Introduction
Manuka honey is produced predominantly in New Zealand by European honey bees (Apis mellifera) feeding on the Manuka tree flower (Leptospermum scoparium) nectars (Adams 2009). According to the New Zealand Ministry for Primary Industries, Manuka-type honey has the following, naturally produced, characteristics (NZMPI 2015):
A color greater than 62 mm pfund.
A conductivity range of 347-867 µS/cm.
A flavor typical of Manuka-type honey (mineral, slightly bitter).
An aroma typical of Manuka-type honey (damp earth, heather, aromatic).
Presence of Manuka-type pollen.
Presence of dihydroxyacetone (DHA) and methylglyoxal (MG)
Manuka Honey Production
Manuka honey is mainly produced in New Zealand by approximately 3800 bee keepers and over 400,000 hives (Leake 2013). Figure 2 illustrates a typical Manuka honey apiary, found adjacent to heavily wooded areas. Some farmers use helicopters to find areas with dense Manuka bushes to aid in hive placement (Leake 2013). According to Unique Manuka Factor™ Honey Association (UMFHA) data, approximately 1,700 tons of Manuka honey are produced annually in New Zealand, representing almost all of the world's production (Leake 2013). However, approximately 10,000 tons are being sold internationally as Manuka honey, including 1,800 tons in the UK alone (Leake 2013). Counterfeit issues have become a significant concern with Manuka honey; in one publication, 41 of 73 Manuka honey samples tested from Britain, China and Singapore showed no non-peroxide activity, which is a key feature of Manuka honey (Leake 2013).
Assessing Manuka Honey’s Purity and Potency
There are various methods reported for determining and documenting the potency and purity of Manuka honey. Unfortunately, there is a lack of standardization in regards to labeling of Manuka honey, making interpretation and comparison very confusing. In order to better standardize labeling of Manuka honey, new interim guidelines have recently been released by New Zealand’s Ministry for Primary Industries (http://archive.mpi.govt.nz/food/food-safety/manuka-honey ; NZMPI 2015). Most Manuka honeys are currently labeled with a grading system that include a numerical value and a potency descriptor. Examples of potency descriptors include: active, bioactive, total activity, total peroxide activity, total non-peroxide activity, unique Manuka factor™ (UMF™), and methylglyoxal levels (NZMPI 2015). Third party test results to substantiate label claims are typically not readily available. Recent guidelines have suggested that terms such as “Non-Peroxide Activity”, “Total Peroxide Activity”, “Peroxide Activity”, “Total Activity” and “Active” should be removed from labels and advertising, as these terms involve a therapeutic claim that suggest an antimicrobial effect (NZPMI 2015). Methylglyoxal is thought to be the major contributor to Manuka honey’s non-peroxide antimicrobial activity (Adams 2009). Levels in honey are typically reported in mg/kg. References to methylglyoxal are permitted on honey labels provided they’re not used to imply antibacterial effects (NZPMI 2015).
One of the most well-known grading systems for Manuka honey is the Unique Manuka Factor® (UMF®) grade, which is a trademark of the Unique Manuka Factor Honey Association. The UMFHA is a group of certified licensees (currently 67) that can use the UMF trade mark on their products. To receive a UMF® grading, a honey must have the presence of DHA (dihydroxyacetone), methylglyoxal, and leptosperin. Values typically range from UMF® 5+ to UMF® 28+ that vary with the methylglyoxal level in the honey (see Table 1, UMFHA 2015).
Table 1 – Methylgloxal level and correlating UMF® grading (UMFHA 2015)
Methylgloxal Level
UMF™ Grade
≥83 mg/kg
5+
≥263 mg/kg
10+
≥514 mg/kg
15+
≥573 mg/kg
16+
≥696 mg/kg
18+
≥829 mg/kg
20+
≥1200 mg/kg
25+
≥1449 mg/kg
28+
Melissopalynology is the study of pollen in honey. Manuka honey can contain Manuka and Kanuka pollens, which can be difficult to differentiate. Recent research by NZPMI indicates that although morphological differences between Manuka and Kanuka pollens may be subtle, the two can be differentiated using direct light microscopy and Classifynder™ (NZMPI 2015). In order for Manuka honey to be classified as a monofloral honey, it must contain >70% Manuka pollens (NZPMI 2015). Manuka pollens alone are not sufficient for the identification of Manuka honey as high pollen counts do not always correlate with contribution of Manuka nectar to the honey (NZPMI 2015).
