Antibacterial Activity of Greek and Cypriot Honeys Against Staphylococcus aureus and Pseudomonas aeruginosa in Comparison to Manuka Honey

Abstract

The antibacterial activity of 31 Greek and Cypriot honeys against Staphylococcus aureus and Pseudomonas aeruginosa was initially screened using an agar-well diffusion assay in comparison with manuka honey. The minimum inhibitory concentration (MIC) was determined in broth using a spectrophotometric-based assay. The MIC of treated honeys with catalase or proteinase K was determined and compared with those of untreated honeys. All tested honeys demonstrated antibacterial activity against S. aureus on agar-well diffusion assay. MICs of tested honeys were determined as 3.125–25% (v/v), compared with manuka honey at 6.25% (v/v). Similarly, 21 of 31 tested honeys demonstrated antibacterial activity on agar-well diffusion assay against P. aeruginosa. Their MICs ranged from 6.25% to 25% (v/v) compared with 12.5% (v/v) for manuka honey. Antibacterial activity of tested honeys could be largely attributed to hydrogen peroxide formation and in some cases to unidentified proteinaceous compounds. In conclusion, Greek and Cypriot honeys demonstrated significant but variable antibacterial activity against P. aeruginosa and especially S. aureus. To the best of our knowledge this is the first study that has thoroughly examined the antibacterial activity of Greek and Cypriot honeys compared with manuka honey. The high antibacterial activity exerted by some tested honeys warrants further investigation.

Introduction

H oney has been used as a traditional remedy due to its antimicrobial activity and wound-healing properties since ancient times.¹ In the current clinical environment, antibiotic-resistant strains of bacteria are increasingly widespread so alternative therapeutic agents are urgently needed.

Honey demonstrates strong antibacterial activity against clinically important pathogens; thus its use in modern medicine has been re-introduced.^2

–7 The antibacterial activity of honey is attributable to different factors such as hydrogen peroxide, low pH, and high osmolarity.⁸ Methylglyoxal and the antibacterial peptide bee defensin-1 were recently identified as important factors of the antibacterial activity exerted by certain honeys. Moreover, it has been shown that antibacterial factors in honey have overlapping activity.^9
–11 The antibacterial potency among the different honeys is variable, mainly depending on its botanical, seasonal, and geographical source, although harvesting, processing, and storage conditions might influence honey's antibacterial properties.^5,7

Manuka honey has been approved by medical regulatory authorities as a wound care agent in the European Union, the United States, Canada, Australia, and other countries. Manuka honey originates from the manuka bush (Leptospermum scoparium), an endemic plant grown in New Zealand. There are many studies documenting the antibacterial activity of manuka honey against clinically important bacteria such as methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia, or oral bacteria causing dental caries.^2,3,12,13 Recent studies on the antibacterial activity of tualang and ulmo honeys reported similar or superior antibacterial efficacy compared with manuka honey, suggesting that further research could identify medically useful honeys around the world that might have distinct advantages over manuka honey.^4,5 In this study, the antibacterial activity of Greek and Cypriot honeys from diverse botanical sources and geographical locations was examined against two major nosocomial pathogens and compared with that of manuka honey for the first time.

Materials and Methods

Bacterial strains and growth conditions

The antibacterial activity of honeys was tested against methicillin-resistant S. aureus 1552 and carbapenem-resistant P. aeruginosa 1773. Both clinical strains (kindly provided by Dr. Spyros Pournaras, School of Medicine, University of Thessaly, Larissa, Greece) were identified and characterized by standard laboratory methods. Bacteria were routinely grown in Mueller–Hinton broth or Mueller–Hinton agar (both from Lab M, Heywood, Greater Manchester, United Kingdom) at 37°C.

Honey samples

In total, 31 Greek and Cypriot honeys were provided by individual beekeepers, beekeeper associations, and honey companies. Each sample was assigned a unique reference number, and details regarding the botanical source, the geographical location, and date of harvest were recorded (Table 1). Honey samples were stored in glass or plastic containers at room temperature in the dark. Identification of the botanical source of each honey was performed by the providers based on the flora availability during the harvest season, location of the apiary, and in some cases pollen analysis. MGO™ Manuka honey 550⁺ (Manuka Health, Saint Johns, New Zealand), equivalent to UMF 25+, was used as a positive control throughout this study, while laboratory-synthesized honey was as a negative control. Laboratory-synthesized honey was prepared by dissolving 3 g of sucrose, 15 g of maltose, 80.1 g of fructose, and 67 g of glucose (all supplied by Sigma-Aldrich, Athens, Greece) in 34 mL of sterile deionized water. The solution was heated to 56°C in a water bath to aid dissolving.⁵

Table 1.

