Honey is one of the natural products whose usage has become a popular approach in both food preservation and medical treatments as a result of its low toxicity and potent activity. This has been attributed to the ineffectiveness of antibiotic drugs in curing diseases which have triggered the need for alternative antimicrobial control strategies in re-evaluation of therapeutic usage of ancient therapies including honey [1]. World Health Organization (WHO) statistics indicate that approximately 80% of the total population in various countries has used natural products in their principal health care [2]. For instance, in Ethiopia, China and India, nearly 80 percent of the total population depend on traditional therapies as a prime health care source [3] [4]. These natural products can be applied in the discovery and development of novel antimicrobial drugs which can be used in treating infectious diseases. Researchers have shown that these natural materials are normally more suitable and effective to consumers, hence leading to reduction on reliance to synthetic substances [4]. Moreover, the study of the natural compounds may result to active components’ discovery that could prevent environmental hazards or ameliorate disease process in human cells [5]. Natural products as opposed to synthetic drugs experience less resistance from the target infectious agents [6].

These include medicinal plants of non-antibiotic drugs with antibacterial potential which have been discovered by scientists having the ability to combat such bacterial resistances towards antibiotics [7] and acting as alternative natural remedies for disease management. In addition to the medicinal plants, honey, a naturally available product, has been proven to possess antibacterial activity against numerous life-threatening bacteria [8], being effective against a variety of inflammatory cases and bacterial infections [9]. It has been used traditionally in treatment of gastrointestinal infections, respiratory diseases, and other various infections such as skin ulcers, burns and wounds [1]. Previous studies on its physicochemical parameters, chemical composition and antimicrobial activity has demonstrated its therapeutic potential [10] [11]. Its high viscosity provides barrier to inhibit infection while its low pH level and high sugar content prevents microbes’ growth [12]. Its moisture content determines its ability to persist stability and to resist decay by yeast fermentation [13]. Besides, its electrical conductivity is related to organic acids, mineral content, some complex sugars, etc. [14]. Honey’s color intensity shows presence of pigments such as flavonoids and carotenoids, which are well-known to contribute to its antioxidant activity [15]. This in return contributes to its antibacterial activity. Honey possesses glucose oxidase and osmotic properties particularly presence of polyphenols and defensin-1, which contributes to its bactericidal effect [16] [17]. Honey also exhibits both bactericidal and bacteriostatic effects against gram-negative as well as gram-positive bacteria and shows antifungal activity [18].

According to Lusby et al. [12], antimicrobial activity of honey varies depending on hydrogen peroxide present. The variation also results from the bee species, concentration and type of honey, floral and entomological origin in different seasons, pathogen tested and external factors such as type of soil, climate, and the maturation period [19]. In western part of Kenya, only antibacterial activity of Apis mellifera honey has been documented and nothing on stingless bee honey has been reported therefore the study was set to scientifically evaluate the in vitro antibacterial activity (bactericidal & bacteriostatic effects) of honeys produced by stingless bees Plebenia hylderbrandii and Meliponula bocandei against E. coli and S. aureus. These bacteria are commonly involved in causing urinary tract infections, diarrhea, septicemia, wound infections, and community acquired and nosocomial infections in humans. Besides, this is the first time these two honeys are being investigated for antibacterial activity.

The results of the in vitro susceptibility of the test microorganisms to the honey samples were varying as shown in Tables 1-4. Plebenia hylderbrandii honey had a bactericidal activity at a concentration of 4.71% (v/v) and 2.35% (v/v) for E. coli and S. aureus, respectively. Meliponula bocandei honey, on the other hand, had a bactericidal activity at a concentration of 16.47% (v/v) for S. aureus but had no bactericidal activity for E. coli since none of the concentrations in this study showed inhibition. From the results in Tables 1-4, it is evident that both honeys had a higher antimicrobial activity against S. aureus than E. coli (Tables 1-4).

