Currently, antibiotic resistance is widespread among bacteria. This problem requires greater awareness because bacterial resistance increases, reducing antibiotic use effectiveness. Consequently, new alternative treatments are needed because the treatment options for these bacteria are limited. This work aims to determine the synergistic interaction and mechanism of action of Boesenbergia rotunda essential oil (BREO) against methicillin‐resistant Staphylococcus aureus (MRSA). Gas chromatography‐mass spectrometry identified 24 BREO chemicals (GC‐MS). The main components of BREO were β‐ocimene (36.73%), trans‐geraniol (25.29%), camphor (14.98%), and eucalyptol (8.99%). BREO and CLX inhibited MRSA DMST 20649, 20651, and 20652 with a minimum inhibitory concentration (MIC) of 4 mg/mL and 512 µg/mL, respectively. The checkerboard method and the time‐kill assay revealed synergy between BREO and CLX with fractional inhibitory concentration (FIC) 2log10 CFU/mL at 24 hours compared to the most effective chemical. BREO inhibited biofilm formation and increased membrane permeability. Exposure alone to BREO or in combination with CLX inhibited biofilm formation and increased cytoplasmic membrane (CM) permeability. The scanning electron microscopy (SEM) and transmission electron microscopy (TEM) results revealed that alterations in the cell walls, cytoplasmic membrane, and leakage of intracellular components of MRSA DMST 20651 after treatment with BREO alone and in combination with CLX were observed. These results indicate that BREO synergizes and could reverse the antibacterial activity of CLX against MRSA strains. The synergy of BREO may lead to novel drug combinations that increase the effectiveness of antibiotics against MRSA.

Increasing antibiotic resistance in enterococci is among the most serious public health problems worldwide. The new naturally occurring antibacterial agents were explored. This study, therefore, investigated the antibacterial potential of Stephania suberosa extract (SSE) and its synergism with ampicillin (AMP) or vancomycin (VAN) against AMP- and VAN-resistant Enterococcus faecium. Disc diffusion assay revealed that SSE inhibited E. faecium DMST 12829, 12852, 12970, and a reference strain of Enterococcus faecalis ATCC 29,212 in a dose-dependent manner. The minimum inhibitory concentration (MIC) of SSE against all E. faecium isolates was 0.5 mg/mL. E. faecium DMST 12,829 and 12,852 were highly resistant to AMP, as indicated by high MIC values, and E. faecium DMST 12,829 and 12,970 were resistant to VAN. Enterococcus spp. were killed by SSE at the minimum bactericidal concentrations (MBCs) ranging from 0.5 to 4 mg/mL. Checkerboard determination showed that SSE plus AMP and SSE plus VAN combinations exhibited synergistic interaction against E. faecium isolates. The killing curve assay of E. faecium isolates confirmed the antibacterial and synergistic activities of combined agents by dramatically reducing the viable counts compared to a single agent. Scanning electron microscope elucidated the cell damage and abnormal cell division. Enterococcal proteases were also inhibited by SSE. These findings support that SSE could reverse the activity of AMP and VAN. Moreover, it can synergistically inhibit AMP- and VAN-resistant E. faecium. Our combined agents could be attractive candidates for developing new combinatorial agents to resurrect the efficacy of antibiotics for treating AMP- and VAN-resistant E. faecium infections.