Evaluation of the Sensitivity of Staphylococcus aureus Isolated from Nasal Swabs to Natural Honey

Introduction : Bacterial resistance to antibacterial agents is a very serious threat to public health. Where some antibacterial agents prove ineffective, the antibacterial properties of honey have been shown to be highly efficacious against several human bacterial pathogens. The purpose of this study is to investigate the sensitivity of Staphylococcus aureus isolated from the nursing staff of a hospital to natural honey. Methods : In this study, 35 strains of methicillin-resistant S. aureus samples were selected from hospital staff’s nasal swabs. Two strains were vancomycin-resistant. The serial dilution tube test methodwas used to determine minimum inhibitory concentration (MIC). The susceptibility of each strain of staph bacteria to natural honey without wax was determined and compared with that of a glucose solution with the same density. Results : In all strains, except for the two strains resistant to vancomycin, MIC level was <8.3% (v/v). The MIC of glucose as dense as honey was four times higher. The two vancomycin-resistant strains were completely resistant to natural honey. Conclusions : This study has therefore demonstrated that inhibiting bacterial growth is not merely done by purely natural honey not because of osmolality, but vancomycin-resistant bacteria are not sensitive to natural honey. 35 different types of S. aureus with antibiotic properties were collected. The study investigates the sensitivity of S. aureus to natural honey. The honey used in this study was obtained from a farm in a mountainous area. Bees produce honey from the dominant plant species. The honey samples used in this investigation were refined and without wax. Sugar was used in the study as control for comparison with honey, in the form of glucose.


Introduction
Increased use of complementary medications has led to a lot of interest in traditional treatments. One treatment that has attracted a lot of interest is honey [1]. Research shows that honey has functional properties in promoting human health, which can be attributed to its osmolarity and antibacterial characteristics [2]. Honey collected from different geographical areas has shown different activities [3], and it has traditionally been used to treat burns, infected and untreated wounds, stomach ulcers, pimples, peptic ulcers, [4], and decrease cough symptoms [5]. It has also been reported that honey has anti-inflammatory, antibacterial, antifungal, and antioxidant effects and is effective against several human bacterial pathogens including Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus [6].
Scientists first reported the ability of honey to eliminate microbial diseases in the late 1800s, but with the emergence of antibiotics in the early 1900s, scientists' interest in honey diminished [7]. However, as resistant pathogens developed, the effect of antibiotics reduced. This kind of bacterial resistance to antibacterial agents is a very serious public health threat [8]. The number of newly developed antibiotics is limited when compared with the increase in the preponderance of bacterial resistance, and there is an increasing need for the development of alternative antimicrobial strategies [9]. Therefore, our ability to treat disease effectively depends on the development of new drugs and one potential source of new drugs is traditional medicine [10].
Honey has long been offering a wide range of antibacterial properties [11,12], although the specific anti-bacterial mechanism is unclear [13] and perhaps hydrogen peroxide, organic acids, Flavonoids, nectar, bee wax, pollen are the important chemical factors for this property. Regardless, it can be considered as an appropriate and safe source of medicinal treatment for humans [14]. Therefore, the aim of this study was to investigate the sensitivity of S. aureus to natural honey in samples taken from nursing staff.

Methods
In this laboratory-controlled study, 35 different types of S. aureus with antibiotic properties were collected. The study investigates the sensitivity of S. aureus to natural honey.
The honey used in this study was obtained from a farm in a mountainous area. Bees produce honey from the dominant plant species. The honey samples used in this investigation were refined and without wax. Sugar was used in the study as control for comparison with honey, in the form of glucose.

