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Microsoft word - 7in vitro.doc

Original article
In vitro assessment of the antimicrobial potential of honey on
common human pathogens

Andargarchew Mulu1, Belay Tessema1, Fetene Derbie2
Abstract
Background: Honey produced by honeybees (Apis mellifera) is one of the ancient traditional medicines used for
treatment and prevention of various illnesses.
Objective: To assess the antimicrobial potential of honey on some common bacterial pathogen.
Methods: This experimental study was conducted in Jimma University, from February 10 – March 14, 2003. The
Minimal Inhibitory Concentrations/ MIC and Minimal Bactericidal Concentrations/ MBC of two honey samples on
Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, Shigella shiga, Klebsiella
aerogenes, Proteus vulgaris
and Proteus mirabilis was investigated by an agar dilution technique.
Results: The MIC of honey for 90% of test organism was 6.25% and 7.5% (V/V) for P.aeruginosa. The MBC of
honey for 70% of the test organisms was again 6.25% (V/V). The MBC of honey for S.shiga (Standard test organism)
and P. aeruginosa (both clinical isolates and control strain) was 7.5% (V/V).
Conclusions: Honey produced by honeybees (Apis mellifera) has both bacteriostatic and bactericidal activity when
tested in vitro. However, Pharmacological standardization and clinical evaluation on the effect of honey are essential
before using honey as a preventive and curative measure to common diseases related to the tested bacterial species.
[Ethiop.J.Health Dev. 2004;18(2):107-111]

Introduction

In developing countries all over the world especially in Recently, many researchers have reported the Africa, large number of people die daily of preventable antibacterial activity of honey against S.aureus, P. and curable diseases because of lack of even simple aeuruginosa, E.coli, P. mirabilis, S. pyogenus, S. flexneri health care (1). Despite the enormous advance in health and S .typhi (9-11). It has been documented that honey care made during the last half century, infectious diseases has a bacteriostatic and bactericidal effect against various still account for 25% of mortality worldwide and 45% in species of both gram positive and gram negative bacteria, low-income countries. Anti-infective drugs are critically as well as an anti-fungal effect (9, 12). important in reducing the global burden of infectious diseases. However, as resistant microbes develop and The ability of honey to kill microorganisms has been spread, the effectiveness of the drugs is diminished (2). attributed to its high osmotic effect, high acidic nature This type of resistance to antimicrobial agent is an (pH being 3.2-4.5), hydrogen peroxide concentration and increasing problem in many areas of the world especially its phytochemical nature, i.e. its content of tetracycline derivatives, peroxides, amylase, fatty acids, phenols, ascorbic acid, flavonides, streptomycin, sulfathiazole, The use of traditional medicine to treat infection has been trepens, benzyl alcohol, and benzoic acids (9,13,14). practiced since the origin of man kind (1), and in past it However the production and type of honey produced by was the only method available. Currently, due to the honeybees is dependent on the natural vegetative flowers absence of sufficient modern health care system, blooming in different seasons. Thus the flowers from particularly in rural areas, people prefer to visit which bees gathered nectar to produce the honey may traditional healers and herbal medicines (5-6). The contribute to the difference in the antimicrobial activities integration traditional and modern medicine is gaining The purpose of the present study was therefore to Honey produced by honeybees (Apis mellifera) is one of evaluate scientifically the in vitro antimicrobial potential the oldest traditional medicines considered to be (bacteriostatic and bactericidal effect) of honey produced important in the treatment of respiratory ailment, by honeybees (Apis mellifera) against eight bacterial gastrointestinal infection and various other diseases. It is species among those commonly involved in causing being used effectively as a dressing for wounds, gastroenteritis, pneumonia, wound and urinary tract (including surgical wounds), burns, and skin ulcers to 1Gondar University College, P.O.Box 196 Gondar, Ethiopia E-mail: andargachewmulu@yahoo.com; 2Faculty of Medical Sciences, Jimma University, P.O.Box 378, Jimma
Materials and methods
