Pharmacognosy Comparison of Antimicrobial Potential of Honey Samples from Apis mellifera and Two Stingless Bees from Nsukka, Nigeria

The antimicrobial activity of honey depends on many factors, including its botanical origin, geographical and entomological source. The aim of this study was to evaluate and compare the antimicrobial potential of honey varieties from Apis mellifera, Hypotrigona sp. and Melipona sp. against MDR Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa ATCC 25783 , Candida tropicalis, Candida albicans SC 5314 and Cryptococcus neoformans . By using standard microbiological procedure, the agar-well diffusion and broth microdilution methods were used to evaluate honey samples for their antimicrobial and non-peroxidase activity. Different concentrations of the honey samples showed inhibition zones diameter (mm) against the test isolates . The Minimum Inhibitory Concentrations (MICs) of the honey varieties from A. mellifera, Hypotrigona sp. and Melipona sp. ranged from 6.3–25.0%, 3.1–12.5% and 6.3–25.0% (v/v) respectively. There were no statistically significant differences between the mean MICs of honey varieties against E. coli, P. aeruginosa (ATCC 25783) and C. neoformans . Hypotrigona sp. honey had the least mean MICs (4.15 ± 1.58–11.11 ± 2.76 % v/v) against most of the test organisms. The Minimum Biocidal Concentration (MBC) of the honey varieties from A. mellifera, Hypotrigona sp. and Melipona sp. against the test organisms varied from 6.3–50%, 3.1–25% and 12–50% (v/v) respectively. There were no significant differences between the mean MBCs of the honeys against MDR S. aureus ( p =0.179), E. coli ( p =0.564), P. aeruginosa (ATCC 25783) ( p =0.846), and C. albicans (SC5314) ( p =0.264). The honeys had some levels of non-peroxidase activity against E. coli, P. aeruginosa (ATCC 25783) and C. neoformans. This study has scientifically authenticated the potential use of stingless bee honeys from “Okotobo and Ifufu” as complementary therapeutic agents.


Introduction
Antimicrobial agents are for now the world's only hope of getting rid of infectious diseases. However, the change in pattern of resistance of pathogenic microbes to essential antibiotics, especially multidrug resistant once has diminished the effectiveness of known antibiotics [1]. As the frequencies of resistance are increasing worldwide, this poses a very serious danger to promotion of good health and all kinds of antibiotics, including the major last-ditch drug [2]. Therefore, there is need for evaluating alternative potential therapeutic agents with antimicrobial properties. Honey is bees' natural product, made up of complex mixture of sugars such as, fructose and glucose. It has been used as a medicine in many cultures for centuries. In more recent times, the insight in the use of honey as a therapeutic substance has increased and it is gaining acceptance as a remedy for treatment of a wide variety of ailments caused by pathogenic microbes [3][4][5]. It is widely used as a topical antibacterial agent for treatment of wounds, burns and skin ulcers as reported in a review by Lusby [6]. The ability of honey to kill microorganisms has been attributed to factors such as high osmotic effect, acidity, hydrogen peroxide (produced enzymatically in especially diluted honey), phytochemical components, antimicrobial peptide (defensin-1), and the induction of increased lasted between 18-24 h at 37°C. The assay was carried out in triplicate and the diameter of zones was recorded as mean ± standard deviation.

