## 3 Results  
### Antibacterial Activities of MLH  
The MLH was more effective as an inhibitory agent than the muiprocin ointment. The MLH and muiprocin inhibited the growth of all bacteria tested. The results indicated that the MIC and MBC of MLH were 0.31 \(\upmu\)g mL\({}^{-1}\) and 2.44 \(\upmu\)g mL\({}^{-1}\), respectively (Table 1). In addition, the MIC and MBC of MLH from flow cytometry were 0.30 \(\upmu\)g mL\({}^{-1}\) and 2.44 \(\upmu\)g mL\({}^{-1}\), respectively. The live-cell counts obtained within 24 h showed a decrease in the number of viable bacteria. At 6 h after treatment, the MLH showed a greater decrease in the number of viable bacteria than muiprocin only, but at 24 h after treatment both compounds gave similar results. The results were similar to the treated muiprocin at the MICs. At 24 h, the viable bacteria for the MIC and MBC were 44-49% and 0.2-1.8%, respectively (Figure 1). Based on the flow cytometric analysis, it was found that the MBC of muiprocin and MLH killed the MRSA bacteria at 24 h.  
The effect of the MIC (0.30 \(\upmu\)g mL\({}^{-1}\)) concentration of MLH on MRSA is shown in Figure 2. Different sampling times were chosen to observe the inhibition of isoleucyl-tRNA synthetase in MRSA by MLH (5, 6, and 12 h). The mid-exponential phase was at 5 h of the growth curve [18] for all group samples. MRSA growth was inhibited after treatment with muiprocin for 1 h. Moreover, the control cells were in the exponential phase, but the treatment cells showed the beginnings of reduced growth during the stringent response after 6 h of the growth curve. The control cells and the treatment cells were in the stationary phase at 12 h. The effect on MRSA growth up to 24 h was significant between control cells and treatment cells. For OD600 measurements, the control cells at 5 h approached 0.3, whereas the treatment cells with MIC of muiprocin and MIC of MLH remained constant at 0.40 and 0.41, respectively, which was not a significant difference (\(p\leq 0.005\)). Muiprocin and MLH had significant effects at 6 h of the cultures (\(p\leq 0.005\)). The OD600 value for the control cells reached 0.78, whereas the OD600 values for the treated cells at the MIC of mupirocin and the MIC of MLH were 0.52 and 0.54, respectively. After 12 h, both the control cells and the treated cells were in the stationary phase. Muiprocin and MLH had highly significant effects (\(p\leq 0.0005\)). The OD600 for the control cells reached 1.358, whereas the OD600 values of the treated cells at the MICs of muiprocin and MLH were 0.49 and 0.44, respectively (Figure 2). \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Antibacterial Activities**} & \multicolumn{5}{c}{**Sample**} \\ \cline{2-6}  & & **MLH** & **Muiprocin** & **Vancomycin** & **Mupirocin** \\  & & & **Ointment** & **Ointment** & **(Pure Drug)** \\ \hline MIC & - & 0.31 & 0.61 & 1.56 & 0.31 \\ MBC & - & 2.44 & 9.76 & 1.56 & 2.44 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: MIC (\(\upmu\)g/mL) and MBC (\(\upmu\)g/mL) of muiprocin nanoparticle-loaded hydrogel, muiprocin ointment, and vancomycin. Figure 1: Flow cytometric analysis of methicillin-resistant _Staphylococcus aureus_ (MRSA) treated with a minimum inhibitory concentration (MIC) of muiprocin, MIC of MLH, MBC of MLH, and MBC of muiprocin at 24 h with fluorescein diacetate (FDA, 10 \(\upmu\)g/mL in PBS at pH 7.4), along with controls; MRSA stained with FDA. Flow cytometry used an FL1 detector and 515–545 nm fluorescence of FDA. Figure 2: Growth curve of (**a**) MRSA (control) and (**b**) MRSA treated with a MIC of muiprocin and (**c**) MRSA treated with a MIC of MLH at mid-exponential growth (5 h) (mean \(\pm\) SD, \(n=3\)). The inhibition of isoleucyl-tRNA synthetase in MRSA at the MICs of muiprocin and MLH was recorded using confocal laser scanning microscopy (Figure 3). The sample stained with Alexa Fluor(r) showed the isoleucyl-tRNA synthetase in green and the DAPI staining showed the cell nuclei in blue (Figure 3).