Non-peroxide Antibacterial Activity Research Articles

EFFECT OF THE PEROXIDE ACTIVITY OF BASHKIR LINDEN HONEY ON ANTIBACTERIAL ACTIVITY

Bashkir linden honey is known not only for its unique aroma and delicate taste, but also for its medicinal antibacterial properties. One of the components responsible for the bactericidal properties of honey is hydrogen peroxide, which is formed as a result of enzymatic oxidation of glucose to gluconic acid by glucose oxidase, the enzyme produced by bees. However, there are no data in the literature on the studies of the peroxide antibacterial properties of Bashkir honey. The present study deals with the quantitative determination of the hydrogen peroxide content in Bashkir linden honey collected from various natural and agricultural zones of the Republic of Bashkortostan. According to the results of the melissopalinological analysis, all the studied 39 honey samples are floral monofloral linden honey. The concentration of hydrogen peroxide in all samples ranges within 0-51.97 mg/kg•h. The high diversity in the sensitivity of the investigated strains of opportunistic microorganisms Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa to the tested samples of linden honey suggests the presence of both peroxide and non-peroxide mechanisms of antibacterial activity. Non-peroxide antibacterial activity can be represented by the honey components of bee, plant and microbial origin: organic acids, antimicrobial proteins and peptides, as well as bacteriocins.

Intrinsic Raman signal amplification for rapid identification and detection of methylglyoxal in manuka honey

Methylglyoxal (MGO) is the primary material basis for the non-peroxide antibacterial activity (NPA) of manuka honey from New Zealand. Therefore, it is necessary to identify the quality or discriminate the grade of honey because no all manuka honeys on the market display the NPA. The current routine method employed for the detection of MGO involves high-performance liquid chromatography (HPLC) test. However, it requires long time (∼8 h) for sample derivatization. Herein, we report an intrinsic Raman signal amplification strategy for the rapid identification and detection of MGO by using silver-coated gold nanoparticles (Au@Ag NPs) along with a high selective surface-enhanced Raman scattering (SERS) probe 8-thioguanosine (8-TG). 8-TG is synthesized via the derivatization of 8-bromoguanosine (8-BG) with thiourea, and its Raman peak assignments were confirmed by computer simulation. The detection is performed through the Raman intensity ratio (I631/I700) variation of N2-(1-carboxyethyl)-thioguanosine (CETG) formed by the reaction between 8-TG and MGO on surface of Au@Ag NPs, where one CETG Raman intensity at 631 cm−1 increases while the other one at 700 cm−1 decreases oppositely. The opposite change not only yields an intrinsic Raman signal amplification, but also provides built-in correction. As a result, the proposed SERS method exhibits high sensitivity and accuracy. In addition, the whole analytical test is achieved within ∼20 min. The method can be used for the fast detection of MGO in manuka honey and discrimination of the honey grade.

Comparative Evaluation of Wound Healing Potential of Manuka and Acacia Honey in Diabetic and Nondiabetic Rats.

ABSTRACTBackground:Manuka honey has attracted the attention of the scientific community for its antimicrobial and antioxidant properties. The active compounds of manuka honey to which its myeloperoxidase activity inhibition is owed are methyl syringate (MSYR) and leptosin (a novel glycoside of MSYR). The non-peroxide antibacterial activity is attributed to glyoxal, 3-deoxyglucosulose, and methylglyoxal. These properties make it an inexpensive and effective topical treatment in wound management. This study has focused on the evaluation of the effect of manuka honey and acacia honey on wound healing in nondiabetic and streptozotocin-induced diabetic rats.Materials and Methods:This study was conducted on a total of 42 rats (six rats in each group) and respective drug/substance was topically applied once daily on the excision wound for 21 days. Induction of diabetes was carried out in rats in groups IV, V, VI, and VII only. Measurement of wound contraction was carried out on days 3, 6, 9, 12, 15, 18, and 21 after operation. Time taken for the complete epithelization was recorded along with a histopathological examination of the healed wound bed.Results:Topical application of manuka honey achieved ≥80% wound contraction on day 9 after operation in both the nondiabetic and diabetic group. Complete epithelization was achieved 2 days earlier than the normal epithelization time in the manuka group. Histopathological examination showed well-formed keratinized squamous epithelium with normal collagen tissue surrounding hair follicles.Conclusion:This study provides good outcome with respect to wound healing (especially in diabetic condition) when manuka honey was compared to acacia honey and standard treatment.

