References
Introduction
Future Directions
Department of Molecular, Cellular, and Developmental Biology
University of Colorado Boulder
Emily Macdonald, Jasmine Plancarte, and Abi Stonefelt
The Efficacy of Valproic Acid on Salmonella Typhimurium Growth
Abstract
Michaelis, M., Doerr, H. W., & Cinatl, J., Jr (2007). Valproic acid as anti-
cancer drug. Current pharmaceutical design, 13(33), 3378–3393.
Pao, P., & Tsai, L (2022). Histone deacetylases 1 and 2 in memory
function. ACS Chemical Neuroscience, 13(7), 8848-858. https://
doi.org/10.1021/acschemneuro.1c00775
Singh, D., Gupta, S., Verma, I., Morsy, M. A., Nair, A. B., & Ahmed, A. F.
(2021). Hidden pharmacological activities of valproic acid: A new
insight. Biomedicine pharmacotherapy, 142, 112021. https://
doi.org/10.1016/j.biopha.2021.112021.
Results
Methods
Acknowledgments
Conclusions
This study aimed to determine if Valproic Acid (VPA) could be used to treat Salmonella Typhi by inhibiting or killing Salmonella Typhimurium. The results from the dose curve demonstrated that Valproic Acid (VPA) is not successful in inhibiting Salmonella Typhimurium, and the results from the bactericidal or bacteriostatic experiment show VPA fits into neither category. The results in Figure 2 demonstrate that Ampicillin (AMP) did not produce expected absorbance values, which potentially explains why 0.001 M of VPA did not display bacteriostatic or bactericidal characteristics in this experiment. Although these results do not demonstrate VPA as a successful antibiotic, it is essential that VPA continues to be tested given the opposing conclusions of previous research.
The golden age of the mid-twentieth century produced many antibiotics that are still used today, but this rapid rate of discovery has ceased and no new classes have been found since 1987; pharmaceutical companies have no incentive to test potential antibiotics because of their low investment return and quick resistance development. Projections show that antibiotic- resistant infections will be the leading cause of
death by 2050, killing ten
million people each year.
It is urgent that new
antibiotics are discovered;
evaluating potential
antimicrobial compounds
is a critical first step.
Typhoid fever in mice serves as a model to determine if novel compounds have any antibiotic characteristics. Salmonellae are Gram-negative, having a peptidoglycan layer surrounded by a lipopolysaccharide-containing membrane. Salmonella was partly chosen because of its Gram-negative properties, making it challenging for antibiotics to penetrate. The Typhimurium serovar was used instead of Typhi because it is cheap, easy, and fast; additionally, it causes food poisoning in humans and typhoid fever in mice, making it a great model to work with.
This study examines the antibiotic potential of Valproic Acid (VPA). VPA is a novel chemotherapeutic treatment because of its histone deacetylase activity, inhibiting the growth of cancerous tumor cells
(Michaelis et al., 2007).
Furthermore, VPA has
exhibited previous antibiotic
activity against
Staphylococcus aureus and
Proteus vulgaris bacteria
Singh et al., 2021). This
experiment draws on prior
findings and applies them to
the S. Typhimurium model.
S. Typhimurium was cultured at 37°C and incubated for 24
hours before being placed in M9 media.
Valproic Acid (BioGems International, Westlake Village, CA)
was stored as a salt at 20-25°C. A stock solution of VPA was
prepared using the salt and dimethyl sulfoxide (DMSO)
before being used in the following experiments:
b. Bacteriostatic vs Bactericidal: 0.001M VPA was added in � triplicate to wells containing S. Typhimurium. DMSO and
AMP were added as controls. After incubating for 24 hours, fresh S. Typhimurium was added to a new plate; contents from the previous plate were transferred into the fresh media.
Hypothesis
Given that Valproic Acid (VPA) has demonstrated previous antibiotic characteristics, VPA can be used to treat Salmonella Typhi, either by inhibiting cell growth or killing the cells of Salmonella Typhimurium.
