|Original Research (Original Article)|
JOURNAL OF APITHERAPY, 2019
VOL 5, NO. 2 PAGE 10–17
Effect of Trigona honey on Escherichia coli cell culture growth: In vitro study
Mohammad Abdulraheem Al Kafaween1, Abu Bakar Mohd Hilmi1, Rao Sanaullah Khan2, Mabrouka Bouacha3, Malik Amonov4
1Faculty of Health Sciences, University Sultan Zainal Abidin, Terengganu, Malaysia
2School of Food and Agricultural Sciences, University of Management and Technology, Lahore, Pakistan
3Laboratory of Biochemistry and Microbiology, Department of Biochemistry, Faculty of Sciences, University of Badji Mokhtar, Annaba, Algeria
4Faculty of medicine, Universiti Sultan Zainal Abidin, Terengganu, Malaysia
Contact Mabrouka Bouacha
Laboratory of Biochemistry and Microbiology, Department of Biochemistry, Faculty of Sciences, University of Badji Mokhtar, Annaba, Algeria.
Received April 07, 2019 | Accepted July 16, 2019 | Published August 01, 2019
This study aimed to investigate the antibacterial activity of Trigona honey against Escherichia coli.
The antibacterial activity of honey was examined by agar well diffusion assay, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-kill curve assay. Bacterial strains were cultivated in microtiter plates with varying concentrations of honey (10%, 20%, 30%, 40%, and 50% w/v) for specific incubation time (24, 48, and 72 hours) at 37°C.
Agar well diffusion assay showed that Trigona honey had the highest antibacterial activity against E. coli with 18.2 ± 0.6 mm. The MIC value against E. coli was 10% (w/v) and MBC was 30% (w/v). In time-kill curve, Trigona honey has inhibited E. coli in a 4 log10 at 18 hours, and total viable counts were killed after 24 hours. It was found that even ≥30% Trigona honey dilution interfered significantly with E. coli cell culture growth. Moreover, it was found that a difference of more than 10% honey concentration between the treatments was considered significant to produce inhibitory effects. This study has shown that Trigona honey has significant inhibitory effects on E. coli growth in vitro. Trigona honey may be used as an alternative to antibiotics in controlling infections caused by E. coli. However, further investigation is required to strengthen this argument.
Trigona honey; Escherichia coli; inhibitory effects; minimum inhibitory concentration; minimum bactericidal concentration; time-kill curve
Antibiotics have significantly reduced the mortality associated with infectious diseases during the 20th century. Unfortunately, their massive and repeated use has led to the emergence of bacteria resistant to these drugs. Today, bacterial resistance to antibiotics has become a worrying reality; the increasing evolution of bacterial resistance associated with a decrease in the stock of antibiotics is one of the essential motivations for research and the introduction of new antibacterial agents . Alternative antimicrobial strategies are urgently needed and thus this situation has led to a re-evaluation of the therapeutic use of ancient remedies, such as honey [2,3]. Honey has a long history of medicinal use that continues to prevent microbial infections to this day. It is also recognized as a topical antimicrobial agent effective in the treatment of burns and infected wounds [4–7]. The therapeutic properties of honey could be due to various factors, and the floral source of honey plays an important role in the biological properties of honey [8,9].
Honey has been used for thousands of years as a food, as a medicine, and has been incorporated into cosmetic products. A large number of different cultures have widely used honey as a medicine for many disorders such as chronic wounds and ulcers [10–14].
Trigona honey is produced by Trigona bees without stings grown in uncultivated bushland in Malaysia and Indonesia. Trigona honey is generally brighter in appearance than ordinary forest honey and has a distinctive “bush” taste, i.e., a mixture of sweet and sour with a hint of fruity taste . It has been reported that honey has more than 100 distinct compounds with different biological functions , the main antibacterial compound in most types of honey is hydrogen peroxide [17,18]. However, perhaps the antibacterial activity of Trigona honey is due to phenolic compounds [15,19,20]. These compounds have non-sticky properties that could be used to control bacterial growth and biofilm formation [21–23].
