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Rationale: Antibiotic Resistance

The gut microbiome has an important role in maintaining good health

(Ohland and Jobin, 2015)

Many cases of antibiotic resistance and side effects have been recorded

(CDC, 2020)

About 35,000 people in the U.S. are dying from antibiotic resistant infection

(CDC, 2020)

Natural alternatives to antibiotics are increasingly being used because rise of antibiotic resistance and side effects

(Ohland and Joblin, 2015)

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Rationale: Antibiotics

Penicillin

Treat infections: gastrointestinal infections and skin infections
Treat most Gram-positive bacterial infections (Lobanovsk, 2017)
Side effects: fever, chills, nausea, and vomiting (Lobanovsk, 2017)

Gentamicin

Treat most Gram-negative bacteria infections (Aronson and Meyler, 2016)
Side effects: upset stomach, vomiting, and fatigue (Aronson and Meyler, 2016)

Chosen due to cost and availability

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Rationale: Garlic

Garlic (Allium sativum) is one of the most common alternative being used (Bayan, 2014)

Garlic holds many probiotic components that help promote a healthy gut (Bayan, 2014)

Experimenting the effectiveness of the treatments on gut microbiome (bacteria)

Created by: Bio-botinica.com

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Rationale: Bacteria

Escherichia coli (E. coli)

Reside in the intestines (Bentley and Meganthan, 1982)
Gram-negative bacteria (Bentley and Meganthan, 1982)
Most are non-pathogenic, but few strains are pathogenic (CDC, 2015)

Lactobacillus acidophilus (L. acidophilus)

Reside in the intestines (Ozogul and Hamed, 2016)
Gram-positive bacteria (Ozogul and Hamed, 2016)
They are very abundant in the human gut

Chosen due to cost and availability

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Research Questions

How do alternative remedies affect Escherichia coli (a gut bacterium) growth compared to standard antibiotics?

How do alternative remedies affect Lactobacillus acidophilus (a gut bacterium) growth compared to standard antibiotics?

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Hypotheses

If I use penicillin, then it will create a larger zone of inhibition for Lactobacillus acidophilus when compared to gentamicin and garlic.

Null:There will be no difference in growth of Lactobacillus acidophilus when treated by either penicillin or gentamicin when compared to garlic.

If I use gentamicin, then it will create a larger zone of inhibition for Escherichia coli when compared to penicillin and garlic.

Null: There will be no difference in growth of Escherichia coli when treated by either penicillin or gentamicin when compared to garlic.

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Variables

Independent Variables: Penicillin, Gentamicin, & Garlic

Dependent Variable: Zone of inhibition

Constants: Type of agar, type of antibiotic/remedy, type of bacteria, number of discs, time in the incubator (120 hours), incubation temperature (37ºC)

Control: Disk with no treatment

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Experiment Design

Escherichia coli - Nutrient Agar

Created by: Karihn Handy

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Experiment Design

Lactobacillus acidophilus - Lactobacilli MRS Agar

Created by: Karihn Handy

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Disk Diffusion Assay Procedure

Species swabbed onto respective agar
1-2 discs placed onto agar
Inoculated plates placed into incubator at 37 ºC
Zones of inhibition were measured and recorded in mm every 24 hours for 5 days

Created by: http://download.systemsbiology.nl/~jasperk/koen/Practical/WetLab/Week4/Monday/AntibioticResistanceTest/

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Photos taken by: Karihn Handy

Zone of Inhibition

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Analysis of Data

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Data table created by: Karihn Handy

Average Zones of Inhibition for Different Treatments on Escherichia coli Over 5 Days
	Day 1	Day 2	Day 3	Day 4	Day 5
Penicillin	5.00 mm	5.44 mm	6.63 mm	7.02 mm	6.26 mm
Gentamicin	23.26 mm	22.54 mm	23.46 mm	22.96 mm	21.74 mm
Garlic	29.88 mm	31.48 mm	31.23 mm	30.63 mm	30.66 mm
Control	0.00 mm	0.00 mm	0.00 mm	0.00 mm	0.00 mm