Figure 1. 1:10 Dilution Series of VPA Against S. Typhimurium
The effect of each treatment on Salmonellae bacterial growth. The absorbance of the samples was observed after being incubated for a minimum of 24 hours at 37°C to mimic conditions within the human body. The mean of the DMSO-treated samples is displayed with a dashed line at an absorbance value of 0.2160. The ∓2 standard deviation range from the mean of the DMSO-treated samples is displayed with solid lines at absorbance values 0.0747-0.3572. The absorbances from all doses fall within the range, demonstrating no dose of Valproic Acid (VPA) is a statistical hit.
3. Measure the absorbance.
The absorbance of the S. Typhimurium was analyzed to
determine bacterial growth.
4. Verify growth.
Mean absorbances from the wells were compared against
the mean, class-wide value of DMSO. The standard
deviation of DMSO was calculated and multiplied by 2; any
absorbances that fell below 2 standard deviations of the
mean were statistical hits.
5. Compare data.
Data were compared to previous research.
Figure 2. Determining if VPA is Bacteriostatic vs. Bactericidal
The effect of 0.001M Valproic Acid (VPA) on Salmonellae bacterial growth. The absorbance of the samples was observed after being incubated for 24 hours at 37°C to mimic conditions within the human body. The absorbance of the samples was observed once more after another 24 hours to determine growth over an extended period of time. The mean of the DMSO-treated samples for this plate is displayed with a dashed line at an absorbance value of 0.2160. The ∓2 standard deviation range from the mean of the DMSO-treated samples is displayed with solid lines at absorbance values 0.0747-0.3572. The average absorbance of the 0.001M VPA treatment was 0.1842, meaning that this treatment exhibits no statistical significance as a bacteriostatic or bactericidal antibiotic.
Antibiotic resistance poses an imminent threat to global health. With projections predicting antimicrobial resistant infections to overtake cancer as the leading cause of death by 2050 and the absence of novel antibiotic class discovery since the 1980s, a significant focus must be placed onto the discovery of new antibiotic classes. Valproic Acid (VPA) is commonly used to treat seizures, bipolar disorder, and migraines and has been used as a recent chemotherapy. This lab studied the potential antibiotic characteristics of VPA on Salmonella Typhimurium. VPA was studied in a dose response curve using Ampicillin as a known inhibitor of S. Typhimurium (positive control) and dimethyl sulfoxide (DMSO) as a solvent (negative control). 0.001M of VPA was successful at inhibiting bacterial growth, although this dose did not demonstrate bactericidal or bacteriostatic properties. Ultimately, while VPA exhibits some potential antibiotic characteristics, more experimentation must be conducted.
Valproic Acid (VPA) was unsuccessful at killing or stopping the growth of S. Typhimurium. Continued testing at low doses should be conducted to determine if a more diluted sample of VPA is consistently effective. A different approach to diluting VPA to lower doses should be used since the method used in this experiment was ineffective and potentially lead to errors. Additionally, the cellular mechanism of VPA should be determined. This could be done through medicinal chemistry and understanding how each of VPA’s functional groups contributes to attacking S. Typhimurium’s cell membrane. Lastly, combining VPA with an allosteric modulator could highlight VPA’s mechanism of action and enhance its efficacy. Ultimately, a significant amount of testing must be conducted before VPA is used as an antibiotics in humans.
Gratitude is extended to Corrie Detweiler, PhD, for her dedication as the principal investigator, Howard Hughes Medical Institute for its funding, and the CU Boulder Dept. of Molecular, Cellular, and Development Biology for its funding, resources, and facilities. An utmost thanks to Pamela Harvey, PhD, and Vinny Scarato, Lab Manager, for their guidance and insight. Lastly, we warmly acknowledge our teaching and learning team for their patience, encouragement, and support: Emma Tilley, Zahra Thathey, and Cogan Cogan.