Partial inhibition for Plebenia hylderbrandii honey was observed from 1.18% - 2.94% (v/v) for E. coli and 1.18% (v/v) for S. aureus while complete inhibition was stronger for S. aureus than E. coli at Minimum Inhibitory Concentrations (MIC) of 1.76% (v/v) and 3.53% (v/v) respectively as presented in Table 5. On the other hand, for Meliponula bocandei honey, partial inhibition was observed at a concentration of 10.59% - 17.65% and 9.41% - 15.29% (v/v) for E. coli and S. aureus, respectively; while complete inhibition was observed at a Minimum Inhibitory Concentration (MIC) of 16.47% (v/v) for S. aureus while for E. coli no complete inhibition was detected since none of the concentrations from the study showed inhibition (Table 5). The Minimum Bactericidal Concentration (MBC) value for Plebenia hylderbrandii honey was 4.71% (v/v) and 2.35% (v/v) for E. coli and S. aureus respectively (Figure 1(b) and Figure 1(c)) while for Meliponula bocandei honey the MBC value was 16.47% (v/v) for S. aureus (Figure 1(a)) but it wasn’t detected for E. coli since none of the tested concentrations showed inhibition as presented in Table 5.

+ means minimum growth; _ means no growth; _ indicates MIC values.

+ means minimum growth; ++ means moderate growth; _ means no growth; _ indicates MIC values.

N/D means not detected; + means minimum growth; _ means no growth; _ indicates MBC values.

N/D means not detected; + means minimum growth; _ means no growth; _ indicates MBC value.

N/D means not detected.

The positive control had a wide PIC range between 0.118% - 0.5882% (w/v) against E. coli while for S. aureus was only at 0.05882% (w/v). It had a higher MIC value of 1.176% (w/v) against E. coli than for S. aureus which had a lower value of 0.088% (w/v). Similarly, its MBC value was 1.176% for E. coli compared to 0.118% (w/v) for S. aureus as presented in Table 6. This indicates that the positive control had a higher bactericidal activity for S. aureus than for E. coli. On the other hand the in vitro antibacterial activity of negative control indicated heavy growth (Table 7) compared to the positive control.

The physicochemical parameters are presented in Table 8. The analysed honey samples were acidic in character. Meliponula bocandei honey had higher pH (3.99 ± 0.006^a) than Plebenia hylderbrandii honey (3.54 ± 0.012^a). The moisture content of the honey samples ranged from 24.54% to 27.79%. Plebenia hylderbrandii honey had higher moisture content (27.79% ± 0.023%) than Meliponula bocandei honey (24.54% ± 0.042%). The honey samples’ electrical conductivity (EC) ranged between 0.42 to 1.00 mS/cm. Meliponula bocandei honey had EC of 0.42 ± 0.002^a mS/cm while Plebenia hylderbrandii honey had EC of 1.00 ± 0.001^a mS/cm. The color intensity of the honey samples ranged between 1.11 ± 0.002^a to 1.2 ± 0.004^a mAU. Meliponula bocandei honey had a higher color intensity of 1.2 ± 0.004^a mAU while Plebenia hylderbrandii honey had 1.11 ± 0.002^a mAU. There was no significant difference between the two samples in all the physicochemical parameters.

The number of replicates (N) for all the physicochemical properties in each honey type was 3.

From the results in this table, similar letter “a” accompanying each mean indicate that there is no significant difference between the physicochemical properties in the two samples under study.

Honey has various characteristics that are believed to contribute to its total antimicrobial activity. These comprise of pH (being 3.2 - 4.5), moisture content, electrical conductivity, color intensity, H₂O₂ concentration, phytochemical factors, and high osmotic effect [22]. The acceptable range for pH of honey is between 3.2 and 6.1, low enough to inhibit pathogens [25]. The acidity nature is

+++ means heavy growth.