Influence of genotype, floral stage, and water stress on floral nectar yield and composition of mānuka (Leptospermum scoparium).

Background and AimsFloral nectar can be variable in composition, influencing pollinator behaviour and the composition of honey derived from it. The non-peroxide antibacterial activity of mānuka (Leptospermum scoparium, Myrtaceae) honey results from the chemical conversion of the triose sugar dihydroxyacetone (DHA), after DHA accumulates for an unknown reason in the nectar. This study examined variation in nectar DHA, glucose, fructose and sucrose content with floral stage of development, between mānuka genotypes with differing flower morphology, and in response to water stress.MethodsSix mānuka genotypes were grown without nectar-feeding insects. Stages of flower development were defined, nectar was harvested and its composition was compared between stages and genotypes, and with floral morphology. Water stress was imposed and its effect on nectar composition was examined.Key ResultsNectar was present from soon after flower opening until the end of petal abscission, with the quantity of accumulated nectar sugars rising, then stabilizing or falling, indicating nectar secretion followed by reabsorption in some genotypes. The quantity of DHA, the ratio of DHA to other nectar sugars and the fructose to glucose ratio also varied with stage of development, indicating differences in rates of production and reabsorption between nectar components. Nectar composition and yield per flower also differed between genotypes, although neither was positively related to nectary area or stomatal density. Drying soil had no effect on nectar composition or yield, but variation in nectar yield was correlated with temperature prior to nectar sampling.ConclusionsMānuka nectar yield and composition are strongly influenced by plant genotype, flower age and the environment. There were clear stoichiometric relationships between glucose, fructose and sucrose per flower, but DHA per flower was only weakly correlated with the amount of other sugars, suggesting that accumulation of the triose sugar is indirectly coupled to secretion of the larger sugars by the nectary parenchyma.

Role of Honey in Topical and Systemic Bacterial Infections.

The development of bacterial resistance to antibiotics has made it more difficult and expensive to treat infections. Honey is getting worldwide attention as a topical therapeutic agent for wound infections and potential future candidate for systemic infections. The purpose of this review was to summarise different antibacterial bio-active compounds in honey, their synergistic interaction and their clinical implications in topical and systemic infections. In addition, contemporary testing methods for evaluating peroxide and non-peroxide antibacterial activity of honey were also critically appraised. MEDLINE, EMBASE, Cochrane Library, Pub Med, reference lists and databases were used to review the literature. Honey contains several unique antibacterial components. These components are believed to act on diverse bacterial targets, are broad spectrum, operate synergistically, prevent biofilm formation, and decrease production of virulence factors. Moreover, honey has the ability to block bacterial communication (quorum sensing), and therefore, it is unlikely that bacteria develop resistance against honey. Bacterial resistance against honey has not been documented so far. Unlike conventional antibiotics, honey only targets pathogenic bacteria without disturbing the growth of normal gastrointestinal flora when taken orally. It also contains prebiotics, probiotics, and zinc and enhances the growth of beneficial gut flora. The presence of such plethora of antibacterial properties in one product makes it a promising candidate not only in wound infections but also in systemic and particularly for gastrointestinal infections. Agar diffusion assay, being used for evaluating antibacterial activity of honey, is not the most appropriate and sensitive assay as it only detects non-peroxide activity when present at a higher level. Therefore, there is a need to develop more sensitive techniques that may be capable of detecting and evaluating different important components in honey as well as their synergistic interaction. Keeping in view the current guidelines for treatment of diarrhea, honey is considered one of the potential candidates for treatment of diarrhea because it contains a natural combination of probiotics, prebiotics, and zinc. Therefore, it would be worthwhile if such a combination is tested in RCTs for treatment of diarrhea.

The Antibacterial Activity of Australian Leptospermum Honey Correlates with Methylglyoxal Levels.

