Collection and Preparation of the Sample Lychee honey (1kg) was collected from a lychee garden situated in Dinajpur district (25.63°N 88.65°E) of Bangladesh. Honey was directly purchased from a beekeeper after breaking a cultivated hive in the early June 2019. This raw honey was centrifugally extracted which was then allowed to pass through a sieve (0.5mm mesh) to remove non soluble materials (egg, pollen, wax) and other coarse particles. It was stored in a hermetically closed glass container at room temperature (250C).
Collection of Bacterial Strains Four gram-positive bacterial strains- Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis and Micrococcus luteus, isolated from feces, cough, nasal mucosa and urine culture respectively. The strains were collected from Center for Medical Biotechnology, Institute of Public Health, Bangladesh. The standard strain of Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 6538), Staphylococcus epidermidis (ATCC 12228) and Micrococcus luteus (ATCC 9341) were obtained and used as references.
Preparation of Inoculums Mueller-Hilton Agar (MHA) plates and Nutrient Broth (NB) tubes were used to sub-culture the collected strain, overnight at 37±1°C. In 5ml of sterile saline water, bacteria could grow. To attain the viable cell count of 107 CFU/ml, the solution was diluted, and absorbance was taken at 580 nm by using a spectrophotometer.
Antimicrobial Susceptibility Test Antibiotic susceptibility test was determined by well diffusion method. Each bacterial strain was streaked over the freshly prepared MHA plate (90mm) by using a sterile cotton swab. Five equal zones were plotted and 6mm well was cut in each zone using a sterile corn borer. 20μl of test agents were poured into wells respectively. Sterile distilled water was used as negative control and a 10μl of 10μg/10μl phenoxymethylpenicillin (Sanofi Aventis (BD) Ltd.) and Amoxicillin/Clavulanic Acid (Sanofi Aventis (BD) Ltd.) were used as positive controls. Then the plates were incubated at 37±1°C for 24h and zone of inhibition was measured.
Minimum Inhibitory Concentration Micro dilution technique is used to measure the Minimum Inhibitory Concentration (MIC) according to Patton et al. with some minor modifications. Two-fold serial dilution was made by using a microplate (96 well) where sterile distilled water was used to produce 50%, 25%, 12.5%, 6.25%, 3.12% and 1.56% (v/v) concentrations from the stock honey (100%). For comparison, phenoxymethylpenicillin and amoxicillin/clavulanic acid were added separately in combination with those concentrations in respective wells. So, each well-constituted 200μl of Nutrient Broth, 10μl of bacterial suspension, 20μl of honey samples and/or 10μl of standard antibiotics. No antibiotic or testing agent was added in the negative control. A reading of the absorbance of the microwells were taken through Biobase-EL10A ELISA Reader (China) to consider the initial value (T0 ). Then the plates were incubated at 37±1°C for 24h. and again absorbance was taken (T24). From the difference, percentage inhibition was calculated using the below formula [18]:
Percentage inhibition = 1 − (OD test/OD control) × 100
The least concentration of well that showed no visual turbidity was taken as the minimum inhibitory concentration (MIC).
Minimum Bactericidal Concentration: Minimum Bactericidal Concentration (MBC) was determined by withdrawing 20μl of suspension from the well which showed invisible growth with the lowest concentration and transferred into MHA plates, having no agent or antibiotic. To observe bacterial growth, plates were incubated for 24h at 37±1°C. The concentration of honey or its combination at which the plates did not show any bacterial growth was considered as MBC.
Statistical Analysis All experiments were done in triplet and all data was expressed as mean±standard deviation (SD). One-way analysis of variance (ANOVA) was used to determine the significance of differences among honey activities as measured by zone of inhibition. <0.05 was considered statistically significant.