contributed by presence of organic acids in honey [26]. In this study, Plebenia hylderbrandii honey had a pH of 3.53667 ± 0.01155 while Meliponula bocandei honey had a pH of 3.98667 ± 0.00577. These values are within the acceptable range hence their contribution to their antibacterial activity. Honey samples from Spanish, Turkish and Brazilian had pH values ranging between 3.63 to 5.01, 3.67 to 4.57 and 3.10 to 4.05 respectively which were consistent with the ones from this study [27]. The Moisture content for honey may range between 13% to 29% [28]. Plebenia hylderbrandii honey (27.79% ± 0.023%) had higher moisture content than Meliponula bocandei honey (24.54% ± 0.042%) which were within that range. The results from this study also corresponded to the ones carried out for stingless bee honeys by Neupane et al. [29] and Jimenez et al. [30] which ranged between 20.12% to 29.1% and 20.61% to 28.04% respectively. However, results of this study were lower compared to the values (28.46%, 30.28%, 31.35% and 31.54%) obtained from a research carried out on Nigerian honey by Oyo, Osun, Ekiti, Lagos and Ogun [31]. The variation might be due to difference in geographical location, climate, bee species, among other factors. The honey samples’ electrical conductivity ranged between 0.42 to 1.00 mS/cm. Meliponula bocandei honey had EC of 0.42 ± 0.002^a mS/cm, which was below the maximum value of 0.8 mS/cm as indicated in European Union and Codex directive while Plebenia hylderbrandii honey had EC of 1.00 ± 0.001^a mS/cm, which was above the maximum value of 0.8 mS/cm. Comparatively, the results from the investigated samples were in agreement with research carried out by Saxena et al. [32] whose honey EC ranged between 0.33 to 0.94 mS/cm. However, EC is closely related to mineral content, proteins, organic acids, polyols, and some complex sugars [32]. Higher acid and ash contents indicate higher conductivity, and hence higher antibacterial activity [33] and this could be the reason for the high activity in P. hylderbrandii. Honey’s color intensity is represented by the ABS₄₅₀. In this study, the honeys’ ABS₄₅₀ values ranged between 1.11 ± 0.002^a to 1.2 ± 0.004^a mAU. A similar research done by Ahmed et al. [34] obtained values ranging between 1.26 and 1.44 mAU. In comparison, the ABS₄₅₀ values were also conveyed to be between 724 and 1188 mAU in Algerian honey samples [35]; 25 and 3413 mAU in Italian honey samples [24]; 70 and 495 mAU in Slovenian honey samples [36]; 524 and 1678 mAU in Indian honey samples [32] and between 254 and 2034 mAU in Bangladesh honey samples [37]. These differences may be due to variation in floral and geographical origin [34]. The color of honey is also dependent on various factors such as ash content, mineral content, storage time and heat [34] [38]. Color intensity correlates with antioxidant activity in that high color intensity implies high antioxidant activity [32].

This study demonstrates that honey from Kenyan stingless bee species Plebenia hylderbrandii and Meliponula bocandei has antibacterial activity. This activity varied between the two honeys and the discrete test organisms were also found to vary in their susceptibilities. On the contrary, a research carried out on honey from Kenyan stingless bee species, Dactylurina schimidti, from Coast did not show any inhibitory effect against both E. coli and S. aureus in all the tested concentrations which ranged between 25% - 100% (v/v) [39]. Another previous study conveyed MICs of honey from various Guatemalan stingless bees including Melipona beecheii, Geotrigona acapulconis, Tetra. Angustula and Scaptotrigona spp. ranging from 2.5% - >10% (v/v), with most of the MICs being 5% (v/v) [40]. For Apis mellifera honeys, the MICs published for medical honey or Manuka honey ranged between 2% - 8% (v/v) for Gram - positive cocci tested [41]. In addition, reports show that Apis mellifera honey inhibits most test organisms at concentrations 2.5% - 7.5% (v/v) [42]. In the current study, Plebenia hylderbrandii honey had a higher antibacterial effect than Meliponula bocandei honey (Table 5). The substantial differences between the antibacterial activities displayed by the two honeys could be because of discrepancies in the stingless bee honeys tested. These discrepancies may have also resulted from their feeding habits since they exploit plant-based resources [43] including pollen, nectar, latex, resin, scents, oil, seed and leaves during their foraging flight [44] which are found in diverse sites on broad diversity of crop plants.

