Study design, study area and period
Experimental study design method was conducted in Mekelle University, College of Health Sciences, Ayder Referral and Teaching Hospital from January to May 2015.
Specimen collection
Swabs from ear discharge and post surgical wounds were collected from patients attended the ear, nose and throat (ENT) clinic and surgical wards. Specimens were then transported to the microbiology laboratory for bacteriological analysis and experimental test.
Isolation and identification of bacteria
Collected swabs were inoculated on MacConkey agar, Blood agar and Manitol Salt agar and incubated at 37 °C for 18–24 h aerobically. Grown isolates were then identified by their colony morphology, Gram staining reaction, and Biochemical tests including Catalase test, Coagulase test, Triple Sugar Iron agar (TSI) (OXOID, UK), Citrate utilization test (BBL™), Urease test (BBL™), Motility Indole Lysine (MIL) [BBL™] and Optochin test using the standard procedure for bacterial identification (Clinical and Laboratory Standards Institute 2013). Accordingly the following bacteria were identified from the clinical specimens isolates S. aureus (ear discharge), P. mirabilis (ear discharge), P.
aeruginosa (wound), S. pyogenes (ear), K.
pneumoniae (wound) E. coli (wound) and Coagulase negative staphylococci(wound) for the experimental study. Control strains [American Type Culture Collection E. coil ATCC 25922, S. aureus ATCC 25923, P. aeruginosa ATCC 27813, K. pneumoniae, P. mirabilis obtained from Ethiopia Health and Nutrition Research Institute (EHNRI)] were used as a quality control.
Identification of multidrug bacterial isolates
Antibiotic susceptibility pattern of the isolated bacteria was done to identify multidrug resistant ones for the experimental study on Muller-Hinton agar (Oxoid, England) using disk diffusion method. Tetracycline (30 μg), penicillin G (10 μg), erythromycin (15 μg), gentamicin (10 μg), ciprofloxacin (5 μg), norfloxacin (10 μg), trimethoprim–sulfamethoxazole (25 μg), nitrofurantoin (300 µg), doxycycline (30 µg), ceftriaxone (30 μg), ampicillin (10 μg) and amoxicillin clavulanic acid (10 μg) (all Oxoid, England) were used to test the resistance pattern. Multidrug resistance was defined as non susceptible to ≥1 agent in ≥3 antimicrobial categories as per the recommendation of Magiorakos et al. (2012).
Honey collection and processing
Six honey samples (three red and three white) from three districts were collected from local markets in sterile screwed-cup container and kept in cool and dry place in the laboratory for processing. Honey samples were first filtered with a sterile mesh to remove the debris and were stored at 2–8 °C for further use.
Preparation of bacterial isolates
From the multidrug resistant colonies, three to five pure colonies were picked from each isolates with an inoculating wire loop, suspended in 4–5 ml of nutrient broth and incubated at 37 °C for 24 h. The bacteria suspension then was diluted with sterile distilled water until it matches the turbidity of 0.5 Mc Far land Standards (105–106 CFU/ml). The resulting suspensions were further diluted 1:100 in sterile nutrient broth to set inoculums density of 1 × 104 CFU/ml according the set procedure (Kacaniova et al. 2011).
MIC determination
The minimum inhibitory concentration of the honeys was determined using broth tube dilution method according to Kacaniova et al. (2011) procedure. Briefly, ten sterile test tubes were placed in rack, labeled each 1 through 8. Honey control tube (HC) and growth control tube (GC) were used as a quality control. One ml of freshly prepared nutrient broth was added to each tube, sterilized and cooled. Then one ml of undiluted honey solution 100 % was added to test tube number 1 and HC with a sterile micropipette and tips. Then twofold serial dilution was performed by transferring 1 ml undiluted honey into the second tube with separate sterile micropipette and tips and vortexed for homogenization. After a through mixing, 1 ml was transferred with another sterile micropipette from tube 2 and tube 3. These procedures continued until eighth tube with a dilution of 1:128 was reached and finally 1 ml was taken and discarded from tube 8. The GC tube received no honey was served as a growth control while the HC tube received no bacterial inoculums was served as a honey control.
Except the HC tube, each tube was inoculated with 1 ml of the culture of respective prepared organism. The whole procedures were repeated for all the organisms tested to each of the honeys. Tubes were then incubated at 37 °C for 24 h and observed by visual inspections for the presence and absence of growth (turbidity).
MBC determination
To determine the MBC, incubated tubes showing no visible sign of growth/turbidity in MIC, were sub-cultured onto sterile nutrient agar plates by streak plate method and incubated at 37 °C for 24 h aerobically. The least concentration of honey that did not show growth of test organisms was considered as the MBC (Kacaniova et al. 2011). Then inoculated plates were scored as bactericidal if no growth; bacteriostatic if there is light to moderate growth and no antibacterial activity if there is heavy growth according the record of Payveld (1986).
Pre test and data quality control
Pretest was conducted to check the method with quality control organisms. Sensitivity test was done against honey of different concentration and bacteria for its reliability and validity before it was used for actual experiment.