<4D6963726F736F667420576F7264202D20E32EE32E20DAE3C7D120D1EDC7D620DEC7D3E3A120E620E32EE32E20E3CDE3CF20CEE1EDDDC920CED6EDD1> Diyala Agricultural Sciences Journal, 2( 2 ) 9 – 19 ,2010 Mohammad and Ammar COMPARATIVE STUDY OF ANTIBACTERIAL ACTIVITY OF TWO DIFFERENT TYPES OF HONEY AGAINST Escherichia coli, Pseudomonas aeruginosa AND Staphylococcus epidermidis. Mohammad, K.K.* Ammar, R.Q. * Biology and Microbiology Dept.- College of Science - Diyala University. ABSTRACT This study aimed to investigate the antibacterial activity of two different types of honey, natural and commercial, by determination the minimum inhibitory concentration (MIC) against isolates of Staphylococcus epidermidis, E.coli and Pseudomonas aeruginosa. In this study, fifty eight (58) human isolates of these organisms were collected from different pathological sources from both males and females with age between (9-50) years and tested for their sensitivity to honey, a natural product that is generating renewed interest for its therapeutic application, during the period from 30/10/2009 to 1/4 /2010. In a tube dilution method, the incidence rate of bacterial inhibition was always significantly higher when natural honey was used compared to commercial honey at the three successive dilutions tested. At the highest concentration tested (1:2 dilution) all the bacterial isolates tested where inhibited (100%) by both types of honey tested. The median MIC was significantly lower for E.coli than Pseudomonas aeruginosa and Staphylococci epidermidis when exposed to commercial honey. The median MIC causing bacterial inhibition and mean rank was always significantly lower for natural honey compared to commercial honey in all the three types of bacterial isolates. ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ Received for publication June 3, 2010. Accepted for publication Sept. 19, 2010. Diyala Agricultural Sciences Journal, 2( 2 ) 9 – 19 ,2010 Mohammad and Ammar Key words: MIC of natural honey, Commercial honey, Staphylococcus epidermidis, E.coli, Pseudomonas aeruginosa. INTRODUCTION The clinical use of honey has been known since ancient times, and in more recent years it has been rediscovered as a therapy for wounds (Molan, 1998). Interest in this approach stems partly from the emergence of antibiotic-resistant pathogens. Honey has been useful in the treatment of infected surgical wounds, burn wounds, and ulcers (Zumla, 1989). Honey maintains a moist wound environment that promotes healing, and its high viscosity helps to provide a protective barrier to prevent infection. In addition, the mild acidity and low-level hydrogen peroxide release assists both tissue repair (Lusby et al., 2005). Many publications attest to honey's antimicrobial properties (Molan, 1992), but the mechanisms by which it acts are incompletely studied. Strong solutions of honey or sugar, and sugar pastes inhibit microbial growth because of their high osmolarity Chirife et al., 1982). The potential of honey as a topical wound dressing is now recognized by the health care community, and there continues to be a search for honeys from different sources with enhanced antibacterial activity (Venkatachalam and Thangam, 2007). Manuka honey, for example, has high antibacterial activity associated with an unidentified phytochemical component (Molan, 1992) .A study by Willix et al., (1992), does specify the type of honey and found that antibacterial activity was primarily due to hydrogen peroxide. In this study, we investigated the antibacterial activity in order to determine the minimum inhibitory concentration (MIC) of two different types of honey against Staphylococcus epidermidis, E.coli, Pseudomonas aeruginosa. Diyala Agricultural Sciences Journal, 2( 2 ) 9 – 19 ,2010 Mohammad and Ammar MATERIALS AND METHODS This study include (58) isolates of multi-drugs resistant organisms isolated from urine, ear, skin, pus, 22(37.94%) of isolates were Staphylococcus epidermidis, 18(31.03%) of isolates were for each E. coli and Pseudomonas aeruginosa; the patients were between (9-50) years of age, the samples collected during the