Antimicrobial properties and isotope investigations of South African honey.

Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Antimicrobial properties and isotope investigations of South African honey F. Khan1, J. Hill2, S. Kaehler3,*, M. Allsopp4 and S. van Vuuren1 1 2 3 4

Department of Pharmacy and Pharmacology, University of the Witwatersrand, Johannesburg, South Africa Department of Zoology and Entomology, Rhodes University, Grahamstown, South Africa Department of Botany, Rhodes University, Grahamstown, South Africa Plant Protection Research Institute, Agricultural Research Council, Stellenbosch, South Africa

Keywords antimicrobial, honey, isotope, protein, wound pathogens. Correspondence Sandy van Vuuren, Department of Pharmacy and Pharmacology, University of the Witwatersrand, 7 York Road, Parktown 2193, Johannesburg, South Africa. E-mail: [email protected] *Present addresses: South African Institute for Aquatic Biodiversity, Somerset St., Grahamstown, South Africa. 2013/2586: received 29 December 2013, revised 11 April 2014 and accepted 21 April 2014 doi:10.1111/jam.12533

Abstract Aims: The therapeutic potential of honey for the treatment of wound infections is well documented. However, South African (SA) honey has been poorly explored as an antimicrobial agent and given the well-established antimicrobial properties of the indigenous plant species from SA, there is the potential that honey from this geographical region may exhibit noteworthy anti-infective properties. In this study, the antimicrobial properties of 42 SA honey samples were determined. Methods and Results: The minimum inhibitory concentration (MIC) agar dilution method was used to determine antimicrobial activity. The MICs of the honeys ranged from 625 to 5000%. Samples 4-(CITYMIX/WC), 12-(BUSHVELD/ KZN), 15-(ONION/WC), 16-(FYNBOS/WC), 17-(AKMS/FS), 19-(CITYMIX/FS), 41-(INDIGENOUS/WC) and 52-(SURBURBANGARDEN/WC) displayed broadspectrum antimicrobial activity. The physicochemical properties including pH, water content and stable isotope analysis (SIA) was analysed. The pH of the honeys ranged between 389 and 509. The SIA revealed strong overall trends between protein concentration and MIC suggesting close links with antimicrobial activity. Conclusion: A number of SA honey samples tested have potential as an effective antimicrobial agent in wound healing. Significance and Impact of the Study: The future of South Africa’s market for medical grade and therapeutic honeys looks promising as the antimicrobial properties of the honeys have some superior activity.

Introduction The continuous use of antibiotics in clinical practice has resulted in the development of multiple resistant microbial strains. This coupled with more recent health trends to adopt a more natural approach to healing has consequently resulted in an increasing need to explore alternative therapies. The treatment of non-healing wounds has also become a global healthcare concern resulting in more laboratory testing, increased hospitalization times and greater treatment options, ultimately resulting in higher financial burdens (Nyasulu et al. 2012). Honey has been utilized therapeutically since ancient times for a wide range of treatments related to infectious 366

diseases. Famous ancient physicians such as Aristotle (384– 322 BC) and Dioscorides (c. 50 AD) commonly used honey as a dressing for wounds and ulcers (Molan 1999a). Other complaints treated with honey include respiratory and gastrointestinal conditions (Molan 1999b; Basualdo et al. 2007; Tan et al. 2009). Honey has consequently shown some potential for the treatment of infectious diseases especially in wound management. Furthermore, there have been no reports documenting microbial resistance to honey to date (Kwakman et al. 2008; Blair et al. 2009; Cooper et al. 2010). This makes honey a lucrative potential therapeutic agent for wounds infected with antibiotic resistant bacteria. This has consequently resulted in increasing efforts to scientifically validate the antimicrobial properties

