Ralf C. Schlothauer

The Effect of New Zealand Kanuka, Manuka and Clover Honeys on Bacterial Growth Dynamics and Cellular Morphology Varies According to the Species

Treatment of chronic wounds is becoming increasingly difficult due to antibiotic resistance. Complex natural products with antimicrobial activity, such as honey, are now under the spotlight as alternative treatments to antibiotics. Several studies have shown honey to have broad-spectrum antibacterial activity at concentrations present in honey dressings, and resistance to honey has not been attainable in the laboratory. However not all honeys are the same and few studies have used honey that is well defined both in geographic and chemical terms. Here we have used a range of concentrations of clover honey and a suite of manuka and kanuka honeys from known geographical locations, and for which the floral source and concentration of methylglyoxal and hydrogen peroxide potential were defined, to determine their effect on growth and cellular morphology of four bacteria: Bacillus subtilis, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. While the general trend in effectiveness of growth inhibition was manuka>manuka-kanuka blend>kanuka>clover, the honeys had varying and diverse effects on the growth and cellular morphology of each bacterium, and each organism had a unique response profile to these honeys. P. aeruginosa showed a markedly different pattern of growth inhibition to the other three organisms when treated with sub-inhibitory concentrations of honey, being equally sensitive to all honeys, including clover, and the least sensitive to honey overall. While hydrogen peroxide potential contributed to the antibacterial activity of the manuka and kanuka honeys, it was never essential for complete growth inhibition. Cell morphology analysis also showed a varied and diverse set of responses to the honeys that included cell length changes, cell lysis, and alterations to DNA appearance. These changes are likely to reflect the different regulatory circuits of the organisms that are activated by the stress of honey treatment.

Synergism between Medihoney and Rifampicin against Methicillin-Resistant Staphylococcus aureus (MRSA)

Skin and chronic wound infections caused by highly antibiotic resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) are an increasing and urgent health problem worldwide, particularly with sharp increases in obesity and diabetes. New Zealand manuka honey has potent broad-spectrum antimicrobial activity, has been shown to inhibit the growth of MRSA strains, and bacteria resistant to this honey have not been obtainable in the laboratory. Combinational treatment of chronic wounds with manuka honey and common antibiotics may offer a wide range of advantages including synergistic enhancement of the antibacterial activity, reduction of the effective dose of the antibiotic, and reduction of the risk of antibiotic resistance. The aim of this study was to investigate the effect of Medihoney in combination with the widely used antibiotic rifampicin on S. aureus. Using checkerboard microdilution assays, time-kill curve experiments and agar diffusion assays, we show a synergism between Medihoney and rifampicin against MRSA and clinical isolates of S. aureus. Furthermore, the Medihoney/rifampicin combination stopped the appearance of rifampicin-resistant S. aureus in vitro. Methylglyoxal (MGO), believed to be the major antibacterial compound in manuka honey, did not act synergistically with rifampicin and is therefore not the sole factor responsible for the synergistic effect of manuka honey with rifampicin. Our findings support the idea that a combination of honey and antibiotics may be an effective new antimicrobial therapy for chronic wound infections.

Manuka-type honeys can eradicate biofilms produced by Staphylococcus aureus strains with different biofilm-forming abilities

Chronic wounds are a major global health problem. Their management is difficult and costly, and the development of antibiotic resistance by both planktonic and biofilm-associated bacteria necessitates the use of alternative wound treatments. Honey is now being revisited as an alternative treatment due to its broad-spectrum antibacterial activity and the inability of bacteria to develop resistance to it. Many previous antibacterial studies have used honeys that are not well characterized, even in terms of quantifying the levels of the major antibacterial components present, making it difficult to build an evidence base for the efficacy of honey as an antibiofilm agent in chronic wound treatment. Here we show that a range of well-characterized New Zealand manuka-type honeys, in which two principle antibacterial components, methylglyoxal and hydrogen peroxide, were quantified, can eradicate biofilms of a range of Staphylococcus aureus strains that differ widely in their biofilm-forming abilities. Using crystal violet and viability assays, along with confocal laser scanning imaging, we demonstrate that in all S. aureus strains, including methicillin-resistant strains, the manuka-type honeys showed significantly higher anti-biofilm activity than clover honey and an isotonic sugar solution. We observed higher anti-biofilm activity as the proportion of manuka-derived honey, and thus methylglyoxal, in a honey blend increased. However, methylglyoxal on its own, or with sugar, was not able to effectively eradicate S. aureus biofilms. We also demonstrate that honey was able to penetrate through the biofilm matrix and kill the embedded cells in some cases. As has been reported for antibiotics, sub-inhibitory concentrations of honey improved biofilm formation by some S. aureus strains, however, biofilm cell suspensions recovered after honey treatment did not develop resistance towards manuka-type honeys. New Zealand manuka-type honeys, at the concentrations they can be applied in wound dressings are highly active in both preventing S. aureus biofilm formation and in their eradication, and do not result in bacteria becoming resistant. Methylglyoxal requires other components in manuka-type honeys for this anti-biofilm activity. Our findings support the use of well-defined manuka-type honeys as a topical anti-biofilm treatment for the effective management of wound healing.

