Effect of United States buckwheat honey on antibiotic-resistant hospital acquired pathogens

Introduction Due to an upsurge in antibiotic-resistant infections and lack of therapeutic options, new approaches are needed for treatment. Honey may be one such potential therapeutic option. We investigated the susceptibility of hospital acquired pathogens to four honeys from Wisconsin, United States, and then determined if the antibacterial effect of each honey against these pathogens is primarily due to the high sugar content. Methods Thirteen pathogens including: four Clostridium difficile, two Methicillin-resistant Staphylococcus aureus, two Pseudomonas aeruginosa, one Methicillin-Susceptible Staphylococcus aureus, two Vancomycin-resistance Enterococcus, one Enterococcus faecalis and one Klebsiella pneumoniae were exposed to 1-50% (w/v) four Wisconsin honeys and Artificial honey to determine their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using the broth dilution method. Results Buckwheat honey predominantly exhibited a bactericidal mode of action against the tested pathogens, and this varied with each pathogen. C. difficile isolates were more sensitive to the Wisconsin buckwheat honey as compared to the other pathogens. Artificial honey at 50% (w/v) failed to kill any of the pathogens. The high sugar content of Wisconsin buckwheat honey is not the only factor responsible for its bactericidal activity. Conclusion Wisconsin buckwheat honey has the potential to be an important addition to therapeutic armamentarium against resistant pathogens and should be investigated further.


Introduction
Antibiotic-resistant pathogens (Methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Klebsiella pneumoniae, Clostridium difficile and Enterococcus faecalis) are major causes of severe infections in hospitalized patients leading to longer hospital stays and higher mortality rates worldwide [1,2]. In the United States, infections associated with antibiotic-resistant organisms occur in over 2 million people and at least 23,000 deaths are recorded annually [3]. According to the WHO report [2], people infected with MRSA are reported to be 64% more likely to die than those infected with antibiotic sensitive strains.
Treatment of antibiotic-resistant associated infection is challenging particularly in healthcare settings due to the increasing trend of antibiotic resistance, side effects of important antibiotics, limited antibiotic options [1,2] and reduction in new antibiotic discovery endeavors by pharmaceutical companies [2,4]. Alternative therapeutic interventions that are effective and without adverse effects are urgently needed. Honey is one such promising option.
Natural honey is obtained from nectar collected by honeybees. Its high sugar content coupled with low pH, bee-derived enzymes, beederived peptides and phytochemical compounds contribute to its antibacterial action [5][6][7][8][9][10][11]. Honey also has antioxidant, antiinflammatory and anti-hyaluronidase properties which vary depending on the nectar source [5,12,13]. The amount of natural phenol in honey plays a significant role in its inhibition activity [12,13]. Honeys with high concentration of phenol are more likely to possess high inhibitory efficacy than those with low or no phenol.
The use of honey as medicinal remedy was initiated many centuries ago, but recent publications have demonstrated the antibacterial efficacy of honey in in vitro and in vivo [14][15][16][17][18][19][20][21][22]. The antibacterial mechanism of honey has gradually been unraveled [23]. Evidence from published studies show that honey disrupts cell walls in P.
aeruginosa [23] and interrupts cell division in MRSA [24]. Honey also stimulates inflammatory cytokines [25] and has been identified to be a strong scavenger of super peroxide anions and highly effective inhibitor of reactive oxygen species (ROS) stimulated from human polymorphonuclear neutrophils (PMNs) [5]. Both medical grade honey and raw honey have been shown to have broad spectrum antibacterial activity against a plethora of pathogens, including antibiotic-resistant organisms and their biofilms [19,26,27]. Furthermore, honey has been shown to heal recalcitrant wounds [28][29][30][31]. Effective application of honey promotes wound healing, prevents cross-infection, and repairs tissue [32]. Unlike some antimicrobial agents (such as fluoroquinolones and clindamycin), honey has no record of adverse side effect on tissues [32] or gut [21].
Public interest in the therapeutic use of natural honey in recent times has greatly increased [31]. Licensed medical grade honeys are available in the medical field globally but the majority are derived from Leptospermum species found in Australia and New Zealand.
The antibacterial property of medical grade Manuka honey is highly recognized in the research field due to its high unique property, Unique Manuka factor (UMF). Though much work has been done on honey, little is known about the antibacterial efficacy of honey from the United States. In this pilot study, our goals were to compare the efficacies of American honeys and artificial honey on antibioticresistant pathogens and then determined if the antibacterial effect of each honey against these pathogensis primarily due to its high sugar content.

Types of honey
Four Wisconsin honeys (Buckwheat honey, Wild honey, Cranberry honey and Orange blossom honey) and an artificial honey (AH) were studied. Wisconsin honeys were locally produced in Wisconsin, USA, and purchased from a local grocery supermarket. AH (sugar solution) 100 g was comprised of 7.5 g D-(+)-maltose monohydrate, 40.5 g D-(-)-fructose, 1.5 g sucrose, and 33.5 g D-(+)-glucose (Sigma-ALDRICH Co., Missouri, USA) dissolved in 17 ml sterile osmotic reverse water [33]. All honeys were stored at 4°C in a dark environment until use. Reference stains were also included to monitor consistency. Wells with the lowest honey concentrations which prevented growth/turbidity under a magnifying mirror were considered as MIC.
For quality assurance purposes, each experiment was run in triplicate at different occasions. The method was validated by using standard antibiotics against the reference strains and the results compared to that in CLSI literature.