Determination of Minimum Inhibitory Concentration (MIC):
Following the initial antimicrobial screening tests, the minimum inhibitory concentration of each honey was determined by using the broth tube microdilution method, a modified method of Andrews [21]. Serial dilutions of each honey sample were made in eppendorf tubes containing 700 µL of Mueller Hinton Broth (MHB) (Oxoid Ltd., UK) and Sabouraud Dextrose Broth (SDB) for bacteria and yeast respectively, to give a final concentrations of 50%, 25%, 12.5%, 6.3%, 3.1% and 1.6% (achieved by adding 700 µL of honey to 700 µL of MHB or SDB and then serially transferring 700 µL from it to the next tube and so on). 700 µL was removed from the last tube. About 10 µL of the standardized test organisms were dispensed into the tubes. Negative control tubes (for MHB or SDA) prepared as described above with different concentrations of each honey samples, were not inoculated with test organism. Positive control tubes contained only 700 µL broth medium and each of the organisms but no honey.
Also, different concentrations of ciprofloxacin and ketoconazole as above, for Pseudomonas aeroginosa ATCC 25783 and Candida albicans SC 5314 respectively were used as positive control drugs. The tubes were incubated in the dark at 37°C for 24 h with constant shaking (at 250 rpm), to prevent adherence and clumping. The MIC was determined by visually inspecting the tubes for turbidity postincubation (matching the mueller hinton broth and sabouraud dextrose broth respectively with the corresponding negative control tube of the same concentration). The MIC was reported as the lowest concentration of test material which results in 100% inhibition of growth of the test organism (the lowest concentration that has the same turbidity with the corresponding negative control). The MIC was determined in triplicates and the values were expressed in % (vol/vol).

Determination of Minimum Biocidal Concentration (MBC)
The Minimum Biocidal Concentration (MBC) of the honey varieties were determined by further sub-culturing from the tubes which showed no visible growth in the MIC assay onto fresh sterile nutrient agar and sabouraud dextrose agar plates respectively. The culture plates were incubated at 37 o C for 24 h. The MBC was therefore taken as the lowest concentration or highest dilution of honey that did not show any visible growth on the sub-cultured NA and SDA plates [20].

Determination of non-peroxide antimicrobial activities
In order to determine non-peroxidase antimicrobial activities of the honey varieties, honey dilutions (50-1.6% v/v) were prepared in MHB/SDB containing catalase solution (Sigma, C-40) at a final concentration of 0.2% (w/v) (2 mg of catalase in 10 mL of MHB/SDB). The assay was conducted similar to the MIC determination as previously described. Control tube received broth, catalase only and containing corresponding honey concentrations (negative control), and bacteria, broth and catalase (positive control) [20]. After incubation, MBCs were also determined as described previously.

Statistical analyses
Results were reported as the mean ± standard deviation of triplicate experiments. One-way ANOVA-Games-Howell Post Hoc Multiple Comparisons and Kruskal Wallis (KW) and Mann Whitney U-test were used for comparison of means using a significant level of p<0.05 (SPSS version 23). despite that, majority of previous studies have been conducted using honey from the Apis species. As there are no studies that have evaluated the antimicrobial activity of honeys from these species of stingless bees, therefore the aim of this study was to compare the antimicrobial and non-peroxidase activity of honeys collected in Nsukka, Nigeria from Melipona sp. (locally called ifufu in South East Nigeria), Hypotrigona sp. (Okotobo) and A. mellifera against eight different human pathogenic microorganisms.

Collection of honey samples
Three honey samples each from Hypotrigona spp. (Okotobo) and Melipona spp. (Ifufu) including Apis mellifera honey (widely known honey) were collected from keepers at Olido, Enugu Ezike, Igbo Eze North Local Government Area of Enugu State, between April and May, 2015. The matured combs, laden with honey, were harvested and aseptically collected in sterile screwed cups, and kept in a cool and dry place before transporting to the laboratory.

Test organisms
The test organisms were obtained from the Department of Microbiology, University of Nigeria, Nsukkka.

Preparation of standard inocula
The inocula were prepared and standardized according to Clinical and Laboratory Standards Institute Approved Standard for bacteria [18]. Stock inoculum suspensions were prepared by taking five colonies (>1 mm in diameter) from 24 h cultures (37°C) into 5 mL sterile saline). Each suspension was shaken for 15 s and density adjusted visually to 0.5 McFarland turbidity standards. The turbidity of each suspension was compared by holding both the standard and the inoculums tubes side by side in front of a white paper with black lines. The colony forming unit per mL (cfu/mL) of each standardized culture was also determined [19].