The properties and compositions of honey depend on its geographical floral origin, season, environmental factors, and beekeeping management [7,24,25]. It is recognized that some chemical changes occur when nectar is transformed into honey. These changes are mainly due to the enzymatic activity of bee enzymes deposited in honey by bees. These enzymes are invertase, which hydrolyzes sucrose into glucose and fructose, amylase or diastase, and glucose oxidase, which generates gluconic acid and hydrogen peroxide from glucose in dilute honey. The other enzymes present in honey are catalase and acid phosphatase [26,27]. Honey is used in some hospitals, particularly for the clinical treatment of ulcers, bedsores, burns, and surgical wounds . The antibacterial properties of honey can be particularly useful against bacteria that have developed resistance to several antibiotics [11,29,30]. The antibacterial properties of honey referred to acidity, the activity of non-hydrogen peroxide, the high osmotic effect, and the presence of phytochemical components [31,32]. The high osmotic effect of honey due to its high sugar content also plays a role in reducing the rate of bacterial growth . In addition to the high osmotic effect of honey, its acidity could also reduce the rate of bacterial growth. The acidity of honey, which is in the pH range 3.2 to 4.5, creates an environment unfavorable to bacterial growth [7,33]. Numerous reports and clinical studies have demonstrated the antimicrobial activity of honey against Escherichia coli, Salmonella entercolitis, Shigella dysenteriae, Mycobacterium, Staphylococcus aureus, Enterococci, Candida albicans, and Streptococcus pyogenes [34–36].
In this study, the antibacterial effect of Trigona honey on E. coli growth was evaluated in several concentrations of Trigona honey. We selected E. coli because it is a common bacterium used in microbiological laboratory tests; its structure and physiology are well-known. In addition, many strains of this bacterium have been implicated in diseases such as gastroenteritis and urinary tract infections. Antibiotic resistance and biofilm formation by pathogenic strains of E. coli are considered a major concern, especially in hosts with weakened immune systems .
Materials and Methods
Medical grade sterile honey from the stingless bee Trigona was obtained from a local pharmacy at Kuala Terengganu, Malaysia. The honey was sterilized using gamma-irradiation 25 kGy). The samples were kept at room temperature and protected from sunlight [23,38,39].
Escherichia coli ATCC 25922 was used for this study. By using a sterile loop, bacteria were streaked across the nutrient agar medium and incubated at 37°C for 24 hours. The bacterial culture was prepared by picking up 1–2 morphologically identical colonies from the stock culture and suspended in 20 mL of sterile Brain Heart Infusion in a sterile conical flask. The inoculum was adjusted to 0.5 McFarland standard (approximately to 1–2 × 108 CFU/ml) and it incubated at 37°C for 24 hours [31,39–43].
The effect of Trigona honey on growth of E. coli
Five different concentrations of honey 50%, 40%, 30%, 20%, and 10% were prepared with inoculums as shown in Table 1, and 150 μl of each concentration were pipetted into a 96-well plate. A 200 μl of honey was used as a corresponding negative control, 200 μl of inoculum was used as a positive control, and 200 μl of broth was used as a sterility control. The plates were incubated for 1, 2, and 3 day at 37°C. Absorbance was measured each day (1st, 2nd, and 3rd day) by using the microtiter plate reader (Tecan Infinite 200 PRO, Austria) at 570 nm. This test was performed in triplicate [38,42,43].
Table 1. Trigona honey diluted with inoculum.
Minimum inhibitory concentration
Micro broth dilution was employed for the determination of the minimum inhibitory concentration (MIC) of the honey. Following the method of [31,39,60], with minor modifications. Working bacteria culture was prepared as previously described, adjusted to be equal to 0.5 McFarland standards. MHB broth was used to prepare 50%, 40%, 30%, 20%, and 10% (w/v) concentrations of honey. Initially, the first well added with 200 μl of every honey dilution was used as dilution sterility controls. 100 μl of bacterial culture was mixed with 100 μl of each honey dilution in other wells. While a well with 200 μl of bacterial culture was used as the assay growth control. Also, a well containing 200 μl broth only was labeled as assay sterility control well. Plates were incubated at 37°C for 24 hours, and the presence/absence of visible growth was noted for each well. Also, the absorbance of the wells was read at 570 nm using microtitre plate reader (Tecan Infinite 200 PRO, Austria) [23,31,38,39,44–46].
