions. This study assessed the antibacterial activity of methylamide-modified rhamnolipids (RLs‑MMA). RLs-MMA was first synthesized through amide condensation of methylamine with the carboxyl group of rhamnolipids. The structure of the derivatives was confirmed using FTIR NMR, and LC‑MS analyses. RLs‑MMA were significantly potent against Gram‑positive bacteria. Against Bacillus cereus , the MIC was as low as 16 mg/L. This value represents a 16‑fold improvement on di‑RLs (256 mg/L) and a 4‑fold improvement on mono‑RLs (64 mg/L). The MIC values of RLs‑MMA against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) were in the range of 64–128 mg/L, significantly lower than values for unmodified RLs (≥256 mg/L). RLs‑MMA alone were ineffective against Gram‑negative bacteria (MIC >512 mg/L). However, combing them with the photosensitizer toluidine blue O (TBO) under red‑light irradiation, the synergy was considerable. Checkerboard assays revealed fractional inhibitory concentration index (FICi) values < 0.375 and < 0.25 for Escherichia coli and Pseudomonas aeruginosa , respectively. According to mechanistic studies, RLs‑MMA permeabilizes bacterial membrane integrity, promotes the uptake of intracellular TBO, and enhances the photodynamic generation of reactive oxygen species (ROS). This combined membrane permeabilization and photodynamic strategy can overcome the outer membrane of Gram‑negative bacteria.