A recent publication found that leptosperin, a novel glycoside that is specific to manuka honey, is stable during storage and its measurement may be applicable for Manuka honey authentication (Kato 2014). Leptosperin testing has recently become available to commercial honey producers as a method of determining Manuka honey purity. PCR testing to detect DNA of L.scoparium is currently being developed and refined to be used on a commercial level for identification of Manuka honey (NZPMI 2015)
Antioxidant Activity:
Manuka honey’s antioxidant effects are due to specific scavenging activity for superoxide anion radicals (Inoue 2005). Methyl syringate has been found to be responsible for part of Manuka honey’s potent antioxidant activity (Inoue 2005)
Antimicrobial Activity of Manuka Honey
Significant research has been conducted on the antimicrobial activity of Manuka honey; these studies are limited to either topical application of the honey or to its in vitro antimicrobial activity. The antimicrobial effects of Manuka honey were first described by Molan and Russell in 1988 and attributed to the honey’s non-peroxide activity. Honeys have been shown to exhibit their antimicrobial effects through various mechanisms including: osmotic effects, honey acidity, peroxide activity, non-peroxide activity, biofilm inhibition and other antimicrobial compounds including phytochemicals (Molan 1992).
Osmotic effects
Osmotic effects from the high level of sugars (including sucrose, glucose, levulose (fructose), and maltose) in honey result in rupture of bacteria (Molan 1992). In one study on Manuka honey against coagulase negative Staphylococci, the antibacterial activity of natural Manuka honey was approximately 8 times more potent than if bacterial inhibition were due to the osmotic effect alone (French and Cooper 2005). Several other studies have also illustrated that the antimicrobial effect of Manuka honey is independent of its sugar content (Cooper and Hasisas et al. 2002, Copper and Molan et al. 2002, Henriques et al. 2010)
Peroxide Activity
Hydrogen peroxide is proposed to be the main antibacterial compound found in most honeys (Molan 1992). Hydrogen peroxide is generated by action of glucose oxidase in honey. With time, glucose oxidase can become inactivated by heat and light. Therefore, peroxide activity has shorter term stability compared to non-peroxide activity (Molan 1992).
Non-Peroxide Activity
Non-peroxide activity is a unique property of Manuka honey and is mainly due to high levels of methylglyoxal (Attrot et al 2012, Mavric et al 2008, Adams 2009). Methylglyoxal originates from the high levels of dihydroxyacetone present in the nectar of Manuka flowers (Adams 2009). Methylglyoxal is unique in that it has long- term stability in honey and its levels typically increase with time (Adams 2009). Methylglyoxal is reported to be the main antimicrobial agent in Manuka honey (Attrot et al 2012, Mavric et al 2008). However, if methylglyoxal is neutralized, the honey still has antimicrobial effects on various bacterial isolates including E.coli, Bacillus subtilis and Pseudomonas aeruginosa, but not S.aureus (Kwakman 2011). Also, non-Manuka honeys with methylglyoxal added do not have the same antimicrobial effects as Manuka honey (Jenkins 2011). Methylglyoxal/non-peroxide activity is not inactivated by irradiation; therefore Manuka honey can be sterilized by irradiation for use as a wound dressing (Maddocks 2013).
Biofilm Inhibition
Biofilm is an assemblage of surface-associated microbial cells that is enclosed in an extracellular polymeric substance matrix (Alandejani et al. 2009). Conventional oral antimicrobial therapies are often ineffective in eliminating bacteria located in the biofilm and topical therapies may also be ineffective depending on type. Biofilm has been shown to play an important role in the pathogenicity of bacteria like Pseudomonas and Staphylococcus that are commonly incriminated in cutaneous/wound infections (Alandejani et al 2009). Manuka honey has been shown to inhibit biofilm in various species of bacteria including Staphylococcus aureus, MRSA, C.difficle, Streptococcus pyogenes, Streptococcus mutans, Proteus mirabilis, and Enterobacter cloacae (Alandejani et al 2009, Maddocks 2012). Methylglyoxal requires other components in Manuka-type honeys for this anti- biofilm activity (Maddocks 2012). Manuka honey have been found to be cidal to 82% of Staph aureus, 67% of MRSA and 91% Pseudomonas biofilms (Alandejani et al 2009).
