Key findings:

Key findings from this study on Nigerian honey samples include: both honey samples from Imo State and Enugu State exhibited good physicochemical properties and high antibacterial activity against Staphylococcus aureus and Escherichia coli isolated from burn injuries, with honey from Enugu State showing particularly strong antibacterial effects at various concentrations.

What is known and what is new?

The known aspect in this abstract is the antimicrobial properties of honey and its potential use in wound management. The new contribution is the investigation of honey samples from specific locations in Nigeria, revealing their high antibacterial activity against Staphylococcus aureus and Escherichia coli, and highlighting the potential for honey to be used as a natural therapeutic agent in treating burn wound infections.

What is the implication, and what should change now?

The implication of this study is the potential use of honey as an effective antimicrobial agent in treating burn wound infections caused by Staphylococcus aureus and Escherichia coli. Changes needed include further research to standardize honey-based treatments, explore optimal application methods, and promote the use of honey as a safe and natural alternative to conventional antibiotics in wound management.

Antibiotics have very important roles in reducing the global burden of diseases. The efficacy of antimicrobials is reduced as resistant bacteria arise and proliferate. Antibiotics resistance in bacteria is a one of major Public Health challenges, and the world is now facing a strong and growing danger from microorganisms resistant to virtually all available antibiotics, even the most powerful last-resort treatments [1]. As a result, new antibacterial approaches are greatly needed, encouraging a re-evaluation of traditional remedies including plants and plant-based products like honey for medicinal purposes [2].

Honey is an ancient traditional medication that has long been used to treat microbiological illnesses. Honey has also been reported in the treatment of wounds, eczema, and inflammation by the ancient Greeks and Egyptians, as well as Persian traditional medicine [3]. This has led to the search for different types of honey with antibacterial activity [4]. Honey is an old-fashioned medicine that has long been used to cure bacterial infections. Honey was also used by the ancient Greeks and Egyptians, as well as Persian traditional medicine, to cure wounds, dermatitis, and inflammation [5]. The floral source of honey plays an important role in it [4]. Honey is being used in the clinical treatment of ulcers, bedsores, burns, injuries and surgical wounds in the hospital and as traditional remedy. The antibacterial properties of honey may be particularly useful against bacteria which have developed resistance to many antibiotics, e.g. Methicillin resistant Staphylococcus aureus (MRSA) is the most common multidrug-resistant pathogen causing nosocomial infections [6].

In surgical infections, burns, and wound infections, honey is an excellent topical wound dressing agent [4]. Honey has been used as a medication since ancient times and is being used now. When tested against bacterial infections, oral bacteria, and food spoilage, raw unadulterated honey has demonstrated to have some broad-spectrum antibacterial activity [7].  Honey's strong osmotic effect and low pH has contributed to its high antibacterial potency. 

The ability to produced hydrogen peroxide which plays a key role in the antimicrobial activity of honey [8], as well as phytochemical components Honey has been shown to have antibacterial efficacy against a wide range of pathogens, including multi-antibiotic resistant strains, in several publications and clinical research. Honey has also been shown to have antimicrobial properties in other investigations [9]

Mycobacterium [10]. Methicillin resistant Staphylococcus aureus and Vancomycin resistant Enterococci [11], Streptococcus pyogenes biofilm formation and common gastrointestinal pathogenic microorganisms [12]. Honey's antifungal action, particularly anti-Candida activity, has also been observed [13]. 

SAMPLE COLLECTION: The honey samples under investigation were obtained from local commercial producers in Okwudor in Njaba local government area of IMO state and Mbu Isi-Uzo local government area of Enugu state. The honey samples are pure as they do not contain diluents or additives and were also not subjected to any form of heat.

PREPARATION OF HONEY SAMPLE:

The honey first went through filtration with a sterile metal sieve and placed in a sterile universal container in a water bath at 60^oC for 30 mins. The honey solution was handled aseptically and protected from bright light to prevent photo-degradation of the glucose oxidase enzyme.

