1.1. Isolation of Pseudomonas aeruginosa. Multidrug-resistant clinical isolates of Pseudomonas aeruginosa were obtained from the hospital waste of Islamic University, Kushtia, Bangladesh. Isolates were identified as P. aeruginosa by Gram staining, its pearlescent appearance on Pseudomonas agar followed by morphological tests, staining as well as biochemical tests such as Methyl red, Voges Proskauer, Indole, Catalase and Oxidase.
1.2. Antibiotic sensitivity Test. The susceptibility of the isolates to commercially used antibiotics was determined by the agar diffusion techniques according to CLSI guideline. Single colony of each isolates were grown in LuriaBertani (LB) broth separately for 18-24 h and then used to prepare the bacterial inoculums with the turbidity of 0.5 McFarland standard (equal to 1.5×108 colony forming units (CFU)/ml). Turbidity of the bacterial suspension was measured at 600 nm. The bacterial inoculum was spread onto Mueller-Hinton agar (MHA) plates (150 mm diameter) using sterile cotton swabs as a lawn culture. Up to Nine commercially used antibiotic disks were placed on the inoculated agar surface. Plates were incubated for 24 h at 37°C prior to determination of results. The diameters of zones of inhibition around the discs were measured in millimeter (mm). Multi-drug resistant (MDR) was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories. The experiment was performed in triplicates.
1.3. Biofilm formation Assay. Biofilm formation assay was carried out by the modified method of crystal violet staining assay in a test tube made with glass. Fresh single colony of the bacterial strain was picked up and grown in LB broth medium for 18-20 h. Bacterial suspensions were added to 5 ml fresh LB broth by maintaining initial absorbance 0.02 at 600 nm and incubated at 37 °C for 36 h in static condition. Only LB broth was also incubated as a negative control. Following 36 h of adhesion and biofilm formation, planktonic cells were removed from the test tube followed by rinsed (twice) with 5 ml distilled water. 5 ml of crystal violet solution (1% w/v) was added to stain the biofilm and incubated at room temperature for 30 min. Excess stains were then washed with 5 ml distilled water twice. The test tubes were then dried in air for 20 to 30 min. The attached dye was solubilized with 95% ethanol and the adherent biofilm was determined by measuring the optical density at 490 nm. LB broth was used as a negative control (background absorbance). All isolates were tested at least three times in triplicate. For interpretation of the biofilm results, the isolates were classified as follow: non-producing, weak, moderate and strong-producing, based on the following optic density (OD) average values: OD(isolate) ≤ OD(control) = non biofilm producing; OD(control) ≤ OD(isolate) ≤ 2OD(control) = weak producing; 2OD(control) ≤ OD(isolate) ≤ 4OD(control) = moderate producing; 4OD(control) ≤ OD(isolate) = strong producing.
1.4. Plants and Spices material. Leaves of plants Centella Asiatica (Thankuni), Mentha spicata (Mentha), Azadirachta indica (Neem) and Psidium guajava (Guava) were obtained from local region of Kushtia, Bangladesh. Spices Syzygium aromaticum (Cloves), and Cinnamomum zeylanicum (Cinnamon) were bought from local market of Kushtia, Bangladesh. Plants and spices were identified by prominent botanist Dr. Nilufa Akhter Banu, Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia.
1.5. Preparation of plants and spices extract. All collected plants and spices material was air-dried at room temperature under shade and then ground with a grinder into fine powders. The powder materials were macerated with methanol and ethyl acetate solvent separately using a ratio of 1 g (plant material) and: 10 ml (solvent) for 72 h. Flasks were agitated daily. Re-extraction was done for a further 24 h. The individual extracts were filtered through Whatman no. 4 filter paper. The filtrate, each case, was concentrated using a rotary evaporator at 40°C. The stock concentration of 2mg/ml of each dry extract in the excipient Dimethyl sulfoxide (DMSO) were prepared, sterile filtered with 0.22 µm micro filter (Millex R filter, Carl Roth, Karlsruhe, Germany) and then stored in the dark at 4°C.
1.6. Antibacterial Assay of Extracts. Well Diffusion method was used to test the antibacterial activities of different extracts. Selected multi-drug resistant bacterial isolates were grown in LB broth for 18-20 h and prepared the bacterial inoculums by maintaining turbidity of 0.5 McFarland standard (equal to 1.5×108 colony forming units (CFU)/ml). Mueller-Hinton agar (MHA) plates (150 mm diameter) were prepared and the bacterial suspensions were spread over the surface. The wells (9 mm diameter) were made by using cork borer in MHA plates. Each well was loaded with 100 µl of different crude extracts and 100 µl DMSO (without extract) was loaded as negative control. Plates were incubated at 37 0C for 24 h. The diameters of zones of inhibition around the wells were measured in millimeter (mm). The experiment was performed in triplicates.
1.7. Antibiofilm Assay of Extracts. A modified crystal violet assay was employed to test the effect of plant extract on bio-film formation. Here, 200 µl of different crude extract was added separately with bacterial suspension and incubated at 37 °C for 36 h. Biofilm formation was determined by following the biofilm formation assay.
1.8. Determination of Minimum Inhibitory Concentration (MIC). Antibacterial activities of the extracts were first screened by agar-well diffusion method as described previously. The MIC testing was performed against selected five MDR Pseudomonas aeruginosa strains by agar-well diffusion method. After preparing the bacterial inoculums by maintaining 0.5 McFarland standard, it was spreaded on MHA plate. The wells (9 mm diameter) were made by using cork borer in MHA plates. Each well was loaded with 100 µl of different concentration crude extracts ranged from 50 to 2000 µg/ml and 100 µl DMSO (without extract) was loaded as negative control. Plates were incubated at 37 0C for 24 h. MIC was regarded as the lowest concentration that produce a visible zone of inhibition.
1.9. Statistics. Statistical analysis was performed using SPSS version 16.0 software 2007 (SPSS Inc., Chicago, IL). All experiments were performed in triplicates. For antibiofilm activity studies, mean values between extract-treated and untreated samples were tested for significance by Student’s t-test. The significant difference in biofilm reduction by different extracts was compared with the control (strain without the extract was normalized as 100%). The significant level was set at <0.5.