In Vitro Investigation of the Antibacterial Activity of Nigella sativa Oil on Some of the Most Commonly Isolated Bacteria in Otitis Media and Externa.

Objective
This study aimed to evaluate the antibacterial efficacy of Nigella sativa (NS) seed oil against the most frequently isolated infectious bacteria of the middle and external ear.


Materials and Methods
The in vitro antibacterial activity of NS oil was evaluated against 34 clinical isolates of Streptococcus pneumoniae, 32 clinical isolates of Moraxella catarrhalis, 32 clinical isolates of Haemophilus influenzae, and 32 clinical isolates of Pseudomonas aeruginosa. Staphylococcus aureus, Escherichia coli, and P. aeruginosa were also evaluated for their sensitivity to the NS oil. The minimum inhibitory concentration (MIC) of the NS oil was determined via a broth dilution technique. Serial solutions were prepared in a Mueller Hinton-F broth to achieve an ultimate concentration of NS oil within the microplate wells ranging from 256 μg/mL to 0.25 μg/mL. The growth control wells and medium were used for each bacterial strain, and the microplates were incubated at 35°C for 24 h. Those wells having no visible growth and the lowest concentration of NS oil were accepted as showing the MIC.


Results
In this study, a comparison was made between NS oil and the various antibiotics known to be effective against the bacterial strains mentioned above. The NS was shown to have bactericidal activity against H. influenzae, M. catarrhalis, and S. pneumoniae. However, the NS was not found to be effective against P. aeruginosa at any concentration.


Conclusion
The results of this laboratory-based study support the use of NS oil as an alternative treatment for ear infections. However, it is necessary to conduct clinical studies to evaluate the antibacterial efficacy of NS oil on patients with ear infections.


Introduction
Acute otitis media (AOM), otitis media with effusion (OME), and otitis externa (OE) are the most commonly seen diseases in otorhinolaryngology clinics [1,2]. Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are the most frequently isolated bacteria in AOM and OME cases [3][4][5][6]. These three bacterial species are responsible for 95% of all AOM cases [7]. OE is an inflammatory disease of the external ear canal and auricula [8]. Fungi and other bacteria may play a role in the etiology, but the two most frequently isolated bacterial species are Pseudomonas aeruginosa and Staphylococcus aureus [9].
In recent years, due to the increase in bacterial resistance, there has been some debate about the selection and use of antibiotherapy in the treatment of AOM and OME. Based on the current guidelines, the use of systemic antibiotics in OME is not recommended [10]. To combat bacterial resistance, it has been suggested that the dose of amoxicillin used as the first step of treatment in AOM should be increased from 40-50 mg/kg to 80-90 mg/kg [11,12]. The overall emergence of antibiotic resistance has led to investigations into different options for treatment. Priority in medical treatment of otitis externa is directed to pain. In most cases, full cure is achieved with analgesic and topical agents [13]. Topical fluroquinolone group agents known to be non-toxic with acidifying agents in the external ear canal are recommended for use with steroidal drops [14].
Nigella sativa (NS), which is a member of Ranunculaceae family, is also known as black seed or black cumin [15]. It is an annual flowering plant that grows in Southern Asian countries, and it has been In Vitro Investigation of the Antibacterial Activity of Nigella sativa Oil on Some of the Most Commonly Isolated Bacteria in Otitis Media and Externa Esra Kocoglu 1 , M. Tayyar Kalcioglu 2 , Lokman Uzun 2 , Ferhat Zengin 1 , Serdal Celik 2 , Serkan Serifler 2 , Hanife Gulbay 3 , Nevriye Gonullu 3 used for treatment in various branches of medicine for over 2000 years [16]. It has been shown that most of the biological activities of the seeds are dependent on thymoquinone, the main component of the volatile oil found in the seed oil. Its antibacterial efficacy against bacteria, especially Gram-positive bacteria, has been previously demonstrated [17][18][19]. In their study, Hariharan et al. [20] proved the efficacy of thymoquinone against the frequently seen methicillin-resistant S. aureus. However, as far as we know, there are no specific studies in the literature about the most frequently isolated bacteria in AOM, OME, and EO cases. In this study, we aimed to investigate the antibacterial efficacy of NS in a laboratory environment on the most frequently isolated bacteria in AOM, OME, and OE, which are S. pneumoniae, P. aeruginosa, M. catarrhalis, and H. influenzae.

Materials and Methods
This study began after receiving permission from the local ethics committee (4 April, 2017; 2017/0120).

Bacterial Isolates
A total of 34 strains of S. pneumoniae, 32 of M. catarrhalis, 32 of H. influenzae, and 32 of P. aeruginosa were isolated from patients with respiratory tract infections. The identification of isolated bacteria was studied using VITEK 2 automated systems (bioMerieux, France), and they were stored at -80°C until they were used in this study. Before this research began, the M. catarrhalis and H. influenzae strains were cultivated on chocolate agar, and the S. pneumoniae and P. aeruginosa were cultivated on 5% Sheep blood agar.