PHYSICOCHEMICAL CHARACTERIZATION OF HONEY SAMPLE

The samples of honey were analyzed according to the official methods of the Association of Official Analytical Chemists to determine the moisture content, sugar composition, ash content, diastase activity, pH, acidity (Free and total acidity), viscosity, electrical conductivity (EC) and the total protein.

Moisture Content:

Moisture content in honey was determined with a Shibuya refractometer reading at 20°C obtaining corresponding percentage moisture from the Chataway table.

Sugar Compositions:

The sugar composition was determined by Gas-liquid Chromatography with flame ionization detector (GC-FID). Trimethylsilyl derivatives of sugar oximes were baseline separated and quantified in a gas chromatograph HP 5890 series II and an HP 33964 integrator under the following conditions: 3 mm stainless-steel column (II8-in o.d.) packed with 4% SE-52 on chromosorb WAWDNCS 100/120 mesh, carrier gas flow 25 mL N₂ min^-1, FID with H₂ at 30 mL min^-1 and O₂ at 400 mL min^-1, temperatures (°C): injector 280, detector 290 and column 205, rate 20°C/min to 280°C, held for 20 min, internal standard calibration with xylose. All standard sugars were analytical grade. Results were expressed as grams of each sugar in 100 g of honey (Sugar compositions in percentages) 

pH Value:

The value was measured, using a Jenway micro pH meter: 3510 model, from a solution containing 10 g honey in 75 mL of CO₂-free distilled water.

Ash Content

Ash percentage was determined by calcinations overnight at 600°C in a furnace to a constant weight. It was allowed to cool over calcium oxide in a desiccator and weighed. The percentage ash was calculated.

Determination of Acidity Content:

Free and total acidity were determined by the titrimetric method using a solution containing 10 g of sample dissolved in 75 mL of water in a 250 mL beaker. The solution was titrated with 0.05 N NaOH at a rate of 5.0 mL min^-1. Immediately the pH read 8.50; the addition of NaOH stopped. Ten mL of 0.05 N NaOH was then pipetted and was back-titrated with 0.05 N HCL from 10 mL burette to pH 8.30. The blank was equally determined as above. The total acidity was determined by adding free acidity and lactone acidity. Results were expressed as meq kg^-1.

Viscosity:

The viscosity of honey was determined by using U-table Viscometer (300BS/IP/CF 4414). The Viscometer was calibrated against water, the flow time of which represented 100% viscosity reduction:

Viscosity (v) = k X t

Where, k is constant (k = 0.25), T = time (second), the unit for viscosity is mm²/s or Cst (AOAC, 1990).

Total Protein Content:

Total protein content was measured using the Kjeldahl method as described in (AOAC, 2005), based on the conversion of the organic nitrogen present in the sample to (NH₄)₂SO₄. Dried sample (1 g) was subjected to two processes: digestion and distillation. The sample was mixed with a selenium catalyst and H₂SO₄ (15 ml, 95–98%). The resulting solution was distilled after adding NaOH, and the distillate was collected in a flask with H₃BO₃ (4%) and mixed indicator. Finally, the mixture was titrated with HCl (0.1 N). The percentage of nitrogen quantified was transformed into protein content by multiplying by a conversion factor of 6.25.

THE ISOLATION OF TEST ORGANISMS (BACTERIAL ISOLATES):

The isolation of test bacteria was obtained from swabs of 205 burnt wound patients attending Federal Medical Center Owerri Imo State. The study took place within the period of seven months. The burn wound swabs were inoculated onto sheep blood agar, mannitol salt agar, MacConkey agar and nutrient agar (Oxoid), plates and incubated at 37^oC for 48 hours. After the end of the incubation period, grown isolates were afterward identified by their colony morphology and thereafter taken for Grams staining and biochemical characterization.