period of October 2009 to the April 2010. The organisms were subcultured on nutrient or blood agar according to species of bacteria. The type of honey that used was standard grade. The standard grade of honey had the following properties; Specific gravity (1-37 at 27 degrees), moisture (25% max), reducing sugar (65% min), sucrose (5% max), fructose and glucose (1.0 min), acidity (0.2% max) (SeeEfem, 1988). Two types of honeys were used a natural honey and a honey of commercial source. Their antibacterial activity was determined by tube dilution method. Different concentrations of honey were prepared in nutrient broth to give double fold dilution of 1:2, 1:4, 1:8, 1:16, 1:32 according to Pamela (1978). One tube containing 1ml of undiluted honey was also used (Pamela, 1978). Honey were measured out and diluted in sterile deionized water to prepare a 50% stock honey solution from which further dilutions in broth could be readily prepared. Appropriate volumes of stock honey and deionized water (totaling 2ml) were dispensed aseptically into 1ml samples of sterilized nutrient broth (Oxide) to produce a dilution series between 1:2 and 1:32 (v/v) of each type of honey in broth. Twenty-four hours old cultures of S. epidermidis, E.coli, P. aeruginosa were inoculated in saline. The turbidity was adjusted to 0.5 McFarland’s standard, and (5µl) of culture was inoculated in the dilutions and incubated at 37oC for 24 hours and were observed for growth. MIC was recorded as the lowest concentration of honey that prevented growth. Diyala Agricultural Sciences Journal, 2( 2 ) 9 – 19 ,2010 Mohammad and Ammar Statistical analysis Analysis were computer aided using SPSS version 13. The statistical significance of association between type of honey and bacterial inhibition was tested by chi-square test of independence. The minimal inhibitory concentration was measured on an ordinal level (4 consecutive dilutions), therefore it was described by median and the non-parametric test of significance was used (Kruskall-Wallis and Mann-Whitney). The mean rank is a product of the test of significance used, it is useful in ordering the groups when their median seems to be almost equal. RESULTS AND DISCUSSION As shown in table 1, the incidence rate of bacterial inhibition was always significantly higher when natural honey was used compared to commercial honey at the 3 successive dilutions tested. At the highest concentration tested (1:2 dilution) all the bacterial isolates tested where inhibited (100%) by both types of honey tested. The incidence of inhibition obviously increases with increasing concentration (decreasing dilution) of tested honey. The same observation holds true when each type of isolated bacteria was tested separately, tables 2 to 4. These tables showed that the effect of natural honey was more than commercial type. E. coli was more sensitive to both types of honey used in this study than P. aeruginosa and S. epidermidis ,these results agreed to Wilkinson and Cavanagh (2005) who found that. E. coli was more susceptible to inhibition by the honeys used when compared with P. aeruginosa , and Adebolu (2005) who explain that the inhibitory effect was highest on E. coli and followed by Salmonella enterocolitis and Shiglla dysenteriae. The reason for this exception is not clear because Gram negative bacteria, of which this organism is one, have been reported to be more sensitive to action of honey than Gram-positive bacteria (El- Diyala Agricultural Sciences Journal, 2( 2 ) 9 – 19 ,2010 Mohammad and Ammar Sukhon et al., 1994). Honey works quicker than many antibiotics because it is easily absorbed in the blood stream (Singh and Suryanarayan, 1988). Table 1. The difference between natural and commercial honey in the incidence rate of bacterial inhibition at three dilutions when tested with all three types of bacterial isolates. Overall Honey dilution 1:4 1:8 1:16 N % N % N % Natural honey (n=58) 43 74. 1 19 32. 8 5 8.6 Commercial honey (n=58) 23 39. 