Study area and sampling. For detection of antibacterial activity of some natural products, five clinical bacterial isolates (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella spp.) were selected. Two local fruit samples pineapple (Ananas comosus), and pomelo. (Citrus maxima), were collected from different markets of Dhaka city, Bangladesh for detection of antibacterial activity against the selected clinical bacterial isolates. Both fruit and the peel of the fruit have been used for this study. The experiment was carried out during the time span of September 2020 to December 2020 in the microbiology laboratory of the Department of Microbiology, Stamford University Bangladesh.
Antibiotic susceptibility test of the pathogenic isolates. For the detection of antibiotic resistance traits of the clinical isolates, twenty-eight antibiotics commonly in use were selected. Meropenum (MME 10µg), Ceftazidime (CAZ 30µg), Cefuroxime (CMX 30 µg), Amoxyclav (AMC 30 µg), Amoxicillin (AX 25 µg), Azithromycin (AZM 15 µg)), Cefixime (CFM 30 µg), Ciprofloxin (CIP 5 µg), Colistin (CO 30 µg), Doripenum (DOR 10 µg), Doxycycline (DO 30 µg), Fusidic acid (10 µg), Gentamycin (GN 10 µg), Amikacin (AK 30 µg), Cephradine (CE 30 µg), Vancomycin (VA 30 µg), Teicoplanin (TEC 30 µg), Cotrimazole (COT 30 µg), Piperocillin/Tazobactam (PTZ, PIT 30 µg), Nitrofurantion (F 300 µg), Nalidixic acid (NAL 30 µg) Impenem (IPM 10 µg), Levofloxacin (LE 5 µg), Linezolid (LZD 30 µg), Clindamycin (CN 10 µg), Cefepime (CPM 30 µg), Tigecycline (TGC 15 µg) and Ceftriaxone (CRO 30 µg). Kirby Bauer disc diffusion method (22) was followed for the antibiotic drug resistance test. Using CLSI guidelines (23) the zone sizes were measured and determined the strains as sensitive or resistant.
Sample processing. The fruit samples were washed vigorously first with tap water and then with distilled water several times to wash out all kinds of impurities. Crude extracts were prepared by blending 10 g of the raw fruits and fruit peel separately with 90 ml saline. Before extraction, raw samples were shed dried for a week after cutting into small portions to make it all dry followed by blending to get a fine powder. The dried powder samples were then further processed for extract preparation.
Preparation of solvent extracts. About 20 g of each dried and powdered fruit and peel samples were mixed with 80 ml of 95% ethanol, methanol, and water separately in sterilized glass bottles followed by incubation at 37oC for 48 hours in shaking condition. After 48 hours, the ethanol, methanol, and aqueous extracts of all of these fruit and peel extract samples were filtered through sterilized cheesecloth and then through Whatman filter paper. Extracts were then concentrated by keeping them in evaporator and kept at 4oC until use as stock solution.
Determination of antibacterial activity of the extracts (crude, ethanolic, methanolic , and aqueous extracts). Bacterial suspensions were prepared until they reach McFarland turbidity standard (108 CFU/ml) and bacterial lawn was made using sterile cotton swab on the Muller Hinton agar media. Crude, ethanol, methanol, and aqueous extracts (100 µl each) of pineapple, pineapple peel, pomelo, and pomelo peel were placed into the well made in the media. Plates were then kept in the refrigerator in an upright position for better absorption for 20 to 30 minutes and then incubated at 37oC for 24 hours (25). Plates were observed for the presence of zone of inhibition after incubation and measured in mm.
Determination of MIC and MBC. Extracts of the samples were diluted in the concentrations of 500 µg/ml, 250 mg/ml, and 125 mg/ml with sterile nutrient broth followed by addition of 0.2 ml bacterial suspensions in each tube. After incubated at 37oC for 24 hours, tubes with no visible growth will be considered for determining MIC using the following equation, MIC=(lowest concentration of extract inhibiting growth+highest con. that allow growth)/2. To detect the concentration of extracts, loop fool samples from the visibly clear tubes were inoculated onto fresh nutrient agar plates where no bacterial growth occurs. The complete absence of visible growth on the agar plate after streaking onto the medium was determined as the MBC.