References
Introduction
Results
Future Directions
Future steps may include:
If procyanidin B1 does not show any other health benefits, the next step would be to inform and caution the public of its probiotic properties.
Jada Sherrill, Kylee Whittington, Maddie Langan, Sam Ghushchyan
The Effects of Procyanidin B1 on Salmonella Typhimurium Growth
Abstract
Hypothesis
Acknowledgments
Methods
Conclusions
We believe that procyanidin B1 will show antibiotic properties and effectively kill Salmonella Typhimurium. Our hypothesis will be proven correct if the absorbance levels shown on the spectrophotometer are low and comparable to our positive control.
We set up a series of different experiments to test varying concentrations of our compound against Salmonella Typhimurium to determine if a specific concentration of procyanidin B1 had antibiotic properties. For each of these tests, we used Salmonella Typhimurium, procured from the Su Lab at the University of Colorado, Boulder, at 10^5 cells/mL kept in M9 minimal media. Our negative control is 50% DMSO, which comes from Fisher bioreagents Fair Laun, NJ. Our positive control is 50mg/mL ampicillin from research products international corp. Mount Prospect, IL. After each test, we incubated the plate at 37 degrees Celsius for 24 hours.
Max Dose
Dose Response
Purpose:
To see how well the compound performs at the maximum dose allowed to administer in humans
Purpose:
To make a final conclusion with a different test.
Dose Response
Mueller- Hinton
10uM = concentration of compound
We added 90uL of Salmonella and 10uL of compound solution at 10uM.
Antibiotic resistance is becoming a globally significant health concern, and the discovery of new classes of antibiotics is crucial for the long-term health of the human population. We believed that procyanidin B1 would show antibiotic properties against Salmonella Typhimurium. However, our results from various tests, including the 1:2 dilutions, 1:10 dilutions, and the solid media plate, showed that the bacterial growth was comparable to our negative control. Therefore, it is not viable as an antibiotic.
Initially, we wanted to test proanthocyanidins, anthocyanins, and an extract from Cornelian cherries to compare the results, but we could not obtain any other compounds. In future experiments, we would test different compounds present in the Cornelian cherry to determine if this Eastern medicinal remedy could be a viable antibiotic candidate or if it is causing bacterial growth.
Breijyeh, Z., Jubeh, B., & Karaman, R. (2020, March 16). Resistance of gram-negative bacteria to current antibacterial
agents and approaches to resolve it. Molecules (Basel, Switzerland). Retrieved November 4, 2022, from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7144564/
�Hosseinpour-Jaghdani F;Shomali T;Gholipour-Shahraki S;Rahimi-Madiseh M;Rafieian-Kopaei M; (n.d.). Cornus mas: A
review on traditional uses and pharmacological properties. Journal of complementary & integrative medicine.
Retrieved October 13, 2022, from
rasitic,fantastic%20value%20of%20this%20plant
Ma, Y., Ding, S., Fei, Y., Liu, G., Jang, H., & Fang, J. (2019, June 13). Antimicrobial activity of anthocyanins and catechins
against foodborne pathogens escherichia coli and salmonella. Food Control. Retrieved October 13, 2022, from
https://www.sciencedirect.com/science/article/pii/S0956713519302932
Rauf, A., Imran, M., Abu-Izneid, T., Iahtisham-Ul-Haq, S., Patel, X., Pan, S., Naz, A., Silva, F. S., & Saeed, H. A. (2019,
May 27). Proanthocyanidins: A comprehensive review. Biomedicine & Pharmacotherapy. Retrieved October
13, 2022, from https://www.sciencedirect.com/science/article/pii/S0753332219305359
Seladi-Schulman, J. (2019, August 30). Can you die from salmonella? yes, but it's rare. Healthline. Retrieved November 4,
2022, from https://www.healthline.com/health/food-safety/can-you-die-from-salmonella#salmonella
1:10
1:2
We took 10uL of our previous dilution/stock and added 90uL of 50% DMSO to each subsequent dilution.
