Honey MEDICAL SCIENCES (2000), 2, 75−79 © 2000 SULTAN QABOOS UNIVERSITY 1Department of Microbiology & Immunology, College of Medicine, Sultan Qaboos University, P O Box 35 Al-Khod, Muscat 123, Sultanate of Oman. 2Department of Surgery, Umm Al-Qura University, Makkah, Saudi Arabia *To whom correspondence should be addressed. E-mail: basil@squ.edu.om 75 Antimicrobial potential of honey on some microbial isolates *Nzeako B C1, Hamdi J2 الجراثيم بعض ضد حيوي آمضاد العسل صالحية حمدي جمال، آوانزي باسل مكة منطقة في تباع العسل من نوعا عشرة احدى لصالحية دراسة البحث هذا في تم لقد :الطريقة .للبكتيريا المضادة العسل فعالية تقييم :الهدف: الملخص :النتائج .الزنجارية والزائفة المعوية أألشريكية البرتقالية، العنقودية آورات22الم ضد حيوية آمضادات ) نامحلي انونوع مستوردة أنواع تسعة ( المكرمة آما العسل نوع حسب تختلف الفاعلية أن البحث وأثبت. المختبر في عزولةالم والفطريات البكتيريا من أنواع عدة على العسل من أنواع ستة فاعلية دراسة تمت درجة في حفظه أو دقيقة عشرة خمس لمدة غليه بعد حتى حيوي آمضاد فاعليته يفقد ال العسل أن البحث أثبت أيضا. العسل لمفعول مقاومة الجراثيم بعض أن زهور وعسل الترآي العسل يتبعه فاعلية أعلى على يحتوي األلماني السوداء الغابة عسل أن تبين آما. أشهر ستة ولمدة مئوية درجة 8 � 2 بين حرارة فبعض أألخرى، الحيوية للمضادات آما للحيوية مضادة خواصا للعسل أن البحث أظهر :الخالصة .الصيف زهور عسل وأخيرا الغابة وعسل البرتقال .العسل نوعية على إعتمادا الحساسية هذه وتختلف له، مقاوم غيرها بينما له حساسة الحية الكائنات ABSTRACT: ��������� – To assess the antimicrobial potential of honey against certain microbial isolates. �� �� – Samples of commercial honeys sold in Makkah area of Saudi Arabia were checked for their antimicrobial activities using standard organisms, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The minimal inhibitory concentration end points of six honey samples found to possess antimicrobial activities were used to determine the sensitivity patterns of some isolates from the laboratory. The tem- perature stabilities of the honey samples were also determined. ������ – The six honey samples had differing levels of antimicrobial activities with the standard organisms and with the laboratory isolates. Black Forest honey showed the highest activity followed respec- tively by Turkish, Orange Flower, Forest Honey and Summer Flower. The antimicrobial activities of the samples were stable after stor- ing at 2–8° C for six months and after boiling for 15 minutes. ���������� – The study shows that honey, like antibiotics, has certain organisms sensitive to it while others are resistant, and the sensitivity varies depending on the source of the honey.�� KEY WORDS: Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, honey, antibiotics, sensitivity, antimicrobial. oney was found by some workers to possess antibacterial activity where antibiotics were inef- fective.1,2,13,14 Some chronic debilitating condi- tions resulting from pressure sores, infected wounds, burns and fournier’s gas gangrene have been found to respond favourably to honey treatment.1,2,3 Radwan4 at- tributed this antibacterial activity to specific chemicals in honey. The nature of these chemicals and the mecha- nisms of their action are not fully understood even though thin layer chromatography (TLC), polyacrylamide gel electrophoresis (PAGE) or high performance liquid chromatography (HPLC) have shown that honey con- tains seven tetracycline derivatives, fatty acids, lipids, amy- lases and ascorbic acid.5,6,7,8 Allen9 showed that there are many types of honey with and without antibacterial activity and postulated that the type of the flower that was the source of the nectar determines the nature of the antibacterial activity of the honey. While the empirical application of honey on open wounds, burns or use of honey in syrups does show that it stops the growth of many microorganisms, the latter have seldom been isolated and identified.2,3 Efem10 found that undiluted honey stopped the growth of Candida species while Pseudomonas aeruginosa, Clostridium oedematiens, Streptococcus pyogenes remained resis- tant. Wellford11 found that some species of Aspergillus did not produce aflotoxin in various dilutions of honey. Radwan4 observed that honey stopped the