Did you know that in the last 30 odd years approximately 73% of all small molecule antibacterial drugs are from or derived from natural sources including plants? In recent years there has been increasing awareness for the need to develop new drugs which are able to combat drug-resistant bacteria. Thus, our very own biodiversity still plays a crucial role with respect to drug discovery.

In order to find new and effective drugs, an extract containing natural, naturally derived, semi-synthetic, or synthetic chemical constituents (compounds) are screened using a bioassay. A bioassay is a scientific approach used to measure the concentration or potency of a substance by observing its effect on a biological system. For many years, bioassays were performed by injecting animals with plant extracts and observing their effects. This is otherwise known as “Hippocratic screening”. Although this method has been widely employed and helped to facilitate the discovery of many biologically active compounds, economic and social pressures have led to the development of cell, enzyme and receptor based high throughput screening  programs in the 1980s. Consequently, automated screening for hundreds if not thousands of extracts using in vitro approaches is now standard protocol.

Despite the application of high throughput screening for over three decades, discovery of new drugs from natural sources remains low and can likely be attributed to the following : 1) biologically active compounds are in low abundance within complex mixtures and consequently are not detected; 2) false positives or negatives are observed due to the presence of compounds which display non-specific activities; 3) interference is caused by compounds which interact with light-based detection methods; 4) adequate drug targets are unavailable or yet to be developed; 5) synergy of multiple active compounds;  and 6) elaborate and costly methods are required to isolate biologically active constituents. Thus, new and innovative approaches for screening plant extracts are still needed.

Devanshi Shukla a high school student from Guelph, Ontario hopes to one day discover new plant antimicrobials as well as develop new screening techniques. Below she shares her experience of working within a research environment for the first time during a recent visit to the Gosling Research Institute for Plant Preservation (GRIPP).

It is not very common to see a high school student working in a university lab amongst post-doctoral fellows and research technicians. However, a normal day for me entailed finishing school and going to the University of Guelph to do research for my high school science project. Being able to carry out my research while still in high school has had immense effects on my student and personal life. I have been able to acquire invaluable skills, deepen my knowledge in the scientific field and grow as an individual. This has all been possible because of GRIPP, which allowed me access to resources and the chance to learn from intelligent scientists, in order to expand my knowledge and delve into the rich and interesting field of plant biology.

Antibiotic resistance in bacteria is on the rise, with hospitals combating deadly infections caused by commonly treated bacteria. As an alternative to conventional antibiotics, scientists hope to develop new and effective antimicrobials through the examination of our very own plant biodiversity. Traditionally, plants have been playing an important role amongst various cultures for treating bacterial infections and in more recent years have led to the development of various antimicrobial drugs. Still, there is a need for new ways to determine the effectiveness of antibacterial properties in medicinal plants and their metabolites. In other words, it is important to screen for which plant chemicals (compounds) are most effective in treating bacterial infections in order to discover new treatments.

Fig 2. Bioluminescences

Bioluminescence of V. fischeri

In order to investigate the antimicrobial efficiency of plant derived compounds – I developed a bioassay involving the use of the brilliantly bioluminescent marine bacteria Vibrio fischeri. In the case of V. fischeri, quorum sensing allows for the bacteria to glow (bioluminescence) once a certain population level has been reached. Quorum sensing, in other words bacterial communication, allows for certain group behaviours to occur. It is only when bacteria are able to communicate with each other, that they are able to express genes that dictate group behaviours such as virulence, antibiotic resistance and bioluminescence such as in V. fischeri.

A bacterium will naturally release hormone molecules called autoinducers into its surroundings. As cell density increases, so does autoinducer production. As the concentration of autoinducers increases, bacterial cells recognize autoinducers produced by other bacteria. An autoinducer from another bacterium can bind to a protein complex of separate bacterium and induce the expression of certain genes. Thus, these genes are known as group behavioural genes because they are only expressed when there is a certain cell density, and this is how quorum sensing works.

Fig. 2 Qorum sensing

The most important tool of this research involved using the bioluminescence of V. fischeri to indicate the effectiveness of the treatment. Lowered luminescence was attributed to an efficient antimicrobial, and vice versa. This was due to the fact that it indicated the inhibition of bacterial communication which is crucial in treating a bacterial infection.

