**1. Introduction**

The ongoing healthcare problems attributed to multidrug-resistant (MDR) bacterial infections that are difficult to treat continue to plague healthcare systems globally. Methicillinresistant *Staphylococcus aureus* (MRSA), one bacterium causing such an infection, remains a pathogen in various regions of the world [1]. Like all MDR bacterial infections, MRSAlinked infections can be associated with extended patient hospitalization, high morbidity, and mortality, especially in immunocompromised patients [1–3]. β-lactam antibiotics were the preferred drugs for the treatment of infections resulting from methicillin-resistant *S. aureus*. Vancomycin, a glycopeptide, still serves as the drug of choice in the treatment of MDR *S. aureus* infections. However, the bacterium has developed resistance to the β-lactams as well as vancomycin [4,5]. Thus, there is a decrease in the number of exogenous antibiotics for treating drug-resistant infections [3]. With a diminishing number of antimicrobials of choice, researchers have called for rationalization on the use of available antibiotics. This is intended to help reduce the rate at which MDR and pan-resistant bacterial superbugs are evolving to prevent the world from being taken back to the pre-antibiotic era [6,7]. Other suggestions are to look into alternatives to antimicrobial treatment due to

**Citation:** Badger-Emeka, L.I.; Emeka, P.M.; Ibrahim, H.I.M. A Molecular Insight into the Synergistic Mechanism of *Nigella sativa* (Black Cumin) with β-Lactam Antibiotics against Clinical Isolates of Methicillin-Resistant *Staphylococcus aureus*. *Appl. Sci.* **2021**, *11*, 3206. https://doi.org/10.3390/app11073206

Academic Editor: Hari Prasad Devkota

Received: 15 March 2021 Accepted: 31 March 2021 Published: 2 April 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

bacterial resistance to systemic monotherapy that has overtaken the rate of production of new antibiotics [8]. One such alternative is the use of a combination of antibiotics [9]. It is postulated that such combined therapeutic measures could lead to successful managemen<sup>t</sup> of patients with extreme drug-resistant (XDR) bacterial infections [10–12]. However, the double and triple combined antibiotic therapies suggested by the researchers were all colistin-based [13]. Presently, colistin is a last-line therapeutic option in the treatment of difficult bacterial infections. Besides the toxicity of the drug, there is a growing resistance to this last-line drug, as reported globally [14–17]. These reports necessitate the continued search for alternative antibiotic combinations that will exclude colistin in combined therapeutic measures [18]. There are also suggestions that such antibiotic synergistic combinations might not necessarily be the optimal options because it is thought that, as treatment progresses, there will be diminishing strength in antibiotic synergism [19]. However, as the problem of difficult-to-treat bacterial superbugs persists, the attention of researchers is currently focused on the use of herbal derivatives with antimicrobial properties, with suggestions that such plants could be synergistic enhancers [20,21]. It is thought that such combined therapy can help with expanding the spectrum of antibiotics as well as being less toxic while preventing the emergence of resistant bacteria strains [22].

Due to the expected significant advantages associated with phytotherapy, several researchers are now looking into the effectiveness of combinations of conventional antimicrobials and plant derivatives with antimicrobial properties [22–24]. This is expected to provide less toxic antimicrobial herbal alternatives that could be used as combined therapies in the treatment of XDR bacterial infections. One such plant reported by researchers in different regions of the world due to its impressive antimicrobial properties is *Nigella sativa* (NS), or black cumin [25–27]. In our previous study, we combined *N. sativa* with chloroquine in the treatment of malaria induced in mice with *Plasmodium berghei* [28]. The obtained results showed significant parasite clearance over a significant period, indicating that NS potentiated the effect of chloroquine when compared with chloroquine alone. Middle Eastern and Far Eastern countries have used *Nigella sativa* oil as a natural remedy or as a condiment/spice in food and food products from ancient times. As documented earlier, there is abundant evidence that *Nigella sativa* oil has been used in combination with synthetic medicines to treat various diseases due to its strong anti-inflammatory and antioxidant properties [28]. Studies have shown that *Nigella sativa* essential oil contains both volatile and nonvolatile bioactive compounds. It also contains alkaloids, saponins, and terpenoids, which are reported to possess antimicrobial activity [27]. Thymoquinone and thymol are the most reported active volatile constituents, possessing antitumor, antihistaminic, antidiabetic, antihypertensive, anti-inflammatory, and antimicrobial abilities. Numerous studies have reported the antimicrobial and antiparasitic activities [28].

