**3. Results**

### *3.1. Antimicrobial Susceptibility of the MRSA Isolates*

The MRSA isolates in this investigation were highly resistant to other antibiotics, with the exception of tigecycline and gentamicin, with resistance of 17% and 33%, respectively (Figure 1). The minimum inhibitory concentration (MIC) results are shown in Table 1. Differences in antimicrobial susceptibility and MIC values show that the isolates differ phenotypically. This difference is also seen in the antimicrobial susceptibility to the dilutions of NS, as shown in Figure 2 for all the MRSA isolates. All the isolates were inhibited by the various concentrations of NS, including the lowest concentration of 0.1 μg/mL, as shown in Figure 2. The least effective of the NS concentrations against the MRSA isolates was 0.1 μg/mL, which exhibited the lowest inhibitory effect among the isolates, with the exception of MRSA 2. Overall, there was no specific pattern in antimicrobial inhibition, as all the isolates responded differently to the NS dilutions. The mean zone of inhibition for MRSA 5 and 6 was more with the 10 μg/mL NS concentration, while MRSA 2 and 3 were inhibited more by the 7.5 and 5.0 μg/mL NS concentrations. For all the isolates, a comparison of differences in mean zones of inhibition between 10.0 μg/mL NS and 7.5, 5.0, and 2.5 μg/mL was not found to be statistically significant.

**Figure 1.** Antimicrobial resistance of methicillin-resistant *Staphylococcus aureus*. Augmentin (AUG), benzyl penicillin (BENZ), oxacillin (OXA), cefuroxime (CEF), cefuroxime/axetil (CEF/AXE), clindamycin (CLD), amikacin (AK), imipenem (IMP), ciprofloxacin (CIP), levofloxacin (LEVO), erythromycin (ERY), sulfamethoxazole/trimethoprim (SXT), tigecycline (TG), tetracycline (TET), rifampicin (RIF), and gentamicin (GEN).

However, comparisons of the mean zones of inhibition for all the isolates with a 10 μg/mL NS concentration and with 1.0 μg/mL as well as 0.1 μg/mL showed statistically significant differences, with *p*-values of 0.034 and 0.0001, respectively. Additionally, comparisons of the mean zones of inhibition resulting from 7.5 μg/mL, when compared with those resulting from the lower NS concentrations, were statistically similar to those at

10.0 μg/mL NS. Differences in zones of inhibition between this 7.5 μg/mL NS concentration and the 1.0 μg/mL were significant (*p*-value: 0.0012).



**Figure 2.** Showing the zones of mean ± SD inhibition of methicillin-resistant *Staphylococcus aureus* (MRSA), MRSA 1–6 bacterial isolates against *Nigella sativa* (NS) dilutions. Comparison of 10 μg/mL and other concentrations. \* *p*-value: 0.034; \*\* *p*-value: 0.0001.

MRSA 1 was the isolate that was most inhibited by the 5.0 μg/mL NS dilution. As with the higher NS concentrations, differences in mean zones of inhibition were significant when compared with those of the 1.0 and 0.1 μg/mL NS, with *p*-values of 0.045 and 0.0002, respectively. For the 2.5 μg/mL NS concentration, statistical differences in inhibition zones were significant when compared to the lower NS dilutions (Figure 2).

### *3.2. Time–Kill Kinetics*

Results on the effect of NS alone, and NS in combination with oxacillin (OXA), augmentin (AUG), and/or cefuroxime (CEF) are shown in Figure 3A-F. There were variations in antimicrobial effects based on strains of MRSA, as well as the type of antibiotic tested. A rapid rate of killing was seen more often with combinations of NS and antibiotics than with NS alone.

**Figure 3.** Time–kill assay curves of 7.5 μg/mL concentration of *Nigella sativa* (NS) alone and in combination with antibiotics against (**A**) MRSA 1, (**B**) MRSA 4, and (**C**) MRSA 5 bacterial isolates. (**A**) \*\*\* Represents significant values at *p* < 0.0039 for NS + OXA, *p* < 0.0016 for NS + AUG and *p* < 0.0142 for NS + CEF; (**B**) \*\*\* Represents significant values at *p* < 0.0113 for NS + OXA, *p* < 0.0061 for NS + AUG and *p* < 0.0053 for NS + CEF; (**C**) \*\*\* Represents significant values at *p* < 0.0179 for NS + OXA, *p* < 0.0336 for NS + AUG and *p* < 0.0180 for NS + CEF. Time–kill assay curves of 5.0 μg/mL concentration of *Nigella sativa* (NS) alone and in combination with antibiotics against (**D**) MRSA 1, (**E**) MRSA 4, and (**F**) MRSA 5 bacteria isolates. (**D**) \*\*\* Represents significant values at *p* < 0.0408 for NS + OXA, *p* < 0.0200 for NS + AUG and *p* < 0.0494 for NS + CEF; (b) \* Represents significant values at *p* < 0.0331 for NS + OXA, *p* < 0.0083 for NS + AUG, and *p* < 0.0254 for NS + CEF; (**F**) \* Represents significant values at *p* < 0.0161 for NS + OXA, *p* < 0.0019 for NS + AUG and *p* < 0.0022 for NS + CEF.