Microorganisms Killed by Manuka Honey
Manuka honey has been shown to have antimicrobial effect against a variety of organisms including Staphylococcus aureus, MRSA, Pseudomonas, E.coli, Streptococcus, Campylobacter, Clostridium dificile and various fungi (dermatophytes and Candida) (Molan 1992, Lin et al 2009, Hammon and Donkor 2013). The reported MIC (and MBC when available) levels for Manuka honey against various microorganisms are reported below in Table 2.
Table 2: MIC/MBC levels for Manuka honey against various microorganisms
Organism
MIC (% v/v unless indicated)
MBC (% v/v unless indicated)
Reference
Clostridium dificle
6.25%
6.25%
Hammon and Donkor 2013
Pseudomonas aeruginosa
<10%
Cooper et al. 2002
Pseudomonas aeruginosa
5.5% - 8.7%
Cooper and Molan 1999
Pseudomonas aeruginosa
12.5%
12.5%
Sherlock et al 2010
Pseudomonas aeruginosa
9.5% w/v
12% w/v
Henriques 2010
Pseudomonas aeruginosa
12% w/v
16% w/v
Roberts 2012
Pseudomonas aeruginosa
15.3% w/v
15.7 w/v
Cooper 2010
Campylobacter jejuni/coli
1%
Lin et al 2009
E.coli
3.7%
Wilkinson and Cavanagh
E.coli
12.5%
12.5%
Sherlock et al 2010
E.coli
6.87%
7.48%
Lin et al 2011
E.coli
16.2% w/v
18% w/v
Cooper 2010
Enterobacter aerogenes, Enterobacter cloacae
10.65-11.89%
16.6%
Lin et al 2011
Yersinia enterocolitica
4.79%
5.45%
Lin et al 2011
Salmonella typhimurium
6%
Wilkinson and Cavanagh
Salmonella typhimurium, Mississippi, enteritidis
6.8%
8.5%
Lin et al 2011
Proteus mirabilis
7.3%
Wilkinson and Cavanagh
Staphylococcus aureus
2-3%
Cooper 1999
Staphylococcus aureus
1.8%
Wilkinson and Cavanagh
Coagulase negative Staphylococci (methicillin resistant and sensitive)
2.7-5%
French and Cooper 2005
S. epidermidis
5.7% w/v
8.3 w/v
Cooper 2010
MRSA
12.5%
12.5%
Sherlock et al 2010
MRSA
6% w/v
Jenkins and Cooper 2012
MRSA
3%
Cooper 1999, Cooper 2002
MRSA
5.83% w/v
8.5% w/v
Cooper 2010
Streptococcus spp and Enterococcus spp
5-10%
Cooper 2011
Manuka Honey’s Anti-Staphylococcal Activity
The Minimum inhibitory concentration of Manuka honey for Staph aureus varies with the strain and publication (see table 2); Cooper et al.1999 first reported the MIC of 58 Staphylococcus aureus isolates from infected wounds to be 2 - 3% (v/v). For Staphylococcus aureus, Manuka honey has a bactericidal mode of inhibition (Henriques 2010). Manuka honey prevents cell division of Staph bacteria, resulting in a failure of progression through the cell cycle and accumulation of fully formed septa within the bacteria visible with transmission electron microscopy; the staphylococcal target site of Manuka honey involves the cell division machinery (Henriques 2010).
Manuka honey has also been shown to be active against coagulase negative Staphylococci, with inhibitory concentrations ranging from 2.7-5% v/v (French 2005). In that study, there was no significant difference in susceptibility to honey between antibiotic sensitive and antibiotic resistant isolates (French 2005). In vitro clinical isolates of methicillin-susceptible and methicillin-resistant staphylococci were shown to be equally susceptible to Manuka honey with MICs reported as 3% (v/v) [equivalent to 41000 mg/L or 4.1% (w/v) (Cooper 1999, Cooper 2002); however, other publications report higher MIC levels for MRSA (see table 2). A decrease in expression of virulence genes has been demonstrated in MRSA isolates exposed to Manuka honey (Jenkins et al. 2013). Exposure of MRSA to inhibitory concentrations of Manuka honey has been shown to down-regulate mecR1 (Jenkins and Cooper 2012). Interestingly, sub inhibitory concentrations of honey in combination with oxacillin restored oxacillin susceptibility to MRSA (Jenkins and Cooper 2012). MRSA isolates exposed to Manuka honey all had impaired cell division similar to what is reported in methicillin sensitive isolates (Jenkins 2011).