Most commercially available therapeutic honey is derived from flowering Leptospermum scoparium (manuka) plants from New Zealand. Australia has more than 80 Leptospermum species, and limited research to date has found at least some produce honey with high non-peroxide antibacterial activity (NPA) similar to New Zealand manuka, suggesting Australia may have a ready supply of medical-grade honey. The activity of manuka honey is largely due to the presence of methylglyoxal (MGO), which is produced non-enzymatically from dihydroxyacetone (DHA) present in manuka nectar. The aims of the current study were to chemically quantify the compounds contributing to antibacterial activity in a collection of Australian Leptospermum honeys, to assess the relationship between MGO and NPA in these samples, and to determine whether NPA changes during honey storage. Eighty different Leptospermum honey samples were analysed, and therapeutically useful NPA was seen in samples derived from species including L. liversidgei and L. polygalifolium. Exceptionally high levels of up to 1100 mg/kg MGO were present in L. polygalifolium honey samples sourced from the Northern Rivers region in NSW and Byfield, QLD, with considerable diversity among samples. There was a strong positive relationship between NPA and MGO concentration, and DHA was present in all of the active honey samples, indicating a potential for ongoing conversion to MGO. NPA was stable, with most samples showing little change following seven years of storage in the dark at 4°C. This study demonstrates the potential for Australian Leptospermum honey as a wound care product, and argues for an extension of this analysis to other Leptospermum species.

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.

Analytical method development using FTIR-ATR and FT-Raman spectroscopy to assay fructose, sucrose, glucose and dihydroxyacetone, in Leptospermum scoparium nectar

The carbohydrate dihydroxyacteone (DHA) occurs in significant levels in Leptospermum scoparium (mānuka) nectar and is the precursor of methylglyoxyl (MGO), the unique non-peroxide antibacterial activity (NPA) component in mānuka honey. The nectar of ten different cultivars of L. scoparium was assayed quantitatively for fructose, glucose, sucrose, and DHA with high pressure liquid chromatography (HPLC) for comparison with FT-Raman and FTIR-ATR spectroscopic methods. FT-Raman spectroscopy and ATR-FTIR spectroscopy, alongside chemometric methods using principal component analysis (PCA) and partial least squares (PLS) prediction were shown to be useful techniques to quantify and compare the nectar composition in a range of cultivars of L. scoparium.

Biomedical Activity and Related Volatile Compounds of Thai Honeys from 3 Different Honeybee Species.

This study investigated the effect of 3 factors (floral source, honeybee species, and postcollection processing) that influence the antibacterial activity, free radical reduction, and other biochemical compositions of different honey types typical of Thailand. Honey samples from 3 honeybee species (Apis mellifera, Apis cerana, and Apis dorsata) were obtained from 9 floral sources (longan, wild flower, lychee, coffee, sunflower, sesame, bitter bush, para-rubber, and manuka as a control) in different regions of Thailand. These samples were evaluated for both their total and nonperoxide antibacterial activity against 10 human pathogens by agar incorporation technique. Honey samples were further analyzed to evaluate the capacity for free radical-scavenging activity, total phenolic content, and the total flavonoid contents by the 2,2-diphenyl-1-picrylhydrazyl assay, Folin-Ciocalteu method, and aluminum chloride colorimetric assay, respectively. Furthermore, the volatile organic compounds (VOCs) of Thai honey samples were investigated by headspace solid-phase microextraction and gas chromatography-mass spectrometry analysis. Findings of this study suggest a strong correlation between floral origin and honeybee species on one hand, and differences in %Brix, total acidity, protein content, antimicrobial activities, free radical reduction, phenolic, and flavonoid contents on the other hand. Moreover, VOCs of wild and coffee honey types were remarkably different, depending on the floral source. Both honeys contained characteristics of VOCs, some of which are involved in antibacterial and antioxidant activities. Different origins and processing (floral source, honeybee species, and postcollection processing) of Thai honeys result in different antibacterial activities, physico-chemical properties, and aroma. Based on these findings, consumers of honey could select the type of honey based on their needs and preferred aroma.

Evaluation of the antibacterial activity of selected Pakistani honeys against multi-drug resistant Salmonella typhi.

