Testing the Susceptibility of E.

Coli to Penicillin Using Various Bioenhancers

(herbal extracts from the Zingiberaceae Family and the Caprifoliaceae Family)

Julia Takla

taklajulia52@gmail.com

Marine Academy of Technology and Environmental Science

Research Proposal

08/02/19
Introduction:

The Zingiberaceae family and the Honeysuckle family are clades of numerous species of flower

plants containing extracts with several benefits. These plants have been used in the past as

anti-inflammatories and antioxidants, both causing many health benefits. The caprifoliaceae family was

introduced in the early 1800’s whereas the zingiberaceae has been used as far back as 500 b.c. The ginger

family is native to South-Eastern Asia and Africa whereas the honeysuckle perennials originate in Japan,

East Asia, and Korea.

In the research, herbs and extracts from both flower plant families are tested for their antimicrobial

effectiveness against E. Coli, S. Epidermidis, and Rhodospirillum rubrum using the disc diffusion method.

Extracts from the ginger family include a galangal organic infusion and a curcumin solution. From the

honeysuckle family, abelia grandiflora and Japanese honeysuckle. E. coli ( or Escherichia Coli ) is a gram

negative bacteria that can be a pathogen that can involve different systems of the body. It can different

diseases based on the system which it attacks such as pneumonia if it affects the respiratory tract,

gastroenteritis if it affects the gastrointestinal tract, and UTI when it affects the urinary tract. By

completing this experiment, I will be able to determine which extract from either of the botanical families

can inhibit bacteria which is useful for people who have developed antibiotic resistance from constantly

relying on medication when it is not needed. The widespread use of antibiotics and the development of

antibiotic resistance has been a global concern and that is why more studies and attention has to be paid in

exploring different antimicrobial properties of other natural plants and herbs.

Hypothesis:

If Zingiberaceae and Caprifoliaceae plant extracts are tested for an antimicrobial effect as bioenhancers I

believe that the curcumin solution from the Zingiberaceae family will be most effective because it is the

most bioactive ingredient in turmeric and is used as an ingredient in antiviral gels. Curcumin has been

thought to contain properties similar to antibiotics according to some theories.

Objective:

Determining which flowering plant family extract is more effective in inhibiting E. Coli with penicillin

using the disc diffusion test (Kirby-Bauer Method).

Methodology:

Materials:

-Japanese Honeysuckle Extract -Tweezer

-Abelia Grandiflora -Bunsen Burner (or any sterilizer)

-Curcumin Capsules -Incubator

-Safety Goggles -E. Coli bacteria.

-Organic Galangel Solution (alpinia galanga herb) -Gloves

-Three Petri Dishes with Pre-poured Agar -Cotton Swabs

-Rhodospirillum Rubrum bacteria. - S. Epidermidis

- (about 50) Neomycin discs -pipette

Procedure:

1. Put on lab apron, gloves, and safety goggles for protection while experimenting.

2. Gather all materials before starting and make sure you are working in a clean environment.

3. Decant the nutrient agar in a petri dish and wait about 15 minutes for the agar to harden.

4. Apple the E. Coli culture on a petri dish using a cotton swab and applying in a zig- zag motion.
 5. Label the penicillin disks 1-3 (1 being the Japanese honeysuckle, 2 being the valerian root extract,

and 3 being the alpinia galanga).

6. Place the three disks into the agar with a tweezer and place a fourth unlabeled disk without any

bioenhancer (this will be the control).

7. Drops of each extract will be combined with the penicillin discs using a pipette according to the

labels on each disk.

8. Steps 3-7 were repeated about 30 times over the course of two days. The more trials, the more

accurate the results.

9. Place the dish in an incubator and set the temperature to 37℃. Leave for about 72 hours or more.

Check the dishes frequently to see if there is any growth.

10. Take the Petri dishes out of the incubator and observe the plate. Around each disc, there should be

a ring around it with a clear area in which the bacteria streaks are not visible. This is called the

zone of inhibition.

11. The diameter of the zone of inhibition will be measured in millimeters for each disk on the plates.

12. Record all the data in a notebook or a journal. It is suggested that you make a table with each

bioenhancer and record the diameter.

13. Before the disposal of the culture plates, sterilize the dishes by autoclaving so the plates are

completely decontaminated, then discard them.

14. Clean up the workspace and turn off the incubator.

Estimated Timeline:
September- Research Proposal should be edited and finalized. Start a journal that will include an account

of the progression of the project.

October- All materials will be purchased. Continue to research and expand knowledge on the honeysuckle

family and ginger family. And additional important info learned will be recorded in the journal.

November- Grow the bacteria and record the results of the disc diffusion test. Take pictures of the

experiment and include the visual images in the journal.

December- Analyze recorded results and plan and design a visual representation of the research

experiment. Add a data analysis section in the journal.

January- Practice oral presentation finalize the journal with any ideas of further research ideas. Include a

conclusion and thoughts on the results.

Resources:
Works Cited

Staphylococcus Epidermidis, web.uconn.edu/mcbstaff/graf/Student presentations/S

epidermidis/sepidermidis.html.

