Antibacterial Activities of Selected Samples of Honey in South-Western Nigeria against Bacteria in Wound Infections

The global challenges resulting from the emergence of numerous resistant pathogenic bacteria have led to a search for alternative therapy ethnomedically. As a result, honey is increasingly valued for its antibacterial activity. In this study, the antibacterial activity of different honey samples was assessed in vitro using agar dilution at different concentration of 5%, 10%, 20%, 30%, 40%, 50%, 75% and 100% (v/v) dilution. The antibacterial activity was also determined by spectrophotometric assay at 620 nm. The result showed that KG honey sample inhibited the different bacterial isolates at a concentration of 5% and LB honey sample inhibited the isolates at 20% while other honey samples inhibited the bacterial isolates at concentrations ranging between 10% and 30% (v/v) dilution of each honey samples. Thus, the minimum inhibitory concentrations ranged between a concentration of 5% and 30% (v/v) dilution even though antibacterial activities concentration ranged from 5% 100% (v/v). Ps. aeruginosa ATCC 15442 was considered the most susceptible of all the test isolates. The increases in the absorbances showed that the bacterial populations in each of the honey samples were reduced as the exposure time increases. The study shows that honey being sold in Nigeria may be used as a therapeutic agent to treat infections as they may be an excellent alternative to curtail the further spreading of multi-drug-resistant microorganisms in Nigeria. Running Title: Antibacterial activities of honey against bacteria


INTRODUCTION
Honey produced by Apis mellifera is one of the oldest traditional medicines considered important in the treatment of several human ailments (Mondo et al., 2004). The ancient Greeks believed that consuming honey could result in living longer (Kwakman et al., 2002). While many pathogenic organisms have developed resistance to many antibiotics (Patton et al., 2006) and multidrug resistance is on the increase, efforts are being intensified to develop antimicrobial agents from natural sources for better therapeutic effects (Gills et al., 1992). Consequently, despite the fact that researchers have focused on the bactericidal potential of honey against many pathogenic aerobic and anaerobic, Gram-negative and Gram-positive bacteria (Haffejee et al., 1985;Hannan et al., 2004), Basson et al. (1994) and Lusby et al. (2005) indicated that natural unheated honey has some broad-spectrum antibacterial activity against pathogenic, oral and food spoilage bacteria.
Although the chemical component of honey is dependent on the type of honey, geographical location, season, flower source from which the final product is derived and production methods ( Nisbet et al., 2010), honey contains enzymatic and non-enzymatic antioxidants (Bogdnov, 1989), numerous types of sugar, acids, vitamins, protein and minerals (Al-Waili et al., 2004) as well as alkaloids, antaquinone, glycosides, cardiac glycosides, flavonoids and reducing compounds (Chute et al., 2010). High sugar concentration, low pH, hydrogen peroxide generation, proteinaceous compounds or other unidentified components present in the honey may provide antimicrobial activity (Mundo et al., 2004 (Marvic, 2008;Adams et al., 2008).
Since honey has been used from ancient times as a method of accelerating wound healing (Van der berg et al., 2008), its possibility to heal wound has constantly been demonstrated (Molan, 2006;Simon et al., 2008). The antibacterial properties of honey increase the growth of new tissue to heal the wound (Lusby et al., 2002). When applied topically, it rapidly clears wound infection to facilitate healing of deep surgical wounds with infection (Ahmed et al., 2003). Its application can promote the healing of peptic ulcers and gastritis (Molan et al., 2001) and methicillin-resistant S. aureus infected wounds (Natarajan et al., 2003). In cognizance of its therapeutic potential, the antimicrobial activities of diluted honey from different countries have been reported (Voidarou et al., 2011;Moussa et al., 2012). In Nigeria, there is a need to validate the therapeutic potentials and the antibacterial activities of different honey samples commonly sold for consumption. The aim of this study, therefore, was to investigate the antibacterial activities of five different honey samples against bacteria in wound infections.

Honey Samples
Five different honey samples locally produced honey from apiary were bought from different pharmaceutical outlets in the Southwest part of Nigeria. The honey samples were stored at 23-25 o C in the dark. For antibacterial assays, each of the honey samples was used undiluted and at 5%, 10%, 20%, 30%, 40%, 50% and 75% v/v dilution by weighing a required amount of each of the honey and diluting to a final volume of 100 ml with sterile deionized distilled water (Cooper et al., 2002a). Incubation was carried out in the dark because both hydrogen peroxide and glucose oxidase are light sensitive (White et al., 1963). Colonies from fresh overnight growth cultures of the different bacterial strains were suspended in 3 -4 ml sterile nutrient broth and incubated for 2 -3 h at 37 o C. These isolates were diluted with distilled water and adjusted to 0.5 McFarland's standard yielding approximately 1.0 ×10 6 CFU/ml before being used in this study.