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of honey. It is evident that this area of research has received a plethora of attention, where investigations have provided strong evidence for the antimicrobial properties of honey. Studies have focused on a wide range of geographical regions including Dubai (Al-Waili 2004), New Zealand (Brady et al. 2004), Australia (Lusby et al. 2005; Irish et al. 2011), Portugal (Henriques et al. 2005), Argentina (Basualdo et al. 2007), Wales (Cooper et al. 2008), Spain (Gallardo-Chacon et al. 2008), Iran (Khosravi et al. 2008), Ireland (Maeda et al. 2008), India (Mandal et al. 2010), Pakistan (Gulfraz et al. 2010), Chile (Sherlock et al. 2010), Cuba (Alvarez-Suarez et al. 2010) Malaysia (Khoo et al. 2010), Greece (Voidaroua et al. 2011), Algeria (Moussa et al. 2012) and Nigeria (Anyanwu 2012). These studies provide an important global perspective as it is evident that the antimicrobial activity of honey varies according to composition which is in turn dependent on geographical location, botanical origin, bee sub-species, season and post-harvest treatment (Kaskoniene and Venskutonis 2010). Moreover, not all honeys are equally effective for wound healing as the antimicrobial activities of honey can demonstrate 100-fold variances (Sherlock et al. 2010). South Africa (SA) possesses a significant floral biodiversity with many unique, indigenous plants. There are over 30 000 plant species in SA, of which the Cape boasts one of the most diverse temperate flora on earth, including over 3000 plant species that are used therapeutically (Goldblatt and Manning 2002; Van Wyk et al. 2009). The antimicrobial efficacies of SA plants have been studied extensively with many promising antimicrobial properties (Van Vuuren 2008). In contrast, the value of SA plants as nectar and pollen sources for indigenous honeybees (Apis mellifera capensis and A. m. scutellata) is relatively poorly studied (Johannsmeier and Mostert 2001). Nonetheless, there is in excess of a 100 significant nectar and pollen producing plants in SA, all of which contribute significantly to honey production, and at least 38 of these species are indigenous (Johannsmeier and Mostert 2001). There is therefore, the potential for some SA honeys to have superior or similar antimicrobial properties to the commonly utilized manuka honey, native to New Zealand. Research has indicated that these honeys, derived from the Leptospermum spp., have significant antimicrobial activity against various wound pathogens as well as antibiotic resistant strains. Leptospermum-derived honey is currently marketed in Australia as Medihoneyâ, a therapeutic honey suitable for use on ulcers, infected wounds and burns (Brady et al. 2004; Irish et al. 2011). Despite this, relevant searches on globally accepted scientific data bases Scopus, ScienceDirect and PubMed (search date – 16 November 2013) show that there are limited studies involving the antimicrobial investigations of SA honeys been undertaken (Theunissen et al. 2001; Basson and Grobler 2008; Manyi-loh et al. 2010,

Antimicrobial & isotopes of SA honey

2012). Manyi-loh et al. (2010), investigated the activity of commercially purchased honeys, Goldcrest, Pure Honey (floral sources of Citrus limon and Citrus sinesis) and Citrus Blossom (floral source of berry orchards) honeys against Helicobacter pylori. All the honeys demonstrated activity against H. pylori at honey concentrations of 1000% (v/v) and greater. Manyi-loh et al. (2012), further investigated the fraction responsible for antibacterial activity in Goldcrest (n-hexane extract) honey. Goldcrest mobile phase 3 fraction (5 mg ml1) displayed the best antibacterial activity. Basson and Grobler (2008) investigated the activity of a variety of honeys from Eucalyptus cladocalyx (sugar gum), Leucospermum cordifolium (pincushion), a mixture of heather shrubs mainly Erica species (fynbos) and Leptospermum scoparium (manuka) against a number of Streptococcus strains as well as Candida albicans, Escherichia coli and Staphylococcus aureus. These honeys demonstrated antimicrobial activity at concentrations of 5000% (v/v). Theunissen et al. (2001) examined the antifungal activity of honey commonly found in the Western Cape region of SA against C. albicans. The floral types of honey investigated were Eucalyptus cladocalyx (sugar gum), Myrica cordifolia (wax berry) and fynbos (a mixture of many heather shrubs mainly derived from the botanical origin of Erica species). Research showed that an increased honey concentration resulted in a greater inhibition of C. albicans. It was also reported that M. cordifolia honey (2500% w/w) produced the greatest inhibition of C. albicans. While these studies demonstrate the antimicrobial potential of SA honeys, they unfortunately focused on a limited number of honey samples. Furthermore, none of the studies addressed the fact that honey has historically been associated with wounds and as such, pathogens associated with wound healing could have been given priority. The market demand for selected honey of high therapeutic value has increased, creating the need for honey to be standardized and authenticated. The adulteration of honey with low cost sugar syrup or artificial honey and/or the mislabelling of honeys from different geographical or botanical regions pose significant challenges to this process (Anklam 1998; Fairchild et al. 2003; Wang and Li 2011). A study published by White and Doner (1978) identified d13C ratios of honey as a useful tool for identifying honey adulteration via C4 sugar additives. This technique has become increasingly popular in recent years (Chesson et al. 2010) and is now recognized by the Association of Official Analytical Chemists (AOAC) as the established official method for testing honey’s authenticity (AOAC, 1999). This study aimed to investigate the antimicrobial properties of 42 SA honeys, in comparison with the commonly utilized manuka New Zealand honey, to assess their potential as a therapeutic honey. Three fresh manuka samples, marked with various levels of Unique Manuka Factor


