ANTIMICROBIAL ACTIVITIES FOR HADHRAMI HONEY ON GROWTH OF SOME PATHOGENIC BACTERIA

The majority of the Yemeni honey varieties are characterized by low moisture content, in addition to the various flora of Yemeni plant, which may not be found in many countries, which makes them of high medicinal importance and high monetary value. This study was carried out the effect of three types of Hadhrami honey: Somur, Sidr (Baghya) and Meria against the growth of some pathogenic bacteria (Pseudomonas aeruginosa, Proteus vulgaris, Escherichia coli, Enterobacter sp., Staphylococcus aureus, and Klebsiella sp.). The results revealed that that Sidr honey gave the highest antibacterial activity against all bacteria tested, whereas the Somur and Meria honey were not recorded activity for growth Klebsiella sp. and Meria honey was not recorded activity for growth E. coli, respectively. When the antibiotics compared to types of honey antimicrobial activity, it was observed that the antimicrobial effect of Sidr honey was better than Imipenem antibiotic effect against P. aeruginosa. The inhibition of the studied strains was dependent on the type of honey source. It is concluded that Yemeni honey could potentially be used as therapeutic agents against bacterial infection particularly to the tested microorganisms.


INTRODUCTION
Honey is the product of beekeeping that has great market potential. Honey contains more than 200 compounds comprising approximately 38% fructose, 31% glucose,10% other sugar types, 18% water and 3% of other compounds. However, precisely the great mixture of compounds in this 3 % is the product's greatest feature, with special reference to phenolic and carotenoids compounds 1 . Honey is one of the most complete foods for humans, due to its therapeutic 2 , antioxidant 3 , antimicrobial 4,5 , antitumoral 6 , anti-inflammatory 7 , antiviral 8 , and antiulcer activities 5 . Most studies on the effects of honey are concentrated on the activities of bioactive compounds, especially phenolic compounds, in the human organism. The most relevant are those widely distributed in nature, including the phenolic acids and flavonoids 9 .
Carotenoids were found in small concentrations in the dark honey (10 mg b-carotene•Kg _1 ) but they were not found in light colored honey. This fact reveals the effect that carotenoids 1,10 and phenolic compounds have in the honey color 11 . The natural ingredients of honey show different activities against various microorganisms. Its activity is likely to be dependent on the grazing grounds and the weather conditions where the bees were raised, and on the natural structure of the blossom nectar 12 . Honey has an increasing effect on the levels of anti-oxidants, iron and rare elements in blood 13 . The antibacterial activity of honey has never been reported nor any toxicity or side effects, low cost of maintenance, and local availability confer valuable advantages to using honey as an alternative antimicrobial therapy 14 . There are numerous reports of the antimicrobial activity of honey against a wide range of bacterial and fungal species 8,12,14,15 19 .
In vitro studies support the antimicrobial effect of honey against an extensive range of pathogens including β-haemolytic streptococci, methicillinresistant S. aureus and Pseudomonas sp 20 .
In vivo studies are less conclusive but honey has been used to treat burns 21 and meningococcal lesions. 22 Subrahmanyam 23 compared between honey and silver sulphadiazine on the treatment of patients with burns and found less inflammation, lower infection rates and faster healing in patients treated with honey. This study aimed to investigate the antibacterial activities of three types of Yemeni Hadhrami honey against some pathogenic microorganisms (gram positive and gram negative bacteria) isolated from patients and compared between them with the effect of antibiotics.

Bacterial strains
The bacteria strains that used in this study are most commonly involved in causing gastroenteritis, wound and burn exudates, urinary tract infection and ear secretions. Six bacteria strains (P. aeruginosa, P. vulgaris, E. coli, S. aureus, Enterobacter sp., and Klebsiella sp.) were isolated from different patients attending Al-Mukalla's Hospitals in Hadhramout-Yemen, and used throughout this study. The isolated bacteria were subcultured on Nutrient agar (Difco) and incubated aerobically at 37°C for 24 hours. Organisms were maintained in the laboratory on nutrient agar slopes at 4°C 24 .