BackgroundThe development of resistance to conventional anti-typhoid drugs and the recent emergence of fluoroquinolone resistance have made it very difficult and expensive to treat typhoid fever. As the therapeutic strategies become even more limited, it is imperative to investigate non-conventional modalities. In this context, honey is a potential candidate for combating antimicrobial resistance because it contains a broad repertoire of antibacterial compounds which act synergistically at multiple sites, thus making it less likely that the bacteria will become resistant. The in vitro antibacterial activity of 100 unifloral honey samples against a blood culture isolate of multi-drug resistant (MDR) Salmonella typhi were investigated.MethodsAll honey samples were evaluated for both total (acidity, osmolarity, hydrogen peroxide and non-peroxide activity) and plant derived non-peroxide antibacterial activity by agar well diffusion assay at 50% and 25% dilution in sterile distilled water and 25% in catalase solution. Manuka (Unique Manuka Factor-21) honey was used for comparison. The phenol equivalence of each honey sample from 2% to 7% (w/v) phenol was obtained from regression analysis. The antibacterial potential of each honey sample was expressed as its equivalent phenol concentration. The honey samples which showed antibacterial activity equivalent to or greater than manuka honey were considered therapeutically active honeys.ResultsNineteen honey samples (19%) displayed higher hydrogen peroxide related antibacterial activity (16–20% phenol), which is more than that of manuka honey (21-UMF). A total of 30% of the honey samples demonstrated antibacterial activity between 11 and 15% phenol similar to that of manuka honey while 51% of the honey samples did not exhibit any zone of inhibition against MDR-S. typhi at 50% (w/v) dilution. None of the indigenous honey samples displayed non-peroxide antibacterial activity. Only manuka honey showed non-peroxide antibacterial activity at 25% dilution (w/v) in catalase solution.ConclusionsThe honey samples which displayed antibacterial activity equal to or greater than manuka honey may be useful in the clinical conditions where higher hydrogen peroxide related antibacterial activity is required. Manuka honey, which is known to possess non-peroxide antibacterial activity, warrants further evaluation in a suitable typhoid animal model.

Plausible authentication of manuka honey and related products by measuring leptosperin with methyl syringate.

Manuka honey, obtained from Leptospermum scoparium flowers in New Zealand, has strong antibacterial properties. In this study, plausible authentication of the manuka honey was inspected by measuring leptosperin, methyl syringate 4-O-β-D-gentiobiose, along with methyl syringate. Despite a gradual decrease in methyl syringate content over 30 days at 50 °C, even at moderate 37 °C, leptosperin remained stable. A considerable correlation between nonperoxide antibacterial activity and leptosperin content was observed in 20 certified manuka honey samples. Leptosperin and methyl syringate in manuka honey and related products were analyzed using HPLC connected with mass spectrometry. One noncertified brand displayed significant variations in the leptosperin and methyl syringate contents between two samples obtained from different regions. Therefore, certification is clearly required to protect consumers from disguised and/or low-quality honey. Because leptosperin is stable during storage and specific to manuka honey, its measurement may be applicable for manuka honey authentication.

Evaluation of the effects of manuka honey on salivary levels of mutans streptococci in children: a pilot study.

There has been much debate in the past about whether honey is harmful to the teeth, mostly as part of the debate about raw sugar versus refined sugar and the results have been equivocal. However, what has not been taken into account is that honey varies markedly in the potency of its antibacterial activity. Manuka (Leptospermum scoparium) honey from New Zealand has been found to have substantial levels of non-peroxide antibacterial activity associated with an unidentified phytochemical component, denoted as Unique Manuka Factor (UMF). Considering the potential antimicrobial effects of manuka honey, the present study attempted to investigate effects of twice daily use of manuka honey with UMF 19.5 on salivary levels of Mutans streptococci in children. The investigation was a stratified comparison of two parallel groups of children who either used manuka honey with regular tooth brushing regimen or continued only with regular tooth brushing regimen twice daily under professional supervision for a 21-day period. A total of three salivary samples were taken from each individual at baseline, day 10, and day 21; colony counts of Streptococcus mutans (S. mutans) were determined. All data was subjected to paired T-test and Wilcoxon's signed ranks sum for intra- and intergroup comparisons respectively. Children using manuka honey showed statistically significant reductions in salivary S. mutans after 10 and 21 days. Manuka honey with UMF 19.5 may be considered as an effective adjunctive oral hygiene measure for reducing colony counts in children.

Analysis of the flavonoid component of bioactive New Zealand mānuka (Leptospermum scoparium) honey and the isolation, characterisation and synthesis of an unusual pyrrole

The flavonoid components of New Zealand mānuka (Leptospermum scoparium) honey have been quantified in a series of 31 honeys of varying non-peroxide antibacterial activity to clarify discrepancies between previous studies reported in the literature. Total flavonoid content was 1.16mg/100g honey. The principal flavonoids present were pinobanksin, pinocembrin, luteolin and chrysin and together these represented 61% of the total flavonoid content. 1, 2-formyl-5-(2-methoxyphenyl)-pyrrole, which was weakly correlated with the non-peroxide antibacterial activity, was isolated from the flavonoid fraction and separately synthesised. 1 did not display inhibitory activity against Staphylococcus aureus in vitro and thus the origin of the correlation, which is still unknown, is not a direct contribution.