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(UMF) activity (Cramp 2013) were included in the same assay to demonstrate activity of other popular therapeutic honeys. This work was followed by stable isotope analysis to provide information on potential adulteration and investigate any isotopic honey trends linked to microbial activity. Materials and methods Honey samples Samples of unheated, non-irradiated, raw (unprocessed) honey from varied botanical sources were collected in 2011 from all eight SA provinces (Eastern Cape, Western Cape, Kwa-Zulu Natal, Mpumalanga, Gauteng, North West, Limpopo and Free State). The perceived nectar source(s) of the honey were recorded as identified by the beekeeper providing the samples. Beekeepers were asked to provide honey samples which represented either monofloral or multifloral sources. In addition, eight processed honey samples were purchased in order to provide samples of some honey with forage plants not represented in the raw honey samples. Honey samples were stored away from light and at room temperature (200 200°C). The samples were numbered, and coded to reference and trace the respective samples. Honey samples were labelled to indicate the order of receipt, as well as to record the perceived floral source, the province of origin, and whether the sample was raw or processed honey (Table 1). Antimicrobial assay A selection of pathogens most commonly responsible for wound microbial colonization and proliferation were selected to test the antimicrobial efficacy of the honey samples utilizing the agar dilution method. These included six Gram-positive strains: Staphylococcus aureus American type culture collection (ATCC) 25923; Staph. aureus, clinical strain 6438300; methicillin-resistant Staph. aureus ATCC 43300, methicillin-resistant Staph. aureus clinical strain 43300, methicillin and gentamycin-resistant Staph. aureus ATCC 33592, Staphyloccocus epidermidis ATCC 2223. One Gram-negative strain, Pseudomonas aeruginosa, National culture type collection (NCTC) 9027 was included as well as two yeast (Candida albicans) strains (ATCC 10231 and clinical CA9B). Stock cultures for ATCC strains were obtained from Davies Diagnostics and clinical strains were obtained from Dr M Patel (Department of Oral Microbiology, University of the Witwatersrand). As per National Committee for Clinical Laboratory Standards (NCCLS, 2003), three to five isolated colonies were selected from a Tryptone Soya agar (TSA) plate and transferred into Tryptone Soya broth (TSB). Purity of cultures were 368

ensured by streaking onto an TSA agar plates and incubating at 37°C for 24 and 48 h for bacterial and yeast species respectively. The minimum inhibitory concentration (MIC), in this study, is defined as the lowest concentration of honey required to inhibit microbial growth. The MICs of the honey samples were determined by the agar dilution method adapted from the National Committee for Clinical Laboratory Standards, 2003. Briefly, honey dilutions were added to molten TSA that was allowed to equilibrate in a water bath at 55°C. The agar and honey at various concentrations (5000%, 2500%, 1250%, 625% and 313%; v/v) were pipetted into sterile test tubes and vortexed to ensure a homogenous mixture. The agars and honey solutions were then poured into Petri dishes to achieve a depth of 3 mm (20 ml per plate). Cultures were inoculated utilizing a multipointelite replicator. After inoculation with 1 9 104 colony forming units (CFU) per spot, the agar dishes were incubated for 24 h at 37°C for bacteria. After examining the presence or absence of growth at all dilutions, the plates were further incubated for an additional 24 h to assess yeast growth. The MIC was read as the lowest concentration of honey-agar dilutions tested that completely inhibited visible growth. TSA plates without honey were inoculated as a control to ensure growth of the pathogens tested. An artificial honey solution (3900% w/v d-fructose, 3100% w/v d-glucose, 800% w/v maltose, 300% w/v sucrose and 1900% w/v water) with a sugar content similar to honey was included in the assays as a control to determine antimicrobial effects due to sugar. Previous studies have demonstrated that the sugar in honey is responsible for antimicrobial properties (Molan 2001; Basson and Grobler 2008). The artificial honey solution was autoclaved at 121°C for 15 min and subjected to the agar dilution method at concentrations of 5000, 2500, 1250, 625 and 313% to mimic honey concentrations. Three manuka honey samples marked with various levels of unique manuka factor from New Zealand were utilized as controls with which to compare assay conditions and relative activity of the SA honeys. The three samples are broadly representative of manuka honey. A 20% phenol agar plate was utilized as a positive control in line with studies by Blair et al. (2009), who when investigating Leptospermum-derived honey, revealed that these honeys had a level of activity equivalent to approx. 18% phenol. To ensure accuracy and consistency, all MICs were performed in triplicate (N = 3). Physicochemical properties The water content of whole honey was measured with a honey refractometer (EW-81150-14), and pH was determined via a pH meter, by dissolving 10 g of whole honey


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Table 1 South African raw honey samples (with the exception of those noted by *) collected in various geographical regions Reference code