In the combination of AUG with 7.5 μg/mL NS, the killing rate for MRSA 1 was more rapid than in combinations of NS with CEF and OXA in a time-dependent fashion, as shown in Figure 3A-C. Additionally, differences in these killing times were statistically significant when compared with the control (7.5 μg/mL NS alone). However, for the MRSA 1 time–kill kinetics, although antibiotic combinations with NS displayed a significantly difference in killing time, the time–kill kinetic curve between AUG and OXA did show a statistically significant difference, indicating that time–kill with AUG was more rapid. The results of the MRSA 5 time–kill assay showed significant differences between the NS and antibiotic combinations and NS alone. The killing times for 0-24 h were markedly different from those for 48 h. Additionally, AUG displayed more rapid killing with respect to CEF and OXA. For this group of 7.5 μg/mL NS with antibiotic combination total killing was seen within 24 h for MRSA 1 and 48 h for MRSA 4 and 5 (Figure 3A–C).

The time–kill assay of the 5 μg/mL NS and antibiotic combination is shown in Figure 3D–F for the MRSA isolates 1, 4, and 5. The results showed complete killing for MRSA 1 at 48 h with combinations as compared with the effect of NS alone. The killing was rapid and statistically significant, with AUG showing a more rapid effect as compared to the control. In MRSA 4, the killing was not total but again AUG displayed rapid killing kinetics with CEF, with OXA showing similar rates of killing (Figure 3E). Additionally, for MRSA 5, there was no total killing after 48 h. However, AUG displayed more rapid killing compared to CEF and OXA. In addition, the antibiotic combinations' time–kill kinetics were statistically significant compared to NS alone.

### *3.3. Synergistic Effect of NS and β-Lactams with FIC Index Analysis*

The results presented in Table 2 are those of the FIC index interpretation that defines the time–kill assay synergistically for antibiotics and NS combinations. Combinations of 5.0 μg/mL NS with antibiotics on MRSA 1 displayed synergism. The best effect was with AUG, while the worst was with OXA. Synergism was observed within 4 h on isolates that exhibited extensive resistance to antibiotics. In MRSA 4, a similar pattern was observed, except that synergistic effects were seen for AUG and CEF within 4 h, while with OXA, an additive effect was mostly displayed. For MRSA 5, the combination of NS with CEF had an additive effect compared with AUG and OXA, which displayed synergism within 8 h. Overall, the study with 5 μg/mL combinations clearly indicates a synergistic effect.


**Table 2.** Fractional inhibitory concentration index synergy interpretation of different MRSA isolates measured at time intervals for combinations of 5.0 μg/mL *N. sativa* and antibiotics.

NS = *Nigella sativa*, OXA = oxacillin; AUG = augmentin; CEF = cefuroxime. The interaction was defined as synergistic if the FIC index was less than or equal to 0.5, additive if the FIC index was greater than 0.5 and less than or equal to 1.0, indifferent if the FIC index was greater than 1.0 and less than or equal to 2.0, and antagonistic if the FIC index was greater than 2.0.

The synergistic time–kill assay, as interpreted with the FIC index for a 7.5 μg/mL NS combination with antibiotics, is shown in Table 2. The combination of OXA, AUG, and CEF antibiotics all displayed synergism, with AUG and CEF exhibiting the best synergism up to 12 h. The pattern of synergistic effect on MRSA 4 was different, however. AUG had the best effect, but only for 4 h, which was then followed by additive effects. In MRSA 5, NS OXA combinations only produced additive effects. However, synergism was only observed for 2 h, as indicated in Table 2 for AUG and CEF combination. The overall FIC index ranged from 0.05 to 0.7, indicating synergism and additive effects.

### *3.4. SEM and TEM Assay on Effect of Combined NS, Antibiotic Treatment*

The structural appearance of MRSA treated with concentrations of NS and in combination with antibiotics was analyzed using SEM. Figure 4A shows MRSA 4 clusters displaying well-formed shapes, while bacterial cell surface disruption is seen in 4B after treatment with 5.0 μg/mL NS. In Figure 4C,D, cellular aggregation of bacteria and cell destruction is seen in combined treatments of NS with oxacillin and augmentin. In Figure 4E,F, similar destruction of bacteria cell surface and cellular aggregation were observed in the combined treatment of MRSA 5 with 7.5 μg/mL NS, augmentin, and cefuroxime. Figure 5A–F represent TEM monograph analysis of the different MRSA isolates with NS 5 and 7.5 μg/mL concentrations. Figure 5A shows altered MRSA 5 cell membranes and dead bacterial cells after exposure to 5.0 μg/mL NS. The figure also shows disruption of bacterial cell division. Figure 5B also shows MRSA 4 cell wall damage after exposure to 7.5 μg/mL NS with oxacillin, as well as vacuoles created in the cytoplasm and perhaps the accumulation of NS within the cell. Figure 5C shows dead bacteria cells and a damaged cell wall in MRSA 4 treated with 7.5 μg/mL and augmentin, while Figure 5D displays MRSA 4 isolates treated with a combination of 7.5 μg/mL and cefuroxime. Figure 5E shows the effects of MRSA 1 treated with 5.0 μg/mL NS plus oxacillin and Figure 5F shows the effect on MRSA 1 treated with 5.0 μg/mL NS plus augmentin, showing cell wall destruction (black arrows). These micrographs show that NS disrupts the MRSA bacterial cell wall, thereby causing cell death. In combination with β-lactam antibiotics, these effects were potentiated and can be described as a bactericidal effect.

