Annals of Burns and Fire Disasters - vol. XIV - n. I - March 2001

ANTIBACTERIAL ACTIVITY OF HONEY ON BACTERIA ISOLATED FROM WOUNDS

Subrahmanyam M.,* Archan Hemmady,** Pawar S.G.**

* Department of Surgery
** Department of Microbiology, Government Medical College, Miraj and General Hospital, Sangli, Maharashtra, India

Introduction

The medicinal properties of honey have been known since ancient times. Ayurveda (Indian medicine) describes honey as the nectar of life and recommends its use in various ailments. There is renewed interest in honey treatment as evidenced by the number of reports appearing in the scientific literature.' Honey has been useful in the treatment of surgical wounds, burns, and decubitus ulcers, and the antibacterial and antifungal properties of honey have been well documented.` In burns in particular, honey has been found to control wound infection and accelerate wound healing." This study was undertaken in order to determine the minimum inhibitory concentration (MIC) of honey on bacteria isolated from burn wounds in our burn unit.

Material and method

Culture swabs collected from 80 patients with burns selected at random from the Burn Unit at the General Hospital, Sangli, India, formed the material for the study. The honey used was from Syzygium cumini, family Myrtacae, locally known as Jambhul. It did not contain any additives or diluents and had not been heated. The antibacterial effects of honey were studied in vitro. The different concentrations of honey (v/v) were prepared in a Muller-Hinton (MH) medium at 56 °C to give final concentrations of 5%, 10%, 15%, 20%, 25%, and 30%. The standard bacterial/broth cultures of the isolates from the burn wounds were inoculated on MH honey agar medium. The plates were incubated for 16-20 h at 37 °C. After overnight incubation, the plates were observed for inhibition of growth. Bacterial broth cultures inoculated only on MH medium without honey served as control. MIC is the lowest concentration of the honey that yielded no growth.
The comb was equilibrated in boiling water, briefly blotted, and then placed for 30 see without pressure on one side of the rat's back. After re-equilibration of the brass comb in boiling water, a second identical comb burn was made on the other side. This created a standard injury with four areas of full-thickness bum, corresponding to the four teeth of the brass comb. Each area measured 10 x 20 mmi, and created intermediate burns' between and around the full-thickness burn zones, the area studied in the experiment.
To simulate clinical use of the drug (i.e. post-injury), the aspirin treatment group received intramuscular injections of 200 mg/kg of aspirin immediately after burn infliction and 24 and 48 h later. The control group received sham injections of saline.
The total burn area was evaluated on days 1, 3, and 7 using fluorescein fluorescence (15 mg/kg). The area of the burn was copied onto a clear plastic sheet. Burn areas were determined by cutting out the marked area of the burn and then weighing the cut-outs on an analytical scale.
The Student t test was used to determine if there was a statistical difference between the groups.

Results

Table 1 shows the organisms isolated from burn wounds and their frequency. All the bacteria failed to grow at concentrations of 30% honey in MH medium. The MIC of honey is shown in Table II. There was no inhibition of growth in MH medium.

Discussion

The organisms isolated in the present study showed a slight variation compared with those of the earlier study. Organisms of the genera Salmonella, Shiegella, and Streptococci were not encountered in the study, in contrast to other reports. Coagulase-negative Streptococci formed the largest group, whereas in the other studies the most commonly isolated organism was coagulase-positive Staphylococcus.
This might be due to changing trends in the frequency pattern of the organisms isolated from burn wounds in hospitals. It is clear from the present evidence that coagulase-negative Staphylococcus has changed from the status of a non-pathogen to that of an opportunistic pathogen. Although once regarded as an innocuous member of the normal skin flora, coagulase-negative Staphylococcus is now recognized as an important opportunistic pathogen. It is routinely found on the skin and in the hospital environment, with a prevalence on the skin surface of 85-100%. Noble believed that coagulase-negative Staphylococcus was found excessively on damaged skin surface in normal persons.
A burned skin surface is a damaged skin surface, and this may be the reason for the high frequency of coagulase-negative Staphylococcus in the present study. Work on the antibacterial activity of honey has been going on since the eighteenth century. 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 inhibines in honey. These inhibines are hydrogen peroxide, fiavinoids, and phenolic acids, plus many other unidentified inhibines. 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 unfavourable environment with low water activity, thereby inhibiting bacterial growth; and a low pH of 3.6 and the fermentation of honey, producing alcohol in. sitar. Honey acts as a highly viscous barrier preventing bacterial penetration and colonization of the wound surface.
This study has shown that 100% inhibition was observed on MH agar with honey, as shown in Table II (coagulase-negative Staphylococcus at 25% honey concentration, Pseudomonas aeruginosa, eoagulase-positive Staphylococcus, Proteus mirabilis, and Citrobacter diversus at 25% concentration, Klebsiella pneumonia at 30% concentration, Citrobacter freundii, and Proteus vulgaris at 20% (v/v) honey concentration). This study thus shows that 30% (v/v) honey concentration is the in vitro minimum inhibitory concentration of honey for all the organisms isolated that cause burn wound infection.

