As we face more challenges when treating and healing humans, revisiting therapies that fell out of favor has become more common. For example, people with open wounds that are not healing receive judicious applications of maggots to remove necrotic tissue and promote healing. Leeches are used for patients after surgery to prevent blood clotting in swollen tissues and encourage healing. However, not all therapies involve direct application of squirmy creatures to skin; in fact, honey is one item people are willing to have in their homes. Honey has been used as a sweetener for food, for soothing sore throats and coughs and, more recently, for treating wounds unresponsive to traditional antibiotic therapy. In a recent PLoS ONE paper, the authors assessed the properties that are associated with honey’s antimicrobial activity against pathogenic and food-spoiling bacteria.

Kwakman et al. examined two different medical-grade honeys, unprocessed Revamil^® source (RS) honey from the Netherlands and nonsterilized UMF™ 16+ Active manuka honey from the United Kingdom. Using 40% (v/v) as the starting amount, RS and manuka honeys were serially diluted twofold to determine maximal dilution that reduced bacterial survival 1000-fold for methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli and Pseudomonas aeruginosa, pathogens found in wounds, and Bacillus subtilis, a food-spoiling agent. After two hours, RS honey inhibited growth of all bacteria but MRSA at 2.5- to 13-fold dilutions, but manuka honey only showed activity against B. subtilis at 2.5-fold dilution. After a 24-hour incubation, manuka honey had more potent antimicrobial activity against all tested bacteria (e.g., tenfold dilution of RS honey versus 80-fold dilution of manuka honey to kill MRSA). Each honey demonstrated effective killing at both time points but at different levels against each tested microbe.

The known factors responsible for the bactericidal activity of honey include high sugar concentration, H₂O₂, the 1,2-dicarbonyl compound methylglyoxal (MGO), the cationic antimicrobial peptide bee defensin-1 and low pH. In 40% (v/v) honey, H₂O₂ concentration for RS honey was determined to be 3.47 ± 0.25mM after accumulating for 24 hours, while manuka honey had no measurable H₂O₂. Levels of MGO differed as well: 0.25 ± 0.01mM for RS honey and 10.94 ± 1.70mM for manuka honey. Bee defensin-1 protein levels in honey were determined by concentrating the proteins, separating the samples on native acid-urea polyacrylamide gel electrophoresis (AU-PAGE) and visualized by parallel Coomassie-staining and a B. subtilis growth inhibition overlay assay. Only the RS honey showed any significant staining on the gel and inhibited B. subtilis growth on agarose.

Because the two medical honeys tested by Kwakman et al. differed in their bactericidal activities, manuka honey was further assessed in the 24-hour treatment for factors that contribute to its effectiveness against bacteria. Since manuka honey had much higher MGO levels than RS honey and showed no other activity (H₂O₂ and bee defensin-1), researchers neutralized MGO in manuka honey and the same panel of bacteria were assessed. Without MGO, the manuka honey activity against MRSA was reduced to that of a honey-equivalent sugar solution. However, E. coli killing activity had not changed and higher dilutions of honey were needed to kill B. subtilis and P. aeruginosa (two- and eightfold more honey, respectively). To examine what cationic factors contributed to manuka honey’s activity, a polyanionic compound that neutralizes cationic compounds was added. As a result, activity against P. aeruginosa was lost while effectiveness again B. subtilis and E. coli was reduced. Even though manuka honey had no measurable H₂O₂, catalase was added to neutralize H₂O₂ with no change in antimicrobial activity. Finally, the MGO- and cationic-neutralized honey had its pH adjusted to 7.0 and was tested again. Activity against E. coli was abolished while manuka honey retained activity against B. subtilis albeit at a lower level. The dilution and neutralization tests revealed that honey concentration, MGO and additional cationic and noncationic factors are part of the 24-hour antimicrobial activity of manuka honey.

Which factors contribute to the bacterial killing after only two hours? For RS honey, neutralizing antimicrobial bee defensin-1 peptide increased the amount of honey needed to kill B. subtilis from 5% to 40% but did not change the amount of honey needed to kill E. coli or P. aeruginosa. After also neutralizing H₂O₂, RS honey was no more effective at killing B. subtilis and E. coli than honey-equivalent sugar solution; activity against P. aeruginosa with at 40% honey was only one order of magnitude above honey-equivalent sugar solution. Any remaining activity against P. aeruginosa was destroyed when the pH was increased to 7. For manuka honey, two-hour activity against B. subtilis was reduced when MGO was neutralized. However, the remaining antimicrobial activity was unaffected by adjusting the pH to 7 or adding catalase to destroy H₂O₂ and was further reduced but not abolished when treated with polyanionic compound.

Before I read this article, I had no idea that honey’s antibacterial activity was multifactorial and heavily concentration-dependent. Since the two honeys examined came from two different sources, different bactericidal factors are not unexpected, but I was surprised that they differed so greatly. Because of the multiple factors that contribute to the antibacterial activity, microbes have difficulty circumventing the killing effects of honey versus that of antibiotic therapy. Now I have a better appreciation that honey can take care of nasty microbes on our bodies and in our food. And if I had a nonhealing wound and was asked to choose between treatment with honey or with maggots, no contest.

Reference
Kwakman, P., te Velde, A., de Boer, L., Vandenbroucke-Grauls, C. and Zaat, S. (2011). Two Major Medicinal Honeys Have Different Mechanisms of Bactericidal Activity. PLoS ONE, 6 (3) DOI: 10.1371/journal.pone.0017709

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Sara Klink

Technical Writer at Promega Corporation

Sara is a native Wisconsinite who grew up on a fifth-generation dairy farm and decided she wanted to be a scientist at age 12. She was educated at the University of Wisconsin—Parkside, where she earned a B.S. in Biology and a Master’s degree in Molecular Biology before earning her second Master’s degree in Oncology at the University of Wisconsin—Madison. She has worked for Promega Corporation for more than 15 years, first as a Technical Services Scientist, currently as a Technical Writer. Sara enjoys talking about her flock of entertaining chickens and tries not to be too ambitious when planning her spring garden.

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