Antibiotic Efficacy and Minimum Inhibitory Concentration Tests
The antibiotic susceptibilities of the strains included in this study were determined via the disk diffusion method and according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standards [21]. The used antibiotic discs (Thermo Fisher Scientific Oxoid Ltd., Basingstoke, UK) were stored at -20°C until the time of testing.
The media (Merck, Darmstadt, Germany) used for the susceptibility testing and to determine the minimum inhibitor concentration (MIC) of the NS on the antimicrobial activity were prepared under laboratory conditions, in accordance with the strains to be studied and the aforementioned standards. The NS MIC was determined via the microdilution method. For this purpose, a stock solution of 512 μg/mL of NS was prepared using dimethyl sulfoxide. The serial dilutions of the NS were prepared from 256 μg/mL to 0.25 μg/mL in a Mueller Hinton-F (MH-F) broth (with 5% (v/v) mechanically defibrinated horse blood and 20 mg/L NAD). Then, 100 μL of the 0.5 McFarland suspension of the clinical isolates and control strains was added to each well. The final NS concentrations of the wells were in the range of 128-0.125. The microplates were incubated at 35°C for 24 h. The lowest concentration at which no visible reproduction was found was accepted as the MIC value of the NS.

Quality Control of Strains
Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), and P. aeruginosa (ATCC 27853) strains were used for both the antibiotic susceptibility tests and as control strains in the NS susceptibility studies.

Statistical Analysis
Percentages are calculated using the Microsoft Excel 2013 program.

Results
The antibiotic susceptibilities of the bacteria used in this study are shown in Table 1. According to our findings, the susceptibilities of

Discussion
NS oil, which is used for the treatment of many health problems in traditional alternative medicine, has been proven to be effective against many bacteria [17][18][19]. For example, Hariharan et al. [20] reported the antibacterial activity of NS against methicillin-resistant S. aureus. In another study, Salman et al. [22] reported that NS was more effective against Gram-positive bacteria than Gram-negative bacteria. Specifically, they noted that NS oil was effective against Gram-positive bacteria Staphylococcus epidermidis, other coagulase negative staphylococci, and Streptococcus pyogenes. They also reported that Enterococcus faecalis and Streptococcus agalactiae were resistant to NS. Efficacy was only detected for P. aeruginosa in the Gram-negative bacterial group [22]. In another study performed by Chaieb et al. [19], the efficacy of thymoquinone, which is the active ingredient of NS, was evaluated on 11 human pathogenic bacteria, with reported efficacy, especially against S. epidermidis and S. aureus. All these studies suggest that NS may possess antibacterial properties that can be used in various fields.
Antibiotic resistance is an extremely important problem. In particular, the use of inappropri-   ate and unnecessary antibiotics increases the number of resistant bacterial strains and their resistance rates. The fact that this resistance becomes more and more evident each day increases concerns for the future. Therefore, research into new molecules that may be effective against infectious agents continues throughout the world.
Some common infections of the ear, nose, and throat area are middle ear and external ear canal infections, such as AOM, OME, and OE. Some of the most frequently found pathogens in these infections are S. pneumoniae, M. catarrhalis, H. influenzae, and P. aeruginosa [3][4][5][6]. Many studies have shown that these pathogens have developed resistance to certain antibiotics at specific rates.
There has been a steady decline in the susceptibility of S. pneumoniae to the most commonly used beta-lactams. In a current study, Cherazard et al. [23] found S. pneumoniae isolate resistance rates of 20%-40%, 22%, and 35% against macrolides, clindamycin, and trimethoprim/ sulfamethoxazole (TMP/SMX), respectively. In another study, Rosenblut et al. [24] reported resistance rates of 5% against penicillin and 33% against erythromycin. According to Sillanpää et al. [25], 80% of S. pneumoniae isolates were penicillin-sensitive and 20% were moderately resistant. They also reported that none of these resistant strains were susceptible to erythromycin or TMP/SMX [25]. Additionally, the authors reported that the resistance to erythromycin was 40% and the resistance to TMP/SMX was 20% in the overall evaluation of all the isolates. According to Sillanpää et al. [25], the antibiotic resistance of H. influenzae was evaluated, and moderate resistance to erythromycin [47%] was found, with full resistance to TMP/SMX and ampicillin reported as 29% and 24%, respectively. Additionally, the authors reported that M. catarrhalis resistance, it was found that all the strains were resistant to ampicillin and sensitive to the other antibiotics [25]. Rosenblut et al. [24] studied H. influenzae and reported the antibiotic resistance against ampicillin as 14%, cefuroxime as 2%, and cefotaxime as 2%. In another study, P. aeruginosa was reported to be highly resistant to various antibiotics, including ciprofloxacin and tobramycin [26]. The common results of these and similar studies show that antibiotic resistance has reached important levels. These results are clear signs of the need for new and different molecules to fight infection.
In our study, the antibacterial efficacy of NS oil against the most frequently found bacterial isolates in AOM, OME, and OE was evaluated in the laboratory. The MIC values were obtained in all the bacteria except P. aeruginosa. When the efficacy of the NS oil were compared with the other known antibiotics against the pathogens in our study, the NS oil was found to be ineffective against P. aeruginosa, as opposed to the Salman et al. report [22]. These contrary results for the same bacteria should be clarified by different studies.
The limitation of the study is the low number of strains. Further studies with more strains may contribute.
To our knowledge, there have been no studies about the effect of NS against the bacteria known to be particularly active in AOM and OME, such as H. influenzae, M. catarrhalis, and S. pneumoniae. For this research, a comparative study was conducted between NS and various antibiotics known to be effective against these bacteria, and the NS was found to have effects similar to some of these antibiotics, and in some cases, stronger effects.
Because of the increase in bacterial resistance to the antibiotics used in daily practice, it appears that NS oil may be an alternative for the treatment of AOM and OME. More detailed studies will contribute to the clarification of this issue in the future. Financial Disclosure: The authors declared that this study has received no financial support.