Manuka Honey’s Anti-Pseudomonal Activity
The MIC (and MBC) of Manuka honey for various Pseudomonas aeruginosa strains has been reported in several publications (Cooper and Molan 1999, Cooper et al. 2002, Henriques 2010). Manuka honey has bactericidal activity against Pseudomonas as it causes a loss of structural integrity and destabilization of the cell wall resulting in lysis (Henriques 2011). OprF, an outer membrane protein that is involved in cell wall stability, diffusion and virulence, has been implicated as a possible genetic target for Manuka honey’s activity against Pseudomonas and decreased expression of this protein has been found in Manuka honey treated bacteria (Roberts 2012). Additionally, Manuka honey inhibits siderophore production in Pseudomonas, limiting its ability to capture iron (Kronda 2013). Manuka honey has also been shown to decrease the adhesion of Pseudomonas to human keratinocytes (Maddock 2013). Exposure of P. aeruginosa to Manuka honey has also been shown to reduce swarming and swimming motility (Roberts 2015). This decreased motility was found to be due to de-flagellation of the bacterial cell, correlating with decreased expression of the major structural flagellin protein, FliC, and concurrent suppression of flagellin-associated genes, including fliA, fliC, flhF, fleN, fleQ and fleR (Roberts 2015).
Resistance to Manuka honey
A recent study (Henriques 2010) evaluated the possibility of Manuka honey resistant by continuously exposing resistant clinical strains of bacteria (MRSA, Pseudomonas and E.coli) from wound infection cases in people to sub-lethal concentrations of Manuka honey for up to 28 days. No honey resistant mutants developed in any of the isolates and viable bacteria were not recovered above the starting MIC values upon repeat exposure (Henriques 2010).
Summary:
Manuka honey is produced predominantly in New Zealand by European honey bees (Apis mellifera) feeding on the Manuka tree flower (Leptospermum scoparium) nectars. High levels of DHA in Manuka flower nectars are converted into methylglyoxal in Manuka honey; the levels of MG increase with time. Methylglyoxal is the main contributor to non-peroxide activity of Manuka honey, but other compounds and properties of Manuka honey also contribute to its potent antimicrobial effects against a variety of microorganisms commonly encountered in cutaneous infections and wounds.
References:
Adams CJ, Manley-Harris M, Molan PC. 2009. The origin of methylglyoxal in New Zealand manuka (Leptospermum scoparium) honey. Carbohydrate Res; 344: 1050–3.
Alandejani T, Marsan J, Ferris W et al. 2009. Effectiveness of honey on Staphylococcus and Pseudomonas aeruginosa biofilms. Otolaryngology–Head and Neck Surgery 141, 114-118
Atrott J, Haberlau S, Henle T. 2012. Studies on the formation of methylglyoxal from dihydroxyacetone in Manuka (Leptospermum scoparium) honey. Carbohydr Res. 361:7–11.
Blair SE, Cokcetin NN, Harry EJ, Carter DA. 2009. The unusual antibacterial activity of medical-grade Leptospermum honey: antibacterial spectrum, resistance and transcriptome analysis. Eur. J Clin Microbiol Infect Dis 10: 1007.
Blaser G, Santos K, Bode U, Vetter H, Simon A. 2007. Effect of medical honey on wounds colonised or infected with MRSA. J Wound Care 16(8):325–328
Chambers J. 2006. Topical manuka honey for MRSA contaminated skin ulcers. Palliat Med 20:557.
Cooper RA, Halas E, Molan PC. 2002. The efficacy of honey in inhibiting strains of Pseudomonas aeruginosa from infected burns. J Burn Care Rehabil. 23(6):366–370.
Cooper RA, Jenkins L, Henriques AF, Duggan RS, Burton NF. 2010. Absence of bacterial resistance to medical-grade manuka honey. Eur J Clin Microbiol Infect Dis. 29(10):1237-41.