Perceived forage source

Province

1-(CITYMIXA/EC) 2-(CITYMIXB/EC) 4-(CITYMIX/WC) 5-(CITYMIXTUART/WC) 6-(MANGO/WC) 7-(FYNBOS/WC) 8-(ECFYNBOS/WC) 9-(STANDVELD/WC) 12-(BUSHVELD/KZN) 13-(BUFFALOTHORN/NC) 14-(HOOKTHORN/NC) 15-(ONION/WC) 16-(FYNBOS/WC) 17-(AKMS/FS) 18-(MIXEDGUM/FS) 19-(CITYMIX/FS) 20-(FORRESTREDGUM/WC) 21-(SUGARGUM/WC) 24-(STRANDVELD/WC) 25-(BUCFYN/WC) 26-(FYNBOS/WC) 27-(AEF/WC) 31-(MACADAMIA/WC) 35-(FYNBOSEC/WC) 36-(EUCLADFICI/WC) 37-(FYNBOS/WC) 38-(WASBESSFYNBOS/WC) 40-(FYNBOSGUARRI/WC) 41-(INDIGENOUS/WC) 43-(KAROOVELD/EC) 44-(STRANDVELD/WC) 45-(FYNBOS/WC) 47-(SALIGNAGUM/KZN)* 48-(BRE/EC) 49-(CITRUS/EC) 51-(URBANFORAGE/WC) 52-(SUBURBANGARDEN/WC) 53-(LITCHI/MP)* 159-(BUFFALOTHORN/NW) 160-(BOEKENHOUT/LIM) 162-(SALIGNAGUM/KZN) 167-(SUNFLOWER/NW) 54-(MANUKA/NZ) 55-(MANUKA/NZ) 56-(MANUKA/NZ)

Eucalyptus spp., Trichiliaemetica, Erythina spp (inner city A mix) Eugenia spp., Eucalyptus spp., Jacaranda spp., Thymus spp., Lavandula spp., etc. (inner city B mix) City mix Eucalyptus gomphocephala and various gums (city mix) Mangifera indica Ericas spp. (fynbos) Eucalyptus cladocalyx and Erica spp. (sugar gum and fynbos) Strandveld Coastal sandy bushveld Ziziphus mucronata Acacia mellifera Allium cepa Erica spp. (fynbos) Acacia karroo and Medicago sativa Mixed gums City mix Eucalyptus tereticornis (forest red gum) Eucalyptus cladocalyx Strandveld wild flowers Agathosma spp., Erica spp. (fynbos) Erica ericoides (fynbos) Agathosma spp., Eucalyptus ficifolia Macadamia integrifolia Eucalyptus conferruminata/Erica spp. Eucalyptus cladocalyx/Eucalyptus ficifolia Erica spp. (fynbos) Morella cordifolia/Erica spp. Erica spp., Euclea racemosa (fynbos) Acacia spp.,Pyrus spp., Aloe spp., Pelargonium spp., Nymphaea spp.,etc. Karoo veld Strandveld Erica spp. (fynbos) Eucalyptus grandis Bushveld, riverine forests, Euphorbia spp. etc. Citrus spp. Urban forage Suburban gardens Litchi chinensis Ziziphus mucronata Faurea saligna Eucalyptus grandis Helianthus annuus Leptospermum scoparium (manuka) Control Leptospermum scoparium (manuka) Control Leptospermum scoparium (manuka) Control

E. Cape E. Cape W. Cape W. Cape W. Cape W. Cape W. Cape W. Cape KZN N. Cape N. Cape W. Cape W. Cape Free State Free State Free State W. Cape W. Cape W. Cape W. Cape W. Cape W. Cape W. Cape W. Cape W. Cape W. Cape W. Cape W. Cape W. Cape E. Cape W. Cape W. Cape KZN E. Cape E. Cape W. Cape W. Cape Mpumalanga North West Limpopo KZN North West New Zealand New Zealand New Zealand

Reference code: Sample number – (Common name or scientific name/Province abbreviated); WC: Western Cape; NC: Northern Cape; EC: Eastern Cape; KZN: Kwa-Zulu Natal; FS: Free State; MP: Mpumalanga; LIM: Limpopo; NZ: New Zealand.

in 75 ml of distilled water, following the official methods published by the AOAC (1999). Stable isotope analysis Purified honey protein was extracted from whole honey using a modified ver. of the repetitive wash method as

described by Rogers et al. (2010, 2013), which employs a centrifugal and filtration step prior to standard AOAC protocols (AOAC 1999). This is to prevent the inclusion of pollen and other insoluble components with the flocculated protein. These non-protein components may have isotope values which are considerably different from those of the pure protein, and can shift isotopic signatures away