The study further shows that microorganisms may differ in their susceptibilities to honeys; with S. aureus being more susceptible when compared to E. coli. This finding is evidenced by the concentrations of the honeys that inhibited S. aureus, which were much lower than those that inhibited E. coli (Tables 1-4). It is also evident that the positive control was more active against S. aureus than E. coli as evidenced by its higher MIC and MBC values against E. coli. This could have resulted from the fact that Gram-negative bacteria such as E. coli are less sensitive to honey activity compared to Gram-positive bacteria, for instance, S. aureus [45]. This is also in agreement with a study by Boorn et al. [46], which also showed S. aureus as the most sensitive organism to honey having MIC ranges 4% to >10% (w/v) for Gram-positive bacteria and 6% to >16% (w/v) for Gram-negative bacteria.

Honey have been proved to have antibacterial effect against E. coli. A study by Tan et al. [47] reported that the MICs for Tualang honey and Manuka honey for E. coli ranged from 17.5% (v/v) to 22.5% (v/v). On the same study, the MBC values ranged from 17.5% to 25% (v/v). Another study by Mulu et al. [42] demonstrated that the concentration of Apis mellifera honey from Ethiopia which fully prevented E. coli growth was 6.5% (v/v). According to Mandal et al. [1], the bactericidal effect of the Apis mellifera honey tested against E. coli was achieved at concentration 3% (v/v). In addition, a study by [48] reported that honey has antibacterial effects against E. coli having a MIC of 20% (v/v), concentrations tested ranged 0% - 30% (v/v). A research on stingless bee honey from Ethiopia also demonstrates its antibacterial activity against E. coli [49]. Trigona laeviceps honey, a stingless bee honey from Thailand also demonstrated its antibacterial activity against E. coli [50]. Nevertheless, Cruz et al. [51] have displayed the antibacterial activity of stingless bee honey against E. coli with MIC ranging between 10% - 20% (v/v). However, there is scarce information for the antibacterial activity of stingless bee honey compared to Apis mellifera honey. In this study, Plebenia hylderbrandii honey had a bactericidal effect at a concentration of 4.71% (v/v) against E. coli (Figure 1). On the contrary, the bactericidal effect for Meliponula bocandei honey against E. coli wasn’t obtained since growth was observed in the concentration range tested. This calls for further research for its determination.

Generally, honey also demonstrates high in vitro anti-Staphylococcal activity [52] although other studies confirms that this activity vary based on different samples obtained from similar botanical sources, with MIC ranges of 3.12% and 6.25% (v/v) [53]. A study done by Irish et al. [54] reported that honey from Australian stingless bee species Trigona carbonaria possessed antibacterial activity against Staphylococcus aureus. A study by Mama et al. [17] reported that the percentage by volume of honey to completely inhibit MRSA growth was in the range of 18.7% to 37.5% (v/v). The concentrations tested in the study ranged between 25% - 100% (v/v). On the contrary, a study carried out in Ethiopia, found out that the% v/v of Apis mellifera honey that prevented growth of S. aureus was 6.5, which is higher than our result obtained from plebenia hylderbrandii honey whose inhibitory concentration was 1.76% (v/v) and lower than that of Meliponula bocandei whose inhibitory concentration was 16.47% (v/v) for S. aureus. The concentrations from the study ranged between 10% - 100% (v/v) [55]. Another study by Willix also reported that the percentage by volume of Manuka honey to completely inhibit S. aureus growth was 1.8% (v/v) [56]. Similarly, Sherlock et al. [57] reported that inhibitory potential of Manuka honey from stinging bees on MRSA was only at concentrations > 12.5% (v/v) while Ulmo 90 honey was bactericidal on MRSA at concentrations of 3.1% (v/v) and 6.3% (v/v). French et al. [58] also reported that manuka honey inhibits S. aureus growth at concentrations 2.7% - 5% (v/v). In this study, the % v/v of the two types of honey to completely inhibit S. aureus was 1.76% (v/v) and 16.47% (v/v) for Plebenia hylderbrandii and Meliponula bocandei honeys respectively (Figure 1).

Although the tested honey showed antibacterial effect, other studies demonstrate that not all honeys have similar antibacterial activity degree. Numerous previous reports of antibacterial activities of stingless bee honeys are hard to compare with the current study due to differences in methods under studies [39]. In addition, most of these honeys were produced by different bee species other than Plebenia hylderbrandii and Meliponula bocandei and from floral sources and regions discrete from those found in Kenya which has further limited the comparisons.