Antibacterial testing by agar dilution method
The antibacterial activity of the different honey samples was carried out by agar dilution and spectrophotmetric assays (Patton et al., 2006;Tan et al., 2009). For the agar dilution assay, double strength Mueller Hinton Agar (MHA) medium was prepared and incorporated with the different honey samples at concentrations (v/v Honey + MAH) of 5%, 10%, 20%, 30%, 40%, 50% and 75%. Since honey is a very viscous liquid, it was kept at 50 o C before mixing with the agar to achieve uniform homogenization. On mixing the honey with the Mueller Hinton agar maintained at 50 o C, the mixture was vigorously vortexed and dispensed into sterile petri dishes. After being allowed to solidify, each of the test organisms was streaked on the different concentrations of the different honey samples before being incubated at 37 o C for 24 h and observed for growth. The experiment was performed in duplicate to ensure reproducibility of the results. Three agar plates sere set up as controls. One agar plate was inoculated with all test strains to confirm their viability. Second control plate contained medium only and the third plate contained different honey samples to check for the sterility of the medium and the honey. The minimum inhibitory concentration (MIC) was recorded as the lowest concentration of honey at which no visible bacterial growths were observed.

Spectrophotometric testing
The sample was prepared by weighing 30%, mix well and poured into 10 test tubes, after which diluted 100 µl of the test organisms were introduced into the 10 test tubes. Spectrophotometric reading was taken at 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h at 625 nm. These are aimed at determining the level of turbidity after hours of inoculation.

RESULT
In this study, the different honey samples exhibited a varied degree of inhibitory effects against the different bacterial isolates. The minimum inhibitory concentrations of the honey samples varied from each other against the bacterial isolates. While KG honey sample inhibited the different bacterial isolates at a concentration of 5% and LB honey sample inhibited these isolates at 20%, other honey samples inhibited the bacterial isolates at concentrations ranging between 10% and 30% (v/v) dilution of each honey samples. While six of the isolates were inhibited by 20% dilution and four of the isolates were inhibited at 30% of the UB honey dilution and this antibacterial activity was a reverse in NF honey samples, eight of the isolates were inhibited by 10% and two isolates were inhibited by the 20% of the DB honey samples as shown in Table 1. No microbial growths were observed on the agar plates containing honey samples used as sterility test for each of the honey samples. While KG honey was the most effective honey inhibiting bacterial isolates at 5%, Ps. aeruginosa ATCC 15442 was considered the most susceptible of all the test isolates. From Figures 1-5, the spectrophotometric reading of the antibacterial activities of the different honey samples showed that there were increases in the reading of the absorbances at 625 nm over a period of 6 h. The increases in the absorbances showed that the bacterial populations in each of the honey samples were reduced to allow more penetration of light as the exposure time increases.

DISCUSSION
The control of infectious diseases due to antimicrobial resistance is a serious challenge to be addressed (WHO, 2010). In the recent time, the emergence of numerous resistant pathogenic bacterial strains to a wide range of formerly efficient antibiotics has constituted a serious threat to public health ( From this study, the absence of microbial growths on agar plates used as sterility control test may be due to the inhibitory effects of the chemical components of the different honey samples (Souza et al., 2007;Teixeira et al., 2008). If bacteria had been detected in the control samples, the presence of isolates may have indicated contamination from a secondary source. However, the antibacterial activities of the different honey samples showed that the minimum inhibitory concentrations ranged between a concentration of 5% and 30% (v/v) dilution even though antibacterial activities concentration ranged from 5% -100% (v/v). While Nzeako and Hamdi (2000) indicated that honey had no activity at 40% (v/v) dilution, Chanchao et al. (2006) indicated that honey provides the same inhibition ability at 25% and 50% (v/v) dilution but not at a dilution of 75% (v/v). Lusby et al., (2005) reported that honey other than commercially available antibacterial honeys like manuka (Leptopsermum scoparium) honey can have equivalent antibacterial activity on certain clinical isolates. Although the antibacterial activity of different types of honey can vary by a factor up to 100 (Willix et al., 1992), the great variety of antibacterial activity of honey may be attributed to differences in the botanical and geographical origin, and consequently in the chemical composition (Akkol et al., 2007).
In previous studies, the antimicrobial activities of honey have been reported. Zumla and Lulat (1989) reported that honey is a very good inhibitor to E. coli, Salmonella and Shigella. French et al. (2005) reported the effectiveness of manuka honey against pathogenic bacteria such as S. aureus and Helicobacter pylori. Wilkinson (2005)  aeruginosa. These reports are in agreement with the results of the present study. The antibacterial effect of the different honey that was used in this study showed a remarkable effect through the rate at which they inhibited the test bacterial isolates used. The bacterial growths were diversely inhibited at concentrations of 5%, 10%, 20%, 30%, 40% and 50% (v/v) dilution of the honey samples. Although the mechanism of antibacterial action of honey is most likely a combination of a number of different factors, the antibacterial activity in most honey samples is due to the enzymatic production of hydrogen peroxide in addition to unidentified phytochemical components (Cooper et al., 2002b). While its dilution enhances hydrogen peroxide mediated antibacterial activity (Melissa et al., 2004), honey has antioxidant properties and increases cytokine release which may explain its antimicrobial effects (Gheldof et al., 2002;Schramm et al., 2003;Tonks et al., 2003). Since hydrogen peroxide is a major contributor to the antimicrobial activity of honey, the different concentrations of this compound in the different honeys could have resulted in their varying antimicrobial effect recorded in this study.
In conclusion, this study confirms that honey possesses antimicrobial activity and holds great promise as antimicrobial agent against pathogenic microorganisms. It may be used as a therapeutic agent and an effective herbal medicine to treat infections as they may be an excellent alternative to curtail the further spreading of multi-drug-resistant microorganisms in Nigeria.