diameter were observed at different concentrations of This experimental study was conducted in Jimma honey. University, School of Medical Laboratory Technology Following this screening test, further investigation of the antimicrobial effect of honey was carried out using the Honey samples harvested during spring 2002 and winter agar dilution technique, which was done by mixing 2003 were collected from Jimma University, College of molten Mueller Hinton agar [(Oxoid, UK) prepared by Agriculture, Animal Science Department, Bee keeping suspending 38 gram of the powder in 1 liter of distilled Unit in sterile screwed cups. Each honey sample was first water and brought to boil to dissolve the medium filtered with a sterile mesh to remove debris and then completely and sterilized by autoclaving at 121oC for 15 streaked on blood agar plate, and incubated overnight to minutes], and held in water bath (45-50oC) with honey check microbial purity and stored at 2-8 oC until used. (19). Hence a known volume (ml) of honey: 0.5, 0.75, 1, 1.25, 1.5, 2 per 20ml of media were used. These are The following control bacterial strain, standard test equivalent to honey concentrations (percentage by organisms and clinical isolates most commonly involved volume) of 2.5, 3.75, 5, 6.25, 7.5 and 10 respectively. in causing gastroenteritis, pneumonia, wound and urinary Similarly two selected antibiotics (penicillin G and tract infections were used. Control [E. coil American chloramphenicol) and a supersaturated solution of sugar Type Culture Collection /ATCC 25922, S. aureus ATCC of the same proportion as honey (85%W/V) were diluted 25923, P.aeruginosa ATCC 27813]; Standard [S.typhi to get a similar concentration as honey and tested on 127, S.shiga 106, K.aerogenes, P.vulgaris]; Clinical separate plates and compared with the MIC & MBC of isolates [S.aureus (ear discharge), P.mirabilis (ear honey. discharge), P.aeruginosa (wound)]. The test media were incubated at 36-37oC overnight to Bacterial cultures, S. aureus ATCC 25923, and S.shiga check their microbial purity (18). Then, the test plates 106 were obtained from Ethiopia Health and Nutrition which showed no microbial contamination were Research Institute (EHNRI); E. coil ATCC 25922 inoculated with the prepared bacterial cultures (104 CFU/ P.aeruginosa ATCC 27813, and S.typhi 127 were ml) and incubated aerobically at 36-37oC for 20 hours in obtained from Jimma University, School of Medical inverted positions. Mueller Hinton plates with out honey Laboratory Technology; K.aerogenes, P.vulgaris and all were similarly inoculated to control the appropriate the clinical isolates were collected from Jimma growth of the organisms. University, Microbiology Department. The clinical isolates were identified based on the standard The Partial Inhibitory effect /the lowest concentration microbiological technique (16) and drug susceptibility that retarded growth/ and Complete Inhibitory effect of test for each clinical isolate was done following the different concentration of honey were examined by standard agar disc diffusion method (17). These placing plates on a dark background and observing organisms were maintained in the laboratory on nutrient macroscopically for the lowest concentration that retarded and completely inhibited growth (in comparison with the control plate) respectively. Thus the Partial Morphologically identical colonies from overnight Inhibitory Concentration /PIC was reported as the lowest growth were picked with an inoculating loop and concentration that retarded growth as compared to the suspended in 3-4 ml of nutrient broth and incubated for control plate and the MIC was reported as the lowest 2-3 hours at 36-37oC and diluted with sterile normal concentration of honey that completely inhibited visible saline to a turbidity that matches 0.5 McFarland standard growth, and the MBC was determined by further sub (106 Colony Forming Unit (CFU)/ml), and further diluted culturing the last plate which showed visible growth and 1:100 in sterile nutrient broth to set an inoculum density all the plates in which there was no growth in Mueller of 1x104 CFU/ml which was used for the test (18, 19). Hinton agar. The MBC was therefore the lowest concentration of honey required to produce sterile culture Preliminary investigation had been carried out by using agar diffusion technique to test the activity of honey against control bacterial strains following the standard A stability test was also conducted as follows: Honey single disc diffusion method developed by Bauer et al samples were first divided into two aliquots. The