Minimum bactericidal concentration determination
Minimum bactericidal concentration (MBC) was determined by plating 10 µl of content from all the MIC wells without visible growth/turbidity onto antibiotic-free or honey-free BA plates in duplicate and incubating aerobically and anaerobically as required.
Positive and negative control wells were included. The lowest honey concentration that killed the organism was considered the MBC.
MBCs were determined on three separate occasions.

Statistical analysis
Data was entered into Microsoft Excel® 2010 and analyzed. The

Results
The selected pathogens for the study are listed in Table 1. The MICs and MBCs of the selected pathogens are shown on (Figure 1).
Generally, WBH exhibited bactericidal mode of action against all the tested organisms with MBC/MIC ≤ 4. WBH exhibited a broad spectrum antimicrobial property but Gram-positive organisms were slightly more susceptible than Gram-negative organisms (Figure 1).
There was a dose response relationship between the tested honey and the pathogens. The MICs of the natural honey was very low as compared to that of the AH. For the aerobic organisms, AH inhibited growth at 50% (w/v) whiles that for anaerobic organisms was 40% (w/v). These high concentrations failed to kill any of the organisms.
However, WBH demonstrated bactericidal activity with MIC range between 6.25-19% (w/v) and MBC range between (6.25-50% (w/v) ( Figure 1). The effect of WBH on VRE 002 and VRE 51559 were high with MBCs of 50 and >50 (% w/v) respectively. There was a significant difference in susceptibility between the WBH and AH for all the tested isolates (p<0.05). The cranberry honey, wild honey and orange blossom honey did not exhibit bactericidal activity at 50% (w/v).

Discussion
The rise in multidrug and extreme antibiotic-resistant pathogens in the healthcare settings is so alarming that it has become necessary to find alternative and effective natural therapeutic agents. In this study, we tested the potency of four local honeys against standardized pathogens (MRSA, P. aeruginosa, MSSA, VRE, E. faecalis, K. pneumoniae and C. difficile). Honeys that did not exhibit bactericidal activity at 50% (w/v) during the study were excluded.
Our study demonstrated that MICs of WBH for the four C. difficile isolates were the same (6.25 % w/v) with MBC ranging between 6.25-12.5% (w/v). Despite the different nectar sources of the honeys, these results were congruent with previous work by Hammond and Donkor [17] who demonstrated that Woundcare  [15,16,19,22,33,37,39].
To investigate the role of high sugar content of selected honeys on antibacterial activity, we exposed test pathogens to various concentrations of AH (10-50% w/v) to mimic the main sugar composition in natural honeys. Our findings indicated that a high AH concentration (50% w/v) failed to completely prevent any of the pathogens from growth, whereas WBH exhibited bactericidal activity at very low honey concentrations (6.25% w/v). A similar conclusion was reported by [16]. Likewise at 50% (w/v), cranberry honey, wild honey and orange blossom honey supported the growth of all the studied pathogens. This further confirms that bactericidal activity of honey is not solely due to the presence of high sugar content and that varying potent antibacterial compounds in honey may work synergistically to extensively disrupt cells and lysis of pathogens as reported by Henriques et al. [23]. This study suggests that WBH may have a complex composition that could effectively resist multiple antibiotic-resistant pathogens to thrive.
WBH remarkably displayed broad spectrum antimicrobial activity in this study. Other published research have demonstrated that phenolic compound in buckwheat honey is high and this influences its antibacterial property [5,12,13]. Based on this, we can deduce that the phenol content in Wisconsin honey may be high, and hence could be a major factor contributing to its antibacterial activity against these important hospital acquired pathogens. The potent bactericidal effect of WBH suggests that more honeys need to be tested to supplement existing medical grade honeys and standard antibiotics to effectively control the increasing antibiotic-resistant pathogens during therapy. If processed, WBH could be employed therapeutically. Furthermore, buckwheat honey is common in the Page number not for citation purposes 5 United States and may be cost effective than imported medical grade Manuka honey but further study is required.
Our findings suggest that it is important to carry out further investigations to determine the rate and concentrations at which WBH inhibits pathogens. Investigating the effect of WBH on C.
difficile spores could be advantageous since C. difficile infections mode of transmission is also through spores which can survive for a long time in the contaminated environment. Since microbial biofilm delays wound healing, it is important to determine if WBH prevents or disrupts biofilm formed by pathogens in the study that could possibly cause wound infections. Furthermore, it will be essential to determine if the antibacterial efficacy of the selected buckwheat honey is representative of all buckwheat honeys in the whole state of Wisconsin or the United States.

Conclusion
We provide the first data on antibacterial efficacy of buckwheat honey from Wisconsin, USA against nosocomial or hospital acquired pathogens. Our data demonstrate that antibiotic and multiple drug resistant pathogen(s) were susceptible to WBH. We also deduced that the antibacterial effect of WBH on pathogens including C.
difficile was not mainly due to its high sugar content. Future work should include in vivo studies to examine efficacy and mechanism of action.  Wisconsin buckwheat honey has the potential to treat nosocomial associated infections.

Competing interests
The authors declare no competing interests.   Table 1: Antibiogram characteristics of pathogens