Determining MIC
In this study, the minimum inhibitory concentration (MIC) of honey was investigated using the test tube serial dilution method [15]. Data were collected through tube medium, adding different concentrations of honey and sugar solutions into the tubes and assessing the bacterial growth. Each sample comprised of three series of 10 tubes.
When the concentration was created, a bacteria culture was produced. This allowed us to determine the MIC for bacterial growth in the presence of honey and sugar solution.
The test tube serial dilution method is very accurate and sensitive, but it is not routinely used in clinical laboratories. This procedure is done in two micro tubes, in which the MIC of a certain bacteria can be calculated. Using this method, 10 sterile tubes (dimensions mm 100 × 13) were numbered and one of the tube was considered as the control group. In each of the tubes from 2 to 10, and control tube, 1cc of Muller-Hinton broth was poured into a sterilized medium and then a sterilized solution made from honey (1 g per 1 ml) (sterilized disposable filter with a pore diameter of 0/45 µm) was added. In tube no 1, 2 ml of culture medium was poured and from tube 1, 1 ml was removed and then added to tube 2 to make 5.0 dilutions; similarly serial dilutions were prepared (twice less than the previous tube); 1 ml of solution was removed from the last tube and thrown away. The control tube had no honey inside. All tubes and pipes were then provided with 1 cc of suspension of bacteria (S. aureus). The complex was incubated for 24 hr at 37ºC. After 24 hr, the tubes were examined.

Determining MBC
The MBC (minimum bactericidal concentration) is obtained in relation to the MIC. MBC is usually two to four times as much as MIC. The last tube in which bacteria did not grow was considered as the MIC. Then from the MIC tube and the three tubes before that

Data Analysis
Data were analyzed using the SPSS 18 software and the Chi-square test.

Results
The staphylococcus species used in this study were all resistant to glucosacchillin; 33 species of these bacteria were resistant to methicillin and 2 to vancomycin (Table 1).      Table 4.
Based on the results, 69/7% of MIC species had 8/3% (v/v%). Two types of samples of Staphylococcus used in the study previously had antibiotic resistance to vancomycin.
They showed full resistance to natural honey and grew at all concentrations. In a study of 33 strains of S. aureus which were susceptible to natural honey from the results (Table 4), 17 species of Staphylococcus (51/8%) had 8.3% MBC (v/v%). In comparison of MIC and MBC of Staphylococcus species in a medium containing honey and glucose, a significant difference was seen. MIC and MBC of honey was at least a quarter of glucose (Table 5).

Discussion
Based on this study, 94.3% of the tested strains did not grow in 66.7% (v/v%) of honey, and the MIC of honey as an antibacterial solution (v/v%) was 8.3%. In the Shimaa T.
Omara study, honey had antibacterial activity and the MIC (v/v%) was 5 and 2.5 [16]. In a study by Aamer and colleagues, all tested honey showed antibacterial activity against multi-drug-resistant bacterial strains [17]. Due to the fact that in the concentration of 8.3% of honey, 94.2% of the bacteria did not grow, but in the case of glucose, all bacteria have grown in this concentration, and even in the concentration of four times this concentration there has been growth of the bacterium in glucose, this study concludes that the lack of growth of the bacterium in the concentration (v/v%) of 8.3% honey is not related to the effect of the concentration and osmotic properties of honey, because in the same concentration of glucose, despite the presence of osmotic, bacterial growth occurred. Instead, this study demonstrates that the limited bacterium growth is based on the antibacterial properties of honey, which, according to the AF Henriques study, can affect cell division in S. aureus [18]. In the study of R. A. Cooper et al., honey bacterial inhibition was not exclusively related to honey osmolarity [19]. In a study by Mulhouse Vebert, the MIC of honey in the case of Staphylococcus coagulase positive 25% (v/v) has been reported [20], which is not consistent with our study. This difference can be due to other honey or honey-producing plant material [21].
In our study, the two strains that had a complete resistance to vancomycin also completely resisted natural honey. However, in the study of Mulan and colleagues, which compared antibiotic susceptible strains with antibiotic-resistant strains, there was no significant difference in the presence of honey [19]. The results of the present study indicate that honey has an antibacterial effect similar to vancomycin, and 33 species of S. aureus are susceptible to honey from amongst the 35 species susceptible to