first (17). In brief, a loop full (4mm in diameter) of the aliquot was stored at -10oC for one month and the second prepared control bacterial suspensions (1x104CFU/ml) aliquot was autoclaved at 121oC for 15 minutes and were separately applied to the center of a sterile Mueller allowed to cool. Then each aliquot was tested for Hinton plate and spread evenly using a sterile dry cotton antimicrobial activity as before, and finally comparisons wool, then 50 micro liter of different concentrations of honey were dispensed and incubated at 37oC for 20 hours. Various inhibition zones, more than 5mm in A single colony or a faint haze left by the initial inoculum was not regarded as growth. In plates with no Ethiop.J.Health Dev. 2004;18(2) In vitro assessment of the antimicrobial potential of hone 109 growth at lower concentration but growth at a higher of 3.75% (V/V) and 7.5%(v/v) of honey respectively. concentration, test organisms were sub cultured to Therefore, the Partial Inhibitory Concentration (PIC), the confirm purity, and the test was repeated. Minimum Inhibitory Concentration (MIC) value for 90% of the tested microorganisms was found to be 2.5 and The antimicrobial substances in honey were not assessed 6.25% (V/V) and for P.aeruginosa which was found to and determined. However, PH was tested and the PH of be 3.5 and 7.5% (V/V) respectively. The Minimum honey (undiluted) and media (with honey) were Bactericidal Concentration (MBC) value for 70% of measured using a digital PH meter. All tests were done in tested microorganisms was found to be similar to the triplicate and with appropriate controls at each step. MIC value of the 90% of tested organisms, i.e. 6.25% (V/V). But the MBC value for S.shiga (standard test organism) and P. aeruginosa (control strain and clinical The results of the in vitro susceptibility of the test isolates) was 7.5% (V/V). microorganisms to honey samples were similar. Of all the microorganisms tested, 90% were sensitive to honey This study also assessed the antibacterial activity of at a concentration of 6.25% (V/V) of honey. P. honey after autoclaving at 121oC for 15 minutes and aeruginosa (clinical isolate and control strain) was deep-freezing at -10oC for one month on control bacterial sensitive at a concentration of 7.5% (V/V) of honey. strains and honey samples retained their antimicrobial Both the control and clinical isolates of P.aeruginosa activity. However, PIC and MIC of honey on all control were the least sensitive of the test microorganism to strains after heat treatment increased by 1.2%, i.e. the honey (Table 1). PIC and MIC value for all control strains were 3.75 and 7.5% (V/V) respectively and the MBC value for E.coli Partial Inhibition for 90% of the test microorganisms was and S.aureus was 7.5% (V/V) and that of P.aeruginosa observed starting from 2.5% (V/V) and Complete 10% (V/V). On the other hand, the PIC, MIC and MBC Inhibition was observed at 6.25% (V/V) of honey and values of honey on control bacterial strains after deep- Partial Inhibition and Complete Inhibition for clinical freezing at -10oC for one month were similar to untreated isolates of P. aeruginosa was observed at a concentration
Table 1: The in vitro antimicrobial activity: PIC, MIC and MBC% (V/V) of honey produced by honeybees (Apis millifera)
in Mueller Hinton agar by agar dilution method against various control strains, standard test organisms and clinical
isolates

Bacterial strains with inoculums density of 104 CFU/ml Antimicrobial activity of honey % (V/V)
PIC MIC
Key: PIC-Partial Inhibitory Concentration MIC-Minimum Inhibitory Concentration MBC-Minimum Bactericidal
Concentration
Table 2: Comparisons of the in vitro antimicrobial activity: PIC, MIC, and MBC of honey produced by honeybees (Apis
mellifera
) in Mueller Hinton agar by agar dilution method before and after autoclaving at 121oC for 15 minutes and
deep freezing at -10oC for one month on control bacterial strains

