Introduction: This study aimed to characterize silver nanoparticles-kaempferol (AgNP-K) and its antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA). Green synthesis method was used to synthesize AgNP-K under the influence of temperature and different ratios of silver nitrate (AgNO3 and kaempferol). Methods: AgNP-K 1:1 was synthesized with 1 mM kaempferol, whereas AgNP-K 1:2 with 2 mM kaempferol. The characterization of AgNP-K 1:1 and AgNP-K 1:2 was performed using UV–visible spectroscopy (UV–Vis), Zetasizer, transmission electron microscopy (TEM), scanning electron microscopy-dispersive X-ray spectrometer (SEM-EDX), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) spectroscopy. The antibacterial activities of five samples (AgNP-K 1:1, AgNP-K 1:2, commercial AgNPs, kaempferol, and vancomycin) at different concentrations (1.25, 2.5, 5, and 10 mg/mL) against MRSA were determined via disc diffusion assay (DDA), minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) assay, and time-kill assay. Results: The presence of a dark brown colour in the solution indicated the formation of AgNP-K. The UV–visible absorption spectrum of the synthesized AgNP-K exhibited a broad peak at 447 nm. TEM, Zetasizer, and SEM-EDX results showed that the morphology and size of AgNP-K were nearly spherical in shape with 16.963 ± 6.0465 nm in size. XRD analysis confirmed that AgNP-K had a crystalline phase structure, while FTIR showed the absence of (-OH) group, indicating that kaempferol was successfully incorporated with silver. In DDA analysis, AgNP-K showed the largest inhibition zone (16.67 ± 1.19 mm) against MRSA as compared to kaempferol and commercial AgNPs. The MIC and MBC values for AgNP-K against MRSA were 1.25 and 2.50 mg/mL, respectively. The time-kill assay results showed that AgNP-K displayed bacteriostatic activity against MRSA. AgNP-K exhibited better antibacterial activity against MRSA when compared to commercial AgNPs or kaempferol alone.

Methicillin-resistant Staphylococcus aureus (MRSA), a multidrug resistant strain, is known to cause a threat to public health due to its limited therapeutic treatment. Kaempferol (K) is a natural flavonoid that shows antibacterial activities toward MRSA, but its effectiveness is limited due to its low water solubility. However, poorly aqueous soluble drugs displayed better solubility through nano formulation. Hence, kaempferols were incorporated with silver nanoparticles (AgNPs) to enhance their solubility and antibacterial activity. Previous study showed that AgNPs incorporated with kaempferol (AgNPs-K) exhibited antibacterial activity against MRSA. However, the knowledge regarding the mechanism of action AgNPs-K against MRSA is still limited. The objective of the study is to unravel the inhibition mechanism of silver nanoparticles-kaempferol (AgNPs-K) on treated MRSA. The scanning electron microscopy (SEM) result showed significant difference in morphology between treated and non-treated MRSA which suggest the effectiveness of the AgNPs-K. Non-treated MRSA has an oval shape while MRSA treated with AgNPs-K showed a disrupted cell wall with contents leakage. The transcriptomic profile analysis by Next Generation Sequencing (NGS) showed that various genes and pathways related to biofilm, virulent activity and glycolysis pathway are differently expressed, with 581 genes were downregulated and 641 were upregulated. The affected genes of icab, clfa and eno which involved in biofilm, clumping factor A (virulent) and glycolysis pathway were validated by RT-PCR technique. The results were consistent with the NGS outcome. In conclusion, AgNPs-K possesses antibacterial activity against MRSA and its mechanism of action are reflected in the gene expression of biofilm pathway, virulent and glycolysis activity. Therefore, AgNPs-K can be suggested as a potential alternative to combat MRSA infection.