Minimum bactericidal concentration
Minimum Bactericidal Concentration (MBC) was conducted using wells that appeared to have no growth (no turbidity) by visual inspection and were streaked onto nutrient agar plates using sterile 100 μl loops. The plates were incubated at 37°C for 24 hours. After incubation, any growth on the plates was marked as positive and no growth was marked as negative. The plates with the lowest concentration of honey showing no growth were recorded as the MBC [23,31,38,39,44–47].
Agar well diffusion assay
A prepared 0.5 McFarland E. coli suspension was streaked evenly on the surface of Muller Hinton agar (MHA) by using a sterile cotton swab. After that, wells with 7-mm diameter were prepared on the agar with a sterile cork borer. Each well was then filled with 70 μl of 50%, 40%, 30%, 20%, and 10% concentration of Trigona honey. Following 24 hours of incubation at 37°C, the diameters of the zone of inhibition for each sample were then recorded in millimeter (mm). Assays were completed in triplicate and an average value was obtained [31,39,44,45].
An overnight broth culture of E. coli in 5 ml of MHB was prepared by inoculating a colony from pure culture and incubating at 37°C for 24 hours. The first tube was inoculated with 0.6 g of honey and 1.4 ml of a broth culture of the test bacterium in an initial concentration of approximately 107 CFU/ml and the second tube was filled with 2 ml of inoculum used as a positive control. The tubes were incubated at 37°C. Broth aliquots were collected at different time points, serially diluted in saline solution, plated on nutrient agar media, and grown for 24 hours at 37°C to determine the colony-forming units (CFUs) in each tube. Finally, a graph of log10total viable count (TVC) versus incubation time was plotted to allow the exponential growth phase to be identified [39,48].
Repeated measures analysis of variance (ANOVA) was applied on the collected data using “Statistical Package for Social Science version 21” The differences of mean values within groups (time effect) were analyzed by using pairwise comparisons with the assumption of compound symmetry as given by Mauchly’s test of sphericity. A separate ANOVA for checking the treatment effect was performed with Post-hoc multiple comparisons to reveal the differences in mean values among groups.
The level of significance was set at 0.05 with two-tailed fashion. The assumptions of normality and homogeneity of variance were applied to check the fit of the model.
Effect of Trigona honey on E. coli growth
As shown in Table 2, there are no significant differences in mean growth rate values between day one, two, and three (F = 0.30, p = 0.745). It was found that E. coli growth was five times higher in 10% honey compared to the other concentrations (F = 551.37, p < 0.001).
The differences in mean values among groups with regard to time (time-treatment interaction) were analyzed by using ANOVA. There were significant differences between mean growth values in day 1 and day 2 (Table 3). It was also observed that the treatments become non-significant even after 72 hours of incubation (Table 3).
Profile plot (Fig. 1) for the adjusted mean (estimated marginal means) of E. coli cell culture for Days 1, 2, and 3 revealed that at 30% honey concentration, days of incubation become irrelevant.
It can be inferred from this study that honey at a concentration of 30% (w/v) may be the most suitable treatment to inhibit the growth of E. coli cells. Similar findings have been reported by  wherein 20%–40% dilutions have shown a marked inhibitory effect on bacterial growth. Moreover, it is observed that 40% and 50% treatment on the third day (Table 3) becomes non-significant in producing inhibitory effects.
MIC and MBC determination
In Table 4, the results obtained show that Trigona honey inhibited the growth of E. coli at 10% (w/v) and it has a bactericidal effect at the dilution of 30% (w/v).
Agar well diffusion
Agar well diffusion assay was summarized in Table 5. The table shows the zone of inhibition for E. coli after treated with Trigona honey. Trigona honey exhibits greater inhibition on E. coli cultures, which is related to its dilution.