Botanical Source, Geographical Location, and Harvest Date of Honeys

Honey number	Botanical source	Geographical location	Harvest date
1	Heather	Blachokerasia, Peloponnese	May 2009
2	Abies	Levidi, Peloponnese	June 2010
3	Abies	Levidi, Peloponnese	June 2009
4	Abies	Levidi, Peloponnese	June 2008
5	Arbutus and heather	Kaltezes, Peloponnese	November 2010
6	Chestnut	Ano Doliana, Peloponnese	July 2009
7	Pine	Thassos Island	2010
8	Mint, oregano, and ironwort	Mount Olympus	2010
9	Acacia	Mount Olympus	2010
10	Herbs and thyme	Mount Olympus	2010
11	Arbutus	Mount Pelion, Thessaly	2010
12	Knotweed and Crocus	Krokos, Northern Greece	November 2009
13	Polyfloral (herbs)	Krokos, Northern Greece	June 2010
14	Polyfloral (herbs)	Vourikas, Northern Greece	June 2010
15	Knotweed	Kerkini Lake, Northern Greece	October 2010
16	Eucalyptus	Nicosia, Cyprus	August 2010
17	Thyme	Limassol, Cyprus	August 2010
18	Fennel	Nicosia, Cyprus	August 2010
19	Sunflower	Komotini, Northern Greece	2010
20	Orange	Argos, Peloponnese	2010
21	Cotton: 20% other flowers	Serres, Northern Greece	2010
22	Thyme	Crete	2010
23	Chestnut and pine	Mount Athos, Northern Greece	2010
24	Arbutus	Mount Pelion, Thessaly	2010
25	Ironwort and origan	Sourpi, Thessaly	2010
26	Origan and clover	Mount Olympus	2010
27	Chestnut	Mount Pelion, Thessaly	2010
28	Abies	Drakotrypa, Thessaly	2010
29	Polyfloral (herbs)	Vassilitsa, Northern Greece	2010
30	Oak	Mount Pelion, Thessaly	2010
31	Arbutus	Melivia, Thessaly	2010

Agar-well diffusion assay

The assay was performed on the basis of CLSI (former NCCLS) guidelines.¹⁴ In brief, overnight bacterial cultures grown in Mueller–Hinton broth were adjusted to 0.5 McFarland turbidity standard (approximately 1.5×10⁸ colony-forming units [CFU]/mL). Mueller–Hinton agar plates were inoculated with roughly 10⁶ CFU over the entire surface of the plate. Three wells 6 mm in diameter were cut into the surface of the agar using a sterile cork borer. Roughly 100 mg of undiluted tested honey, manuka honey, or laboratory-synthesized honey was added separately to each well. Plates were incubated at 37°C for 16–18 h. The diameter of inhibition zones, including the diameter of the well, was recorded. The diameter of the inhibition zone, if present, in the negative control was recorded and subtracted from inhibition zones of tested honeys as well as manuka honey. Each assay was carried out in triplicate. The SD value of inhibition zones was calculated, and comparison of means was conducted using t test for independent samples. Data were analyzed for significance level α=0.05 using SPSS version 19.0 software (SPSS, Chicago, IL, USA).

Determination of minimum inhibitory concentration

The minimum inhibitory concentration (MIC) of honeys was determined in sterile 96-well polystyrene microtiter plates (Kisker Biotech, Steinfurt, Germany) using a spectrophotometric bioassay.¹⁵ In brief, overnight bacterial cultures grown in Mueller–Hinton broth were adjusted to 0.5 McFarland turbidity standard (approximately 1.5×10⁸ CFU/mL). Approximately 5×10⁴ CFU in 10 μL of Mueller–Hinton broth was added to 190 μL of twofold diluted test honey (honey concentration ranged from 50% to 0.78% [v/v]) in Mueller–Hinton broth. Twofold serial dilutions of the same range of manuka honey were included for comparison. Control wells contained only Mueller–Hinton broth inoculated with bacteria. Optical density (OD) was determined at 630 nm using an ELx808 absorbance microplate reader (BioTek, Winooski, VT, USA) just prior to incubation (t=0) and after a 24-h incubation (t=24) at 37°C. The OD for each replicate well at t=0 was subtracted from the OD of the same replicate well at t=24 h. The growth inhibition at each honey dilution was determined using the following formula: % inhibition=(1 – [OD of test well/OD of corresponding control well])×100. The MIC was determined as the lowest honey concentration that resulted in 100% growth inhibition.