PURIFICATION OF ISOLATES:

The streak plate technique described by (Ogbo 2005) was adopted for the purification of the isolates. A wire loop was flared in Bunsen burner and a loop full of bacterial colonies were picked and inoculated onto the sheep blood agar, mannitol salt agar and nutrient agar after preparation in accordance to manufacturer's direction. The wire loop was sterilized and used to streak from the inoculated spot to make streak 1. The wire loop flamed again and cooled, the streaking continued from the end of streak 1 to 2. This procedure continued until streak 5 was made. The Petri dish lid was replaced and the plate was incubated at 37^oC for 24 hours.

The bacterial discrete colonies were isolated and sub-cultured onto MacConkey agar, blood agar and mannitol salt agar plate and incubated at 37^oC for 24 hours for isolation and purification of the test bacteria.

IDENTIFICATION OF ISOLATES:

Some bacteria species have similar morphological, culture or staining reactions which make exhaustive biochemical test important in bacteria identification. The biochemical tests employed in this study is Gram staining, motility test, catalase test, coagulase test, indole test, Methyl Red-Voges Proskauer (Mr-Vp) Test, Oxidase Test, Voges ProskauerTest,Urease Production, Citrate Utilization Test, Utilization Of Sugars, Production Of Indole From Tryptophan And methyl red.

Gram’s Staining:

A thin smear was prepared on a clean grease free glass slide. The smear was flooded with Crystal violet and allowed to stand for 1 minute. Then the slides were washed with water and then flooded with gram’s iodine and left for 1 minute. The stain was drained and decolorized with 95% ethanol and was washed with water. The smear was counter stained with Safranin for 1 minute. The slide was blot dried and examined under a microscope.

• Catalase Test:

With the aid of a wire loop, the test organism was emulsified with a loopful of hydrogen peroxide on a glass slide. Effervescence caused by the liberation of free oxygen as gas bubbles, indicated the presence of catalase in the test culture.

     H₂0₂     H₂+ 0₂    

• Coagulase Test:

This was carried out using a slide test method. A colony of the test organism was emulsified on a drop of physiological saline on a glass slide to make a thick suspension. A drop of plasma was then added to the suspension and mixed gently.

• Methyl Red-Voges Proskauer (Mr-Vp) Test:

15g of MR VP broth was weighed and dissolved in 1 liter of distilled water. The medium contains:

Peptone    - 7.0g

Dextrose   -5.0g

K₂hp0₄         -5.0g

The medium was autoclaved after distribution into the test tube. After cooling, the isolates were inoculated into the test tubes in duplicates and then incubated for 48 hours at 37⁰c.

• Methyl Red Test:

To about 5 ml of the broth culture, a few drops of methyl red solution were added.

A red color indicated positive methyl red test (which showed that the organism can produce acid from glucose hosphate) while negative test was indicated when the color is yellow.

1. Voges Proskauer Test:

To the remaining portion of the broth culture, 3 ml of 5% alpha- naphthol and 1 ml 0f 40% potassium hydroxide were added, shake and then observed for color formation.

A pink color (or red color) within 2-5 minutes showed a positive VP reaction.

1. Oxidase Test:

This was performed by placing 2 to 3 drops of 1% solution of tetramethyl-P-phenylenediamine dihydrochloride (TMPPEH) onto a filter paper in a Petri-dish. Smear of the test organism was made on the filter paper using an inoculating loop. The appearance of a purple color indicated a positive reaction.

1. Citrate Utilization Test:

This test was carried out using Simmons Citrate Agar method. The medium was prepared according to the manufactured directions. 5-10ml portions were dispensed in test tubes and sterilized at 121⁰C for 15 minutes.  They were kept in slanting positions to set. The slope surfaces were inoculated with test isolates and incubated at 37⁰C for 4-7 days. Utilization of citrate resulted in an alkaline reaction which was indicated by color changing from green to blue and growth of the organism, while negative test retained the green color without any growth of the isolate.