Control bacterial strains with inoculums density of 104CFU/ml %( V/V) The MIC and MBC of two selected common antibiotics, a more marked growth retardation and inhibition on B. penicillin G and chloroamphenicol were assessed on cereus and S. aureus were observed at concentrations of control bacterial strains for control and comparison 10% (20). In contrast to this report honey produced by purposes and the result revealed that the MIC and MBC honeybees (Apis mellifera), in the present study could of penicillin G for S. aureus was less than 2.5% (V/V) or inhibit most of the test organisms at a very low 0.5ml of stock penicillin G (1x106 IU per 2 ml of sterile concentration (2.5-7.5%V/V). This might be due to the water) per 20 ml of media. Again, all control bacterial differences in the species of bees, which in turn results in strains were sensitive to chloroamphenicol, i.e. MIC was difference in the production and type of honey (15) and 6.25% (V/V) or 1.25 ml of stock chloroamphenicol the differences in the test methods and test organisms. (1gm/3ml of sterile water) per 20 ml of media. Control bacterial strains and clinical isolates were resistant to Studies on honey produced by honeybees (Apis meliffera) have shown that honey has antimicrobial activity against S.aureus, P.aeruginosa, E.coli, P.mirabilis, Citrobacter This study also compared the antibacterial activity of ferundi, Streptococcus faecalis, S.flexinari, and S.typhi honey to a super saturated solution of sugar of the same (9,10). It completely inhibits major wound infection sugar proportion as in honey (85% W/V) and the result pathogens including S.pyogenus and S.aureus (11). The showed that this supersaturated solution of sugar results of our study are consistent with the above study. exhibited less degree of antibacterial activity as compared to honey (data not shown). Molan demonstrated the activity of honey against S.aureus, Methicilin Resistance S.aureus and The PH values of undiluted and different concentrations Pseudomona Spp. He also cited that Willix D found the of honey were measured by digital PH meter and these percentage (by volume) of Manuka honey needed to were found to be 6.92, 6.71, 6.5, 6.31, 6.11 and 3.8 for completely prevent growth of each species of bacteria to 2.5%, 3.75%, 5%, 6.25%, 7.5% (V/V) and undiluted be 1.8, 3.6, 3.7, 6.0, 6.3, 7.3, and 10.8 % (V/V) for honey respectively. S.aureus, S. pyogeneus, E.coli, S. typhimurium, P.mirabilis and P.aeruginosa respectively (12). But the Discussion
percentage by volume of honey to completely prevent In our study two honey samples were tested for their growth of E.coli, S.aureus and P.mirabilis in the present antimicrobial activity on selected bacterial species and study was 6.5 and for P.aeruginosa it was 7.5; indicating the antimicrobial effect of these two honey samples on that there is a variation in the antimicrobial potency of test microorganisms were similar. The honey samples were found to have both bacteriostatic and bactericidal properties on both gram-positive and gram-negative Another study by Molan reported the concentration of bacteria. Honey samples used in this study showed partial honey in nutrient agar (% V/V) against various strains of inhibitory (bacteriostatic) and bactericidal activities for bacteria which cause gastroenteritis, and the PIC, MIC all of the test organisms at concentrations 2.5 - 7.25% and MBC were found to be 6, 7, 10 for E.coli; 6, 7, 8 for (V/V). Growth retardation and complete inhibition on S.typhmurim; 6, 7, 10 for S.flexinari and 6, 7, 10 for 90% of the test organisms were observed at a S.sonnei respectively (9). This is in contrast to our study. concentration of 2.5 % [PIC] and 6.25 % [MIC] of honey Here the variation in the antimicrobial potential of honey used in the present study as compared to the previous similar studies highlights that the source of the nectars The highest PIC and MIC were recorded for clinical may have contributed to the difference in the isolates (wound) of P.aeruginosa, i.e. 3.25 and 7.5 % antimicrobial activities of honey; that is, the flowers from (V/V) of honey respectively. The study showed that which bees gathered nectar to produce the honey, since honey has less antimicrobial activity against P. flora source determines many of the attributes of honey, aeruginosa and S. shiga as compared with other test for example flavor, aroma, color and composition. And microorganisms. The reason for this is not clear. Honey being a natural product, the composition of honey is samples also exerted antimicrobial activities on P. highly variable (15). The variation in sensitivity is also aeruginosa, P. mirabilis and other bacteria, which were attributable to differences in growth rate of pathogens, resistant to some common antibiotics discs such as nutritional requirements, temperature, inoculum’s size penicillin, ampicillin, chloroamphenicol, cotrimoxazol, and the test method it self (19). and gentamycin. In the present study, the antimicrobial substances in In Ethiopia, a study by Mogessie Ashenafi (1994) honey were not estimated except for PH reported that ‘tazma mar’ honey