7 2 3.4 0 0.0 P (Chi-square) <0.001 <0.001 0.022 Note: All bacterial isolates were inhibited by the highest concentration (1:2 dilution) of both honey types. Table 2. The difference between natural and commercial honey in the incidence rate of bacterial inhibition at three dilutions when tested with E.coli. E. coli Honey dilution 1: 4 1:8 1:16 N % N % N % Natural honey (n=18) 1 6 88.9 8 44.4 4 22. 2 Commercial honey (n=18) 1 2 66.7 2 11.1 0 0.0 P (Chi-square) 0.11[NS] 0.026 0.034 Note: All bacterial isolates were inhibited by the highest concentration (1:2 dilution) of both honey types. Diyala Agricultural Sciences Journal, 2( 2 ) 9 – 19 ,2010 Mohammad and Ammar Table 3. The difference between natural and commercial honey in the incidence rate of bacterial inhibition at three dilutions when tested with Pseudomonas aeruginosa. Pseudomonas aeruginosa Honey dilution 1:4 1:8 1:16 N % N % N % Natural honey (n=18) 12 66.7 4 22.2 0 0.0 Commercial honey (n=18) 4 22.2 0 0.0 0 0.0 P (Chi-square) 0.007 0.034 ** Note: All bacterial isolates were inhibited by the highest concentration (1:2 dilution) of both honey types. Table 4. The difference between natural and commercial honey in the incidence rate of bacterial inhibition at three dilutions when tested with Staphylococcus epidermidis. Staphylococcus epidermidis Honey dilution 1:4 1:8 1:16 N % N % N % Natural honey (n=22) 15 68.2 7 31 .8 1 4.5 Commercial honey (n=22) 7 31. 8 0 0. 0 0 0.0 P (Chi-square) 0.016 0.004 0.31[NS] Note: All bacterial isolates were inhibited by the highest concentration (1:2 dilution) of both honey types. As shown in table 5, the median MIC was significantly lower for E.coli than Pseudomonas and Staphylococci when exposed to commercial honey. It was also obviously lower for E.coli than Pseudomonas and Staphylococci when exposed to natural honey, but the difference in MIC fail short of statistical significance possibly because of small sample size. In general, whatever was the type of honey the median MIC was smallest with E.coli (0.25), followed by Staphylococci (0.375) and highest for Pseudomonas (0.5). In this study, both natural honey and commercially processed therapeutic honey have been shown antibacterial activity against three bacterial species. Diyala Agricultural Sciences Journal, 2( 2 ) 9 – 19 ,2010 Mohammad and Ammar P. aeruginosa is notoriously resistant to antimicrobial therapy it is protected from host immune effectors and can grow to sufficient levels to elaborate toxins that break down host factors. Hence, Pseudomonas is a major problem for chronic wounds and contributes to delay in healing (Scmidtchen et al., 2003). Various researchers have shown that honey exerts an antibacterial activity against various organisms, including both gram-positive and gram-negative bacteria. The antibacterial activity of honey is mainly due to inhibiters in honey. These inhibiters are hydrogen peroxide, fiavinoids, and phenolic acids, plus many other unidentified inhibiters. A number of reasons for this have been suggested: shrinkage disruption of the bacterial cell wall due to the osmotic effect of the sugar content; induction of an unfavorable environment with low water activity (Subrahmanyam et al., 2001). The median MIC causing bacterial inhibition and mean rank was always significantly lower for natural honey compared to commercial honey in all the three types of bacterial isolates, table 6. The variations recorded in the antibacterial activity of two types of honey tested were consistent with the reports of Jeddar et al. (1985) and Molan et al. (1988) and have been attributed to delayed levels of hydrogen peroxide/ thermal stability of the glucose oxidase enzyme, non-peroxide factors, and the plant/floral source (Willix et al., 1999 ; Moudoi et al., 2005). The development of honey in form of a rubbery gel that can be moulded to conform to any shape will further increase the practicality of use with medical devices beyond that with the honey-impregnated dressings currently available. It remains for further clinical evaluation to be tried (Molan and Betts, 2004). Diyala Agricultural Sciences Journal, 2( 2 ) 9 – 19 ,2010 Mohammad and Ammar Table 5. Difference between 3 types of bacteria in mean