1. Urease Production:

Test isolates were inoculated into slant tubes of Christensen’s urea agar medium and incubated at 35⁰C for 72 hours. They were watched daily for any color change. Urea production led to the hydrolysis of urea to ammonia which increased the pH as indicated by color change in the medium from yellow to pink.

1. Production Of Indole From Tryptophan:

The medium, peptone water (tryptone 2% sodium chloride 0.5%, final pH 7.2) was used for the test. This was dispensed in 5ml amounts in test-tubes and sterilized by autoclaving for 15 minutes at 121⁰C. After sterilization, the medium was inoculated with test organisms and incubated at 37⁰C for 24-72 hours. 0.5ml kovac’s reagent was added and the tubes were shaken gently and allowed to stand. Appearance of red color indicates the presence of indole.

1. Utilization Of Sugars:

The medium used here was a basal medium which was composed of 10g of tryptone, 5g of NaCl, 2.5ml of 1% of bromocresol purple and 1 liter of distilled water. The components were added to the distilled water and dissolved by steaming. The 1% bromocresol purple was also added and the color changed to purple. The solution was then distributed into test tubes each provided with an inverted Durham tube; and care was taken to ensure that no gas was in the inverted vials. The test tubes were then covered with cotton wool and foil paper and sterilized at 121⁰C for 15 minutes. The sugars which included glucose, lactose, fructose and sucrose were weighed out in 1g each and dissolved in 10mls of distilled water respectively. The sugars were sterilized at 121⁰C for 15 minutes. After sterilization, the sugars were distributed aseptically in the test tubes containing the basal medium. After this, the tubes were incubated with the test isolates and then incubated at 37⁰C for about 5-7 days. After incubation, the change of color of the basal medium from purple to yellow indicates acid production and the presence of a gas bubble inside the inverted Durham tubes showed gas production.

Antimicrobial Activity of Honey Sample on Bacterial Isolates from Burn Wound in Vitro:

Agar well diffusion method was used to screen antibacterial activity of honey samples [14]. A representative colony of each isolated bacteria was used for the antimicrobial assay. 0.1ml volume of the species was aseptically introduced into the nutrient agar (Oxoid) plates.  The cultures were uniformly distributed all over the agar plate with the aid of a sterile glass spreader. The inoculated plates were allowed to dry and sterile cork borers were used to bore holes on each agar plate. 0.1ml volume 100% concentrated honey samples were introduced into the holes using a sterile Pasteur pipette. The inoculated plates were allowed to stand for one hour to ensure proper diffusion of the honey into the medium and incubated at 37 °C for 24 hours. After incubation, the plates were observed for inhibition zones around the holes.

Determination of Minimum Inhibitory Concentration (MIC) of Honey Samples:

Disc diffusion method was used as a preference for susceptibility and MIC. Escherichia coli and Staphylococcus aureus were aseptically inoculated onto nutrient agar and sterile glass rod spreader was used to gently spread the inoculums in the agar plates. Sterile disc or blank disc (10mm) was dipped in different concentrations of honey after the honey was subjected to ten-fold serial dilution with sterile water. The dilution factor is 1/2, 1/4 until 1/256 was made as described by (Omoregbe et al., 2007; Ogbo 2005).  Thereafter, the impregnated discs were placed on swabbed plates containing the three bacteria isolates (Escherichia coli and Staphylococcus aureus). All agar plates were thereafter incubated at 37°c for 48hrs for the observation and measurement of zone of inhibition from each paper disc with different honey concentration placed on the three bacteria isolates. 

Statistical Analysis:

Descriptive statistics was done using Microsoft Excel 2016. The data were analyzed using SPSS 24 software package. A one-way ANOVA was done to detect any significant differences (P < 0.05) between parameters.

The result for the physicochemical analysis of the honey is presented in Table 3.1

Table 3.1: Physicochemical characteristics of honey

*Values in row with the same alphabet are not significantly different (p > 0.05)

Out of 205 burnt wounds investigated for the presence of Staphylococcus aureus and E.coli, after culture and biochemical examinations, 94 representing 45.8% showed heavy growth with the pathogens while 111 representing 54.2% showed no growth of the pathogens under investigation. It was also discovered that out of 84 male patients investigated 37 representing 18% were isolated from the pathogens while 121 female patients 57 representing 27.8% were also isolated from the pathogens.