Arabinogalactan proteins contribute to the immunostimulatory properties of New Zealand honeys.

Context: Factors in honey that improve wound healing are poorly understood, but are thought to include lipopolysaccharide (LPS), apalbumin-1 and -2, and a 5.8 kDa component that stimulate cytokine release from macrophages. Objective: To characterize the ability of New Zealand honeys to elicit the release of tumor necrosis factor-α (TNF-α) from monocytic cell lines as a model for early events within a wound site. Materials and methods: The ability of kanuka (Kunzea ericoides), manuka (Leptospermum scoparium), and clover (Trifolium spp.) honeys to stimulate the release of TNF-α from monocytic cell lines THP-1 and U937 was assayed by ELISA. Results: All three honeys stimulated TNF-α release from THP-1 cells, with kanuka honey being the most active. The activity of kanuka honey was associated with a high molecular weight (>30 kDa) component that was partially heat labile and inhibitable with polymyxin B. LPS concentrations in the honeys were too low to adequately explain the level of immunostimulation. The contribution of type II arabinogalactan proteins (AGPs) we recently identified in kanuka honey was tested, as AGPs are known immunostimulators. AGPs purified from kanuka honey stimulated the release of TNF-α from THP-1 and U937 cells. Discussion: Here we demonstrated that AGPs we recently identified in kanuka honey have immunostimulatory activity. We propose that the immunostimulatory properties of individual honeys relate to their particular content of LPS, apalbumins, the 5.8 kDa component and AGPs. Conclusion: The immunostimulatory activity of kanuka honey may be particularly dependent on AGPs derived from the nectar of kanuka flowers.

Fluorescence markers in some New Zealand honeys.

The fluorescence characteristics of various New Zealand honeys were investigated to establish if this technique might detect signatures unique to manuka (Leptospermum scoparium) and kanuka (Kunzea ericoides) honeys. We found unique fluorescence profiles for these honeys which distinguished them from other New Zealand honey floral types. Two excitation-emission (ex-em) marker wavelengths each for manuka and kanuka honeys were identified; manuka honey at 270-365 (MM1) and 330-470 (MM2) nm and kanuka honey at 275-305 (KM1) and 445-525 (KM2) nm. Dilution of manuka and kanuka honeys with other honey types that did not possess these fluorescence profiles resulted in a proportional reduction in fluorescence signal of the honeys at the marker wavelengths. By comparison, rewarewa (Knightia excelsa), kamahi (Weinmannia racemosa), and clover (Trifolium spp.) honeys did not exhibit unique fluorescence patterns. These findings suggests that a fluorescence-based screening approach has potential utility for determining the monoflorality status of manuka and kanuka honeys.

Antibiotic-specific differences in the response of Staphylococcus aureus to treatment with antimicrobials combined with manuka honey