The present work therefore looks at a possible synergistic effect of *N. sativa* with common antibiotics that show treatment failures with bacterial infections due to resistance to these drugs. The use of *N. sativa* with the aforementioned antibiotics could enhance their antimicrobial efficacy. This study aims at evaluating the synergistic effect of combining *N. sativa* with oxacillin (OXA), augmentin (AUG), and cefuroxime (CEF) on different XDR MRSA isolates, as well as looking into the possible mechanism of action of *N. sativa* on the isolates.

### **2. Materials and Methods**

### *2.1. Ethical Considerations*

Approval for the research was given by the Deanship of Scientific Research (approval number: 186388). MRSA clinical isolates were from routine hospital diagnoses for care of patients. They were from the microbial bank at the College of Medicine, Medical Microbiology division, of King Faisal University.

### *2.2. Bacterial Isolates and Antimicrobial Susceptibility Determination*

Six non-replicate clinical MRSA isolates from specimens of blood, sputum, pus, and wound swaps were used for the investigation. They had been stored in the Microbank of the Microbiology division of the College of Medicine, King Faisal University at a temperature of −80 ◦C. They were cultured on blood agar and incubated aerobically at 37 ◦C for 24 h. Isolates were identified with a Vitek compact 2 automated system (BioMerieux, Marcy L'Etoile, France) using the Gram-positive ID cards according to the guidelines of the manufacturer. The antimicrobial susceptibility and minimum inhibitory concentrations were determined using the AST-GN cards of the Vitek 2 compact automated system (BioMerieux, Marcy L'Etoile, France). They were against the following antibiotics: 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). Confirmation of resistance against imipenem (IMP), tigecycline (TG), and amikacin was by disc diffusion method (Oxoid Ltd., Basingstoke UK). All of the MRSA isolates had been confirmed by the presence of the *mecA* gene.

### *2.3. Nigella sativa and MRSA Susceptibility Test*

*Nigella sativa* (NS) black seed oil, a product of Al Hussan Food Products Factory, Riyadh was purchased from a local herbal medicine shop in Al-Ahsa, Saudi Arabia. According to the manufacturer, it is 100% pure organic cold pressed oil. Undiluted and diluted forms of the oil extract were used for the investigation. Oil extract was diluted in 0.2% dimethyl sulfoxide (DMSO) to obtain concentrations of 1000 μg/mL, 750 μg/mL, 500 μg/mL, 250 μg/mL, and 100 μg/mL dilutions, representing 100%, 75%, 50%, 25%, and 10% concentrations. In these, 0.01 mL of each concentration was used to make the actual concentrations 10, 7.5, 5.0, 2.5, and 1.0 μg/mL. The susceptibility of the isolates was tested against these dilutions of NS by the well diffusion method. Mueller-Hinton agar (MHA) was seeded with each bacterial isolate and spread out to cover the entire surface of the agar using sterile cotton swabs, moistened with the bacterial suspension [29,30]. Seven evenly distributed wells were punched into the MHA using a sterile cork borer of 6 mm diameter. The six NS dilutions were introduced into the wells aseptically, while the seventh well, serving as the control, was filled with an equal volume of 0.2% DMSO. All plates were allowed to diffuse under room temperature for 1 h according to the recommendations of the National Committee for Clinical Laboratory standards (NCCLS). All plates were incubated aerobically at 37 ◦C for 24 h in an upright position. Experiments were carried out in three replicates with zones of inhibition measured in millimeters (mm) 24 h post-incubation, and the results are presented as means ± standard deviation.

### *2.4. Determination of Time–Kill Assay*

The CLSI guidelines were used for the determination of the time–kill assay using a nutrient broth (NB) [31]. MRSA bacteria strains 1, 4, and 5, which were randomly selected from six phenotypically different isolates, were inoculated into tubes containing NB to form a 5 × 10<sup>5</sup> CFU/mL bacteria suspension. They were each tested against combinations of NS, OXA, AUG, and CEF and for the time–kill assay. Tubes contained NB, a bacterial strain, and NS, while a second set had the bacterial strain, NB, NS, and one antibiotic. According to the McFarland standard, all bacterial suspensions were diluted to about 10<sup>6</sup> CFU/mL and incubated aerobically at 37 ◦C in an orbital shaker for 48 h. At hours 0, 1, 3, 6, 24, and 48, 2 mL of each suspension were drawn out from each tube and inoculated into blood agar. The inoculum was spread out using sterile inoculating loops, with all plates incubated aerobically at 37 ◦C for 24 h. The viability of the MRSA isolates was evaluated by counting the bacterial colonies or noting the lack of microbial growth [3], while time–kill analysis curves were plotted with synergism, defined as described earlier [32,33].