TABLE 3.2: Bacterial pathogens isolated from patients in relation to sex

TABLE 3.3: Bacteria Pathogens Isolated in Respect to Wound Duration

TABLE 3.4: Minimum Inhibitory Concentration of Okwudor Honey Sample (V/V)

TABLE 3.5: Minimum Inhibitory Concentration of Mbu Honey Sample V/V

TABLE 3.6: Biochemical and Morphological characteristics of bacteria isolates

 TABLE 3.7: Biochemical and Morphological Characteristics of Bacteria Isolates

  Key: + Positive, - = Negative

TABLE 3.8: Zone of Inhibition Diameter in Millimeter (mm)

Figure 1: Bar chart showing the zone of inhibition diameter in millimeter (mm) of honey samples against E. coli and Staphylococcus aureus

Pysicochemical Properties of Honey:

The moisture content of the honey had mean values of 17.23±3.52 % for O. Honey and 14.32±2.02 % H. Honey, which were lower than the recommended limits of < 21 stipulated by Codex Alimentarious (2001). The honey samples showed no significant differences (p > 0.05). The recorded moisture content is comparable to the ones reported in other studies. According to (El Sohaimy et al., 2015), moisture contents of honey samples  were  18.32 ± 0.67 g/100 g  for Egyptian,16.28 ± 0.22 g/100 g for Yemeni, 15.64 ± 0.30 g/100 g for Saudi  and  14.73 ± 0.3 g/100 g  for Kashmiri  respectively. Furthermore, (Fasasi, K. A. 2012) [15] reported that Nigerian honey contains 17.9±2 % of moisture. Moisture content of honey is a limiting factor in the indetermination of its quality, stability and spoilage resistance against yeast fermentation. The higher the moisture content is the higher probability of honey fermentation during storage. Lower moisture limits (<20%), elongates honey shelf life and is accepted by the international regulations for honey quality.

The pH measures the acidity and alkalinity of the honey. The pH recorded was higher in O. honey (4.08±0.15) than M. honey (3.92±0.00). The samples all fell within the acceptable range (3.4 to 6.1) by international standard. The results obtained showed that the honey was acidic. The pH value of the tested honey samples were close to those previously reported in Nigerian, Indian, Algerian, Brazilian, Spanish and Turkish honeys (between  pH  3.49 and  4.70 [15] The high acidity of honey correlates with the fermentation of sugars present in the honey into organic acid, which is responsible for two important characteristics of honey: flavour and stability against microbial spoilage. Furthermore, it might also indicate that the honey samples have high content of minerals.

Ash content is a quality criterion for botanical and geographical origin of honey. In the present study, the ash content was 0.6 ± 0.2  0 % in O. honey while 0.49±0.26 % in M. honey. Only O. honey showed high mean ash content above the international standard (0 to 0.5 %). The obtained result is comparable to (Fasasi, K. A. 2012) [15] for Nigeria honey. Saudi and Kashmiri honey samples showed lower ash content (0.23 ± 0.02 and0.30 ± 0.03 g/100 g) respectively. On the other hand Egyptian and Yemeni samples showed higher values of ash content (1.07 ± 0.02 and 2.33 ± 0.02 g/100 g) respectively. These results referred to the rich content of pollen sources surrounding the apiary yard during honey production. 