Antibacterial activity attributed to hydrogen peroxide and proteinaceous compounds

The MIC of honeys treated with bovine catalase or proteinase K was determined and compared with that of untreated honey.^10,16 In brief, 50% (v/v) honey in Muller–Hinton broth containing 100 μg/mL proteinase K (HT Biotechnology, Dorchester, United Kingdom) or 600 U/mL bovine catalase (Serva, Heidelberg, Germany) was incubated for 16 h at 37°C and then was diluted twofold and tested as described above. An elevated MIC of treated honey compared with untreated honey revealed the presence of hydrogen peroxide and/or proteinaceous compounds that contribute to the antibacterial activity of tested honey.

Results

Agar-well diffusion assay

Initial screening with the agar-well diffusion assay demonstrated that all tested honeys had antibacterial activity against S. aureus. Of the 31 tested honeys, 14 demonstrated significantly larger inhibition zones (P<.05) compared with manuka honey (Table 2). Regarding P. aeuruginosa, antibacterial activity was demonstrated for 21 of all tested honeys. Six of 21 honeys demonstrated significantly larger inhibition zones (P<.05) compared with manuka honey (Table 3). Antibacterial activity of tested honeys (included manuka honey) was higher against S. aureus, as demonstrated by larger inhibition zones, compared with that against P. aeruginosa.

Table 2.

Antibacterial Activity of Honeys Against S. aureus in Comparison with Manuka Honey: Mean Values of Inhibition Zones (Including SD) by Agar-Well Diffusion

	Well diffusion assay (mm)			MIC (% [v/v]) of honey		MIC (% [v/v]) of honey treated with
Honey number	Honey	Manuka 25+	P value<.05 ^a	Untreated	Manuka 25+	Catalase	Proteinase Κ
1	20±1	20±0		6.25	6.25	ND	ND
2	22±0	20±1	.038	6.25	6.25	50	6.25
3	24±2	20±1	.038	6.25	6.25	50	6.25
4	20±2	20±1		6.25	6.25	ND	ND
5	24±1	20±1	.001	6.25	6.25	25	6.25
6	15±1	11±1	.008	6.25	6.25	25	6.25
7	27±1	21±1	.001	3.125	6.25	25	3.125
8	22±.5	20±0		6.25	6.25	ND	ND
9	16±0	18±1		25	6.25	ND	ND
10	20±1	21±1		25	6.25	ND	ND
11	21±0	20±0	.001	6.25	6.25	25	6.25
12	12±2	10±1		6.25	6.25	ND	ND
13	24±1	21±1	.016	6.25	6.25	12.5	12.5
14	9±2	10±2.5		3.125	6.25	25	3.125
15	22±0.6	10±2.5	.032	6.25	6.25	25	6.25
16	15±1	10±1	.001	12.5	6.25	25	12.5
17	15±1	20±1		6.25	6.25	ND	ND
18	10±1	9±1.5		6.25	6.25	ND	ND
19	8±1	11±1		3.125	6.25	12.5	6.25
20	15±1	21±1		12.5	6.25	ND	ND
21	24±1	22±1	.025	6.25	6.25	25	6.25
22	21±1	20±1		6.25	6.25	ND	ND
23	14±2	9±2	.027	3.125	6.25	25	6.25
24	24±1.5	20±1	.013	6.25	6.25	25	6.25
25	23±1	20±1	.003	6.25	6.25	25	12.5
26	26±0	20±0		6.25	6.25	25	6.25
27	24±1.5	21±1	.033	6.25	6.25	50	12.5
28	11±1	10±0.6		6.25	6.25	25	6.25
29	19±1	20±1		6.25	6.25	ND	ND
30	17±7	18±6		12.5	6.25	ND	ND
31	18±2	21±0.6		6.25	6.25	ND	ND

Minimum inhibitory concentration (MIC) values of untreated honey were compared with those of manuka honey and catalase- or proteinase K-treated honeys.

Statistically larger inhibition zones are indicated by a value of P<.05.

