Future research should continue the dose response curve into lower concentrations to determine LD50. There is still potential for a highly tunable dosage range as our investigation stands and determining this value is critical to potential future uses of ellagic acid. An important secondary piece of this testing would be determining the TD50 of vulnerable human cell lines. Given the extreme effectiveness of this compound, as shown by its effectiveness into nM regimes, it is important to ensure that it isn’t also highly toxic. For ease of use as well as to control for potential unrecognized solvent effects, determining if it is possible to transfer the drug into other common solvents and repeating testing to compare properties would lend additional surety to conclusions reported herein.
Long term research depends largely on the relationship of the LD50 and TD50. Given that there is a region where the drug is effective but not toxic, experimentation could continue. An important intermediate step would be to try ellagic acid in a system containing both macrophages and Salmonella, to even more closely mimic ultimate conditions. If this also produced favorable results, beginning to do animal testing in mice would likely be the next step.
Experts predict that, by 2050, antibiotic-resistant bacteria will be the leading cause of death over cancer. Discovery of antibiotics with novel activity has ground to a halt, but having a large toolbox of treatments is essential to address this growing threat. In an effort to address the grave shortage, we investigated ellagic acid’s effects on Salmonella (S.) Typhimurium. Ellagic acid was selected as a target since it functioned effectively as an antibiotic in previous research on gram-negative bacteria. S. Typhimurium was studied as it provides an ideal model organism by producing typhoid fever-like symptoms in mice. We dosed the bacteria with concentrations ranging from 10 uM to past 1 nM in our investigation. There was no appreciable decline in activity in these regions and LD50 was not determined. Additional experimentation, performed by regrowing previously drugged bacteria, suggested that the compound has a bacteriostatic mechanism of action. Our work sets the stage for future investigations into conclusively determining the LD50 of the drug as well as determining toxicity in mammalian cell lines.
Future Directions
Department of Molecular, Cellular, and Developmental Biology
University of Colorado Boulder
Jadon Holliday, Andy Kellam, Leshia Snively, Connor Thomas
Ellagic Acid as a Potential Antibiotic for Salmonella Typhiumurium
As a society, our medical concerns have been incorrectly assessed. There is a lack of concern for what will be our largest concern: antibiotic resistance. As of right now, there have been no new classes of antibiotics since the 1980s which will become a serious problem in the future, as deaths from antibiotic resistance are expected to jump to ten million. A lack of research stems from the fact that it is no longer profitable or as easy to create new antibiotics which creates a lack of incentive. If we continue to use the same classes of antibiotics the resistant bacteria will be able to grow and thrive without any foreseeable threats. To add on, antibiotic resistance can occur in four separate ways, including inherent resistance, acquired resistance, vertical gene transfer, and horizontal gene transfer which in and of itself has three possible mechanisms. This issue of antibiotic resistance is so pressing because in addition to humans there is direct and/or indirect use of antibiotics in agriculture, livestock, aquaculture, and household chemicals. Our entire lives are surrounded by antibiotics, only increasing the potential for antibiotic resistance, and increasing the likelihood of a bleak future. There is an imperative need to reinvent the golden age of drug discovery in our current generation to save the future ones.
Choi, J.-G., Kang, O.-H., Lee, Y.-S., Chae, H.-S., Oh, Y.-C., Brice, O.-O., Kim, M.-S., Sohn, D.-H., Kim, H.-S., Park, H., Shin, D.-W., Rho, J.-R., & Kwon, D.-Y. (2011). In vitro and in vivo antibacterial activity of punica granatum peel ethanol extract against salmonella. Evidence-Based Complementary and Alternative Medicine, 2011, 1–8. https://doi.org/10.1093/ecam/nep105
De, R., Sarkar, A., Ghosh, P., Ganguly, M., Karmakar, B. C., Saha, D. R., Halder, A., Chowdhury, A., & Mukhopadhyay, A. K. (2018). Antimicrobial activity of ellagic acid against helicobacter pylori isolates from India and during infections in mice. Journal of Antimicrobial Chemotherapy, 73(6), 1595–1603. https://doi.org/10.1093/jac/dky079
Derosa, G., Maffioli, P., & Sahebkar, A. (2016). Ellagic acid and its role in chronic diseases. Advances in Experimental Medicine and Biology, 473–479. https://doi.org/10.1007/978-3-319-41334-1_20
Hypothesis: Ellagic Acid will significantly reduce the growth of Salmonella Typhimurium
A current treatable bacterium is Salmonella Typhimurium. The Fall 2021 Drug Discovery lab worked towards identifying novel antimicrobial agents for the treatment of this bacterium. Salmonella Typhimurium is a rod-headed, flagellate, gram-negative bacterium of the genus Salmonella. The name comes from the observation of typhoid fever-like symptoms in mice, although these symptoms are not true of humans. This is especially important because we can easily monitor these typhoid symptoms in mice without putting the researchers at risk all while developing relevant treatments for typhoid-like symptoms. It is important to work within this genus because Salmonella-based diseases will affect around 100 million people worldwide, even with antibiotics available. Using Salmonella Typhimurium is also advantageous because it is inexpensive, easy to screen, and quick to get results (results come in after 24 hours). These factors allow us to easily work with the bacterium and possibly create viable and applicable treatments.