Cooper R, Lindsay E, Molan P. 2011. Testing the susceptibility to Manuka honey of streptococci isolated from wound swabs. Journal of ApiProduct and ApiMedical Scienc 3 (3): 117-122
Cooper RA, Molan PC, Harding KG. 1999. Antibacterial activity of honey against strains of Staphylococcus aureus from infected wounds. J Roy Soc Med. 92: 283–5. 11
Cooper RA, Molan PC, Harding KG. 2002. The sensitivity to honey of Gram-positive cocci of clinical significance isolated from wounds. J Appl Microbiol. 93: 857–63.
French VM, Cooper RA, Molan PC. 2005. The antibacterial activity of honey against coagulase negative staphylococci. Journal of Antimicrobial Chemotherapy 56, 228–231
Hammond E, Donkor E. 2013. Antibacterial effect of Manuka honey on Clostridium difiicle. BMC Research Notes, 6:188
Henriques AF, Jenkins RE, Burton NF, et al. 2010. The intracellular effects of manuka honey on Staphylococcus aureus. Eur J Clin Microbiol Infect Dis, 29(1):45-50
Henriques AF, Jenkins RE, Burton NF, et al. 2011. The effects of manuka honey on the structure of Psuedomonas aeruginosa. Eur J Clin Microbiol Infect Dis. 30(2):167-71
Jenkins R, Burton N, Cooper R. 2011. Manuka honey inhibits cell division in methicillin-resistant Staphylococcus aureus. J Antimicrob Chemother. 66: 2536–42.
Jenkins R, Burton N, Cooper C. 2013. Proteomic and genomic analysis of methicillin-resistant Staphylococcus aureus (MRSA) exposed to manuka honey in vitro demonstrated down-regulation of virulence markers. J Antimicrob Chemother 69(3):603-15
Jenkins R, Cooper C. 2012. Synergy between oxacillin and manuka honey sensitizes methicillin-resistant Staphylococcus aureus to oxacillin. J Antimicrob Chemother 2012; 67: 1405–1407
Kato Y, Fujinaka R, Ishisaka A, Nitta Y, Kitamoto N, Takimoto Y. 2014. Plausible authentication of manuka honey and related products by measuring leptosperin with methyl syringate. J Agric Food Chem. 62(27):6400-7.
Kronda JM, Cooper RA, Maddocks SE. 2013. Manuka honey inhibits siderophore production in Pseudomonas aeruginosa. J Appl Microbio. 115: 86–90.
Kwakman PHS, te Velde AA, de Boer L et al. Two major medicinal honeys have different mechanisms of bactericidal activity. PLoSONE 2011; 6: e17709.
Leake J. 2013, 26 August 2013). "Food fraud buzz over fake manuka honey".The Times (London). Retrieved Aygust 26 2013.
Lin SM, Molan PC, Cursons RT. 2009) The in vitro susceptibility of Campylobacter spp to the antibacterial effect of Manuka honey. Eur J Clin Microbiol Infect Dis (2009) 28:339–34
Maddocks SE, Jenkins RE, Rowlands RS et al. Manuka honey inhibits adhesion and invasion in medically important wound bacteria in vitro. Future Microbiol 2013; 8: 1523–36.
Maddocks SE, Lopez MS, Rowlands RS, Cooper RA. 2012. Manuka honey inhibits the development of Streptococcus pyogenes biofilms and causes reduced expression of two fibronectin binding proteins. Microbiology. 158: 781–790
Mavric E, Wittmann S, Barth G et al. 2008. Identification and quantification of methylglyoxal as the dominant antibacterial constituent of manuka (Leptospermum scoparium) honeys from New Zealand. Mol Nutr Food Res. 52: 1–7.
Molan PC, Russell KM. 1998. Non-peroxide antibacterial activity in some New
Zealand honeys. J Apic Res. 27(1):62–67.
Natarajan S, Williamson D, Grey J, Harding KG, Cooper RA. 2001. Healing of an MRSA-colonized, hydroxyurea-induced leg ulcer with honey. J Dermatolog Treat. 12:33–36
New Zealand Ministry for Primary Industries 2015. Interim Labelling Guide for Mānuka Honey. http://archive.mpi.govt.nz/food/food-safety/manuka-honey. Accessed 1/15/2016
Roberts AE, Maddocks SE, Cooper RA. 2012. Manuka honey is bactericidal against Pseudomonas aeruginosa and results in differential expression of oprF and algD. Microbiology 158: 3005–13.