A convenient new analysis of dihydroxyacetone and methylglyoxal applied to Australian Leptospermum honeys

New Zealand mAnuka (Leptospermum scoparium) honey is known to exhibit non-peroxide antibacterial activity caused by the active ingredient methylglyoxal which arises by chemical conversion of dihydroxyacetone during honey maturation. This study determines whether methylglyoxal and dihydroxyacetone are present in Australian Leptospermumhoneys. This research developed a rapid and sensitive high-performance liquid chromatographic method for the concurrent analysis of methylglyoxal and dihydroxyacetone in honeys. Both compounds were quantified as their O-(2, 3, 4, 5, 6-pentafluorobenzyl) hydroxylamine. HCl derivatives on single run reversed phase high-performance liquid chromatography with diode array detection. Four species of monofloral Leptospermum honeys sourced from Northern Rivers Region, New South Wales, Australia contained methylglyoxal and dihydroxyacetone. The highest methylglyoxal concentrations were found in Leptospermum polygalifolium honeys. Key words: High-performance liquid chromatography, dihydroxyacetone; methylglyoxal,Leptospermum, honey, mAnuka.

The effect of gamma-irradiation on the antibacterial activity of honey.

There is increasing usage of honey as a dressing on infected wounds, burns and ulcers, but there is some concern that there may be a risk of wound botulism from the clostridial spores sometimes found in honey. It is well-established that the antibacterial activity is heat-labile so would be destroyed if honey were sterilized by autoclaving, but the effect of gamma-irradiation on the antibacterial activity of honey is not known. Therefore an investigation was carried out to assess the effect on the antibacterial activity of honey when the honey was subjected to a commercial sterilization procedure using gamma-irradiation (25 kGy). Two honeys with antibacterial activity due to enzymically-generated hydrogen peroxide and three manuka honeys with non-peroxide antibacterial activity were investigated. The honeys were tested against Staphylococcus aureus in an agar well diffusion assay. There was no significant change found in either type of antibacterial activity resulting from this form of sterilization of honey, even when the radiation was doubled (to 50 kGy). Testing of honey seeded with spores of Clostridium perfringens and C. tetani (10000 and 1000 spores g-1 of honey, respectively) showed that 25 kGy of gamma-irradiation was sufficient to achieve sterility.

The Sensitivity of Dermatophytes to the Antimicrobial Activity of Manuka Honey and Other Honey

Honey has been reported to have antifungal activity and so was tested against clinical isolates of the common dermatophyte species which cause tineas in man. A honey with an average level of hydrogen peroxide, and a manuka (Leptospermum scoparium J. R. and G. Forst, family Myrtaceae) honey with an average level of non-peroxide antibacterial activity were used. An agar well diffusion assay was used, the contents of the wells being replaced with freshly prepared honey solutions at 24-h intervals over the 3–4 days of incubation. The lowest concentrations (% v/v, in steps of 5%) of manuka honey with catalase added to remove hydrogen peroxide, and of the other honey (without catalase) showed that inhibition of growth around the wells were, respectively, Epidermophyton floccosum 25%, 10%; Microsporum canis 25%, 15%; Microsporum gypseum 55%, 20%; Trichophyton mentagrophytes var. interdigitale 45%, 15%; Trichophyton mentagrophytes var. mentagrophytes 25%, 15%; Trichophyton rubrum 20%, 5% and Trichophyton tonsurans 25%, 20%. No inhibitory activity was detected with the other honey at 50% (v/v) with catalase added. The results of this investigation show that the common dermatophytes are sensitive to the antimicrobial activity of honey, indicating that clinical evaluation of honey in the treatment of tineas is warranted. This would determine whether the hydrogen peroxide or the non-peroxide antifungal agent diffuses better into the skin.