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from true protein values (Rogers et al. 2010, 2013). Briefly, 4 ml of distilled H2O was added to 10–12 g honey. This honey solution was then heated in a water bath (to aid dissolution) and mixed well. Following dissolution, the samples were centrifuged at 3500 rev min1 (5 min) to remove fine material such as dust and pollen. The supernatant was carefully decanted and vacuum-filtered directly onto Whatmanâ GF/C glass-fibre filters (12 mm pore size) to remove any larger insoluble particulate material (e.g. bee body parts, wax and pollen not removed by centrifuging). An amount of 2 ml of 10% sodium tungstate and 2 ml 067N H2SO4 were mixed together and immediately added to the honey filtrate. The honey filtrate mix was then agitated in a heated (80°C) water bath until a visible protein flocculent formed with a clear supernatant. Further, 2 ml increments of 067N H2SO4 were added if no visible flocculent formed after 5 min. Once floccs were formed, the centrifuge tube was filled with distilled H2O and again centrifuged at 3500 rev min1 (5 min). The supernatant was discarded and the pellet rinsed and centrifuged five more times with 50 ml of water, thoroughly dispersing the pellet each time. The precipitated protein was then oven dried for 24–48 h at 50°C. Approximately, 15 mg of whole honey or protein was used for stable isotope analyses (AOAC 1999). The d13C and d15N values of all samples were determined using a Europa Scientific 20–20 IRMS interfaced to an ANCA SL Elemental Analyser at Isoenvironmental cc, Department of Botany, Rhodes University, Grahamstown, South Africa. All d13C and d15N values were reported as & vs Vienna PeeDeeBelemnite (VPDB) and air, respectively, and normalized to internal standards calibrated to the International Atomic Energy reference materials (IAEA-CH6 for d13C and IAEA-N2 for d15N). Results are expressed in standard delta notation, dX = ([Rsample/Rstandard]1) 9 1000, where X is the element in question and R is the ratio of the heavy over the light isotope. Precision of replicate determinations for both d13C and d15N were approx. 008 and 001 respectively. Apparent % C4 adulteration, where C4 values of greater than 7% are considered questionable, were calculated for each honey (White and Winters 1989), using the formula: Adulterationð%Þ ¼

½ðd13 CðproteinÞÞ ðd13 Cðwhole honeyÞÞ 100 ½ðd13 CðproteinÞ ð97ÞÞ

Results Antimicrobial investigation The mean MIC (% inhibition) of each honey collected from various geographical regions of SA are reported in 370

Table 2. The majority of the samples sourced originated from the Western Cape (6364%) and the mean antimicrobial activity of honey samples for all pathogens tested, ranged from 1042 to 5000%. At a concentration of 5000%, the growth of all nine test pathogens was completely inhibited by all the honey samples. A number of honeys had MICs of 25% and 50% for all pathogens tested. This was consistent with results from artificial honey (Table 3), suggesting that the antimicrobial activity of these samples can be attributed to a hyper-osmolar effects, as the carbohydrate concentration has a vital effect on the antimicrobial activity of honeys above 25% (Basson and Grobler 2008). Hyper-osmolarity prevents the growth of bacteria and promotes wound healing and hyper-osmolar agents like sugar have been identified and utilized in wound healing (Moore et al. 2001). Samples of manuka honey and artificial honey were utilized as positive controls (Table 3). The antimicrobial activity of the manuka samples tested ranged from 1528 to 4167%, with the mean activity of these three samples at 2569% (SD 1404). The manuka sample demonstrating the highest activity was 54-(MANUKA/NZ) with a mean MIC of 1528%. Various antimicrobial potencies have been reported globally. Blair et al. (2009), reported a range of MIC (w/v% honey) of 40–148 against all pathogens tested for Medihoneyâ (Blair et al. 2009). An even earlier study revealed an MIC range of 6–14% for various pathogens tested including that of P. aeruginosa (George and Cutting 2007). Some of the SA honey samples tested [4-(CITYMIX/WC); 12–(BUSHVELD/KZN); 16–(FYNBOS/WC); 17–(AKMS/FS); 19–(CITYMIX/FS); 41-(INDIGENOUS/ WC); 52-(SUBURBANGARDEN/WC)] had a greater antimicrobial activity (MIC values lower than 1528%) than the best ‘gold standard’ manuka sample included in this study, and 24 of 42 (57%) of tested SA honeys had MIC values as low or lower than the average manuka samples tested. It was observed that P. aeruginosa was the most sensitive strain tested with the mean MIC of 1414% (SD 1235). Honey samples 1-(CITYMIXA/EC), 4-(CITYMIX/WC), 9(STRANDVELD/WC), 12-(BUSHVELD/KZN), 13-(BUFFALOTHORN/KZN), 16-(FYNBOS/WC), 17–(AKMS/FS), 18-(MIXEDGUM/FS), 19-(CITYMIX/FS), 20-(FORRESTREDGUM/WC), 26-(FYNBOS/WC), 27-(AEF/WC), 36-(EUCLADFICI/WC), 38-(WASBESSFYN/WC), 41-(INDIGENOUS/WC), 43-(KAROOVELD/EC), 44-(STRANDVELD/ WC), 45-(FYNBOS/WC), 49-(CITRUS/EC), 51-(URBANFORAGE/WC), 52-(SUBURBANGARDEN/WC), 53-(LITCHI/ MP) and 160-(BOEKENHOUT/LIM) demonstrated good efficacy against P. aeruginosa. A proportion of samples (1951%) samples tested resulted in an MIC of 625% and 3171% of samples tested resulted in an MIC of 125% when tested against