Roberts AE, Maddocks SE, Cooper RA. 2015. Manuka honey reduces the motility of Pseudomonas aeruginosa by suppression of flagella-associated genes. J Antimicrob Chemother. 70(3):716-25.
Unique Manuka Factor Honey Association. http://www.umf.org.nz/grading-system. Accessed 2/1/16.
Visavadia BG, Honeysett J, Danford MH (2008) Manuka honey dressing: an effective treatment for chronic wound infections. Br J Oral Maxill Surg 46:55–56
Wilkinson JM, Cavanagh MA. 2005. Antibacterial activity of 13 honeys against Escherichia coli and Pseudomonas aeruginosa. J Med Food. 8(1):100–103
Table 1 – Methylgloxal level and correlating UMF® grading (UMFHA 2015)
Methylgloxal Level | UMF™ Grade |
≥83 mg/kg | 5+ |
≥263 mg/kg | 10+ |
≥514 mg/kg | 15+ |
≥573 mg/kg | 16+ |
≥696 mg/kg | 18+ |
≥829 mg/kg | 20+ |
≥1200 mg/kg | 25+ |
≥1449 mg/kg | 28+ |
Melissopalynology is the study of pollen in honey. Manuka honey can contain Manuka and Kanuka pollens, which can be difficult to differentiate. Recent research by NZPMI indicates that although morphological differences between Manuka and Kanuka pollens may be subtle, the two can be differentiated using direct light microscopy and Classifynder™ (NZMPI 2015). In order for Manuka honey to be classified as a monofloral honey, it must contain >70% Manuka pollens (NZPMI 2015). Manuka pollens alone are not sufficient for the identification of Manuka honey as high pollen counts do not always correlate with contribution of Manuka nectar to the honey (NZPMI 2015).
A recent publication found that leptosperin, a novel glycoside that is specific to manuka honey, is stable during storage and its measurement may be applicable for Manuka honey authentication (Kato 2014). Leptosperin testing has recently become available to commercial honey producers as a method of determining Manuka honey purity. PCR testing to detect DNA of L.scoparium is currently being developed and refined to be used on a commercial level for identification of Manuka honey (NZPMI 2015)
Antioxidant Activity:
Manuka honey’s antioxidant effects are due to specific scavenging activity for superoxide anion radicals (Inoue 2005). Methyl syringate has been found to be responsible for part of Manuka honey’s potent antioxidant activity (Inoue 2005)
Antimicrobial Activity of Manuka Honey
Significant research has been conducted on the antimicrobial activity of Manuka honey; these studies are limited to either topical application of the honey or to its in vitro antimicrobial activity. The antimicrobial effects of Manuka honey were first described by Molan and Russell in 1988 and attributed to the honey’s non-peroxide activity. Honeys have been shown to exhibit their antimicrobial effects through various mechanisms including: osmotic effects, honey acidity, peroxide activity, non-peroxide activity, biofilm inhibition and other antimicrobial compounds including phytochemicals (Molan 1992).
Osmotic effects
Osmotic effects from the high level of sugars (including sucrose, glucose, levulose (fructose), and maltose) in honey result in rupture of bacteria (Molan 1992). In one study on Manuka honey against coagulase negative Staphylococci, the antibacterial activity of natural Manuka honey was approximately 8 times more potent than if bacterial inhibition were due to the osmotic effect alone (French and Cooper 2005). Several other studies have also illustrated that the antimicrobial effect of Manuka honey is independent of its sugar content (Cooper and Hasisas et al. 2002, Copper and Molan et al. 2002, Henriques et al. 2010)
Peroxide Activity
Hydrogen peroxide is proposed to be the main antibacterial compound found in most honeys (Molan 1992). Hydrogen peroxide is generated by action of glucose oxidase in honey. With time, glucose oxidase can become inactivated by heat and light. Therefore, peroxide activity has shorter term stability compared to non-peroxide activity (Molan 1992).