Purpose:
To test the compound’s efficacy at varying doses.
Max Dose 10uM
1uM (1:10) 5uM (1:2)
0.1uM (1:10) 2.5uM (1:2)
0.01uM (1:10) 1.75uM (1:2)
0.001uM (1:10) 0.875uM (1:2)
50% DMSO (negative control)
Ampicillin (positive control)
Figure 4
Figure 5
Figures 1 & 2 depict results from the solid media test of procyanidin B1. Comparison of bacterial growth near our compound (zones C & D) to our positive and negative controls (ampicillin: zone A & DMSO: zone B) showed that our compound did not kill S. Typhimurium.
Figure 1
A
D
B
C
Figure 2
D
C
B
A
Department of Molecular, Cellular, and Developmental Biology
University of Colorado Boulder
Figure 4 represents data from the 1:2 dilution of our compound and figure 5 depicts data from the 1:10 dilutions. None of our dilutions inhibited bacterial growth, meaning we had no hits. We considered a compound a hit if the absorbance was 2 standard deviations above or below the mean absorbance of wells treated with DMSO. The absorbance of each well treated with a different dilution is similar to that of the DMSO-treated wells (negative control), but is a little lower at higher concentrations.
We want to thank the Department of Molecular, Cellular, and Developmental Biology at CU Boulder for funding our research. We also want to express our appreciation to our department chair Lee Niswander for advocating the importance of our research, along with Dr. Corrie Detweiler, the Detweiler Lab, and the Biological Sciences Initiative for supporting us throughout the semester. Finally, we would like to thank Dr. Pamela Harvey and our TAs, Madelyn Maclaughlin, Josh Fandel, and Logan Faberowski, for their support and assistance during our research.
Antibiotic resistance is a rising health risk, predicted to cause more deaths than cancer by 2050 (McCarthy & Richter, 2015). Antibiotic misuse and a lack of antibiotic discovery are much to blame for these high mortality rates. If new bacterial targeting methods are not developed soon, the predicted statistics will become a reality. Our research aims to find new antibiotics through experimentation with gram-negative Salmonella Typhimurium bacteria. We procured this type of bacteria because it is easy to grow in a lab and causes typhoid fever symptoms in rats while inflicting no serious health issues in humans. This is useful in testing antibiotic treatments for typhoid fever without endangering human researchers. The media we use also mimics the inside of a macrophage, making our research likely to translate to in vivo conditions. We decided to research potential antimicrobial compounds found in the Cornelian Cherry, a remedy used in Eastern medicine. Studies on Cornelian Cherries reveal that two active compounds, anthocyanins and proanthocyanidins, exhibit bactericidal behavior against certain kinds of bacteria (Rauf et al., 2019), (Ma et al., 2019). We tested procyanidin B1 through multiple dose-response studies, a max-dose study, and a solid media test (Mueller-Hinton test) to see if the compound had antibiotic effects on Salmonella Typhimurium.
The high absorbance values obtained from our dilution series and the higher bacteria growth observed around procyanidin B1 holes in the Mueller-Hinton plate reveal that this compound has no antibiotic properties. In both tests, bacteria growth when exposed to the compound was comparable to our bacteria-promoting negative control (50% DMSO) and the opposite of our positive control (ampicillin). A time-kill assay, the test required to determine if a compound is bacteriostatic (bacteria growth-inhibiting) or bactericidal (Bacteria-killing), was not required after it was proven that procyanidin B1 does not have strong antimicrobial properties. However, Cornelian cherries could still be useful as an antibiotic, as other proanthocyanidins have not yet been tested in this context.
We took 30uL of our previous dilution/stock and added 30uL of 50% DMSO to each subsequent dilution.
The plate set up for each dilution series
We added 90uL of Salmonella and 10uL of each dilution to each well
Key
We poked 4 holes into the solid media plate with a pipette tip (7.25mm) about 1 cm away from the edge. We then placed ampicillin in one hole (A), DMSO in one (B), and our compound in two (C and D) (see Figure 2).