Staph. aureus ATCC 25923

1250 2500 625 5000 2500 2500 2500 2500 1250 2500 5000 1250 625 1250 625 625 625 2500 2500 2500 1250 2500 2500 2500 1250 1250 1250 2500 1250 1250 1250 1250 5000 2500 5000

Reference code

1-(CITYMIXA/EC) 2-(CITYMIXB/EC) 4-(CITYMIX/WC) 5-(CITYMIXTUART/WC) 6-(MANGO/WC) 7-(FYNBOS/WC) 8-(ECFYNBOS/WC) 9-(STANDVELD/WC) 12-(BUSHVELD/KZN) 13-(BUFFALOTHORN/NC) 14-(HOOKTHORN/NC) 15-(ONION/WC) 16-(FYNBOS/WC) 17-(AKMS/FS) 18-(MIXEDGUM/FS) 19-(CITYMIX/FS) 20-(FORRESTREDGUM/WC) 21-(SUGARGUM/WC) 24-(STRANDVELD/WC) 25–(BUCFYN/WC) 26-(FYNBOS/WC) 27-(AEF/WC) 31-(MACADAMIA/WC) 35-(FYNBOSEC/WC) 36-(EUCLADFICI/WC) 37-(FYNBOS/WC) 38-(WASBESSFYNBOS/WC) 40-(FYNBOSGUARRI/WC) 41-(INDIGENOUS/WC) 43-(KAROOVELD/EC) 44-(STRANDVELD/WC) 45-(FYNBOS/WC) 47-(SALIGNAGUM/KZN) 48-(BRE/EC) 49-(CITRUS/EC)

1250 2500 625 5000 2500 2500 2500 2500 1250 2500 5000 1250 625 1250 625 625 125 2500 2500 2500 1250 2500 2500 2500 1250 2500 1250 2500 1250 1250 2500 1250 5000 2500 5000

Staph. aureus clinical 1250 2500 625 5000 2500 2500 2500 2500 1250 2500 5000 1250 625 1250 625 625 125 2500 2500 2500 1250 2500 2500 2500 1250 1250 1250 2500 625 1250 1250 1250 5000 2500 5000

Staph. aureus (MRSA) ATCC 4330 1250 2500 625 5000 2500 2500 2500 2500 1250 2500 5000 1250 625 1250 625 625 625 2500 2500 2500 1250 2500 2500 2500 1250 1250 1250 2500 625 2500 1250 1250 5000 2500 5000

Staph. aureus (MRSA) clinical 1250 2500 1250 5000 2500 2500 2500 2500 1250 2500 5000 1250 625 1250 625 625 625 2500 2500 2500 1250 2500 2500 2500 2500 2500 1250 5000 1250 1250 2500 1250 5000 2500 5000

Staph. aureus (M & G) ATCC 33592

Table 2 Minimum inhibitory concentration (% inhibition) of South African honey samples collected in 2011

1250 2500 625 5000 2500 2500 2500 2500 1250 2500 5000 1250 625 1250 625 625 625 2500 2500 1250 1250 1250 1250 2500 1250 2500 1250 2500 1250 1250 1250 1250 5000 2500 5000

Staph. epidermidis ATCC 2223 625 2500 625 5000 2500 2500 2500 625 625 625 5000 1250 625 625 625 625 625 2500 2500 1250 625 625 1250 1250 625 2500 625 1250 625 625 625 625 5000 1250 625

Pseudomonas aeruginosa ATCC 9027 5000 5000 5000 5000 5000 5000 5000 5000 2500 2500 5000 2500 2500 2500 5000 5000 5000 5000 2500 5000 5000 2500 5000 5000 2500 5000 5000 5000 2500 5000 5000 5000 5000 5000 5000

Candida albicans ATCC 10231 2500 5000 2500 5000 5000 5000 5000 5000 2500 5000 5000 2500 2500 2500 5000 625 5000 5000 2500 2500 5000 1250 5000 2500 2500 5000 5000 5000 2500 5000 5000 5000 5000 5000 5000

Candida albicans clinical


SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD SD

1318 1102 1491 000 1102 1102 1102 1366 625 1102 000 551 827 625 1929 1458 1869 1102 000 1083 1705 751 1366 977 706 1458 1705 1413 729 1686 1654 1705 000 1250 1458

(Continued)

1736 3056 1389 5000 3056 3056 3056 2847 1458 2569 5000 1528 1042 1458 1597 1111 1736 3056 2500 2500 2014 2014 2778 2639 1597 2639 2014 3194 1319 2153 2292 2014 5000 2917 4514

Mean MIC

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1736SD 1458 SD 4514 SD 2917 SD 2639 SD 3056 SD 2917 SD 2550 SD

MRSA: Methicillin resistant Staphylococcus aureus; M & G: Methicillin and gentamycin resistant Staphylococcus aureus; MIC: minimum inhibitory concentration.