“Benefits of Galangal - Times of India.” The Times of India, The Times of India, 6 July 2017,

timesofindia.indiatimes.com/life-style/health-fitness/diet/benefits-of-galangal/articleshow/8065447

.cms.

Cowan, Marjorie Murphy. “Plant Products as Antimicrobial Agents.” Clinical Microbiology Reviews, vol.

12, no. 4, 1999, pp. 564–582., doi:10.1128/cmr.12.4.564.

Cowan, Marjorie Murphy. “Plant Products as Antimicrobial Agents.” Clinical Microbiology Reviews, vol.

12, no. 4, 1999, pp. 564–582., doi:10.1128/cmr.12.4.564.

“Disk Diffusion.” Disk Diffusion - an Overview | ScienceDirect Topics,

www.sciencedirect.com/topics/immunology-and-microbiology/disk-diffusion.

“E. Coli Information.” Marler Blog, www.marlerblog.com/articles/e-coli-information/.

Hewlings, Susan J, and Douglas S Kalman. “Curcumin: A Review of Its' Effects on Human Health.”

Foods (Basel, Switzerland), MDPI, 22 Oct. 2017,

www.ncbi.nlm.nih.gov/pmc/articles/PMC5664031/.

Munk, A Christine, et al. “Complete Genome Sequence of Rhodospirillum Rubrum Type Strain (S1).”

Standards in Genomic Sciences, Michigan State University, 1 July 2011,

www.ncbi.nlm.nih.gov/pmc/articles/PMC3156396/.

Sharma, Gaurav. “Synthesis, Characterization and Antimicrobial Activity of Abelia Grandiflora Assisted

AgNPs.” Journal of Microbial & Biochemical Technology, vol. 06, no. 05, 2014,

doi:10.4172/1948-5948.1000156.

“Using Medication: Using Antibiotics Correctly and Avoiding Resistance.” InformedHealth.org

[Internet]., U.S. National Library of Medicine, 18 Dec. 2013,

www.ncbi.nlm.nih.gov/books/NBK361005/.

“Medicinal HerbsJapanese HoneysuckleLonicera Japonica.” Medicinal Herbs: JAPANESE

HONEYSUCKLE - Lonicera Japonica,

www.naturalmedicinalherbs.net/herbs/l/lonicera-japonica=japanese-honeysuckle.php.

Avasthi, A. S., Jain, S., Bhatnagar, M., & Ghosal, S. (n.d.). In vitro antibacterial, antifungal,

antioxidant and antihemolytic activities of Alpinia galanga. Retrieved from

https://www.arjournals.org/index.php/ijpm/article/view/1652

The article is a summary of an investigation in which alpinia galanga was tested for

antifungal and antioxidant properties. This pdf provides information on the background of

this perennial and its biological activities.

Littmann, J., & Viens, A. M. (2015, November). The Ethical Significance of Antimicrobial

Resistance. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4638062/

This informative article explains the complication of antibiotic resistance and gives details

on how the issue can be resolved. It further provides more about the raised issue of drug

development.

Medicinal Plant: 1 The Japanese Honeysuckle 1 Caitlyn Scott. (n.d.). Retrieved from

http://mason.gmu.edu/~cscottm/plants.html

Japanese Honeysuckle properties are numerous and my investigation will compare the

antibacterial effect it has to other flower extract. This article provides information on the

chemical structure luteolin- an active compound in japanese honeysuckle that could be

correlated to its effects.

4) Tang, X., Xu, C., Yagiz, Y., Simonne, A., & Marshall, M. R. (2018, July 30). Phytochemical

profiles, and antimicrobial and antioxidant activities of greater galangal [Alpinia galanga

(Linn.) Swartz.] flowers. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/29571480

JULIA TAKLA, BLOCK 4, SCIENCE CLASS, MARINE ACADEMY OF TECHNOLOGY AND

ENVIRONMENTAL SCIENCE (MATES), ADVISOR: DR. JOHN WNEK
Antibiotic resistance is the ability of microbes to resist the effect of medication. This has become a global
concern for the reason that 2.8 million people are affected yearly. A bioenhancer is an agent capable of
enhancing bioavailability and efficacy of a drug with which it is co-administered, without any
pharmacological activity of its own at therapeutic dose used. Several conducted studies showed that
bioenhancers can reduce the development of antibiotic resistance. In this experiment, various
bioenhancing agents were used synergistically with penicillin to test their combined effect against
Echerichia coli. The bacteria was applied onto petri dishes and herbal extracts from the zingiberaceae and
caprifoliaceae families were added to 10 mcg 5 enicillin sensitivity discs. The extracts included Japanese
honeysuckle, valerian extract, and alpinia galanga. These discs were added to the plates and after being
left for 72 hours, developed a ring of clear area. The diameters of the zone of inhibitions were recorded
and then compared to those of the penicillin exclusively. The general trend of the data showed that there
was a slight increase of the zone of inhibition with bioenhancers (specifically the Japanese honeysuckle)
as compared to without it. This indicates that there could be more plant-based antimicrobials that can
serve as a temporary aid against antibiotic resistance.