Moreover, a study carried out by Mohapatra et al. [59] shows that honey is effective against both gram-negative (P. aeruginosa, E. coli and Salmonella typhi) and Gram-positive bacteria (Bacillus subtilis, S. aureus, Bacillus cereus, Micrococcus luteus and Enterococcus faecalis); this effect is either bactericidal or bacteriostatic. A study carried out in Ethiopia on red and white honeys indicate their antibacterial activity with the MIC and MBC of all the isolates (Escherichia coli, proteus mirabilis, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia, Streptococcus pyogenes) ranging between 6.25% - 50% and 12.5% - 100% (v/v) respectively [60]. A study by Ng et al. [60] also found out that stingless bee honeydew honey exhibit antibacterial property against E. coli and S. aureus. In this study, Plebenia hylderbrandii honey had both bacteriostatic and bactericidal effect on S. aureus at concentrations above 1.76% (v/v) and 2.35% (v/v) (Figure 1) respectively and on E. coli at concentrations above 3.53% (v/v) and 4.71% (v/v) respectively. However, Meliponula bocandei honey had both bacteriostatic effect at a concentration of 16.47% (v/v) and bactericidal effect at a concentration of 16.47% (v/v) on S. aureus but had neither bacteriostatic nor bactericidal effect on the growth of E. coli isolate because of the observed growth in all concentrations. In this study, the bactericidal effect was higher than that of Ulmo 90 honey from honeybee, which may have resulted from different geographical origin [57]. However, in this study the honeys had a lower bactericidal activity on E. coli than on S. aureus. This could be attributed by the lower susceptibility of E. coli.

Antibacterial activity of honey varies depending on various factors such as bee species, storage time, honey type and its concentration, type of microbe, test methods used, honey components/characteristics, geographical location, and source of nectar on which the reared bees were fed [61]. The difference may also be attributed to bees’ foraging behavior and feeding habits which vary depending on the plant resources exploited. Tan et al. [47] also indicated that honey has many sources of production and its antimicrobial activity may vary depending on the processing and origin. The antibacterial activity of stingless bee honey differs from that of Apis honey which is attributed to the storage of the specific bees. According to Ewnetu et al. [62], stingless bee honey has higher antibacterial effects than Apis mellifera honeys. In western Kenya, Apis mellifera honey has been reported to possess antibacterial effects against E. coli [63]. However, no stingless bee honey has been explored for comparison purposes hence the purpose for this study. In addition, the efficacy of honey against test microorganisms depends on the honey used, botanical origin variation, honey processing, geographical location, and bee health [57].

In comparison with the formerly published data, it is evident that Plebenia hylderbrandii and Meliponula bocandei honeys have similar activity to the other medical honeys and thus can be used as therapeutic agents.

Stingless bee honey possesses antibacterial activity with both bacteriostatic and bactericidal effects on E. coli and S. aureus. From this study, activity against E. coli on Meliponula bocandei honey was limited since it had neither bacteriostatic nor bactericidal effect on E. coli. Thus, it can be inferred that Plebenia hylderbrandii honey was highly effective than Meliponula bocandei honey. The stingless bee honey samples differed in activity although they are from similar botanical region and the basis for this observation need to be determined.

Our heartfelt thanks go to Masinde Muliro University of Science and Technology (MMUST) for the University Research Fund (URF) which assisted in bridging some financial gap in this research project. The Center for African Medicinal and Nutritional Flora and Fauna is much acknowledged for allowing most of this research was done at the Center’s research facilities and provision of the honey research samples used in this work.

The authors declare no conflicts of interest regarding the publication of this paper.

Wavinya, F., Omolo, M., Malebo, H., Sifuna, A., Nyongesa, P. and Nonoh, J. (2021) Antibacterial Activity of Honey from Wild Species of Stingless Bees; Plebenia hylderbrandii and Meliponula bocandei. Journal of Biosciences and Medicines, 9, 67-84. https://doi.org/10.4236/jbm.2021.97009