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Medical problems have been arising in the management of infectious diseases due to the widespread development of highly resistant microorganisms. This microbial resistance is attributed to the vast use of antibiotics.
The emergence of drug resistance prompted the interest of the researchers in the use of readily available alternative medicinal therapy to control the infection in order to prevent the over use of antibiotics.
Recently, there are reviews and researches conducted on the use of honey. Its use in promoting wound healing has long been known since ancient times. In fact, it is mentioned in one of the verses of Holy Qur'an attributing therapeutic values to it.
Nowadays, there are several investigations conducted regarding its antibacterial activity. However, at present, there has been no local study yet in evaluating if there is an antibacterial effect of the locally available organic floral honey in Toril, Davao City on the common pathogens in acute and chronic bacterial sinusitis. An in-vitro investigation of the potency of honey is thought to be a necessary step before its application in the treatment sinonasal infections.
Review of Related Literature: "What is already known about the topic?"
Honey is prepared by bees from plant nectars, from plant secretions and from excretions of plant sucking insects ("honeydew"). The Food Standards Code defines it as "the nectar and saccharine exudations of plants gathered, modified and stored by the honey bee". The British pharmacopeia (1993) defines purified honey as being "obtained by purification of the honey from the comb of the bee, Apis mellifera L, and other species of Apis (Office of Complementary Medicine, 1998).
Honey is a rich source of carbohydrates mainly fructose and glucose. The chemical composition of honey varies depending on the plant source, season and production methods. Therefore the color, concentration and compounds vary depending on the floral sources. Other compounds which can be found in honey include proteins and acids such as gluconic acid (C6H11O7, also known as 2,3,4,5,6-pentahydroxyhexanoic Acid), Minerals and Anti-Oxidants such as hydrogen peroxide (H202) and Vitamins (B6 and B12) (BD Yates et al, 1996).
Honey has a low pH and a low moisture content, which is usually on average about 17 percent. The gluconic acid in honey is produced when bees secrete glucose oxidase, while processing the nectar, this give honey a low pH. There are many varieties of honey from around the world which come in three main types which are liquid, whipped and comb honey (Office of Complementary Medicine, 1998).
Honey has long been known for its medicinal properties since ancient times (Dustmann, 1979). Its healing purposes are mentioned in the Bible, the Koran, and the Torah. Its medicinal application was known but nothing much on its antibacterial activity.
The antibacterial action of honey was recognized for the first time in 1892 by Van Ketel. It has been assumed that this is due entirely to the osmotic effect of its high sugar content (Molan, 2001). Recently, its antibacterial properties have been extensively explored. The mechanism of antibacterial activity of honey is attributed to its following natural properties: low pH, hydrogen peroxide, and low water activity. A low pH is inhibitory to most bacteria since mostly they live in environments around pH 7. In effect it causes the protein to denature. Hydrogen peroxide is an oxidizing agent which has the ability to damage cells and allows the formation of other free radicals. Although the amount of H2O2 present in honey is very low it is still effective as an antimicrobial agent. Its amount varies depending on the type of honey. The low water activity of the honey inhibits bacteria through the effect of osmosis which causes plasmolysis of the cell which eventually cause cell death (El-Sabbahy, 2009).
Other largely uncharacterized substances are also present in some honeys which are thought to have antibacterial effects. Some compounds that have been identified include syringic acid, methyl syringate, 3,4,5-trimethoxybenzoic acid, 2-hydroxy-3-phenylpropionic acid and the flavonoid pinocembrin(honey scientific report). Manuka honey from New Zealand has been found to have substantial levels of non-peroxide antibacterial activity and unidentified phytochemical component (Peter Charles Molan-Honey as a topical agent for ). It contains acidic and phenolic substances, the most dominant being 2-hydroxy-3-phenylpropionic acid as demonstrated in gas chromatography by Tan et al (1989). Australian honeys were identified also of having volatile substances which contribute to honey's antibacterial activity (Molan, 2001).
Recently there are researches that have shown that some honeys have an immunologic activity. Honey stimulates the proliferation of peripheral blood B-lymphocytes and T-lymphocytes in cell culture (Abuharfeil et. al., 1999). It also stimulates monocytes in cell culture to release cytokines, tumor necrosis factor-alpha, interleukin-1 and IL-6 (Tonks et. al., 2001).
A study conducted by Sam El-Sabbahy (2009) on the antibacterial effects of different honeys on different bacterial species showed that all honeys exhibited antibacterial activity which increased in increasing concentrations. Honeys used were Manuka honey, forest honey, organic floral honey, Royale honey, and Turkish clover honey. It showed also that different honeys act differently on the same microorganism because the composition of each honey is different. Furthermore, it revealed that the same honey acts differently on different microorganisms.
Robson et al (2007) concluded in their study that the use of honey in wound management following ENT surgery is effective. It enhances rapid healing of the wound.
Another study by Talal et al (2008) found that honey is more effective in killing both planktonic and biofilm-grown forms of bacteria compared with the rate of bactericide by antibiotics commonly used against bacteria.
Research Question: "What is not yet known about the topic?"
The use of honey in accelerating wound healing and its antibacterial effect have been proven and investigated thoroughly. However, little is known as to its antimicrobial effect in cases of head and neck infectious diseases particularly in sinonasal area.
