Oxidative Stress-Mediated Antibacterial Activity of the Total Flavonoid Extracted from the Agrimonia pilosa Ledeb. in Methicillin-Resistant Staphylococcusaureus (MRSA)

Abstract

(1) Background: Methicillin-resistant Staphylococcus&nbsp;aureus (MRSA) is a zoonotic pathogen that causes endocarditis, pneumonia, and skin diseases in humans and livestock. (2) Methods: The antibacterial effect of the total flavonoid against MRSA (ATCC43300) extracted from the Agrimonia pilosa Ledeb. (A. pilosa Ledeb) was evaluated by the microdilution method. The oxidative stresses in MRSA were evaluated by the levels of intracellular hydrogen peroxide (H₂O₂), reactive oxygen species (ROS), and oxidative stress-related genes. The DNA oxidative damage was tested by the 8-hydroxy-2'-deoxyguanosine (8-OHdG) and DNA gel electrophoresis. The differentially expressed proteins were determined by the method of SDS-PAGE and NanoLC-ESI-MS/MS, while the mRNAs of differential proteins were determined by Real-Time PCR. The changes of ultra-structures in MRSA were observed by Transmission Electron Microscope (TEM). (3) Results: The minimum inhibitory concentration (MIC) of the total flavonoid against MRSA was recorded as 62.5 μg/mL. After treatment with the total flavonoid, the levels of intracellular H₂O₂ and ROS were increased and the gene expressions against oxidative stress (SodA, katA, TrxB) were decreased (p < 0.01), while the gene expression for oxidative stress (PerR) was increased (p < 0.01). The level of intracellular 8-OHdG in MRSA was increased (p < 0.01) and the DNA was damaged. The results of TEM also showed that the total flavonoid could destroy the ultra-structures in the bacteria. (4) Conclusions: The total flavonoid extracted from the A. pilosa Ledeb can induce the oxidative stress that disturbed the energy metabolism and protein synthesis in MRSA.

Keywords: Agrimonia pilosa Ledeb. (A. pilosa Ledeb.); methicillin-resistant Staphylococcus aureus (MRSA); oxidative stress; total flavonoid.

Figure 1

The chromatogram of the total flavonoid extracted from the A. pilosa Ledeb at 254 nm.

Figure 2

The time–kill curves for the total flavonoid against MRSA ATCC43300 at different concentrations (4 × MIC, 2 × MIC, 1 × MIC, 0.5 × MIC, and 0.25 × MIC). Ceftiofur sodium was used as the positive control. The normal MHB was set as the negative control (NC).

Figure 3

The levels of intracellular H₂O₂ in MRSA ATCC43300 after the treatment with different concentrations of the total flavonoid extracted from A. pilosa Ledeb. The Ceftiofur sodium was used as positive control. The normal MHB was set as the negative control (NC). ## means p-value < 0.01.

Figure 4

The levels of intracellular ROS in MRSA ATCC43300 after treatment of the total flavonoid extracted from the A. pilosa Ledeb. The normal MHB was set as the negative control (NC). (a) was the NC group, (b) was the 1 × MIC group.

Figure 5

The relative gene expressions of SodA, katA, TrxB, and PerR after treatment of the total flavonoid extracted from the A. pilosa Ledeb. The normal MHB was set as the negative control (NC). ## means p-value < 0.01.

Figure 6

The levels of 8-OHdG in MRSA ATCC43300 after treatment of the total flavonoid extracted from the A. pilosa Ledeb. The normal MHB was set as the negative control (NC). ## means p-value < 0.01.

Figure 7

The DNA gel electrophoresis after treatment of the total flavonoid extracted from the A. pilosa Ledeb. The normal MHB was set as the negative control (NC).

Figure 8

The SDS-PAGE of the total proteins from MRSA ATCC43300 after treatment of the total flavonoid extracted from the A. pilosa Ledeb. Lanes 1–4 were the proteins from the NC group and Lanes 5–8 were the proteins from the 1 × MIC group. Labels (a–e) indicated the differentially expressed protein bands selected for NanoLC-ESI-MS/MS analysis.

Figure 9

The mRNA expressions of crr, rplD, pdhB, fba, and fda after treatment of the total flavonoid extracted from the A. pilosa Ledeb. The normal MHB was set as the negative control (NC). ## means p-value < 0.01 and # means p-value < 0.05.

Figure 10

The ultra-structural changes of MRSA ATCC43300 were observed by TEM. (a1,a2) were the NC group. (b1,b2) were the 1 × MIC group. 1—cell wall; 2—cytoplasm; 3—intermediate layer; 4—nucleoid; 5— “empty” areas; 6—areas filled with grains. ((a1,b1), 25,000×; (a2), 40,000×; (b2) 30,000×).