Potential antibacterial activity of some Saudi Arabia honey

Aim: The aim of this study was to investigate the potential antibacterial activity of some Saudi Arabia honey against selected bacterial strains of medical importance. Materials and Methods: A total of 10 Saudi Arabia honey used to evaluate their antimicrobial activity against some antibiotic-resistant pathogenic bacterial strains. The bacterial strains were Staphylococcus aureus, Streptococcus pyogenes, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa. Results: The antibacterial activity of Saudi honey against five bacterial strains showed different levels of inhibition according to the type of honey. The overall results showed that the potential activity was differing according to the pathogen and honey type. Conclusion: It could be concluded that the Saudi honey inhibit the growth of bacterial strains and that honey can be used as complementary antimicrobial agent against selected pathogenic bacteria.


Introduction
The microbial resistance to antibiotics and chemicals has been increased worldwide against harmful microorganisms [1,2].
Thus, this investigation was evaluated the potential antibacterial activity of 10 Saudi Arabia honey against some bacterial strains of medical importance.

Ethical approval
Experiments were performed according to the Guide for the care and use of Laboratory animals and Ethical Approval of Animal Rights according to Committee, National Research Centre, Egypt.  Available at www.veterinaryworld.org/Vol.10/February-2017/15.pdf Honey Fresh 10 Saudi honey samples (1 kg each) were kindly provided by Alnahal Aljwal Company, 2015 flowering season). The monofloral honey harvested from apiaries (From Authorized proved apiary farm of Alnahal Aljwal, Saudi Arabia), these honey are vended as "monofloral" meaning that the honey must derive from at least 55% of pollen from a single floral source according to Louveaux et al. [37]. The collected honey samples were Shafallah honey (Capparis spinosa), acacia (Acacia nilotica) honey, Astragalus honey (Astragalus pelecinus), Talh honey (Thymus vulgaris), Sidr honey (Ziziphus spina-christi), spring Lena honey (Rhanterium epapposum), large influx honey (Acacia tortilis), olive (Alaatm) honey (Olea europaea), Dahbianh honey (Carduus acicularis), and Citrus honey (Citrus sinensis). Each honey sample was collected in a sterile universal glass container and kept at 2-8°C until tested. Physiological saline PBS pH 7.2 was used for all dilution steps under aseptic condition according to the method described by Nzeako and Hamdi [38]. Evaluations of the antibacterial activity of different honey dilution were performed according to Hegazi and Allah [7,39]. The results of antibacterial activity against different examined bacteria were determined.

Antibacterial assays
Five bacterial strains were used: S. aureus (ATCC 25923), S. mutans (1815 T ), K. pneumoniae (ATCC 27736), E. coli (ATCC 35218), and P. aeruginosa (ATCC 27853). The bacterial suspension was adjusted by comparison of 0.5 Mc-Farland turbidity standards (5 × 10 7 cells/ml). Then, it was further diluted to obtain a final of 5 × 10 6 cells/ml. These bacterial strains were enriched on selective broth for bacterial propagation [40]. In a separate tube containing 40 µl of 21.30% honey [34] concentration mixed with 0.20 µl/10 ml from inarched broth of each propagated S. aureus, S. mutans, K. pneumoniae, E. coli, and P. aeruginosa. These tubes were incubated at 37°C for 24 h. The growths of control bacterial strains as well as inhibitions of the bacterial growth due to mixed with honey were measured by turbidity at 420 nm wavelength. The mean values of inhibition were calculated from triple reading in each test [7].

Statistical analysis
Data were analyzed statistically using student "T" test showing mean + standard deviation. Data were compared using one-way. Statistical significance was accepted at p<0.01 according to Zar [41].

Results
The results of the different Saudi honey induced growth inhibition of S. aureus, S. mutans, K. pneumoniae, E. coli, and P. aeruginosa were illustrated Table-1 and Figure- The highest antibacterial activity was determined in Spring Lena honey (0.381±0.031) against K. pneumonia, while Shafallah honey (0.398±0.001) against E. coli but Citrus honey (0.382±0.003) against P. aeruginosa, where the lowest activity was observed in Dahbianh honey (0.551±0.001) against K. pneumonia, while Olive (Alaatm) honey (0.634±0.014) against E. coli but large influx honey (0.595±0.001). It was clear that all honey types induced an inhibitory activity of the growth of different pathogens. This reduction depends on the type of honey.