ND, not determined.

Table 3.

Antibacterial Activity of Honeys Against P. aeruginosa in Comparison with Manuka Honey: Mean Values of Inhibition Zones (Including SD) by Agar-Well Diffusion

	Well diffusion assay (mm)			MIC (% [v/v])		MIC (% [v/v]) of honey treated with
Honey number	Honey	Manuka 25+	P value<.05 ^a	Untreated	Manuka 25+	Catalase	Proteinase Κ
1	No inhibition zone	10±0.5		12.5	12.5	ND	ND
2	9±1.5	12±1		25	12.5	50	25
3	11±0	10±0		12.5	12.5	25	25
4	10±0	11±0		12.5	12.5	ND	ND
5	10±0.5	12±1.5		12.5	12.5	>50	>50
6	12±1	10±0.1		12.5	12.5	25	12.5
7	14±1	10±0.5	.003	12.5	12.5	25	12.5
8	13±1	12±1		12.5	12.5	ND	ND
9	No inhibition zone	11±0		25	12.5	ND	ND
10	No inhibition zone	12±0		25	12.5	ND	ND
11	11±1	11±0.5		12.5	12.5	25	25
12	13±0.5	11±1		12.5	12.5	ND	ND
13	12±0	10±0.5	.02	12.5	12.5	25	12.5
14	13±1.5	10±1.5		6.25	12.5	25	6.25
15	12±1	10±0.5	.025	12.5	12.5	25	12.5
16	9±1	11±0.5		12.5	12.5	25	12.5
17	No inhibition zone	11±1		25	12.5	ND	ND
18	10±1	9±0.5		12.5	12.5	ND	ND
19	No inhibition zone	10±0		12.5	12.5	12.5	25
20	No inhibition zone	12±1		25	12.5	ND	ND
21	11±0.6	11±0		12.5	12.5	50	12.5
22	No inhibition zone	11±1		25	12.5	ND	ND
23	13±0.5	10±2	.025	12.5	12.5	25	12.5
24	10±0.6	10±0		12.5	12.5	25	12.5
25	10±0	10±0.6		12.5	12.5	25	12.5
26	15±1.5	10±0.5	.01	6.25	12.5	25	12.5
27	11±1	10±0		12.5	12.5	25	12.5
28	16±1	12±0.5	.003	12.5	12.5	25	12.5
29	No inhibition zone	10±0.6		12.5	12.5	ND	ND
30	No inhibition zone	10±2		25	12.5	ND	ND
31	No inhibition zone	11±0		25	12.5	ND	ND

MIC values of untreated honey are compared with those of manuka honey and catalase- or proteinase K-treated honeys.

Statistically larger inhibition zones indicated by P<.05.

Determination of MIC

The MICs of tested honeys against S. aureus was variable (3.125–25% [v/v]). In comparison, the MIC of manuka honey was determined at 6.25% (v/v). Four honeys (7, 14, 19, and 23) demonstrated a lower MIC than manuka honey (Table 2). Similarly MICs against P. aeruginosa were variable (6.25–25% [v/v]), while the MIC of manuka honey was determined at 12.5% (v/v). Two honeys (14 and 26) demonstrated a lower MIC than manuka honey (Table 3). The MIC spectrophotometric-based assay, a more sensitive measure of antibacterial activity, showed that in general S. aureus was more susceptible to honey than P. aeruginosa, thus confirming the data obtained by agar-well diffusion assay.

Antibacterial activity attributed to hydrogen peroxide and proteinaceous compounds

Eighteen honeys (2, 3, 5, 6, 7, 11, 13–16, 19, 21, and 23–28) showed higher antibacterial activity either by agar-well diffusion (significantly larger inhibition zones) and/or by MIC determination (lower MIC) compared with manuka honey. These honeys were further tested in order to investigate the mechanisms of antibacterial activity. All catalase-treated honeys showed a higher MIC against S. aureus, indicating that hydrogen peroxide exerts significant antibacterial activity in all tested honeys. Moreover, five proteinase K-treated honeys (13, 19, 23, 25, and 27) showed a higher MIC against S. aureus, indicating the presence of antibacterial proteinaceous compounds (Table 2). Similarly, all but one (19) catalase-treated honeys showed a higher MIC against P. aeruginosa, whereas five proteinase K-treated honeys (3, 5, 11, 19, and 26) demonstrated a higher MIC, indicating the presence of antibacterial proteinaceous compounds (Table 3).