rank of minimal inhibitory concentration of each type of honey. Concentrati on of honey used Type of bacterial isolate tested Type of honey used E. coli Pseudomo nas aeruginosa Staphyloco ccus epidermidi s P (Kruskal l-Wallis) Group Total Commercia l 0.01 Range (0.125 - 0.5) (0.25 - 0.5) (0.25 - 0.5) (0.125 - 0.5) Median 0.25 0.5 0.5 0.5 N 18 18 22 58 Mean rank 21.06 34.78 32.09 Natural honey 0.11[NS ] Range (0.062 5 - 0.5) (0.125 - 0.5) (0.0625 - 0.5) (0.062 5 - 0.5) Median 0.25 0.25 0.25 0.25 N 18 18 22 58 Mean rank 23.11 33.78 31.23 Group Total 0.006 Range (0.062 5 - 0.5) (0.125 - 0.5) (0.0625 - 0.5) (0.062 5 - 0.5) Median 0.25 0.5 0.375 0.25 N 36 36 44 116 Mean rank 44.92 67 62.66 Diyala Agricultural Sciences Journal, 2( 2 ) 9 – 19 ,2010 Mohammad and Ammar Table 6. Difference between natural and commercial honey in mean rank of minimal inhibitory concentration, stratified by type of bacteria tested. Mean rank E. coli Pseudomonas aeruginosa Staphylo coccus epidermi dis Overall Natural honey 14.5 14.06 17.39 45.39 Commercial honey 22.5 22.94 27.61 71.61 P (Mann- Whitney) 0.014 0.004 0.004 <0.001 REFERENCES Adebolu, T.T. 2005. Effect of natural honey on local isolates of diarrhea causing bacteria in southwestern Nigeria. African Journal of Biotechnology. 4(10):1172-1174. Chirife, J., G. Scarmato and L. Herszage. 1982. Scientific basis for use of granulated sugar in treatment of infected wounds. Lancet; 1: 560-1. El-Sukhon, S.N., N. Abu-Harfeil and A.K. Sallal .1994. Effects of honey on Bacterial Growth and Spore Germination. J. Food Prot.; 57(10): 918- 920. Jeddar, A., A. Kharsany, U.G. Ramsaroop, A. Bhamjee, I.E. Hafejee and A. Moosa, 1985. The antimicrobial action of honey. South Afr. Med. J.; 67:257-8. Lusby, P., A.L. Coombes and J.M. Wilkinson. 2005. Bactericidal activity of different honeys against pathogenic bacteria. Arch. Med. Res.; 36:464–46. Molan, P.C. 1992. 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Thangam . 2007. Bactericidal Activity of Different Types of Honey Against Clinical and Environmental Isolates of Pseudomonas aeruginosa.;13(4):439–441. Wilkinson, J.M. and H.M. Cavanagh. 2005. Antibacterial activity of 13 honeys against Escherichia coli and Pseudomonas aeruginosa. J. Med. Food. ;8(1):100-3. Willix, D.J., P.C. Molan and C.G. Harfoot 1992. A comparison of the sensitivity of wound-infecting species of bacteria to the antibacterial activity of manuka honey and other honey. J Appl Bacteriol;73:388-94. Willix, D.J., P.C. Molan and C.G. Harfoot. 1999. A comparison of the sensitivity of wound-infecting species of bacteria to the antibacterial activity of manuka honey and other honeys. J. Appl. Bacteriol.;73: 388-94. Zumla, A. 1989. Honey—a remedy discovered. J. R. Soc. Med.;82:384– 385. Diyala Agricultural Sciences Journal, 2( 2 ) 9 – 19 ,2010 Mohammad and Ammar االيشيريشيا نوعين مختلفينِ من العسِل ضد ل بكتيريادراسة مقارنة للنشاط المضاد لل . القولونية، الزائفة الزنجارية والمكورات العنقودية الجلدية * عمار رياض قاسم* محمد خليفة خضير . جامعة ديالى - آلية العلوم - المجهرية واألحياءقسم علوم الحياة * الخالصة ،) الطبيعـي والتجـاري (نوعين مختلفين من العسل اختبار نشاط إلىهذه الدراسة تهدف Staphylococcusكتريـا ضـد ب األدنـى عن طريق تحديد التركيز المثبط كمضاد حيوي epidermidis, E.coli, Pseudomonas aeruginosa . عـتْ ثمانيـةمج ،في هذه الدراسة المختلفة مـن كـال الجنسـين تتـراوح لمرضيةعزلة بكتيريا من المصادرِ ا) 58(وخمسون وتـم فحـص .2010 /1/4ولغاية 30/10/2009سنة خالل الفترة من ) 50-9(من أعمارهم الذي أعيـد االهتمـام بـه فـي و التجاري حساسية العزالت البكتيرية للعسل المنتج الطبيعي .التطبيقات العالجية كال النوعين خفف حيثالمستخدمة في هذه الدراسة، األنابيبطريقة التخفيف باستخدام تمت دائمـاً معنويـة انتْ نسبة التثبيط البكتيري كَ إنالنتائج أظهرت. 32:1 إلى 2:1من العسل من أعلىعند . عند استعمال العسَل طبيعي مقَارنة بالعسِل التجاريِ في التخفيفات المتعاقبة الثالثة بكـال أنـواع العسـِل %) 100(كُّل العزالت البكتيرية قد ثبط نموهـا ) تخفيف 1:2(تركيز mean rankالبكتيريا وقيمة الذي يسبب تثبيط نمو األدنىمتوسط التركيز المثبط . المستخدمة الثالثة للعـزالت البكتيريـة األنواعقليلة جدا للعسل الطبيعي مقارنة بالعسل التجاري لكل من . لمكوناته الطبيعية النقية وذلك