TEM interpretation of combined treatment of NS and antibiotics is shown in Figure 6A–F. The figure gives TEM monographs of the different MRSA bacterial isolates treated with a combination of 7.5 μg/mL NS concentrations and antibiotics. Figure 6A shows damaged MRSA 4 cell walls, disrupted bacterial replication, and cell death with oxacillin. The figure also shows more cell death due to fewer cells, with similar observations seen with MRSA 5 in Figure 6B. In combination treatments with augmentin on MRSA 1, Figure 6C shows more cell deaths, while Figure 6D also displays cell death and the disruption of cell division in MRSA 5. Additionally, the TEM images in Figure 6E show the effect of treatments with 7.5 μg/mL NS in combination with cefuroxime led to MRSA 1 complete and incomplete cell wall disrupted and bacterial cell death. Figure 6F shows complete destruction by 7.5 μg/mL NS plus cefuroxime in MRSA 5. These effects clearly indicate that the combinations were bactericidal.

### *3.5. Results of GC-MS Analysis*

The GC-MS analysis of volatile compounds contained in *N. sativa* oil used in this study showed that the major components were thymoquinone at 7.85%, *p*-cymene at 5.18%, trans-anethole at 1.52%, and linalool at 1.12%, as presented in Table 3 and Figure 7.


**Table 3.** GC-MS data.

**Figure 4.** SEM micrographs of untreated, NS, and NS plus β-lactam antibiotics treated methicillin-resistant *S. aureus* isolates. (**A**) Control methicillin-resistant *Staphylococcus aureus* (MRSA 4) without any treatment, showing clusters of bacteria. (**B**) MRSA 4 treatment with 5.0 μg/mL *Nigella sativa* (NS), displaying surface alterations and destruction. (**C**) SEM of MRSA 4 treated with a combination of 5.0 μg/mL NS and oxacillin. (**D**) Augmentin combination with the same concentration of NS, showing surface destruction of *S. aureus*. (**E**) MRSA 5 at 2700× magnification showing 7.5 μg/mL NS treatment in combination with augmentin. (**F**) Combination treatment with cefuroxime and 7.5 μg/mL NS at 1800× showing MRSA 5 bacteria cluster surface destruction.

**Figure 5.** TEM micrographs of methicillin-resistant *Staphylococcus aureus* (MRSA) treated with NS and NS plus β-lactam antibiotics. (**A**) 5.0 μg/mL of NS at 25,000× showing MRSA 5 cell wall destruction (black arrow). (**B**) 5.0 μg/mL NS plus oxacillin showing MRSA 4 cell death at 50,000× magnification (blue arrow). (**C**) MRSA 5 treated with NS 5.0 μg/mL plus augmentin at 25,000× showing incomplete cell wall destruction (black arrow) and (**D**) MRSA 4 treated with 5.0 μg/mL NS plus cefuroxime showing cell death (blue arrow) at 40,000× magnifications. (**E**) MRSA 1 treated with 5.0 μg/mL NS plus oxacillin and (**F**) MRSA 1 treated with 5.0 μg/mL NS plus augmentin, showing cell wall destruction (black arrows) at a magnification of 30,000<sup>×</sup>.

**Figure 6.** TEM micrographs of methicillin-resistant *Staphylococcus aureus* (MRSA) treated with NS and NS plus β-lactam antibiotics. (**A**) 7.5 μg/mL NS plus oxacillin at a magnification of 40,000<sup>×</sup>, showing incomplete destruction of MRSA 4 cell wall (black arrow) and (**B**) 7.5 μg/mL NS plus oxacillin, showing incomplete of destruction of MRSA 5 cell wall (blue arrow). (**C**) MRSA 1 treated with 7.5 μg/mL NS plus augmentin, showing dead bacterial cells at a magnification of 30,000× (blue arrow). (**D**) MRSA 5 treated with 7.5 μg/mL NS plus augmentin, showing complete cell wall destruction. (**E**) 7.5 μg/mL NS plus cefuroxime, showing incomplete (black arrow) and complete (blue arrow) destruction of MRSA 1 cell wall at a magnification of 80,000<sup>×</sup>. (**F**) Complete destruction by 7.5 μg/mL NS plus cefuroxime of MRSA 5 cell wall at a magnification of 40,000× (blue arrow).

**Figure 7.** Chromatogram of *N. Sativa* oil characterized by GC-MS analysis.
