Synergistic Antibiotic Activity of Ricini Semen Extract with Oxacillin against Methicillin-Resistant Staphylococcus aureus

Abstract

Resistant bacteria are emerging as a critical problem in the treatment of bacterial infections by neutralizing antibiotic activity. The development of new traditional mechanisms of antibiotics is not the optimal solution. A more reasonable approach may be to use relatively safe, plant-based compounds in combination with conventional antibiotics in an effort to increase their efficacy or restore their activity against resistant bacteria. We present our study of mixing Ricini Semen extract, or its constituent fatty acids, with oxacillin and testing the effects of each on the growth of methicillin-resistant Staphylococcus aureus. Changes in the cell membrane fluidity of methicillin-resistant S. aureus were found to be a major component of the mechanism of synergistic antibiotic activity of Ricini Semen extract and its constituent fatty acids. In our model, changes in cellular membrane fluidity disrupted the normal function of bacterial signaling membrane proteins BlaR1 and MecR1, which are known to detect oxacillin, and resulted in the incomplete expression of penicillin-binding proteins 2a and β-lactamase. Utilizing the mechanism presented in this study presents the possibility of developing a method for treating antibiotic-resistant bacteria using traditional antibiotics with plant-based compounds.

Keywords: Ricini Semen extract; membrane fluidity; methicillin-resistant Staphylococcus aureus; oxacillin; synergistic antibiotic activity.

Figure 1

Effect on the growth of MRSA CCARM 3807 and MRSA CCARM 3820. In (A), cell growth of MRSA CCARM 3807 was tested without compounds as a control (●), with 64 mg/L of oxacillin (○), with 64 mg/L of Ricini Semen extract (■), with 64 mg/L of Ricini Semen extract combined with 64 mg/L of oxacillin (☐), with 32 mg/L of ricinoleic acid (▲), and with 32 mg/L of ricinoleic acid combined with 64 mg/L of oxacillin (△). In (B), cell growth of MRSA CCARM 3807 was tested without compounds as a control (●), with 64 mg/L of oxacillin (○), with 128 mg/L of palmitic acid (■), with 128 mg/L of palmitic acid combined with 64 mg/L of oxacillin (☐), with 128 mg/L of stearic acid (▲), and with 128 mg/L of stearic acid combined with 64 mg/L of oxacillin (△). In (C), cell growth of MRSA CCARM 3820 was tested without compounds as a control (●), with 4 mg/L of oxacillin (○), with 64 mg/L of Ricini Semen extract (■), with 64 mg/L of Ricini Semen extract combined with 4 mg/L of oxacillin (☐), with 64 mg/L of ricinoleic acid (▲), and with 64 mg/L of ricinoleic acid combined with 4 mg/L of oxacillin (△). In (D), cell growth of MRSA CCARM 3820 was tested without compounds as a control (●), with 4 mg/L of oxacillin (○), with 128 mg/L of palmitic acid (■), with 128 mg/L of palmitic acid in combination with 4 mg/L of oxacillin (☐), with 128 mg/L of stearic acid (▲), and with the combination of 128 mg/L of stearic acid and 4 mg/L of oxacillin (△). All components of this experiment were repeated three times, and the mean and standard deviations were calculated.

Figure 2

Evaluating the synergistic activity on S. aureus. To evaluate the synergistic activity, the cell density was measured in colony-forming units (CFU) after 24-h incubation. In (A), the cell density of MRSA CCARM 3807 was measured after 24 h incubation with either 64 mg/L of oxacillin, 64 mg/L of Ricini Semen extract, or 32 mg/L of ricinoleic acid. In (B), the cell density of MRSA CCARM 3807 was measured after 24 h incubation with either 64 mg/L of oxacillin, 128 mg/L of palmitic acid, or 128 mg/L of stearic acid. In (C), the cell density of MRSA CCARM 3820 was measured after 24 h incubation with either 4 mg/L of oxacillin, 64 mg/L of Ricini Semen extract, or 64 mg/L of ricinoleic acid. In (D), the cell density of MRSA CCARM 3820 was measured after 24 h incubation with either 4 mg/L of oxacillin, 128 mg/L of palmitic acid, or 128 mg/L of stearic acid. Each experiment was repeated three times, and the mean and standard deviations were calculated.

Figure 3

Cellular membrane fluidity changes induced by Ricini Semen extract and its constituent fatty acids. Laurdan generalized polarization (GP) decreases when cell membrane fluidity increases and increases when fluidity decreases. In (A), the membrane fluidity of MRSA CCARM 3807 was measured with 64 mg/L of oxacillin, 64 mg/L of Ricini Semen extract, or 32 mg/L of ricinoleic acid. In (B), the membrane fluidity of MRSA CCARM 3807 was measured with either 64 mg/L of oxacillin, 128 mg/L of palmitic acid, or 128 mg/L of stearic acid. In (C), the membrane fluidity of MRSA CCARM 3820 was measured with either 4 mg/L of oxacillin, 64 mg/L of the Ricini Semen extract, or 64 mg/L of ricinoleic acid. In (D), the membrane fluidity of MRSA CCARM 3820 was measured with either 4 mg/L of oxacillin, 128 mg/L of palmitic acid, or 128 mg/L of stearic acid. Each of these experiments was performed three times independently, and the mean and standard deviations were calculated.

Figure 4

Gene expression change in SCCmec element induced by Ricini Semen extract and its constituent fatty acids. The relative expression levels of mecA (black bars), mecR1 (slashed bars), and blaR1 (gray bars) were calculated by comparing them with the expression level of 16S rRNA measured using RT-PCR. In (A), the relative gene expression of MRSA CCARM 3807 was measured after 3-h incubation with 64 mg/L of oxacillin, 64 mg/L of Ricini Semen extract, or 32 mg/L of ricinoleic acid. In (B), the relative gene expression of MRSA CCARM 3807 was measured after 3 h incubation with either 64 mg/L of oxacillin, 128 mg/L of palmitic acid, or 128 mg/L of stearic acid. In (C), the relative gene expression of MRSA CCARM 3820 was measured after 3 h incubation with either 4 mg/L of oxacillin, 64 mg/L of the Ricini Semen extract, or 64 mg/L of ricinoleic acid. In (D), the relative gene expression of MRSA CCARM 3820 was measured after 3 h incubation with either 4 mg/L of oxacillin, 128 mg/L of palmitic acid, or 128 mg/L of stearic acid. Each of these experiments was performed in duplicate.