5000 2500 5000 5000 5000 5000 5000 4092 SD 1340 5000 5000 5000 5000 5000 5000 5000 4492 SD 1038 625 625 625 1250 625 2500 1250 1414 SD 1235 625 625 5000 2500 625 2500 2500 2024 SD 1322 51-(URBANFORAGE/WC) 52-(SUBURBANGARDEN/WC) 53-(LITCHI/MP) 159-(BUFFALOTHORN/NW) 160-(BOEKENHOUT/LIM) 162-(SALIGNAGUM/KZN) 167-(SUNFLOWER/NW) Mean of pathogens

1250 625 5000 2500 2500 2500 2500 2143 SD 1281

625 1250 5000 2500 2500 2500 2500 2217 SD 1251

625 625 5000 2500 2500 2500 2500 2123 SD 1295

625 625 5000 2500 2500 2500 2500 2143 SD 1303

1250 1250 5000 2500 2500 2500 2500 2321 SD 1297

Pseudomonas aeruginosa ATCC 9027 Reference code

Table 2 (Continued)


Staph. aureus clinical

Staph. aureus (MRSA) ATCC 4330

Staph. aureus (MRSA) clinical


Staph. epidermidis ATCC 2223

Candida albicans ATCC 10231


Mean MIC

1869 1466 1458 1250 1555 1250 1018 1051


Staph. epidermidis. Manuka honeys, when tested against Staph. epidermidis resulted in an MIC that ranged from 125 to 25%. Honey samples 4-(CITYMIX/WC), 16-(FYNBOS/WC), 18-(MIXEDGUM/FS), 19-(CITYMIX/FS), 20-(FORRESTREDGUM/WC), 51-(URBANFORAGE/ WC), 52-(SUBURBANGARDEN/WC) exhibited strong efficacy against Staph. epidermidis. Honey samples 4-(CITYMIX/WC), 16–(FYNBOS/WC), 18–(MIXEDGUM/FS), 19–(CITYMIX/FS), 20–(FORRESTREDGUM/WC) and 52-(SUBURBANGARDEN/WC) demonstrated the greatest efficacy against the Staph. aureus strains tested. The SA honey and manuka samples tested against C. albicans had a MIC of 5000% with the exception of one sample (27-(AEF/WC) which inhibited the laboratory strain at 2500% and its clinical counterpart at 1250%. Honey samples 19-(CITYMIX/FS) and 27-(AEF/WC) had a MIC of 625% and 125%, respectively, for the clinical strain of C. albicans. Physicochemical and isotope analysis The water content amongst the SA honeys was found to range between and 1580 and 2160% (Table 4). Research has indicated that an acidic pH (32–45) also contributes to antimicrobial activity (Mandal et al. 2010). In this study, the pH of the honeys ranged between 389 and 509, providing some validation for the antimicrobial efficacies noted here (Table 4). Stable isotope analysis was undertaken according to the AOAC protocols and the results demonstrating the relationship between the d13C ratio of the whole honey and its protein are presented in Fig. 1. Three of the honeys [1-(CITYMIXA/EC) at 1519%; 47-(SALIGNAGUM/ KZN) at 1943% and 53-(LITCHI/MP) at 3360%] collected from SA apiaries and one of the New Zealand golden standard manuka Medihoneyâ [56-(MANUKA/ NZ) at 1454%] were classed as having some apparent impurification levels (>70%). While by current AOAC protocols and SA legislation (DAFF 2000) these samples would be classified as ‘adulterated’, it is highly unlikely that deliberate adulteration did take place. Questionable differences, where variances are given in % between the d13C ratio of whole honey and protein content are most likely due to other reasons (see discussion). The d13C isotope ratios of whole honey and the corresponding honey protein for South African samples ranged from 2784 02& (41-(INDIGENOUS/WC)) to 1772 005& (53-(LITCHI/MP)) and 2890 005& (1-(CITYMIXA/EC)) to 2108 002& (47(SALIGNAGUM/KZN))& respectively. The range of d15N values (flocculated protein only) were between 383 024& (35-(FYNBOSEC/WC)) and 1132 046& Journal of Applied Microbiology 117, 366--379 © 2014 The Society for Applied Microbiology

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Table 3 Minimum inhibitory concentration (% inhibition) of controls

Control Leptospermum scoparium 54-(MANUKA/NZ) Leptospermum scoparium 55-(MANUKA/NZ) Leptospermum scoparium 56-(MANUKA/NZ) Artificial honey Ciprofloxacin (ug/ml)