Discussion

Although the high antibacterial activity of manuka honey against pathogenic bacteria is well documented,^2,3,12,13 other honeys might have comparable or even superior antimicrobial activity, depending on the botanical source, geographical location, and tested microorganism.^4,5,17 In this study initial screening with the agar-well diffusion assay demonstrated that all tested honeys had antibacterial activity against S. aureus. In contrast, 10 tested honeys did not show antibacterial activity (no inhibition zones) against P. aeruginosa. The rest of the honeys demonstrated smaller inhibition zones compared with those against S. aureus, indicating that P. aeruginosa is less susceptible to honey's antibacterial activity. In another study it was similarly observed that P. aeruginosa is less susceptible to honey's antibacterial activity than Escherichia coli.¹⁸ Determination of MIC in broth is generally regarded as a more sensitive and quantitatively precise method to study antimicrobial activity compared with agar-well diffusion assay because diffusion rates of active substances might be slower in agar than in broth.^4,19 Therefore we determined the MIC of honeys in broth using a spectrophotometric-based assay. MIC values showed a significant but variable antibacterial activity of Greek and Cypriot honeys against both tested pathogens, particularly S. aureus, as documented by lower MIC values. The same was observed for manuka honey. The reasons for this differential bacterial susceptibility to honey are not fully understood. A recent study on the effect of manuka honey on P. aeruginosa has shown loss of structural integrity and marked changes in cell shape followed by extensive cell disruption and lysis.²⁰ A similar study on S. aureus has demonstrated that manuka honey targets the cell division machinery because the cells appeared to form normal septa but were unable to separate at the point of cell division.²¹ Further investigation, including transcriptome analysis, could elucidate the global effect of honey on bacteria, thus revealing putative cell targets and antibacterial mechanisms in detail.

Four tested honeys originating from diverse botanical sources (7, 14, 19, and 23) demonstrated lower MIC values against S. aureus compared with manuka honey, indicating higher antibacterial activity. It is interesting that two of these honeys (14 and 19) did not demonstrate higher antibacterial activity in the agar-well diffusion assay. This could be explained by the fact that undiluted honey was tested in the agar-well diffusion assay. It is known that glucose oxidase, the enzyme that catalyzes hydrogen peroxide formation in honey, is sharply activated upon honey dilution.⁸ Catalase treatment is often used to reveal the contribution of hydrogen peroxide in antibacterial activity of tested honeys because catalase efficiently degrades hydrogen peroxide, which is formed by glucose oxidase. The antibacterial activity of two honeys (7 and 14) could be solely attributed to hydrogen peroxide because catalase treatment sharply elevated the MIC values (eightfold). Besides hydrogen peroxide, unidentified proteinaceous compounds contribute to antibacterial activity exerted by the other two honeys (19 and 23) as demonstrated by elevated MIC values after proteinase K treatment. Proteinase K is a broad-spectrum protease that digests proteins and proteinaceous compounds present in honey, thus contributing to its antibacterial activity.

Regarding the antibacterial activity against P. aeruginosa, two tested honeys (14 and 26) had demonstrated lower MIC values than manuka honey. The antibacterial activity of the first honey (14) could be solely attributed to hydrogen peroxide in accordance with data obtained for S. aureus. Besides hydrogen peroxide, proteinaceous compounds contribute to antibacterial activity exerted by the second honey (26). It is interesting that the antibacterial activity exerted by the same honey (26) against S. aureus (comparable but not higher to that exerted by manuka) could be solely attributed to hydrogen peroxide, possibly reflecting altered bacterial susceptibility due to distinct antibacterial mechanisms. In conclusion, the antibacterial properties of Greek and Cypriot honeys, especially those that are comparable or even superior to that of manuka honey, warrant further investigation as this may lead to medical applications.

Footnotes

Acknowledgments

Many thanks to all those who kindly provided honey samples and Dr. Spyros Pournaras (School of Medicine, University of Thessaly) who provided the bacterial strains used in this study. We especially thank Dr. Vassilios Bagiatis (Department of Biochemistry and Biotechnology, University of Thessaly) for his help with statistical analysis. This work was supported by the Postgraduate Programme “Biotechnology—Quality Assessment in Nutrition and the Environment” of the Department of Biochemistry and Biotechnology, University of Thessaly.

Author Disclosure Statement

No competing financial interests exist.

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