The study aims to determine the in-vitro antibacterial effect of two available organic floral honey in Toril, Davao City on the common pathogenic bacteria in acute and chronic sinusitis.
1. Does the locally available organic floral honey possess an antibacterial effect o the common pathogenic bacteria in acute and chronic sinusitis?
2. Which among the two types of organic floral honey has a better antibacterial property against the common pathogens in acute and chronic sinusitis.
3. What level of dilution of honey does it still possess its antibacterial potency to the common pathogens in acute and chronic sinusitis?
4. What common pathogenic bacteria in acute and chronic sinusitis are susceptible to honey?
Significance of the Study: "What will the healthcare be if the answers to the research question will be known?"
Because of the emergence of antibiotic- resistance bacteria the interest in research is now geared towards the use of other treatment options such as alternative medicinal therapies that are effective, readily available and affordable. This study will investigate and explore the in-vitro antibacterial effect of locally produced organic floral honey on the common bacterial pathogens in acute and chronic sinusitis.
Objectives: "What will the study do?"
To determine if the locally available honey in Toril, Davao City has an antibacterial effect on the common bacterial pathogens in acute and chronic sinusitis.
1. To determine whether the available two different types of organic floral honey has an antibacterial property.
2. To compare which of the two different organic floral honey has a better antibacterial activity.
3. To determine up to what level of dilution of honey will it still have an antibacterial effect
4. To determine what common pathogens in acute and chronic sinusitis are susceptible to honey.
Study Design: Experimental Study
Setting: Southern Philippines Medical Center
Two different samples of honey (Type A and B) of organic type coming from different floral sources will be used in the study. These will be obtained from a beekeeper in Toril, Davao City.
There are five different species of bacteria which will be used in the study. These are the common bacterial pathogens causing acute and chronic rhinosinusitis. The pure cultures of these bacteria will be obtained from the laboratory of Southern Philippines Medical Center. The five bacterial species are: Strep. Pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staph. aureus, and Bacteroides spp.
The following procedures are adapted from the previous study by EL Sabbahy (2009).
Sources of Isolates
The cultures of the five different species will be used to separately inoculate the five different nutrient agar plates using an inoculum loop to streak the bacteria onto each plate
Culture Incubation and Storage Condition
The five different bacterial species which will be used in the study will be incubated at room temperatures for up to 48 hrs. The bacterial cultures will then be stored at 4ËšC.
Detection of Antibacterial Activity
Five universal bottles containing 9 ml each of nutrient broth will be inoculated separately with the bacterial species using a loop. The nutrient broth solutions will then be incubated at 37 ËšC for up to 48 hours.
There will be five different concentrations which will be prepared from each of the two types of organic floral honey. The honey sample will be diluted with sterile water to 0% (negative control), 25%, 50%, 75%, and 100%.
10 ml from each dilution from the two types of organic floral honey will be poured into five separate flasks and will be covered with foil. These were stored at room temperature out of direct sunlight.
Disc diffusion assay
Five sets of five Nutrient Agar plate will be set (will be done in triplicates), each agar plate in every set will be incubated separately with the bacterial species, by pipetting 100 uml of each bacteria directly onto the agar surface o each plate of every set. Using the spread plate technique, the bacteria samples will then be spread across the surface using a glass spreader.
The plates will be left to dry for 15 minutes, while sterile absorbent discs will be placed into each honey flask. The absorbent discs will be left in honey for 10 minutes to absorb the honey. An absorbent disc from each honey will be placed on every agar plate in each set.
The plates inoculated by the five bacterial species will then be incubated at 37ËšC for up to 48 hours.
Each experiment will be done in triplicate to have an accurate result.
Detection of antibacterial activity and minimum inhibitory concentration
After 48 hours of incubation, the bacterial growth inhibition will be determined by the visual confirmation of a zone of inhibition, which is a clear area surrounding the absorbent disc. There is positive inhibition (+) if there will be a visible clear ring around the absorbent disc, and there is negative inhibition (-) if there will be no clear ring. The amount of inhibition will be recorded by measuring the diameter of the zone of inhibition, in millimeters (mm), this will be measured using a ruler. The measurement will include the diameter of the absorbent disc.
There will be three statistical methods that will be used in the analysis of the results. These include mean (x), Standard Deviation (SD) and the Percentage of Coefficient of Variation (%CV).
The mean will be used to determine the average inhibition of each honey at each concentration of inhibition. The Percentage Coefficient of Variation will be used to determine the amount of variation of each result.
To determine the amount of antibacterial activity for each honey and to generate a more precise Minimum Inhibitory Concentration measurement dose response curve will be generated by plotting the zone of inhibition- diameter squared (mm square) against the level of concentration. This will be carried out on each type of honey which will be used I this investigation for each of the species of bacteria used.
Data Analysis Plan
The dummy tables below will be used as a guide for data analysis.
Table 1.Bacterial Inhibition at 100% concentration of honey
(+) positive inhibition (-) No visible inhibition
Table 2. Disc Diffusion Assay for the minimum Inhibitory Concentration on each bacterial pathogen. The Average (x). Standard Deviation (SD) and Percentage Coefficient of Variation (%CV).