### *2.5. Antimicrobial Synergistic NS and β-Lactam Antibiotics Assay*

Bacterial strains were tested against the two concentrations of NS (7.5 μg/mL and 5.0 μg/mL) and each with the combination of the three β-lactam antibiotics to evaluate the synergistic activity with oxacillin (OXA), augmentin (AUG), and cefuroxime (CEF). The antimicrobial agents were used at their break points (OXA: 1 mg; AUG: 20/10 mg; CEF: 30 mg). Synergistic assay on MRSA strains 1, 4, and 5 was accessed using a spectrophotometer (BOECO, Hamburg Germany) with the additive/synergy effect calculated as earlier described [32,34,35]. Accordingly, the fractional inhibitory concentration (FIC) of each agen<sup>t</sup> was calculated using the following formula:

OD600 of wells of agen<sup>t</sup> in combination with the drug.

OD600 of wells of agen<sup>t</sup> alone.

### *2.6. Molecular Assay by SEM and TEM Microscopy*

SEM and TEM imaging were performed to evaluate the treatment effects on both the bacterial surface and within bacterial cells. The previously described method was used for the preparation of MRSA bacterial samples for scanning electron microscopy (SEM) [36]. Treated bacterial cultures as well as untreated samples that served as controls were incubated in a shaker incubator at 37 ◦C for 24 h. The resulting samples were centrifuged and prefixed in a 2.5% glutaraldehyde solution at 4 ◦C for 24 h, after which they were rinsed in PBS and post-fixed with 100% acetone applied at the last stage. Gold sputtering was used to obtain a layer 20 nm thick. The images were obtained with SEM (JSM 6390 LA, JEOL, Tokyo, Japan) at a 15 KV accelerating voltage.

Preparation and observation of TEM microscopy samples is as described previously, but with modifications [37,38]. Slices of selected MRSA bacterial samples were fixed by immersion in 2.5% glutaraldehyde (GA) in a 0.1 M sodium cacodylate buffer with pH 7.4 at a temperature of 4 ◦C for 1 h, then washed in cacodylate buffer. All bacterial samples were double-fixed in 1% osmium tetroxide (OsO4) in a cacodylate buffer at room temperature for 90 min. They were then dehydrated in acetone and embedded in Epon-Araldite (502 kit, Pelco, CA, USA). Then, 500–1000 nm sections of bacteria samples were obtained in a Leica EM UC6 (Wetzlar, Germany), ultra-microtome mounted on glass slides, stained with 1% toluidine blue stain. For light microscopy observations, bacterial samples were further dissected to 50-70 nm [37]. Staining of Epon-sectioned samples was carried out with the Leica automated EM stain (0.5% uranyl acetate and 3% lead citrate). All samples' sections were scanned and examined using JEM 1011 (JEOL) electron microscope at 80 kv.

### *2.7. Gas Chromatography/Mass Spectrometry (GC/MS) Analysis*

The extract of 100% pure organic cold pressed oil of *Nigella sativa* was analyzed by gas chromatography/mass spectrometry (GC/MS), and data were recorded on a GCMS-QP2010 Plus (Shimadzu Corporation, Kyoto, Japan). The column used was a RTX-5MS® fused silica capillary (30 m × 0.25 mm i.d and 0.25 μm film thickness); the oven temperature initially was held at 45 ◦C for 2 min and then increased to 280 ◦C at 4 ◦C/min. The carrier gas was helium with a flow rate of 2.0 mL/min; the temperature of the injector and detector were 250 and 300 ◦C, respectively, the split ratio was 1:30, and the injection volume was 1 μL. The ionization energy for the mass spectrometer was 70 eV. Identification of components was confirmed from the mass spectra library.

### *2.8. Statistical Analysis*

Data are presented as the mean ± SD and the susceptibility analysis is presented in percentages. GraphPad Prism version 8.0 (San Diego, CA, USA) statistical software was used for statistical interpretation of the results. Two-way ANOVA, applying Tukey's multiple comparison test, was used to assess the statistical significance of zones of inhibition of various concentrations of NS on the different MRSA isolates. Additionally, one-way ANOVA, multiple comparison was used to compare the time–kill assay results and to

determine the existence of any significant differences. Significance was taken to be *p* < 0.05 and *p*-values were calculated between groups.
