Discovering Novel Antibiotics From Medicinal Plants to Combat The Rise of Super-Bacteria

Raised of $6,330 Goal
Ended on 6/18/17
Campaign Ended
  • $841
  • 14%
  • Finished
    on 6/18/17



Methods To Identify Potential Bioactive Extracts and Eluates:

1.1. Bacterial Isolates

The microorganisms tested were all environmental isolates. They were representative of Gram negative, Gram positive, aerobic and facultative anaerobic categories. They comprised of Bacillus cereus, Escherichia coli, Micrococcus luteus, Mycobacterium avium subsp. avium, Paenibacillus alvei and Aeromonas caviae. They were streak plated on LB agar and maintained at 4 oC in dark. Stock freezer cultures were kept at -80 oC.

1.2. Collection of Plant Material

            The plant materials in this study were whole plant samples, including root and flower for some plants.  They were collected during the late flowering season (June-August) in Thunder Bay region, Ontario. The specimens were identified by Erika North, herbarium curator and contract lecturer at Lakehead University, Thunder Bay, Ontario. The scientific names were based on those proposed by Morton and Venn (1990). All voucher specimens were deposited in -80 oC fridge at the Bio-Refining Institute, Lakehead University.

1.3. Extraction and Separation

            The collected plant samples were separated into root, stem, flower and leaf parts. The plant parts were dried in dark over 24 - 48 hours at 30 oC. The dried plant samples (2 g) were ground up, milled through no. 40 mesh and extracted twice with 50 ml of ethanol (85% v/v) over a period of 48 hours at room temperature. All extracts were filtered through Whatman No. 1 filter paper, evaporated to dryness under rotary evaporator at 34 oC and stored at -80 oC in glass vials.

            The extracts were subjected to manual LC on silica gel (40-60μm; 1 x 14cm) using Hex/ EtOAc/ MeOH 9:1:0, 5:1:0, 0:1:0, 0:9:1, 0:3:2, 5ml/min, in 100 ml aliquots.  All fractions were dried in a rotary evaporator and high vacuum for 24 hours, and stored at -80 oC.

1.4. Hole-Plate Diffusion Assay

            The procedure was followed according to Gallo and Riera (2005) with modifications. In this assay, 100 μl of 2x106 CFU/mL bacterial suspensions were homogenously seeded into 25 ml of liquid broth agar and poured into 100 mm petri dishes. Then, 6 mm holes were aseptically bored into agar and 40 μl of 10 mg/ml methanolic extracts were pipetted into the wells. The plates were kept at 4 oC for 3 hours, then, incubated at 32 oC for 18 - 20 hours in dark. Chloramphenicol (100 μg/ml) and ampicillin (100 μg/ml) were used as positive controls and methanol was used as negative control. All results were recorded as mean of the triplicate and the zones of inhibition were determined as the diameter of inhibition zones around the holes.

1.5. MIC and MBC Assay

            The minimum inhibitory concentration (MIC) assay was performed according to Sarkar et al. (2007) with modifications. First and second fractions from each extract were dissolved in EtOAc/ MeOH 6:1, whereas, the rest of fractions were dissolved in methanol (99.85 % v/v). The extracts were re-suspended to a concentration of 20 mg/ml.  Then, 50μl of 20 mg/ml extract was aseptically added to 230 μl of LB broth in the first well of a sterile 96 well micro-plate. To each subsequent well, 180 μl of LB broth was added. Two fold serial dilutions, in 100 μl aliquots, were performed using a multichannel pipette to achieve a concentration gradient of 5.00 - 0.04 mg/ml. Prior to each subsequent dilution, precaution was taken to maximize homogeneity of the solution. Then, 20 μl of 2 x 106 CFU/ml bacterial suspensions in 1X PBS buffer were inoculated in their respective wells (to a final volume of 200 μl), the plate was wrapped with parafilm and placed in a plastic bag with mildly wet paper towel to prevent dehydration of bacteria. The plates were incubated at 32 oC for 18 hours in dark. After incubation, 10 μl of 0.04% resazurin in 1 X PBS buffer was added and the plate was further incubated in dark at 32 oC for 90 - 180 minutes. Micro-plates were viewed under short wavelength UV lamp for fluorescence and color change. The lowest concentration in which color changed from blue to pink, and displayed fluorescence, was taken as MIC. The MBC value was determined by plating 15 μl aliquots from wells without visible growth.  Wells exhibiting complete annihilation of bacteria after incubation of LB agar plates at 32 oC for 18- 24 hrs were taken as MBC. A two-fold serial dilution of chloramphenicol (100 μg/ ml) and ampicillin (100 μg/ml) were used as positive controls, and EtOAc/ MeOH 6:1, 0:1 as negative control.  The tests were performed in duplicates.

1.6. Time-Kill Analysis

            Time-kill analysis was performed according to Imazatol et al. (1999). Bacterial suspensions prepared in 1 X PBS buffer were added in 2 ml of LB broth to a final concentration of 2x106 CFU/ ml. The LB broth was prepared with a certain concentration of plant extracts and incubated at 30 oC with shaking at 200 rpm. After 30 min, 1 hr, 2 hr, 4 hr, 8 hr, 12 hr and 24 hr of incubation, 40 μl aliquot was acquired, serially diluted and plated on LB agar. The plates were incubated at 32 oC for 18 - 24 hrs and the numbers of viable cells were determined by counting the colonies. The test was performed in duplicates. 

1.7. Statistical Analysis

            The results were expressed as means ± standard deviation (SD) of two or three replicate measurements. The standard error was calculated in MS Excel v. 2011.


The challenges for this campaign will be to match our findings from the panel of 6 bacteria to a panel of super-bacteria. In addition, the bioactivity in our promising extracts could be caused by either one single compound or a series of pharmacophores that act in concert. For pharmaceutical reasons, its beneficial that the bioactivity be traced to a single compound, which I will try and identify. 

Pre Analysis Plan

The analyses will be performed with appropriate negative and positive controls. 

Negative Controls: Solvents that the extracts or eluates are dissolved in (etc. ethanol or methanol for crude extracts) 

Positive Controls: Ampicillin and Chloramphenicol at the dosage its used in clinic  


This project has not yet shared any protocols.