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Graph created by: Karihn Handy

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Photos taken by: Karihn Handy

Garlic Extract Treatment

Gentamicin Treatment

Penicillin Treatment

Control Group

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Data table created by: Karihn Handy

Average Zone of Inhibitions for Different Treatments on Lactobacillus acidophilus Over 5 Days
	Day 1	Day 2	Day 3	Day 4	Day 5
Penicillin	0.00 mm	57.36 mm	52.79 mm	53.31 mm	55.46 mm
Gentamicin	0.00 mm	29.26 mm	26.22 mm	23.80 mm	22.81 mm
Garlic	0.00 mm	22.09 mm	19.24 mm	0.00 mm	0.00 mm
Control	0.00 mm	0.00 mm	0.00 mm	0.00 mm	0.00 mm

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Graph created by: Karihn Handy

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Photos created by: Karihn Handy

Penicillin Treatment

Control Group

Gentamicin Treatment

Garlic Extract Treatment

Garlic Extract Treatment w/ bacterial growth

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Statistical Analysis

Escherichia coli

Day 1 - p < 0.00001

Day 2 - p < 0.00001

Day 3 - p < 0.00001

Day 4 - p < 0.00001

Day 5 - p < 0.00001

Lactobacillus acidophilus

Day 1 - p < 0.00001

Day 2 - p < 0.00001

Day 3 - p < 0.00001

Day 4 - p < 0.00001

Day 5 - p < 0.00001

On each of the 5 days, statistically significant data was observed, indicating the treatments did impact the growth of E. coli and L. acidophilus

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Conclusion

Garlic Extract proved to be the most effective against E. coli
Gentamicin had the second largest zone of inhibition on E. coli
Penicillin had little to no zone of inhibition on E. coli
Penicillin proved to be the most effective against L. acidophilus
Gentamicin had the second largest zone of inhibition on L. acidophilus
Garlic was effective on L. acidophilus to an extent

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Limitations/Problems

A few discs were contaminated
Measurements and pictures were not taken at the same time
Had to redo garlic trial on L. acidophilus

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Future Research & Significance

More types of treatments could be tested
More trials can be conducted
Testing on other probiotic species
Experiment shows different types of treatments that could be used

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Acknowledgments

I would like to give my thanks to:

Karisa Boyer

LaHeather Fisher

Adam Bennett

Whitmire Foundation (funding)

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Work Cited

Aronson, J. K., and L. Meyler. Meyler's Side Effects of Drugs: the International Encyclopedia of Adverse Drug Reactions and Interactions. Elsevier, 2016.

Bentley, R, and R Meganathan. “Biosynthesis of Vitamin K (Menaquinone) in Bacteria.” Microbiology and Molecular Biology Reviews, American Society for Microbiology Journals, 1 Sept. 1982, mmbr.asm.org/content/46/3/241.

Bayan, L., Koulivand, P. H., & Gorji, A. (2014). Lactobacillus acidophilus. Retrieved February 19, 2021, from https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/lactobacillus-acidophilus

Centers for Disease Control and Prevention (CDC). (2015). General Information-E.coli. Retrieved October 18, 2020, from https://www.cdc.gov/ecoli/general/index.html

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Work Cited (Continued)

CDC. “Antibiotic / Antimicrobial Resistance.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 20 July 2020, www.cdc.gov/drugresistance/index.html.

Lobanovska M, Pilla G. “Penicillin's Discovery and Antibiotic Resistance: Lessons for the Future?” The Yale Journal of Biology and Medicine, U.S. National Library of Medicine, pubmed.ncbi.nlm.nih.gov/28356901/.

Ohland, C. L., & Jobin, C. (2015). Microbial Activities and Intestinal Homeostasis: A Delicate Balance Between Health and Disease. Cellular and Molecular Gastroenterology and Hepatology, 1(1), 28-40.

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