growth of Salmonella, Escherichia coli, Aspergillus niger and Penicillium chrysogenium. However, these investigations were not con- ducted with standard organisms of known sensitivity to common therapeutic agents. Conclusions were mostly H N Z E A K O & H A M D I 76 drawn from the results from one sample of honey.9 Since honey is used extensively in the Arabian Gulf region, the authors felt it desirable to scientifically deter- mine the antimicrobial activities of the honeys in com- mon use. This study was conducted to assess the antimicrobial potential of a few commercial honey sam- ples on some laboratory isolates of known sensitivities to common antibiotics. The investigation hoped to determine the tempera- ture stability of the active agent(s) in honey since the am- bient temperature might affect both its shelf life and its killing potential. This knowledge could be effectively util- ised in hospital practice and in primary health care where open skin lesions are routinely treated. METHOD Six brands of commercial honey available in Saudi Arabia were used in the study: Black Forest, Orange Flower and Summer Flowers produced by Biophar, Germany, Black Forest and Forest Honey produced by Langaneza, Germany, and Turkish from Turkey (figure 1). TABLE 1 Examined commercial honey samples and their sources. Name Source 1 Black Forest Germany (Biophar) 2 Orange Flower Germany (Biophar) 3 Black Forest Germany (Langaneza) 4 Forest honey Germany (Langaneza) 5 Summer Flowers Germany (Biophar) 6 Turkish Turkey The samples were collected and marked randomly by one investigator while the experiments were performed blindly by the other. Each honey sample was collected in a sterile universal container and kept at 2–8°C until tested. Each sample was checked for purity on blood agar plates and was diluted to 75, 50, 40, 20, and 10% of its original concentration using physiological saline. Three control organisms, Staphylococcus aureus (ATCC25923), Escherichia coli (ATCC 25922) and Pseudo- monas aeruginosa (ATCC 27853) were used to determine the antimicrobial activity of each sample of honey. Three colonies (1.1×106 organisms per ml, equivalent to Brown’s opacity tube 3) of each standard organism were emulsi- fied in 4ml of distilled water and used to swab Mueller Hinton sensitivity agar plates. Fifty microlitres (50µl) of each honey dilution were applied on each plate using 1ml sterile syringe without the needle. Each dilution was done in triplicate. The plates were left at room temperature till the honey seeped into the agar. After incubation, the in- hibition zones were measured in millimetres (mm) and the average of the inhibition zones recorded. The end point of antimicrobial activity of each honey was defined as the highest dilution (lowest concentration) producing an inhibition zone with the control organisms. Using Stokes12 method, some multiresistant organ- isms isolated from hospital patients were subjected to sensitivity test using the honeys at their antimicrobial ac- tivity end points (lowest concentration end points). The sensitivities of Proteus spp. to honey samples were read after 4 hours up to 12 hours duration to check for swarming activities. Organisms showing inhibition zones equal to or greater than that of the control organisms were regarded as sensitive to honey samples. A stability test was also conducted as follows: Each honey sample was divided into two aliquots. The first aliquots were stored for six months at 2–8° C while the second aliquots were boiled for 15 minutes and allowed to cool. Each aliquot was retested for antimicrobial activ- ity as before. RESULTS Table 2 shows the zones of inhibition of the six honey samples with the standard organisms. These de- pended on the species of the control organism. Turkish honey (sample 6) had highest activity with Staph. aureus and least with Pseudomonas aeruginosa while Black Forest honey (sample 3) had highest activity with Pseudomonas and least with Esch. coli. All the samples showed zones of inhibition of 10 mm or more at 50% dilution with Staph. aureus, Ps. aeruginosa and Esch. coli except samples, 4 and 5 with Staph. aureus and Esch. coli