Non-Peroxide Activity
Non-peroxide activity is a unique property of Manuka honey and is mainly due to high levels of methylglyoxal (Attrot et al 2012, Mavric et al 2008, Adams 2009). Methylglyoxal originates from the high levels of dihydroxyacetone present in the nectar of Manuka flowers (Adams 2009). Methylglyoxal is unique in that it has long- term stability in honey and its levels typically increase with time (Adams 2009). Methylglyoxal is reported to be the main antimicrobial agent in Manuka honey (Attrot et al 2012, Mavric et al 2008). However, if methylglyoxal is neutralized, the honey still has antimicrobial effects on various bacterial isolates including E.coli, Bacillus subtilis and Pseudomonas aeruginosa, but not S.aureus (Kwakman 2011). Also, non-Manuka honeys with methylglyoxal added do not have the same antimicrobial effects as Manuka honey (Jenkins 2011). Methylglyoxal/non-peroxide activity is not inactivated by irradiation; therefore Manuka honey can be sterilized by irradiation for use as a wound dressing (Maddocks 2013).
Biofilm Inhibition
Biofilm is an assemblage of surface-associated microbial cells that is enclosed in an extracellular polymeric substance matrix (Alandejani et al. 2009). Conventional oral antimicrobial therapies are often ineffective in eliminating bacteria located in the biofilm and topical therapies may also be ineffective depending on type. Biofilm has been shown to play an important role in the pathogenicity of bacteria like Pseudomonas and Staphylococcus that are commonly incriminated in cutaneous/wound infections (Alandejani et al 2009). Manuka honey has been shown to inhibit biofilm in various species of bacteria including Staphylococcus aureus, MRSA, C.difficle, Streptococcus pyogenes, Streptococcus mutans, Proteus mirabilis, and Enterobacter cloacae (Alandejani et al 2009, Maddocks 2012). Methylglyoxal requires other components in Manuka-type honeys for this anti- biofilm activity (Maddocks 2012). Manuka honey have been found to be cidal to 82% of Staph aureus, 67% of MRSA and 91% Pseudomonas biofilms (Alandejani et al 2009).
Microorganisms Killed by Manuka Honey
Manuka honey has been shown to have antimicrobial effect against a variety of organisms including Staphylococcus aureus, MRSA, Pseudomonas, E.coli, Streptococcus, Campylobacter, Clostridium dificile and various fungi (dermatophytes and Candida) (Molan 1992, Lin et al 2009, Hammon and Donkor 2013). The reported MIC (and MBC when available) levels for Manuka honey against various microorganisms are reported below in Table 2.
Table 2: MIC/MBC levels for Manuka honey against various microorganisms
Organism | MIC (% v/v unless indicated) | MBC (% v/v unless indicated) | Reference |
Clostridium dificle | 6.25% | 6.25% | Hammon and Donkor 2013 |
Pseudomonas aeruginosa | <10% |
| Cooper et al. 2002 |
Pseudomonas aeruginosa | 5.5% - 8.7% |
| Cooper and Molan 1999 |
Pseudomonas aeruginosa | 12.5% | 12.5% | Sherlock et al 2010 |
Pseudomonas aeruginosa | 9.5% w/v | 12% w/v | Henriques 2010 |
Pseudomonas aeruginosa | 12% w/v | 16% w/v | Roberts 2012 |
Pseudomonas aeruginosa | 15.3% w/v | 15.7 w/v | Cooper 2010 |
Campylobacter jejuni/coli | 1% |
| Lin et al 2009 |
E.coli | 3.7% |
| Wilkinson and Cavanagh |
E.coli | 12.5% | 12.5% | Sherlock et al 2010 |
E.coli | 6.87% | 7.48% | Lin et al 2011 |
E.coli | 16.2% w/v | 18% w/v | Cooper 2010 |
Enterobacter aerogenes, Enterobacter cloacae | 10.65-11.89% | 16.6% | Lin et al 2011 |
Yersinia enterocolitica | 4.79% | 5.45% | Lin et al 2011 |
Salmonella typhimurium | 6% |
| Wilkinson and Cavanagh |
Salmonella typhimurium, Mississippi, enteritidis | 6.8% | 8.5% | Lin et al 2011 |
Proteus mirabilis | 7.3% |
| Wilkinson and Cavanagh |
Staphylococcus aureus | 2-3% |
| Cooper 1999 |
Staphylococcus aureus | 1.8% |
| Wilkinson and Cavanagh |
Coagulase negative Staphylococci (methicillin resistant and sensitive) | 2.7-5% |
| French and Cooper 2005 |
S. epidermidis | 5.7% w/v | 8.3 w/v | Cooper 2010 |
MRSA | 12.5% | 12.5% | Sherlock et al 2010 |
MRSA | 6% w/v |
| Jenkins and Cooper 2012 |
MRSA | 3% |
| Cooper 1999, Cooper 2002 |
MRSA | 5.83% w/v | 8.5% w/v | Cooper 2010 |
Streptococcus spp and Enterococcus spp | 5-10% |
| Cooper 2011 |
Manuka Honey’s Anti-Staphylococcal Activity
The Minimum inhibitory concentration of Manuka honey for Staph aureus varies with the strain and publication (see table 2); Cooper et al.1999 first reported the MIC of 58 Staphylococcus aureus isolates from infected wounds to be 2 - 3% (v/v). For Staphylococcus aureus, Manuka honey has a bactericidal mode of inhibition (Henriques 2010). Manuka honey prevents cell division of Staph bacteria, resulting in a failure of progression through the cell cycle and accumulation of fully formed septa within the bacteria visible with transmission electron microscopy; the staphylococcal target site of Manuka honey involves the cell division machinery (Henriques 2010).