Antimicrobial activity
Agar well diffusion method: The agar diffusion technique was employed according to method used by Allen et al. [20]. The honey samples were first inoculated separately on standard nutrient media (Oxoid Ltd., UK), to test for sterility. A micropipette was used to introduce 30 µL of the standard inoculum of the previously prepared bacterial and yeast isolates onto Nutrient Agar (NA) and Sabouraud Dextrose Agar (SDA) plates respectively, and spread with a sterile glass spreader. The plates were allowed to dry for 20-30 minutes. With the aid of sterile cork borer, 6 radial wells of 6 mm diameter were punched equidistantly at different sites on the plates (three wells per plate). Fifty microlitre of each of the honey concentrations (100% (undiluted honey), 80%, 60%, 40%, 20% and 10%, (v/v) were placed onto the bored wells. Sterile distilled water and different concentrations of commercial antibiotics (500-31.3 μg/mL of ciprofloxacin and 400-12.5 μg/mL of ketoconazole) served as negative and positive controls respectively against Pseudomonas aeruginosa ATCC 25783 and Candida albicans SC 5314 respectively. The plates were left on the bench for 30 minutes for pre-diffusion to take place followed by an overnight incubation that

Antimicrobial activity screening of the honey varieties
It was observed that all organisms tested showed clear zones of inhibition in response to different concentration of the honey varieties. Ten percent (v/v) and above of the honey samples showed inhibition zones against E. coli (Figure 1a). Twenty percent (v/v) and above showed inhibition zones against B. cereus (Figure 1b All the three Hypotrigona sp. honey samples showed antimicrobial activity against the tested organisms at a concentration range of 10-40% (v/v). Except for C. albicans SC5314, the three honey samples inhibited all the test organisms at a concentration of 10% (v/v) and above (Figures 1a, 1b, 1d and 2a-2d). Hypotrigona sp. honey samples showed inhibition zones against C. albicans SC5314 at concentrations range of 20-40% (Figure 1c).
The Melipona sp. honey samples showed activity against all the tested organisms at a concentration range of 10-40% (v/v). The honey samples at 10% and above showed inhibition zones against B. cereus ( Figure 1b As shown in Table 1, there were statistically significant differences between the mean inhibition zone diameters (mm) of Apis Mellifera,  In addition, positive control drugs i.e., ciprofloxacin (500-15.6 μg/ mL) and ketoconazole (400-12.56 μg/mL) produced respectively 20 ± 0.88-10 ± 0.29 and 22 ± 0.87 -9 ± 0.87 mm mean inhibition zone against reference strains respectively.

Minimum inhibitory concentration of investigated honey samples
The Minimum Inhibitory Concentrations (MICs) of the honey varieties were determined using micro-dilution methods. Apis Mellifera honey samples (I-III) inhibited all isolates tested at MIC range between 12.5 and 25.0% (v/v) ( Melipona sp. honey samples (I-III) also inhibited all the tested isolates at concentration range of 6.3-25.0% (v/v) ( Table 2). The three honey samples have MIC of 6.3% against B. cereus, C. tropicalis, and C. neoformans. Except for P. aeruginosa (ATCC 25783) and E. coli that were inhibited at MIC of 6.3%, the rest of the test isolates were inhibited at MIC of 12.5% (v/v). Table 3, Kruskal-Wallis (KW) test revealed that there were statistically significant differences between the mean MICs of the honey varieties against B. cereus (p=0.029), S. aureus (p=0.018), MDR S. enterica (p=0.018), C. albicans SC5314 (p=0.030) and C. tropicalis (p=0.032). Hypotrigona sp. honey had the least mean MICs against B. cereus, S. aureus, MDR S. enterica, C. albicans SC5314 and C. tropicalis. There were no significant differences between the mean MIC of the honeys against E. coli (p=0.102), P. aeruginosa ATCC 25783 (p=0.846) and C. neoformans (p=0.102) ( Table 3).
The MICs for the control drugs were 15.63 and 12.5 (µg/mL) against the P. aeruginosa (ATCC 25783) and C. albicans (SC5314) respectively. While the MBCs for the control drugs were 125 and 200 (µg/mL) against the P. aeruginosa (ATCC 25783) and C. albicans (SC5314) respectively.