respectively and sample 2 with Esch. coli and Pseudomonas aeruginosa respectively. All the laboratory isolates were found sensitive to the honey samples except Proteus mirabilis, Aspergillus niger, Aspergillus fumigatus, Enterococcus faecalis and Streptococcus pyogenes (Table 3). Some Pseudomonas aeruginosa and Acineto- bacter species found resistant to amikacin, ceftriaxone, tobramicin, aztreonam, gentamicin and imipenem were also found sensitive to all the honey samples. The honey samples retained their antimicrobial ac- tivities with the control organisms even after storage at 2– 8°C for six months and after boiling for 15 minutes, though the activity on Esch. coli was destroyed at 50% dilution of honey, but retained at neat (undiluted honey). DISCUSSION The six commercial samples of honey showed differing antimicrobial activities with organisms isolated from the laboratory. Commercial Black Forest honey, followed by Turkish honey (sample 6), had the highest antimicrobial activity (Tables 2 and 3). A N T I M I C R O B I A L P O T E N T I A L O F H O N E Y The antimicrobial effects of the honey samples w more with Pseudomonas and Acinetobacter species than w the other bacteria tested. The reason for this is not cl It is possible that the low redox potential of asco acid5 in honey affects aerobic organisms such as the P domonas and Acinetobacter species. Jeddar15 found ho inhibitory to the growth of microorganisms at 40% d tion. This observation is not in conformity with our sults; some honey samples tested by us had no activit 40% dilution. Our findings also disagree with Radw who found Aspergillus niger sensitive to honey and Efe who found Pseudomonas aeruginosa resistant. The reason Staphylococcus aureus being sensitive to honey and Strept cus spp. resistant is not understood. However, it is kno that Streptococcus spp. are lactic acid bacteria while staph cocci are not. If lactic acid accumulates in the areas c taining honey samples as one of the microbial metab products of streptococcal growth, the activity of ho may be altered since high acidity affects the inhibit zones produced by various antibiotics.16 Our findi agree with Obaseiki Ebor17 who found Candida albi sensitive. Our honey samples also exerted antimicro activities on Pseudomonas and Acinetobacter species, wh were resistant to some antibiotics. The ability of honey to kill microorganisms has b attributed to its high content of tetracycline derivati peroxidases, fatty acids, phenols, ascorbic acids and a lases.1,5,18–20 In this study, the antimicrobial substance the honeys were not estimated. However, the fact Black Forest honey had more activity than Turkish other honeys, highlights the finding that the sources Minimal inhibitory concentration o Staphylococcus aureus� Honey dilution Honey samples & zones of inhibition (mm) � Hone Sample 1 2 3 4 5 6 1 Neat 22.00 21.00 18.00 13.00 15.00 15.00 20.00 75 % 17.00 15.00 14.00 11.00 11.00 12.00 19.00 50 % 12.00 11.00 12.00 9.00 11.00 10.00 14.00 40 % 6.00 0.00 6.00 0.00 0.00 8.00 9.00 20 % 0 0 0 0 0 0 0 10 % 0 0 0 0 0 0 0 TABLE 2 f various honey samples with control organisms Escherichia coli� Pseudomonas aeruginosa� y samples & zones of inhibition (mm] � Honey samples & zones of inhibition (mm) 2 3 4 5 6 1 2 3 4 5 6 15.00 20.00 19.00 15.00 20.00 25.00 14.00 20.00 15.00 18.00 18.00 11.00 15.00 15.00 12.00 17.00 16.00 11.00 16.00 14.00 13.00 14.00 9.00 13.00 12.00 9.00 13.00 13.00 10.00 13.00 11.00 11.00 12.00 0.00 3.00 0.00 0.00 9.00 8.00 0 12.00 6.00 7.00 5.00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 77 ere ith ear. rbic seu- ney ilu- re- y at an5 m10 for ococ- wn ylo- on- olic ney ion ngs cans bial ich een ves, my- s in that or of the nectars may have contributed to the differences in their antimicrobial activities.9 The experiment showed that the antimicrobial sub- stances in honey can withstand refrigeration temperatures for six months and are heat stable at 100°C. This shows that its antimicrobial activity is not dependent alone on its tetracycline derivatives, ascorbic acid, peroxidase or amy- lase activities as claimed by other workers, for these agents are heat labile. Takeba19 