Staph. aureus clinical

Staph. aureus (MRSA) ATCC 4330

Staph. aureus (MRSA) clinical


Staph. epidermidis ATCC 2223

P. aeruginosa ATCC 9027

Candida albicans ATCC 10231


Mean MIC

1250

1250

1250

1250

1250

2500

1250

2500

1250

1528

625

1250

625

625

2500

125

1250

5000

5000

2017

5000

5000

5000

2500

5000

2500

2500

5000

5000

4167

5000 008

5000 008

5000 008

5000 125

5000 N/A

2500 270°C (Rogers et al. 2014a,b). Although in this study all honeys were stored at ~20°C, the possibility of higher storage temperatures prior to arrival at the laboratory cannot be excluded. Deliberate adulteration on the other hand is the addition by the beekeeper, processer or retailer of any lower cost C4 sugar syrups (cane syrup and/or high fructose corn syrup) in order to ‘stretch’ the honey for the purpose of greater profits (Padovan et al. 2003; Rogers et al. 2010). The addition of sucrose is easily traceable through carbohydrate analysis, and more sophisticated adulteration is now detectable through stable isotope analysis, where the d13C ratios of whole honey are


F. Khan et al.

compared with those of the isolated honey protein. Interestingly, perceived unadulterated New Zealand manuka honeys routinely fail the AOAC adulteration test, especially manuka honeys with high antimicrobial activity (Rogers et al. 2010, 2013) and similar situations have been reported in other European honeys (White 1992; Cotte et al. 2007). To date, the factors underpinning this phenomenon are largely unexplained; however, work done by Rogers et al. (2010, 2013, 2014a,b) has clearly shown that in many cases the current official methods (AOAC, 1999) are not sufficiently sensitive for C4 level detection in some honeys and have called for a revision to the accepted protocols. In conclusion, the in vitro antimicrobial efficacy results from this study highlight the potential for using selected SA honey samples as an effective antimicrobial agent in wound healing. Some SA honeys further demonstrate the potential to be utilized to heal potentially problematic burn wounds in patients that may lead to systemic sepsis as it has been anticipated that honeys which produce MIC values between 1000 and 2000% could be effective in eradicating Pseudomonas related infections (Mullai and Menon 2007). Hydrogen peroxide-dependent honeys have been reported to be more effective against fungal organisms than non-peroxide honeys like manuka honey. Further research is required to determine if SA honeys are hydrogen peroxide dependent. The overall inverse relationships, seen between % impurification (White and Winters 1989) and protein concentration as well as protein concentration and MIC, suggest that protein concentration and estimates of honey authenticity are linked with antimicrobial activity. However, the underlying causes of these relationships and the perceived impurification of some of the honeys requires further investigation. Finally, there is no doubt that this study, highlighting antimicrobial activities which have some superior activity, and in some cases superior to the manuka honey, puts South Africa on the map towards providing high standard therapeutic honeys. The results presented herewith are the initial steps in identifying SA honey with superior activity and investigations regarding the pollen analysis for highly active antimicrobial honeys are recommended. Furthermore, before definite comparative efficacies are to be made with commercial samples, the hydrogen peroxide activities should be evaluated. Acknowledgements The authors acknowledge Kim Morgado (beekeeper in Johannesburg) for his enthusiastic conceptualizations to the study. Furthermore, our grateful thanks go to all the honey farmers from various locations in SA for generously donating honey samples. The laboratory component of the


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Antimicrobial activity and chemometric modelling of South African propolis.

A 1000-Year Carbon Isotope Rainfall Proxy Record from South African Baobab Trees (Adansonia digitata L.).

A Regional Stable Carbon Isotope Dendro-Climatology from the South African Summer Rainfall Area.

Genome Sequence of Hafnia alvei bta3_1, a Bacterium with Antimicrobial Properties Isolated from Honey Bee Gut.

Bloodstream infections and antimicrobial resistance patterns in a South African neonatal intensive care unit.

The antimicrobial spectrum of honey and its clinical significance.

Black South african lesbians.

Bilingualism--South African style.

Mechanistic investigations of the hydrolysis of amides, oxoesters and thioesters via kinetic isotope effects and positional isotope exchange.

Phytochemical, Antimicrobial and Antiplasmodial Investigations of Terminalia brownii.

Antimicrobial effect of different types of honey on Staphylococcus aureus.

Evaluation of Physicochemical Properties of South African Cashew Apple Juice as a Biofuel Feedstock.

Antimicrobial activities of Saudi honey against Pseudomonas aeruginosa.

Honey: A realistic antimicrobial for disorders of the skin.

Environmental health and the South African community.

Antimicrobial properties of cumin.

Disability and masculinity in South African autosomatography.

Histopathological and biochemical investigations of protective role of honey in rats with experimental aflatoxicosis.

African National Congress drafts blueprint for South African science.

Professionalization of South African nursing: who benefits?.

[Antimicrobial properties of ceftaroline fosamil].

Chemical composition and temperature influence on honey texture properties.

Eye gaze technology: a South African perspective.

Multiple myeloma in South African blacks.