The fructose and glucose mean values were 37.40±2.23 and 29.77±1.56 %, respectively for O. honey and 31.26±0.92 and 34.09±3.41 % for M. honey. The concentrations of studied honey samples varied slightly from the reported results of 36.6±0.6 fructose and 31.2±0.4 % glucose in Aragon, Spain (Perez-Aguillue et al., 2010) but compared with low values of 32.9±0.4% for fructose and 25.0±0.5% for glucose obtained in Libya and Nigeria [15]. The mean proportion of sucrose for O. honey (1.8 %) was lower while 2.12±2.40 % for M. honey was higher than the 1.97 % sucrose report.. Also, maltose was found at lower levels (4.60±0.53 % for O. honey and 6.72±1.31 % for M. honey) than the 7.2±0.2% for 27 samples in Aragon, Spain and 5.7 % reported in Spain [15]. The sum total obtained from the percentages of fructose, glucose and maltose of the honey samples was above 65%, the minimum limit set by EEC regulations (EEC, 1974) for reducing sugars. The mean percentages of sucrose of the studied honey were all below 5% which the maximum limit is proposed by FAO /WHO standards of honey.

Acids are an important component of the flavor and aroma of monofloral honeys. The total and free acidity of the honey samples were all below the recommended limits (≤ 50 and 10.6 to 26.9 meq/kg respectively). The acid aspect of honey may be due to the presence of aromatic acids, aliphatic acids, and mainly gluconic acid, which is considered as the main organic acid responsible for the acidity of honey [16]. Its presence in honey is especially due to the glucose oxidase activity that bees add during ripening [16]. 

The protein content of honey samples was 2.25±0.50 mg/g for O. honey and 3.05±1.04 mg/g for M. honey, which were lower than those reported in other studies. It is well known that honey contains a trace amount of protein usually originated from pollens which is a natural and protein-rich food source and some enzymes such as glucose oxidase, invertase and diastase [16]. The variability in protein content of different types of honey might refer to the origin of honey and the type of pollen.

Antibacterial Activity of Honey Samples:

The aim of this study was to investigate the possible antibacterial activity of honey sample from two different location (Okwudor in Njaba LGA IMO state and Mbu Enugu state) against Staphylococcus aureus and Escherichia coli isolated from burn wounds. The result of the antimicrobial effect of honey on the bacterial isolates using 100% concentration of honey samples from Okwudor IMO state and Mbu Enugu state inhibited the growth of the two bacterial isolates. 

The inhibitory properties of the honey samples were established and these antimicrobial activities on the isolates are due to the presence of a number of properties inherent in honey which might contribute to its ability to fight infection and promote healing. Its high sugar content allows it to draw infection and fluid from wounds by a process called osmosis. Honey prevents bacterial growth through its low acidic pH and through the work of an enzyme (glucose oxidase) that produces small amounts of hydrogen peroxide, responsible for antimicrobial activity. Honey may contain components from the specific plant used by the bees in their production, and it is speculated that some of these components might further add to the antibacterial effects of certain honey. These compounds include tannins, saponins, flavonoids, alkaloids, phenolic acid, and low acidic pH. The minimum inhibitory concentration of the honey samples are very significant with Okwudor honey showing MIC of 0.16v/v for Staphylococcusaureus and Escherichia coli respectively while Mbu honey showed MIC of 0.16v/v for Staphylococcus aureus.

On the comparative analysis of the different honey samples being used according to location, it was discovered that the honey sample from Okwudor IMO had a high rate of microbial inhibition thereby showing more sensitivity than that of Mbu Enugu, which is  as a result of  environmental differences.

The honey samples collected from Okwudor (in Imo state) and Mbu (in Enugu state) showed good physicochemical properties as they conform to international standards, which indicates the good beekeeping practices used by beekeepers during honey harvesting. The honey samples further showed antibacterial potential against E. coli and S. aureus isolated from burn wounds with minimum inhibitory concentrations of 0.16 for S. aureus and 0.32 and 0.65 for E. coli by Okwudor and Mbu honey samples respectively. Overall, we can conclude that the analyzed honey samples showed variability in their antibacterial potential and physicochemical parameters and the changes in the parameters analyzed seem to be due to the geographical differences, weather, soil composition, or additional floral plant sources.

No funding sources..

None declared.

The study was approved by the Institutional Ethics Committee of Nekede

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