Skin infections caused by antibiotic resistant Staphylococcus aureus are a significant health problem worldwide; often associated with high treatment cost and mortality rate. Complex natural products like New Zealand (NZ) manuka honey have been revisited and studied extensively as an alternative to antibiotics due to their potent broad-spectrum antimicrobial activity, and the inability to isolate honey-resistant S. aureus. Previous studies showing synergistic effects between manuka-type honeys and antibiotics have been demonstrated against the growth of one methicillin-resistant S. aureus (MRSA) strain. We have previously demonstrated strong synergistic activity between NZ manuka-type honey and rifampicin against growth and biofilm formation of multiple S. arueus strains. Here, we have expanded our investigation using multiple S. aureus strains and four different antibiotics commonly used to treat S. aureus-related skin infections: rifampicin, oxacillin, gentamicin, and clindamycin. Using checkerboard microdilution and agar diffusion assays with S. aureus strains including clinical isolates and MRSA we demonstrate that manuka-type honey combined with these four antibiotics frequently produces a synergistic effect. In some cases when synergism was not observed, there was a significant enhancement in antibiotic susceptibility. Some strains that were highly resistant to an antibiotic when present alone become sensitive to clinically achievable concentrations when combined with honey. However, not all of the S. aureus strains tested responded in the same way to these combinational treatments. Our findings support the use of NZ manuka-type honeys in clinical treatment against S. aureus-related infections and extend their potential use as an antibiotic adjuvant in combinational therapy. Our data also suggest that manuka-type honeys may not work as antibiotic adjuvants for all strains of S. aureus, and this may help determine the mechanistic processes behind honey synergy.

Isolation, Structural Elucidation, and Synthesis of Lepteridine From Ma̅nuka (Leptospermum scoparium) Honey

Ma̅nuka honey, made from the nectar of Leptospermum scoparium, has garnered scientific and economical interest due to its nonperoxide antibacterial activity. Biomarkers for genuine ma̅nuka honey are increasingly in demand due to the presence of counterfeit ma̅nuka honey. This work reports the identification of a compound previously unreported in ma̅nuka honey by HPLC, and determination of the structure of the as 3,6,7-trimethyllumazine using NMR, MS, IR, and UV/vis spectroscopy. This assignment was confirmed by total synthesis. The natural product, renamed lepteridine, was only observed in ma̅nuka honeys and could potentially serve as a biomarker for genuine ma̅nuka honey.

Honeybee apisimin and plant arabinogalactans in honey costimulate monocytes

Here we determined whether immunostimulatory plant-derived arabinogalactan proteins (AGPs) and the honeybee-derived protein apisimin are present in varieties of New Zealand honey. Apisimin is a protein of unknown function secreted from the glands of honeybees into Royal Jelly, forming a complex with apalbumin1 capable of stimulating lymphocyte proliferation. AGPs were abundant in kanuka honey with lesser amounts in manuka, kowhai and clover honeys, but absent from Royal Jelly. Apisimin was present in all honeys, as well as Royal Jelly. We report that apisimin shares with honey AGPs the ability to stimulate the release of TNF-α from blood monocytes. Further, it synergizes with AGPs to enhance the release of TNF-α, via a mechanism not involving the formation of a complex with AGPs. In summary, this study provides evidence that AGPs and apisimin are commonly present in different floral varieties of honey, and hence contribute to their immunostimulatory properties.

Leptosperin is a distinct and detectable fluorophore in Leptospermum honeys.

New Zealand manuka (Leptospermum scoparium) honey exhibits two unique fluorescence signatures that distinguish it from other honey types. One of these is the MM1 fluorescence marker (270-365nm excitation-emission) which we show is due to a Leptospermum nectar-derived compound, leptosperin. Synthetic or honey-purified leptosperin not only displayed an identical fluorescence spectrum, but supplementation of leptosperin into clover or artificial honeys generated the MM1 fluorescence signature. There was a quenching effect of the honey matrix on leptosperin fluorescence but otherwise leptosperin was chemically stable over prolonged storage at 37°C. Leptosperin was also present in the woody-fruited Australian Leptospermum species at elevated concentrations but virtually absent in Leptospermum subtenue suggesting its elevated expression developed following the mid-Miocene separation of the genus. These findings suggest that fluorescence spectroscopy could offer a rapid and high-throughput screening method for identification of Leptospermum honeys using the MM1 fluorescence marker.

Honey Production and Compositional Parameters

Abstract Manuka honey is produced from the nectar of Leptospermum scoparium and is of high therapeutic and commercial value due to its non-peroxide antibacterial activity, known as unique Manuka factor (UMF). These properties are attributable to the presence of methylglyoxal (MGO), which is derived from the precursor molecule dihydroxyacetone (DHA) present in the plant nectar, and potentially other components such as phenolic compounds. Despite the therapeutic importance of Manuka honey, relatively little is known about how its molecular composition and the MGO concentration are affected by aging and storage conditions. Here we describe findings using mass spectrometry showing that honey contains a diverse array of small-molecule compounds and that these vary depending on floral origin and storage conditions. We also describe how MGO levels change as Manuka honey matures as well as other components that might be useful as floral markers of this honey.