To continue in the antibiotic research for Salmonella Typhimurium, our drug discovery group tested ellagic acid. Ellagic acid is a natural phenol antioxidant. It is easily accessible being found in numerous fruits and vegetables and is shown to have a number of medicinal properties. Aside from results specific to our bacterium, this acid caught our interest as it is found to be promising in treatments of disorders ranging from Crohn’s disease to cancer (Derosa et al., 2016) which helps highlight its possible medical benefits. More specifically, it has been shown to inhibit the growth of 55 different strains of H. Pylori (Ronita et al., 2018) which is another gram-negative bacterium, which gives our group the inclination that it will be able to inhibit the growth of Salmonella Typhimurium. Moreover, other studies researched the effect of ellagic acid on Salmonella found it to be effective against 16 strains of Salmonella serotypes and even found it to inhibit the growth of E. Coli and Staphylococcus (Jang-Gi et al., 2011).
We would like to Acknowledge and thank Dr. Corrie Detweiler for her lab work, Biological Sciences Initiative (BSI) and the Howard Hughes Medical Institute for the funding and opportunities. We would also like to show our appreciation towards Pam Harvey and the teaching assistants, Alejandro Salmeron and Pauline Nguyen for their guidance, experience, and enthusiasm towards the research material.
Ellagic acid was tested as an antibacterial agent on S. Typhimurium and impaired bacterial growth comparably to ampicillin under all conditions tested, indicating strong antibacterial activity. However, ellagic acid acted as a poison (i.e. had non modulatable activity) for doses ranging as low as 0.125 nM. This may imply that the antibacterial activity could not be modulated sufficiently for safe action. Assays were also conducted to determine whether the mechanism of action was bacteriostatic or bactericidal in nature. As the compound exceeded the statistical bar on the second and third days of treatment, bacteria appear to have survived and reproduced-indicating a bacteriostatic mechanism of action.
This study was limited by a number of factors. We did not have access to a scale for sufficient resolution to make a stock solution at the desired concentration, instead necessitating many iterated dilutions. This combined with the low concentration needed for our dose response curves makes it fairly likely that the low concentrations of compound are not as precise as would be desirable. Furthermore, there is a possibility that the compound separates from solution at long time scales and reproduces antibiotic activity via creating anoxic conditions in the well.
D30
Figure 2: Bacterial growth represented as A260 over three portages following dosing. Positive control is represented by growth following ampicillin treatment. Statistical cutoff between being considered bacteriostatic and bactericidal is indicated as the green line. Treatments that are below the line are considered to have significant growth reduction. Error Bars are 土 1 SD.
Figure 1: Bacterial growth represented as A620 over 4 dose-response experiments. The blue bars represent the average absorbances of dose concentrations ranging from 10 uM to 0.000125 uM. The negative control (DMSO) represents the absorbance of Salmonella Typhimurium in a compound that has no effect on the bacteria, which is shown by the single green bar. The positive control (ampicillin) shows the absorbance of Salmonella Typhimurium in a compound that inhibits and/or kills the growth of the bacteria, which is represented by a single red bar. The dotted orange line represents 2 standard deviations below the mean DMSO absorbance, which depicts the statistical cutoff for antibiotic action. Error Bars are 土 1 SD.
Acknowledgements
Conclusion
Results
Methods
Introduction
Abstract
References