A Comparison Between Medical Grade Honey and Table Honeys in Relation to Antimicrobial Efficacy

Rose A. Cooper, PhD; Leighton Jenkins, BSc

Disclosures

Wounds. 2009;21(2) 

In This Article

Materials and Methods

Honey Samples

During the spring of 2007, 17 samples of table honey were purchased from British supermarkets and one was purchased from a market stall in rural France ( Table 1 ). Sample 19 was sterile, medical-grade manuka honey (Manukacare 18+™, Comvita UK, Ltd, Berkshire). This honey is marketed in Canada and the United States as Medihoney™ (Derma Sciences, Princeton, NJ). An artificial honey solution was used to determine whether inhibitory effects were due to the sugar content of the honey samples–100 g was prepared by dissolving 1.5-g sucrose, 7.5-g maltose, 40.5-g D-fructose, and 33.5-g D-glucose in 17-mL of sterile, deionized water. Honey samples were stored in the dark at 4°C until tested (all tests were completed within 6 months of purchase). Each sample was mixed thoroughly with a sterile spatula to remove a representative sample for each test.

Determination of Antibacterial Activity

The Allen et al[18] bioassay was utilized to determine total and non-peroxide activity of all honey samples, except that the test organism used was S aureus NCTC 6571 instead of S aureus ATCC 9144. Each sample was assayed in quadruplicate, and dilutions were prepared either with deionized water (to determine total antibacterial activity) or with 2 mg/mL catalase solution ([Sigma, Poole, UK] to determine non-peroxide antibacterial activity). Zones of inhibition were measured and compared to those obtained using reference solutions of phenol (Fisher Scientific, Fair Lawn, NJ), so that activity was expressed as phenol equivalent %(w/v). The inhibitory potential of each honey sample was estimated by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against 6 bacterial cultures.

Minimum Inhibitory Concentration

Using aseptic technique, a stock solution of each honey (1 g/mL) was prepared by weighing 10 g and making up to 10 mL in a volumetric flask using sterile iso-sensitest broth ([ISB], Oxoid, Cambridge, UK). Each stock solution was used to prepare a serial doubling dilution series to 31.25 mg/mL using iso-sensitest broth and 200-µL aliquots were transferred to a 96 well microtiter plate. Apart from the wells in column 1 (negative control: ISB only) and in row A (honey sterility control), all wells were inoculated with 1-μL of an overnight culture of test organism (approx. 106 cfu/mL). The following organisms were used: S aureus NCTC 6571 (Oxford Staph), Escherichia coli NCTC 10418, E-MRSA 15 NCTC 13142, Staphylococcus epidermidis (clinical isolate), Streptococcus pyogenes (clinical isolate) and Pseudomonas aeruginosa (clinical isolate). Wells in column 2 were positive controls (no honey). Plates were incubated overnight at 37°C and absorbance determined at 490 nm using a plate reader (Dynex, West Sussex, UK). Bacterial growth (turbidity) was determined visually and confirmed with the plate reader printouts. The lowest concentration of honey to prevent growth of each organism was recorded as the MIC. All wells with no apparent growth were streaked to nutrient agar plates (Oxoid, Cambridge, UK) and incubated at 37°C for 24 hours to determine whether viable bacteria had persisted. The MBC was recorded as the lowest concentration of honey to prevent the survival of viable bacteria. These tests were repeated with honey samples prepared with ISB containing 2-mg/mL catalase.

Characterization of microbial flora of the natural honey samples. With the exception of the artificial honey sample, solutions of each honey were prepared according to the details provided in Table 2 and either plated directly onto the media specified or cultured (enriched) in broth and streaked onto selective media. Hence, the presence and number of mesophilic aerobic bacteria, mesophilic anaerobic bacteria, coliforms, salmonellae, clostridia, yeasts, and fungi were determined. Representative colonies were identified as far as possible using rapid identification kits for Gram-positive bacteria, Gram-negative bacteria, and anaerobes supplied by BBL and bioMériuex according to the manufacturers' instructions.

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