Manuka honey has also been shown to be active against coagulase negative Staphylococci, with inhibitory concentrations ranging from 2.7-5% v/v (French 2005). In that study, there was no significant difference in susceptibility to honey between antibiotic sensitive and antibiotic resistant isolates (French 2005). In vitro clinical isolates of methicillin-susceptible and methicillin-resistant staphylococci were shown to be equally susceptible to Manuka honey with MICs reported as 3% (v/v) [equivalent to 41000 mg/L or 4.1% (w/v) (Cooper 1999, Cooper 2002); however, other publications report higher MIC levels for MRSA (see table 2). A decrease in expression of virulence genes has been demonstrated in MRSA isolates exposed to Manuka honey (Jenkins et al. 2013). Exposure of MRSA to inhibitory concentrations of Manuka honey has been shown to down-regulate mecR1 (Jenkins and Cooper 2012). Interestingly, sub inhibitory concentrations of honey in combination with oxacillin restored oxacillin susceptibility to MRSA (Jenkins and Cooper 2012). MRSA isolates exposed to Manuka honey all had impaired cell division similar to what is reported in methicillin sensitive isolates (Jenkins 2011).
Manuka Honey’s Anti-Pseudomonal Activity
The MIC (and MBC) of Manuka honey for various Pseudomonas aeruginosa strains has been reported in several publications (Cooper and Molan 1999, Cooper et al. 2002, Henriques 2010). Manuka honey has bactericidal activity against Pseudomonas as it causes a loss of structural integrity and destabilization of the cell wall resulting in lysis (Henriques 2011). OprF, an outer membrane protein that is involved in cell wall stability, diffusion and virulence, has been implicated as a possible genetic target for Manuka honey’s activity against Pseudomonas and decreased expression of this protein has been found in Manuka honey treated bacteria (Roberts 2012). Additionally, Manuka honey inhibits siderophore production in Pseudomonas, limiting its ability to capture iron (Kronda 2013). Manuka honey has also been shown to decrease the adhesion of Pseudomonas to human keratinocytes (Maddock 2013). Exposure of P. aeruginosa to Manuka honey has also been shown to reduce swarming and swimming motility (Roberts 2015). This decreased motility was found to be due to de-flagellation of the bacterial cell, correlating with decreased expression of the major structural flagellin protein, FliC, and concurrent suppression of flagellin-associated genes, including fliA, fliC, flhF, fleN, fleQ and fleR (Roberts 2015).
Resistance to Manuka honey
A recent study (Henriques 2010) evaluated the possibility of Manuka honey resistant by continuously exposing resistant clinical strains of bacteria (MRSA, Pseudomonas and E.coli) from wound infection cases in people to sub-lethal concentrations of Manuka honey for up to 28 days. No honey resistant mutants developed in any of the isolates and viable bacteria were not recovered above the starting MIC values upon repeat exposure (Henriques 2010).
Summary:
Manuka honey is produced predominantly in New Zealand by European honey bees (Apis mellifera) feeding on the Manuka tree flower (Leptospermum scoparium) nectars. High levels of DHA in Manuka flower nectars are converted into methylglyoxal in Manuka honey; the levels of MG increase with time. Methylglyoxal is the main contributor to non-peroxide activity of Manuka honey, but other compounds and properties of Manuka honey also contribute to its potent antimicrobial effects against a variety of microorganisms commonly encountered in cutaneous infections and wounds.
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