Results

Bioassay Results

Various medicinal plants were tested for their inhibition of quorum sensing, including Holy Basil (Ocimum sanctum L.), Cumin (Cuminum cyminum L.), Coriander (Coriandrum sativum L.), Turmeric (Curcuma longa L.), Nutmeg (Myristica fragrans Houtt.) and Artemisia (Artemisia annua L.). First off, various concentrations of the medicinal plant extracts were added to liquid cultures of V. fischeri and revealed that suppression of bioluminescence occurred in liquid bacterial cultures which contained medicinal plant extracts. Holy basil extract was the most efficient in reducing the luminescence and cell population (Optical Density readings) of V. fischeri. This suppression of luminescence indicated that there was quorum sensing inhibition as the bacterial communication allows for the bacteria to bioluminesce.

Fig 2. Holy Basil

Holy Basil Plant Growing in the Greenhouse

The known compounds of Holy basil essential oil: linalool and eugenol, were identified as powerful antibacterial agents, not only through their direct contact but through their volatile effects (ability to travel in the air) as well. This was tested first by placing freshly cut leaves of Holy basil inside of a Petri plate streaked with V. fischeri and by preventing direct contact of the leaves.Through the above, I observed lowered bioluminescence and cell growth in the Petri plate, indicating that the volatile compounds found in Holy basil had an antimicrobial effect. In addition to the above, liquid bacterial cultures were treated with linalool and eugenol without direct contact. This was done by placing a sterile plastic pipette tip containing a cotton plug soaked in either eugenol or linalool inside an Erlenmeyer flask containing liquid bacterial culture. Again, suppression of bioluminescence and cell growth was seen in the liquid bacterial cultures. To further examine the quorum sensing inhibition through volatile effects, seven different essential oils and six different volatile compounds were tested.

All the compounds tested showed varying degrees of quorum sensing inhibition (seen through bioluminescence suppression). Thus, V. fischeri served to highlight the anti-microbial efficiency of the various plant compounds tested.  From my experiments, I concluded that various

Fig 5. VOC exp

Pipette Tip Containing a Cotton Swab Soaked in Essential Oil and Placed in a V. fischeri Bacterial Culture. .

plant compounds actively inhibit quorum sensing in V. fischeri, meaning that they have the potential to be effective treatments for bacterial infections. As discussed previously, if quorum sensing is inhibited, the bacterial population cannot express genes that cause virulence – making it a target mechanism to disrupt for efficient treatment. Therefore, many of the plant compounds tested in these experiments have promising applications for the healthcare field.

At this young age, I am already very invested in the field of research, and I feel lucky to be aware of the importance and specialities of plants.

Fig. 6 Award

Fig 6. Devanshi Receiving the GRIPP Award for Scientific Excellence

Through my own experiments, I was able to discover the extent of the effectiveness of plant compounds as anti-bacterial agents, specifically in inhibiting bacterial communication. I have been inspired to continue researching in my future, and have had my eyes opened to some of the secrets held by plants. For this work, I was honoured to be recognized with the Scientific Excellence Award presented by GRIPP. I earned the Best in Division and Award of Merit at the Waterloo Wellington Science and Engineering Fair and a Gold Medal and Challenge Award for Innovation at the Canada Wide Science Fair. I also placed First Place at the Science and Plants Fair, held at the University of Guelph. I would like to express my sincere gratitude to Professor Praveen Saxena for giving me the opportunity to do research work at GRIPP. My research helped me uncover a few special things about plants, but there is a plethora of things still waiting to be discovered. In essence, plants are nature’s treasure chests.

References

Hastings, J.W., Greenberg, E.P. (1999). Quorum Sensing: the explanation of a curious phenomenon reveals a common characteristic of bacteria. Journal of Bacteriology 181(9), 2667-2668.

Kováts, N., Ács, A., Gölöncsér, F., & Barabás, A. (2011). Quantifying of bactericide properties of medicinal plants. Plant Signaling & Behavior, 6(6):777-779.

Miller, J.S. (2011). The discovery of medicines from plants: A current biological perspective. Economic Botany, 65, 396-407.

Mishra, K.P., Ganju, L., Sairam, M., Banerjee, P.K., & Sawhney, R.C. (2008). A review of high throughput technology for the screening of natural products. Biomedicine & Pharmacotherapy, 62, 94-98.

Newman, D.J., & Cragg, G.M. (2016). Natural products as sources of new drugs from 1981 to 2014. Journal of Natural Products, Doi: 10.1021/acs.jnatprod.5b01055.

Potterat, O., & Hamburger, M., (2013). Concepts and technologies for tracking bioactive compounds in natural product extracts: generation of libraries, and hyphenation of analytical processes with bioassays. Natural Products Reports, 30, 546-564.

Stromstedt, A.A., Felth, J., & Bohlin, L., 2014. Bioassays in natural product research – Strategies and methods in the search for anti-inflammatory and antimicrobial activity. Phytochemical Analysis, 25, 13-28.

Posted by Christina Turi