and Joerg21 attributed the antibacterial effect of honey to its phenolic content. Phe- nol is heat stable and may be an active agent but its con- centration in honey appears too low (1.3–5.0µg/l) to be solely responsible. The antimicrobial agent therefore may depend on the integrity of a particular honey sample. CONCLUSION In this experiment, we attempted to assess the value of honey as an antimicrobial therapeutic agent. We have found some samples to have high broad-spectrum antim- icrobial activity, even after the honey has been exposed to boiling or refrigerating temperatures. This makes honey unique since many topical antibiotics used in open skin lesions are heat labile. Among our samples, Black Forest honey had the highest antimicrobial activity followed re- spectively by Turkish, Orange Flower, Forest Honey and Summer Flower. The study also shows that some organ- isms are sensitive to some types of honey while others are resistant. However, much remains unknown, which makes this a fertile field for further research. N Z E A K O & H A M D I 78 REFERENCES 1. Molan PC. The antibacterial activity of honey: the na- ture of the antibacterial activity. J Bee World 1992, 73, 5– 28 2. Efem SE. Recent advances in the management of fournier’s gangrene: Preliminary observations. Surgery 1993, 113, 200–4. 3. Efem SE. Clinical observation on the wound healing properties of honey. Br J Surg. 1988, 75, 679–81. 4. Radwan S, El-Essawy A, Sarhan MM. Experimental evidence for the occurrence in honey of specific sub- stances active against microorganisms. Zentral Mikrobiol 1984, 139, 249–55. 5. 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Evaluation of inhibitory action of honey on fungal growth, sporula- tion and aflotoxin production. Z Lebensm Unters Forsch 1978, 166, 280–3. 12. Stokes ES, Ridway GI, Wren MW. Clinical Microbiol- ogy (7th ed.) Arnold London 1993. 13. Subrahmanyam M. Topical application of honey in treatment of burns. Br J Surg 1991, 78, 497–8. 14. Al Somal N, Coley KE, Molan PC, Hancock BM. Susceptibility of Helicobacter pylori to the antibacterial ac- tivity of manuka honey. J R Soc Med 1994, 87, 9–12. 15. Jeddar A, Kharsan YA, Ramsaroop UG, Bhamjee A, Haffejee E, Moosa A. The antibacterial action of honey: an in vitro study. S Afr Med J 1985, 67, 257–8. 16. O’Grady FW, Lambert HP, Finch RG, Greenwood D. Antibiotic and chemotherapy. Anti Infective Agents and Their Use in Therapy (7th ed), Churchill Livingstone, New York 1997. 17. Obeseiki-Ebor EE, Afonya TC. In-vitro evaluation of the anti-candidiasis activity of honey distillate (HY- 1) compared with that of some antimycotic agents. 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Honey samples with Average Inhibition Zones (mm) Organism No. tested 1 2 3 4 5 6 Pseudomonas aeruginosa 31 19.00 17.00 18.00 17.00 18.00 18.00 Klebsiella pneumonia 13 14.00 16.00 20.00 15.00 15.00 16.00 Acinetobacer spp. 4 16.00 15.00 20.00 22.00 18.00 19.00 Candida albicans 3 12.00 10.00 19.00 12.00 12.00 13.00 Staphylococcus aureus 15 10.00 17.00 20.00 15.00 19.00 18.00 Escherichia coli 14 22.00 21.00 20.00 18.00 20.00 14.00 Serrratia spp. 4 14.00 15.00 16.00 12.00 14.00 10.00 Enterobacter cloacae 4 17.00 18.00 17.00 18.00 14.00 16.00 Proteus mirabilis 4 0 0 0 0 0 0 Aspergillus niger 5 0 0 0 0 0 0 Aspergillus fumigatus 4 0 0 0 0 0 0 Streptococcus faecalis 10 0 0 0 0 0 0 Strep pyogenes 5 0 0 0 0 0 0 A N T I M I C R O B I A L P O T E N T I A L O F H O N E Y 79 Determination of phenol in honey by liquid chroma- tography with amperometric detection. J Assoc Anal Chem 1990, 73, 602–4. 20. Willix DJ. Molan PC. Harfoot CG. A comparison of the sensitivity of wound infecting species of bacteria to the antibacterial activity of manuka honey and other honey. J Appl Bacteriol 1992, 73, 388–94. 21. Joerg E, Sontag G. Multichannel coulometric detec- tion coupled with liquid chromatography for determi- nation of phenolic esters in honey. J Chromatogr 1993, 635, 137–42. N Z E A K O & H A M D I 80 Antimicrobial potential of honey �on some microbial isolates METHOD Table 1 Examined commercial honey samples and their sources. RESULTS DISCUSSION CONCLUSION REFERENCES