The time-kill curve clearly shows an increase in the number of E.coli cells without honey treatment (Fig. 2). However, a reduction in the number of E. coli was observed, which showed the decreased 2 log10 reductions in TVCs at 6 hours (Table 6). At 12 hours, E. coli was decreased 3 log10reduction in TVCs, and at 18 hours, it was decreased by 4 log10 reductions. Escherichia coli was killed after 24 hours (log10CFU/ml = 0, p = 0.001).
Table 2. Overall mean difference of growth rate (cell culture) among groups (Treatment effect).
F-stat (df) = 551.37 (5), p-value < 0.001.
The time-kill curve is used to determine the bactericidal or bacteriostatic activity of antimicrobials. It is analyzed by plotting log 10 CFU/ml versus time. Total cell count is defined as the total number of both dead and living cells in the sample, whereas the TVC is defined as the number of living cells . To maintain and minimize the impact of time-kill variables, several factors should be considered when performing time-kill studies. These variations affect the results and its interpretation. These factors are first, the initial or starting inoculum of 104 to 107 CFU/ml should be applied. Second, the samples should be incubated at 37°C. Third, the assay should be continued up to 24 hours . In this study, all these conditions were applied in the time-kill assays. The log10 CFU/ml for treated E. coli was noticed at 12 hours almost half of E. coli was killed (log10 CFU/ml = 3.2). Also, at 24 hours, almost 100% of E. coli was killed.
Table 3. Comparison of mean cell culture among different groups based on time (time-treatment).
*Non-significant (p > 0.001).
Figure 1. Mean values of E. coli cell culture (measured as OD) incubated at 37°C.
Table 4. MIC and MBC determination.
Table 5. Inhibition zone (mm) (Mean ±SD), n = 3.
In this study, we have demonstrated that all concentrations of Trigona honey were able to decrease the growth rate of E. coli. This study demonstrated that there was considerable variation between the concentrations of honey and that indication of activity derived solely from the mean size of the zone of inhibition to determine the relative activity of honey. Antibacterial activities of honey have been broadly discussed among researchers worldwide. It is strongly related to several factors such as osmolarity, pH, and other major constituents such as phenolic acids and flavonoids . The MIC value of Trigona honey against E. coli was 10%, whereas the MBC value was 30% (w/v). A study by Zainol et al.  showed that MIC and MBC of Kelulut honey against E. coli were at 20% concentration of honey. Previous studies showed that the MIC of stingless honey was 4% to >10% (w/ v) for Gram-positives bacteria, 6% to >16% (w/ v) for Gram-negative bacteria, and 6% to >10% (w/ v) for Candida spp. [55,56]. Currently, the studies conducted by [12,57] have been reported that MIC of Manuka honey against S. pyogenes was at 20% concentration and MBC was at 25% concentration. Previous studies showed that MIC of Kelulut honey, Algerian honey, and Manuka honey against Pseudomonas aeruginosa was at 20% concentration and MBC was at 25% concentration [31,39,58,59]. These variations affect the results and its interpretation. Firstly, the initial or starting inoculum of 104 to 107 CFU/ml should be applied. Secondly, the samples should be incubated at 37°C. Thirdly, the assay should be continued up to 24 hours . Previous studies showed that stingless bee Trigona carbonaria decreased 1–3 log for S. aureus, and >3 log for P. aeruginosa after treated with 20% of honey . Four major antibacterial properties of honey including acidity, non-hydrogen peroxide activity, high osmotic effect, and the presence of phytochemical components . The high osmotic effect of honey due to the high contents of sugar in honey also plays a role in reducing biofilm mass . Besides the high osmotic effect of Trigona honey, the acidity of honey is assumed to play a role in reducing biofilm mass as well. The acidity of Trigona honey which is within the range of pH 3.2 to 4.5, creates an unfavorable environment for bacterial growth whereas their optimum pH for growth is about pH 7.2 to 7.4 .
Figure 2. Time-kill curve showing in vitro bactericidal effect of Trigona honey on E. coli.
Table 6. Log reduction (LR) for E. coli after 24 hours of exposure to 30% of Trigona honey.