Non-peroxidase activities of the honey varieties
The antimicrobial activity of the honey samples generally decrease after treatment with catalase. The MICs and MBCs of catalase treated Apis mellifera honey samples were within the range of 12.5-50.0% (v/v) and 25-50% (v/v) respectively (  I  II  III  I  II  III  I  II  III   MIC I  II  III  I  II  III  I  II  III   MIC  MBC  MIC  MBC  MIC     and Salmonella sp. (8-18 mm) [22][23][24][25]. There are similar reports on the antifungal activity of A. mellifera honey from Nigeria against C. albicans (4-16 mm) [26]. This is the first report on antimicrobial activity of Nigerian stingless bee honeys. Through well diffusion assay, the antimicrobial activities of stingless bee honeys especially from Melipona sp. and Trigona sp. (3-22 mm) have been reported in Ethiopia [27], Australia [28], Germany [29], Thailand [30] and Brazil [31].
Almost all the honey varieties used in this study especially Hypotrigona sp. honey, inhibited most of the test isolates at a lower MIC. The honey varieties had similar inhibitory effects against E. coli, P. aeruginosa (ATCC 25783) and C. neoformans. Recently, similar findings were reported by Ewnetu et al. [27], Boorn et al. [28] and Fahim et al. [32], who showed that MIC of A. mellifera honey against some isolates did not exceed 40%. There are reports on MIC values for Melipona sp honeys (MIC range of 11.1-50%) [31] and Trigona sp. honeys (MIC range of 4->16%) [28] against bacterial and fungal isolates.
All tested honey samples were biocidal to all test isolates, except against MDR S. enterica. The MBC of the investigated honey samples corroborated with the findings of Oyeleke et al. [33], who also reported MBC range between 6.25% and >50%. The present findings are supported by Othman [34] who showed that MBC values of Yemeni honey samples were in the range of 20-40% and that E. coli was the most susceptible to antimicrobial activity of honey. Zainol et al. [35] also reported the MBC of selected Malaysian honey to range between 6.25 and 50% similar to our findings. Anwanwu [26] reported that the minimum fungicidal concentration of Nigerian honeys ranged between 12.5 and 50% (v/v) against Candida albicans. Similarly, Ewnetu et al. reported stingless bee honeys to be more effective than A. mellifera honey against all isolates they tested (MBC of 12.5%) [27]. On the contrary, there are reports on MBCs of Melipona sp. honeys (≥ 50%) [31] and Trigon asp honeys (1 ≥ 32%) [28] against some bacterial and fungal isolates.
When the honey samples were treated with catalase to eliminate the effects of hydrogen peroxide, the results showed that MIC and MBC values generally increased. In the absence of hydrogen peroxide, some of honey sample varieties were effective against B. cereus, E. coli, P. aeruginosa (ATCC 25783) and C. neoformans. This is the first report on non-peroxidase antimicrobial activity of Nigerian honey. These results were similar to findings of Fahim et al., who investigated the nonperoxidase activity of honeys indigenous to Pakistan against similar organisms (MBC range between 15% and >50%) [32]. Brudzynski reported similar results against some isolates, in which he showed that residual hydrogen peroxide was responsible for the antimicrobial activity of honey [15]. Even in the absence of hydrogen peroxide, other physicochemical properties of the honey maybe responsible for the antimicrobial activity of honey.

Conclusion
This research has shown that the honey varieties varied significantly in their antimicrobial potentials. Hypotrigona sp. and Melipona sp. honey varieties have shown to possess antimicrobial properties similar to widely used A. mellifera honey. This study scientifically authenticates the potentials use of these stingless bee honeys as an alternative therapeutic agent.
Hypotrigona sp. (Okotobo) and Melipona sp. (Ifufu) honeys that are not consumed as widely as regular bee honey have shown to have antimicrobial properties similar to those of regular bee honey.