produced by sting- less the media at which MIC and MBC observed were 6.3 and bee (Apis mellipodae) was found to be effective against 6.11 respectively, which is low enough to be inhibitory to some food-borne pathogens of humans. Growth many pathogens; the PH for growth of these pathogens Retardation and inhibition on S.typhimurim, S.enteritidis and E.coli were noted at 15 and 20% concentration, while Ethiop.J.Health Dev. 2004;18(2) In vitro assessment of the antimicrobial potential of hone 111 The experiment also showed that antimicrobial substance 5. Andrews. J.A Bibliography on herbs, herbal in honey could withstand deep freezing at -10oC for one medicine, “Natural” foods, and unconventional month. However, the MBC of honey on all tested medical treatment. 1982; Libraries unlimited, Inc., microorganisms decreased by 1.2% after autoclaving of honey at 121oC for 15 minutes. This shows that its 6. Abebe D. The Development of Drug Research. antimicrobial activity is not dependent alone on its Ethiopian Health and Nutrition Research Institute phytochemical nature, i.e. tetracycline derivatives, ascorbic acid, peroxidase or amylases, streptomycin, 7. Ministry of Health. Health Policy of Traditional sulfonamides which are claimed as heat labile (14). On the other hand, the antimicrobial effect of honey is 8. World Health Organization. Drug information. attributed to its phenolic acid, flavonides, benzyl - alcohol, 2-hydroxy benzoic acid which are heat stable 9. Molan P.C. The antibacterial activity of honey. Bee and may be active agents but their concentration in honey appears too low to sorely responsible (14). 10. Nzeako and Hamdi. Antimicrobial potential of Again, the experiment showed that, supersaturated 11. Kingsley A. Supplements. The use of honey in solution of sugar of the same proportion as honey, i.e. treatment of infected wounds. Case studies. BJ of 85% [W/V](10) did not have the same degree of antibacterial activity as honey, indicating that while the 12. Molan P.C., Betts J. Using honey dressings: The removal of water from bacteria is important; other factors are operating to provide the observed antibacterial effect. 13. Bogdanov S. Charactrization of antibacterial In conclusions, honey produced by honeybees (Apis substance in honey. Lebensm Wiss Technol. mellifera) has both a bactreiostatic and bactericidal activity when tested in vitro. However, pharmacological 14. Heerng. W et al. Immunochemical screening for standardization and clinical evaluation on the effect of antimicrobial drug residue in commercial honey. honey are essential before using honey as a preventive and curative measure to common diseases related to the 15. National Honey Board. Honey Definitions tested bacterial species. The wider availability of honey Document. American bee Journal. Feb 1994. in rural areas provides its utilization for certain diseases 16. Cheesbrough M. Medical Laboratory Manual for Acknowledgements
Tropical Countries 1998, Vol II Microbiology. 196- We gratefully acknowledge the Research and Publication Office of Jimma University for its financial support. We 17. Bauer, A.W, Kirby, W.M.M., Sherirs, J.C. and are also thankful to School of Medical Laboratory Turck, M. Antibiotic susceptibility testing by Technology, Jimma University for its material support standard single disk method. American Journal of and College of Agriculture, Jimma University for providing us pure honey samples. Finally we would like 18. Mackie McCartney. Practical Medical Microbiology. to thank Ethiopian Health and Nutrition Research In: R.S. Miller, S.G.B. Amyes, 4th ed. Laboratory Institute, Addis Ababa for providing test organisms. Control of Antimicrobial Therapy. 1999;151-178. 19. Gaill Woods and Jon A. Washington. Antimicrobial Reference
susceptibility test; dilution & disk diffusion methods. 1. Sofowora, A. Medicinal plant and traditional Manual of Clinical Microbiology 1995:6th Ed; 1327– medicine in Africa. 1987; Chapter 1 and 2. 2. World Health Organization. Drug information. 20. Mogessie A. The in vitro Antibacterial activity of ‘Tazma mar’ honey produced by sting less 3. Shears P. Antimicrobial resistance in the tropics. bee. Ethiopian Journal of Health Development. 4. Assefa A. and Yohannes G. Antibiotic Sensitivity of 21. Cooper R. Molan P. The use of honey as an S. aureus and E.coli strains isolated in Gondar, antiseptic in managing pseudomonas infection. Ethiopia. Tropical Doctor. 1997;27(2):121-126. Journal of Wound Care. 1999;8(4):161–164.

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