This study has provided evidence to show that Trigona honey can significantly inhibit the growth of E. coli cell culture in vitro. Further 30% honey dilution is found to be the most appropriate concentration for getting significant results in inhibiting the growth. Therefore, it can be concluded that Trigona honey in its diluted form can be effectively applied in controlling E. coli infections. However, further investigation is needed to understand the mechanism of this inhibition, which was not the scope of this study. Further study with a scanning electron microscope followed by quantification of active compounds would help to understand the mechanism of inhibition.
This important piece of research would have not been possible without the generous sport of faculty of health sciences at University Sultan Zainal Abidin, Terengganu, Malaysia.
|How to Cite this Article|
Kafaween MAA, Hilmi ABM, Khan RS, BOUACHA M, Amonov M, . EFFECT OF TRIGONA HONEY ON Escherichia Coli CELL CULTURE GROWTH: IN VITRO STUDY. J Apither. 2019; 5(2): 10-17. doi:10.5455/ja.20190407083601
Kafaween MAA, Hilmi ABM, Khan RS, BOUACHA M, Amonov M, . EFFECT OF TRIGONA HONEY ON Escherichia Coli CELL CULTURE GROWTH: IN VITRO STUDY. http://www.japitherapy.com/?mno=39729 [Access: September 21, 2019]. doi:10.5455/ja.20190407083601
AMA (American Medical Association) Style
Kafaween MAA, Hilmi ABM, Khan RS, BOUACHA M, Amonov M, . EFFECT OF TRIGONA HONEY ON Escherichia Coli CELL CULTURE GROWTH: IN VITRO STUDY. J Apither. 2019; 5(2): 10-17. doi:10.5455/ja.20190407083601
Kafaween MAA, Hilmi ABM, Khan RS, BOUACHA M, Amonov M, . EFFECT OF TRIGONA HONEY ON Escherichia Coli CELL CULTURE GROWTH: IN VITRO STUDY. J Apither. (2019), [cited September 21, 2019]; 5(2): 10-17. doi:10.5455/ja.20190407083601
Kafaween, M. A. A., Hilmi, . A. B. M., Khan, . R. S., BOUACHA, . M., Amonov, . M. & (2019) EFFECT OF TRIGONA HONEY ON Escherichia Coli CELL CULTURE GROWTH: IN VITRO STUDY. J Apither, 5 (2), 10-17. doi:10.5455/ja.20190407083601
Kafaween, Mohammad Abdulraheem Al, Abu Bakar Mohd Hilmi, Rao Sanaullah Khan, Mabrouka BOUACHA, Malik Amonov, and . 2019. EFFECT OF TRIGONA HONEY ON Escherichia Coli CELL CULTURE GROWTH: IN VITRO STUDY. Journal of Apitherapy, 5 (2), 10-17. doi:10.5455/ja.20190407083601
Kafaween, Mohammad Abdulraheem Al, Abu Bakar Mohd Hilmi, Rao Sanaullah Khan, Mabrouka BOUACHA, Malik Amonov, and . "EFFECT OF TRIGONA HONEY ON Escherichia Coli CELL CULTURE GROWTH: IN VITRO STUDY." Journal of Apitherapy 5 (2019), 10-17. doi:10.5455/ja.20190407083601
MLA (The Modern Language Association) Style
Kafaween, Mohammad Abdulraheem Al, Abu Bakar Mohd Hilmi, Rao Sanaullah Khan, Mabrouka BOUACHA, Malik Amonov, and . "EFFECT OF TRIGONA HONEY ON Escherichia Coli CELL CULTURE GROWTH: IN VITRO STUDY." Journal of Apitherapy 5.2 (2019), 10-17. Print. doi:10.5455/ja.20190407083601
APA (American Psychological Association) Style
Kafaween, M. A. A., Hilmi, . A. B. M., Khan, . R. S., BOUACHA, . M., Amonov, . M. & (2019) EFFECT OF TRIGONA HONEY ON Escherichia Coli CELL CULTURE GROWTH: IN VITRO STUDY. Journal of Apitherapy, 5 (2), 10-17. doi:10.5455/ja.20190407083601