Honey samples
In this study , three Hadhrami honey samples were taken from Yemeni mountain nature were used: {Somur, Sidr (Baghya) and Meria}, and stored in the dark at room temperature. The physical characterizations of honey samples such as pH which was measured using a pH meter (JeNWAY-3505), while the appearance was assessed in each sample by visual examination to determine the color.

Determination the minimal inhibitory concentration of the honey
The minimum inhibitory concentration (MIC) of honey was determined by using a different dilution for each type of honey that diluted with sterile distilled water. It was weighed 7gm/7mL from honey and the following concentration was prepared (1:4, 1:8, 1:10, and 1:20) 25 .

Antibacterial activity
The well diffusion technique under aerobic condition was employed as previously described by Harris et al. 26 About 20 mL of the sterilized medium was poured into each sterile Petri-dish (9 cm diameter) and allowed to solidify. Bacterial suspension for each strain tested was adjusted at 3 x 10 8 CFU/mL by McFarland scale which prepared by mixing 0.1 mL of 1.0% dehydrate barium chloride with 9.9 mL of 1.0% Sulfuric acid H 2 SO 4 as described by McFarland. 27 0.1 mL of the prepared bacterial suspension was spread evenly onto the agar surface using a cotton swab and kept in a refrigerator for 2 h. Wells (7 mm) were cut into the plates using sterile cork proper and different concentrations of each honey that were placed into each well. Thereafter, all prepared plates were incubated at 37 o C for 24 h 28 . After that, the diameter of inhibition zone around the well was measured in mm.

Antibiotic susceptibility test
Antibiotic susceptibility tests were carried out by the Kirby-Bauer disk diffusion technique according to Clinical Laboratory Standard Institute guidelines. 29 Mueller Hinton agar was used for growing the lawn of culture of the strains by spreading the culture onto the agar plate 30,31 .
In this study, eight of different antibiotics disks (OXOID and HIMEDIA) were used against investigated bacteria. These antibiotics disks were:

