�This study was designed to explore the effect of flufenamic acid as an antibiotic through testing its interactions with the gram-negative bacteria Salmonella Typhimurium. The compound was tested through various trials: a maximum physiological possible dose, a serial dilution to outline the dose response curve, and a bacteriostatic/ bactericidal test. Ultimately, flufenamic acid was not found to statistically qualify as a significant "hit" or demonstrate antimicrobial properties. Due to these findings, it can be suggested that more testing is needed for flufenamic acid to be considered a possible antibiotic in vivo.
Though our methods of testing came short of promising results, the compound should not be completely dismissed. One long-term option would be to chemically modify by changing the functional groups. This may allow for better absorption, cell interactions, and fix the solubility issues from the beginning. Flufenamic acid could also be combined with hyperpermeability to better represent body interactions. Overall, as a short-term goal, flufenamic acid should be further tested due to the promising qualities listed when the compound was first chosen.
¹Department of Molecular, Cellular, and Developmental Biology; University of Colorado Boulder
Payton Keeler¹, Joshua Lee¹, Luke Hasselback¹, Alex Martidis¹
Antimicrobial Effects of Flufenamic Acid on Salmonella Typhimurium
Abstract
Antibiotic resistance is a rapidly growing problem that is on track to be the leading cause of death by 2050, surpassing cancer with an estimated 10 million deaths per year1. Through random genetic mutations, bacteria can develop resistance to antibiotics, effectively rendering them untreatable; however, no efforts to discover a new class of antibiotic have been successful since the 1980s. This is mostly due to lack of profits for pharmaceutical companies when researching and developing quick cures instead of treatments for chronic conditions. Antibiotic-resistant bacteria will only continue to worsen, instigating the need for more creative compound screens and the identification of new antibiotic classes.
For this study, Salmonella Typhimurium was chosen as the model organism as it has nearly identical qualities to S. Typhi as a gram-negative bacteria while being more accessible and safer to handle. In addition, S. Typhimurium produces typhoid fever-like symptoms in mice allowing for future in vivo testing if our tested compound proves effective.
Flufenamic acid has shown to be promising in a multitude of antimicrobial studies but has not been extensively tested, allowing for more research regarding its effects on S. Typhimurium2,3. Based on molecular simulation studies, the compound seemed promising to hydrogen bond to residues in bacterial DNA gyrase, indicating that it would inhibit DNA replication and therefore cell division4.
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We would like to thank the Howard Hughes Medical Institute and the MCDB department for funding this research. Furthermore, we would like to thank Dr. Pamela Harvey for guiding us through this experiment, as well as our TA’s Michael Shiferaw, Murphy Brasuel, and Sarah Posewitz. We also extend our gratitude to Dr. Niswander, the chair of our MCDB department, for allowing this research to take place. Lastly, we further extend our gratitude to Dr. Corrie Detweiler for supplying the groundwork for our research.
1. O’Neill, Jim. May 2016. The Review on Antimicrobial Resistance. Tackling Drug-Resistant Infections Globally: Final Report and Recommendations. https://amrreview.org/sites/default/files/160518_Final%20paper_with%20cover.pdf
2. Kim, Kyeong Kyu and Kang, Sung Wook. Jan 19, 2016. Novel antibiotic composition comprising flufenamic acid as an active ingredient, US 9.238,016 B2, United States Patent
3. Seong, Y. J., Alhashimi, M., Mayhoub, A., Mohammad, H., & Seleem, M. N. 2020. Repurposing Fenamic Acid Drugs To Combat Multidrug-Resistant Neisseria gonorrhoeae. Antimicrobial agents and chemotherapy, 64(7), e02206-19. https://doi.org/10.1128/AAC.02206-19
4. Preethi, B., Shanthi, V., & Ramanathan, K. 2016. Identification of potential therapeutics to conquer drug resistance in salmonella typhimurium: Drug repurposing strategy. BioDrugs, 30(6), 593-605. doi: https://doi.org/10.1007/s40259-016-0200-7 �
Introduction
� Figure 1. Projected leading causes of death by 2050
Conclusions
Future Directions
Acknowledgments
References
Based on our limited data, there is grounds to reject our hypothesis and dismiss flufenamic acid as a potential antibiotic as a result of the testing performed. The lack of hits at any concentration suggests flufenamic acid does not function as an antibiotic against gram bacteria. In addition, it was difficult to fully dissolve in 50% DMSO, which may have lead to inaccurate concentrations. Replicates under stricter conditions should be performed to ensure that the compound has no antimicrobial properties at the reported doses.
As flufenamic acid is known to inhibit growth in various gram-positive bacteria through the modulation of cellular functions, the addition of this compound to samples of Salmonella Typhimurium is expected to impede the growth of these bacterial cultures, leading to a decrease in absorbance over time.
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Hypothesis
Methods
Results
Figure 2. Max dose trial; 10μM flufenamic acid compared to DMSO (-) and Ampicillin (+). Hit range outlined in red, negative control ± 2 standard dev (0.2456-0.3412).
Figure 3. Dose response curve; serial dilution of flufenamic acid compared to DMSO (-) and Ampicillin (+). Hit range outlined in red, negative control ± 2 standard dev (0.2456-0.3412).
To investigate the potential antimicrobial properties of flufenamic acid (Acros Organics, China, a stock solution and dilution series was created in order to determine the concentration at which flufenamic acid is most effective. 50% dimethylsulfoxide (DMSO) was used as a negative control as it is the compound solvent and served as a static comparison and 50μg/mL ampicillin (Research Products International Corp. Mount Prospect, IL) was used as a positive control as it represented the desired result. For statistical analyses to determine significance, a “hit” was defined as outside of two standard deviations from the negative control mean which indicates that the compound should be further examined as having antimicrobial or growth promoting factors (above vs. below the mean). The desired outcome was S. typhimurium cell death or inhibition of growth.
Max Dose Trial: 10µM
Dose Response Curve: 5µM
Dose Response Curve: 2.5µM
Dose Response Curve: 1.25µM
Dose Response Curve: 0.625µM
Negative Control: DMSO
Positive Control: Ampicillin
Microtiter Plate Key:
Max Dose Trial
Dose Response Curve
Bacteriostatic Bactericidal Test
100µM stock 50% DMSO
10µL of 10 µM compound
90µL of bacteria
Incubator at 37°C
for 24 hours then
spectrophotometer analyses
Serial Dilution:
Various Concentrations
10µL of 10µM, 5µM, 2.5µM, 1.25µM, and 0.625 µM compound
90µL of bacteria
Incubator at 37°C
for 24 hours then
spectrophotometer analyses
Incubator at 37°C
for 24 hours then
spectrophotometer analyses
100µM stock 50% DMSO
10µL of 10 µM compound
90µL of bacteria
10µL from wells
90µL of new bacteria
M9 minimal media
Repeat last step