The controlled in vitro susceptibility of gastrointestinal pathogens to the antibacterial effect of manuka honey

The susceptibility of common gastrointestinal bacteria against manuka honey with median level non-peroxide antibacterial activity (equivalent to that of 16.5% phenol) was investigated by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using a standardized manuka honey with the broth microdilution method. The measured sensitivity of bacteria showed that manuka honey is significantly more effective than artificial honey (a mixture of sugars as in honey), indicating that osmolarity is not the only factor that is responsible for the antibacterial activity of the honey. Most tested gastrointestinal pathogens have MIC and MBC values in the range of 5-10% of honey, other than Enterobacter spp. which was in the range of 10-17%. The difference in efficacy between the honey with and without hydrogen peroxide removed was also studied, and it was found that both hydrogen peroxide and the non-peroxide components contribute to the bacteriostatic and bactericidal activity of the honey. It was also found that treatment against multi-antibiotic resistant microorganisms such as Salmonella typhimurium DT104 and ESBL-producing organisms with manuka honey may be promising.

Determination of royal jelly acids in honey

In the present work we report on the compounds characteristic of larval food (royal jelly, RJ) of the honeybee ( Apis mellifera L.) that were identified in 34 different samples of genuine honey and in 3 sugar-adulterated “herbal honeys” by using solid-phase extraction and gas chromatography–mass spectrometry (SPE/GC–MS). The unique feature of RJ is a set of C 8, C 10 and C 12 hydroxy fatty acids. In all, ten acids characteristic of this bee product were identified in different combinations in the analysed honey samples, namely: 7- and 8-hydroxyoctanoic, 3-hydroxydecanoic, 9-hydroxydecanoic, 9-hydroxy-2-decenoic, 10-hydroxydecanoic, 10-hydroxy-2-decenoic (10-HDA), 3,10-dihydroxydecanoic, 2-octene-1,8-dioic and 2-decene-1,10-dioic acids. The higher relative abundance of these compounds was determined in genuine honeydew and heather honeys, and in “herbal honeys” (23.8–40.8, 18.2–48.5, and 27.0–48.4 μg/g, respectively). Since RJ is known to have strong antibiotic efficacy, our results suggest that a part of the non-peroxide antibacterial activity of honey might be of bee origin.

Isolation by HPLC and characterisation of the bioactive fraction of New Zealand manuka ( Leptospermum scoparium) honey

Using HPLC a fraction of New Zealand manuka honey has been isolated, which gives rise to the non-peroxide antibacterial activity. This fraction proved to be methylglyoxal, a highly reactive precursor in the formation of advanced glycation endproducts (AGEs). Methylglyoxal concentrations in 49 manuka and 34 non-manuka honey samples were determined using a direct detection method and compared with values obtained using standard o-phenylenediamine derivatisation. Concentrations obtained using both the methods were similar and varied from 38 to 828 mg/kg.

Growth inhibition of foodborne pathogens and food spoilage organisms by select raw honeys

Twenty-seven honey samples from different floral sources and geographical locations were evaluated for their ability to inhibit the growth of seven food spoilage organisms ( Alcaligenes faecalis, Aspergillus niger, Bacillus stearothermophilus, Geotrichum candidum, Lactobacillus acidophilus, Penicillium expansum, Pseudomonas fluorescens) and five foodborne pathogens ( Bacillus cereus, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica Ser. Typhimurium, and Staphylococcus aureus) using an overlay inhibition assay. They were also tested for specific activity against S. aureus 9144 and B. stearothermophilus using the equivalent percent phenol test—a well diffusion assay corresponding to a dilute phenol standard curve. Honey inhibited bacterial growth due to high sugar concentration (reduced water activity), hydrogen peroxide generation, and proteinaceous compounds present in the honey. Some antibacterial activity was due to other unidentified components. The ability of honey to inhibit the growth of microorganisms varies widely, and could not be attributed to a specific floral source or demographic region produced in this study. Antibacterially active samples in this study included Montana buckwheat, tarweed, manuka, melaleuca, and saw palmetto. Furthermore, the bacteria were not uniformly affected by honey. Varying sensitivities to the antimicrobial properties were observed with four strains of S. aureus thus emphasizing the variability in the antibacterial effect of honey samples. Mold growth was not inhibited by any of the honeys tested. B. stearothermophilus, a heat-resistant spoilage bacteria, was shown to be highly sensitive to honey in both the overlay and well diffusion assays; other sensitive bacteria included A. faecalis and L. acidophilus. Non-peroxide antibacterial activity was observed in both assays; the highest instance was observed in the specific activity assay against B. stearothermophilus. Further research could indicate whether honey has potential as a preservative in minimally processed foods.