RESULTS AND DISCUSSION
The results of the physical characterizations and the prices of the three types of Hadhrami honey (Somur, Sidr and Meria) were recorded in Table 1. The results of antibacterial activity of different honey types against P. aeruginosa, P. vulgaris, E. coli, S. aureus, Enterobacter sp., and Klebsiella sp. were presented in Table 2. The Sidr honey was highly antimicrobial effective against all tested bacteria, which ranged between 10±0.11 mm to 30±0.1 mm, while the Somur and Meria honey showed no activity for the growth of Klebsiella sp. and Meria honey against E. coli (not inhibition zone). It was found that the Sidr honey has more influence antimicrobial activity, followed by Somur honey and then the Meria honey as the last one. These results are in agreement with reported by Othman 24  The present study showed varying degree of growth inhibition activity of three types of Yemeni honey against the tested microorganisms, these might be due to an advantage for honey like osmotic effect, the effect of low pH, and these organisms are sensitive to hydrogen peroxide which are unsuitable for bacterial growth, represented as an inhibition factor in honey. 21 Another study by Ceyhan and Ugar 36 investigated 84 types of honey against eight bacteria and two fungi. It was shown that the honey has a broad-spectrum activity against the used microorganisms. In addition, these authors found that the antibacterial activity of honey was greater than that which could be attributed to the sugar content of the honey. The antibacterial activity of honey has also been investigated for its potential use in reducing food-borne pathogens, 37 preventing catheter exit/entry site infection, 38 for the treatment of colitis, 39 or even to protect the gastric mucousin Helicobacter pylori induced inflammation 40,41 . The application of honey to wounds to animals in veterinary environments has also been noted. 42 Furthermore, the results of the current study revealed that most bacteria tested were sensitive at 1:4 concentration of all types of honey studied, except Klebsiella sp. was resistance to Somur and Meria honey. P. aeruginosa was more sensitive than others ( Table 3). All bacteria were resistant to 1:20 concentration of all types of honey except P. aeruginosa and P. vulgaris were sensitive to Sidr honey with inhibition zone (10mm and 11mm), respectively. A similar result was previously reported by Shreef et al. 25 who reported that most bacteria tested sensitive to 1:4 concentration more than other concentration (1:8, 1:10, 1:16, 1:20, 1:24) of both natural and industrial honey. Also, Al-Nahari et al. 43 studied antimicrobial activities of Saudi honey against P. aeruginosa. The results indicated that all types of honey tested exerted a full inhibition of bacterial growth at the highest concentration tested of 50% at 24 h of contact. Othman 24 showed that all the different concentrations of both honey samples (10 to 80%) showed growth inhibitory activity against E. coli more than other bacteria tested. It was recorded that all the tested bacteria were sensitive to Isis and Yemeni Sidr honey at 40 to 80% concentrations. The antibacterial activity of Yemeni Sidr honey was higher than those obtained by Isis honey. Variations seen in overall antibacterial activity were due to changes in the level of hydrogen peroxide achieved and in some cases to the level of non-peroxide factors. 24 The content of non-peroxide factors was obviously related to the Yemeni floral source and sometimes accounted for the major part of the antibacterial activity in honey 44 . Molan and Cooper 45 reported that the difference in antimicrobial potency among the different honey can be more than 100-fold, depending on its geographical, seasonal and botanical source. This result was in agreement with those previously reported by Mohammed et al 46 . The different concentrations of the two honey samples had good growth inhibitory effect on the tested microorganisms. A similar result was previously reported by Mohapatra et al. 47 for E. coli and P. aeruginosa, 48 for E. coli, K. pneumoniae and for Haemophilus influenza 49 . The less inhibition effect of the two tested honey against K. pneumoniae and S. aureus was in agreement with Patricia et al. 50 who reported that the overall poor activity of the honey against S. aureus was unexpected as previous reports which recorded that Maunka honey has an excellent activity against this organism. For example, Cooper and Molan 20 who also used an agar dilution method and demonstrated that the minimum inhibitory concentration for Maunka honey against 58 strains of Staphylococcus sp. was 2-3% (v/v) and for pasture honey 3-4% (v/v). In this study, the antibiotic susceptibility was tested and the highest percentage of the sensitivity to Imipenem for most bacteria were recorded. The highest inhibition zone of Imipenem was (22 mm) for P. aeruginosa (Table 4), while the highest inhibition zone of Sidr honey was (30 mm) that indicated the antimicrobial effect of honey was higher than antibiotic effect. A similar result was previously reported by Al-Nahari et al 43 for Manuka honey UMF +10 was the most effect on antimicrobial resistance and had an effect on modulation of Imipenem resistant P. aeruginosa. The resistance of pathogenic microorganisms to antibiotics is a serious global health concern 51 . On another hand, Al-Naama 52 showed that honey, like antibiotics, has certain organisms sensitive to it, and provides alternative therapy against certain bacteria and is also shown to have an antimicrobial action against a broad spectrum of bacteria (both grampositive and -negative bacteria). Honey contains compounds with antioxidant and antibacterial capacities, such as phenolic compounds and carotenoids 53 . Honey bees add an enzyme, called glucose oxidase, to the collected nectar during the honey-making process, which converts the glucose in the honey into hydrogen peroxide (H 2 O 2 ) and gluconic acid. H 2 O 2 is toxic to many microbes 51 . Mohapatra et al. 47 showed that the honey has an antibacterial effect against both gram-positive bacteria (S. aureus, Bacillus subtilis, Bacillus cereus, Enterococcus faecalis, and Micrococcus luteus) as well as anti-gram negative bacteria (E. coli, P. aeruginosa, and Salmonella typhi). This effect was either bacteriostatic or bactericidal depending on the type of honey tested. There are countless varieties of honey being produced worldwide, and some may have   ---  19  10  18  13  15  11  Piperacillin  16  11  15  13  16  15  Amikacin  22  19  17  20  18  17  Imipenem  0  0  15  0  10  11  Cefuroxime  18  19  20  20  12  14  Aztreonam   0  11  18  20  10  13  Amoxicillin +  Clavulanic acid  16  19  17  20  17  10  Gentamicin  0  0  14  12  0  0  Ceftazidime