RESUME. Les Auteurs presentent une etude de 80 cas de brfilures. Les organismes isoles dans les cultures des tampons etaient: Streptococci (56), Pseudomonas aeruginosa (44), Staphylococcus coagulase-positif (28), Klebsiella pneumonia (24), Proteus mirabilis (11), Escherichia coli (9), Citrobacter diversus (8), Citrobacter freundii (7) et Proteus vulgaris (3). Les isoles ont ete soumis a des analyses pour mesurer la sensibilite au miel utilisant le milieu Muller-Hinton avec une concentration de miel de 5%, 10%, 20%, 25% et 30% (v/v). La concentration inhibitrice minimum du miel etait 30% (v/v) pour toutes les bacteries isolees dans les brfilures.

1. Tovey F.: Editorial. Honey and sugar as a dressing for wounds and 8.ulcers. Tropical Doctor, 30: 1, 2000.
2. Armon P.J.: The use of honey in the treatment of infected wounds. Tropical Doctor, 10: 1037-40, 1980
3. Zulma A., Lulat A.: Honey - a remedy rediscovered. J. Soc. Med., 82: 384-5, 1989.
4. Cavanagh D., Beasley J., Ostapowicz F.: Radical treatment of carcinoma of the vulva. A new approach to wound healing. J. Obstet. Gynaecol., 77: 1037-40, 1970.
5. Phurpadit W., Saropla N.: Topical application of honey in treatment of abdominal wound disruption. Aust. N. Z. J. Obstet. Gynaecol., 32: 381-4, 1992.
6. Subrahmanyam M.: Honey dressing for burns. An appraisal. Ann. Burns and Fire Disasters, 9: 33-5, 1996.
7. Philips C.E.: Honey for burns. Gleanings in Bee Culture, 61: 284, 1933.
8. Posterns T., Bosch M.M.C., Butrieux R., Van Barre J., Hoekstra M.J.: Speeding up the healing of bums with honey. An experimental study with histological assessment of wound biopsies. "Bee Products", Marahi and Lensky (eds), Plenum Press, New York, 5763, 1997.
9. Subrahmanyam M.: Topical application of honey in treatment of burns. Br. J. Surg., 78: 497-8, 1991.
10. Williams R.E.: Benefit and mischief from commensal bacteria. J. Clin. Path., 22: 249-53, 1969.
11. Noble W.C.: Skin carnage of micrococcaceae. J. Clin. Path., 22: 249-53, 1969.
12. Allen K.L., Molar P.C., Reid G.M.: A survey of antibacterial activity of New Zealand honeys. J. Pharmacol., 43: 817-22, 1991.
13. White J.W., jr, Subers M., Schepartz A.L: The identification of inhibine, the antibacterial factor in honey as hydrogen peroxide and its origin in a honey glucose oxidase system. Biochem. Biophys. Acta, 73: 57-70, 1963.
14. Jeddar A. Kharsany A., Ramsaroop U.G., Bhamjee A., Maffejee I.E., Moosa: The antibacterial action of honey. An in vitro study. SAMJ, 67: 257-8, 1985.
15. Anand A., Shanmugam J.: Antistaphylococcal properties of natural honey. Biomedicine, 18: 15-18, 1998.