**4. Discussion**

The MRSA isolates examined here were extremely drug-resistant and fit into the classification of XDR, as they were resistant to aminoglycosides, β-lactams, fluoroquinolones, macrolides, and tetracycline [39–41]. However, this study has shown that *Nigella sativa* (NS), a naturally derived herbal product, inhibits the growth of these XDR MRSA isolates, thereby confirming earlier findings. This suggests that NS derivatives could serve as possible alternatives in the managemen<sup>t</sup> of extreme drug-resistant (XDR) MRSA isolates. The inherent antimicrobial potential of NS derivatives had been reported as equally effective as that of clinically available antibiotics [26,32,42]. All these reports demonstrated better inhibition of MRSA isolates by NS as compared to commonly used antibiotics, as also seen in the present study.

In this investigation, the isolates responded differently to various concentrations of NS, as seen in the exhibited zones of inhibition. Additionally, although there were differences among the isolates in zones of inhibition, all NS concentrations, including the lowest concentration of 0.1 μg/mL, were able to hinder bacterial growth, a finding that is similar to earlier reports [32,43]. Differences in zones of inhibition could be attributed to phenotypic differences in the MRSA strains. However, that the lowest concentration of NS exhibited growth inhibition against the isolates in this study points to its effectiveness as a potential antimicrobial.

The study also shows the time–kill kinetics for the MRSA strains used in this study to be more rapid in NS and β-lactam combined therapies than with NS alone. This suggests the possibility of a combined additive effect or potentiation of NS to those of the antibiotics that the MRSA isolates had previously been resistant to. These findings are in agreemen<sup>t</sup> with earlier suggestions for the need to develop a synergistic herbal drug product that could be suitable for use in combined therapeutic measures to combat the scourge of bacterial resistance [44–46]. Phytotherapy combinations with proven synergistic efficacy would not only have a multi-targeted mechanism of action but could be a main tool in delaying, alleviating, or reducing resistance to existing antimicrobials [32,44]. Additionally, our findings show NS synergistic activity in combination with OXA, AUG, and CEF against XDR MRSA isolates, with the best effect seen in the NS-AUG combination as opposed to NS-OXA and NS-CEF, similar to an earlier report [32]. This reported that, in NS βlactam synergism, there was a reversal in resistance to augmentin by MRSA, findings that are similar to those of this investigation. An earlier report on the synergistic effects of combining the by-products contained in the plant with conventional antimicrobial drugs found improved effects compared to antimicrobials alone [47]. This might explain why all NS β-lactam combinations were more effective than NS or the antibiotic alone. On the other hand, that the combination of 0.5 mg/mL NS and β-lactam showed varying results (Figure 3D-F), with complete killing seen with MRSA 1, suggests that differences in bacterial strains could be a contributory factor, as had been reported previously [25].

However, that there was better killing against the same MRSA 4 and 5 isolates (Figure 3E,F) in the 7.5 μg/mL NS and antibiotic combinations could point to a dose-dependent NS concentration, a view expressed earlier [32]. Molecular insight was gained by examining the probable mechanism of action of NS alone and with β-lactam antibiotic combinations using SEM and TEM techniques. A SEM micrograph analysis showed bacterial cell aggregation without the usual well-defined clustering of *S. aureus*. This finding is consistent with previous studies that reported the effect of antibiotics on bacterial cells [48,49]. NS alone disrupted the bacterial cell surface and therefore caused destruction, which was evident in the bacterial growth inhibition. However, in the combinations of NS with OXA and AUG, where there was marked cellular aggregation/cell destruction, we can point to the possible synergistic/additive effects of NS, as shown in the FIC results in Tables 2 and 3, all of which created significant changes in the molecular ultrastructure that Gram-positive bacteria such as MRSA use for survival. It could also mean that binding of β-lactam antibiotics to penicillin binding protein (PBP) sites was significantly enhanced. Note that the isolates used in this study exhibited marked antimicrobial resistance. The TEM micrograph analysis also confirmed this view. It showed cell death and cell surface disruption, similar to the ultrastructural changes in the MRSA isolates from a previous study with NS alone [32]. The molecular structural changes seen in the TEM micrographs could also be due to interference in the transportation or the synthesis of PBP by NS. Such cell wall destruction, either by NS or in combination with β-lactam antibiotics, would be expected to remove the bacterial protective barriers, thus leading to cell lysis, as shown by the TEM micrograph results.

Generally, the mode of action of augmentin as a β-lactam is that it leads to the inhibition of bacterial cell wall peptidoglycan by binding and inhibiting PBPs [50]. It is documented that antimicrobial resistance results from the activation of the Mec A gene and its variants, which leads to the formation of penicillin-binding protein 2A (PBP2a), which binds to β-lactams, thus creating resistance [50]. However, we noted that alkaloid plant components, alone or when combined with conventional antibiotics, could have an effect on bacterial acquired resistance, which could explain the NS and antibiotic combined effects seen in this investigation [3].

*N. sativa* oil is rich in diverse volatile compounds involved in various biological activities [51]. The GC-MS results confirmed that *N. sativa* essential oil is a good source of bioactive components such as p-cymene, linalool, thymoquinone, transanethole, and mthymol, which have been evaluated by various reports and are considered to possess good antimicrobial activity [51–54]. Earlier reports documented synergistic effects of the extracts of black cumin with streptomycin and gentamicin against antibiotic sensitive *S. aureus* and other bacterial isolates [55,56]. Thymoquinone was indicated to exert synergism with the antibiotics against *S. aureus* [56]. Thus, thymoquinone an abundant component of *N. sativa* is reported to be the bioactive compound with multiple pharmacological actions including being a strong antimicrobial [55,57]. Additionally, as other reports show synergistic effects when thymoquinone is combined with synthetic and natural compound, the present study further confirms that the contents of *N. sativa* essential oil are potent at inhibiting bacterial activity [54,58].