Discussion
The investigation into antibacterial activity of Saudi honey from different sources of 5 pathogenic  Table-1 and Figure-1. 20.30% honey concentration from different types showed inhibition of five bacterial growths. The inhibition of these bacteria may depend on the type of honey origin. These results were attributed to the floral source of honey which acts an important role on its biological properties [19]. The antimicrobial activity of honey also return to several factors [17,20,21] as osmotic effect of honey [20,26,42]. Acidity of honey (pH range from 3.2 to 4.5) or activity of glucose oxidase in the ripening of nectar [43]. The presence of hydrogen peroxide [44,45], endogenous hydrogen peroxide content [11,22], inhibin [23] which acts as antibacterial factor other than H 2 O 2 [24], hydrogen peroxide [25], non-peroxide substances [46,47], defensin-1, as well as the presence of phytochemical factors [27,28] and phytochemical components [17,24,29,30]. The antibacterial activity of different honey was studied by several authors [5][6][7][8]20,21,[33][34][35][36]48]. Comparison between Manuka honey with ling heather honey was determined by Lu Hodgeson [49] who found that whereas S. aureus and P. aeruginosa were inhibited by both honey. While, ling heather honey was inhibited E. coli, Proteus mirabilis and Streptococcus faecalis, on the other hand, yet Manuka honey was inhibited E. coli, P. mirabilis and S. faecalis. Media containing various concentrations of honey was evaluated against Gram-positive and Gram-negative bacteria [16] and they found that most pathogenic bacteria failed to grow in honey at a concentration of 40% or above. Hegazi and Allah [8] found that honey samples with different Saudi honey, were effective antibacterial against different examined pathogenic bacteria. Several honey available in the Saudi market especially the locally produced Shaoka, and Taify Sidr, in addition to imported Yemeni Sidr, black seed, clover and orange blossom are as potent as Manuka honey [36]. Furthermore, 10 honey samples collected from different floral areas around Riyadh were investigated [3]. 9 widely used honey in Saudi Arabia (Yemeni Sidr, Taify Sidr, Kashmiri Sidr, Shaoka, Somra, Black Seed, Black Forest, and Clover honey), and Manuka honey against E. coli, P. aeruginosa, Salmonella enterica serovar Typhimurium, Shigella flexneri and K. pneumoniae, S. aureus, and Streptococcus pyogenes were examined by Halawani and Shohayeb [35]. The most sensitive Gram-negative bacterium was P. aeruginosa while the most sensitive Gram-positive bacterium was and S. pyogenes [36]. Honey from some countries as Manuka honey from Australia, heather honey from the United Kingdom, and locally marketed Indian honey was detected their antibacterial activity [50]. Honey obtained from Izmir proved more effective as inhibitors against P. aeruginosa, E. coli and S. aureus, where the honey obtained from Muğla exhibited high anticandidal activity on C. albicans [22].
Finally, we could have concluded that the variations in the activity of different honey were attributed to the previously mentioned factors which influenced the antibacterial activity [7] as osmotic properties of honey [20,39]; honey pH or activity of glucose oxidase [41]; hydrogen peroxide [42,45], non-peroxide substances [46,47], presence of propolis which contain flavonoid [46], and volatile antibacterial substances [40].

Conclusion
From the current results, it concluded that the Saudi honeys inhibit the growth of bacterial strains and that honey can be used as complementary antimicrobial agent against selected pathogenic bacteria.

Authors' Contributions
AGH, give the idea, share as well as supervise on practical work, editing and help publishing of the article. FMA and AFMA, provided some materials and help in editing the article. FMAA, AAS and EF. They make isolation and identification and do the practical work, help during editing and publishing of the article. All authors have read and approved the final manuscript.