References
Antibiotic resistant bacteria have existed as a threat since before penicillin was even on the market. However, as the development of new antibiotics has stagnated and antibiotic usage has massively increased, the threat posed by antibiotic resistance grows everyday. By 2050, antibiotic-resistant bacteria will be responsible for more deaths than cancer.
Typhoid fever is a bacterial infection caused by Salmonella Typhi, affecting an estimated 9 million people per year and responsible for approximately 110,000 deaths in the same period. In the lab, we used Salmonella Typhimurium as an analogous bacterium, which is close to structurally identical to S. Typhi, but causes only food-poisoning-like symptoms in humans while causing typhoid-like symptoms in rodents.
This experiment considered elderberry extract as a potential novel antibiotic compound against S. typhimurium, and by extension S. Typhi. This was predicated upon previous research that indicated Sambucus nigra as a promising compound against both microbial and viral infection.
Elderberry extract was tested at a max concentration of 10 ug/mL, then serially diluted to 75%, 50%, 25%, and 0% of max dose to establish a dose response curve. Using spectrophotometry, we considered absorbance values of wells of S. Typhimurium treated at these concentrations to determine the effectiveness of our antibiotic, comparing absorbance values against a negative control of DMSO (dimethyl sulfoxide) and a positive control of ampicillin.
Our results indicated that elderberry extract was ineffective as an antibiotic, based on our hit criteria of two standard deviations from the mean absorbance of S. Typhimurium wells treated with DMSO. In the future, resources should be shifted from further investigation of this apparently ineffective compound to research on more promising compounds.
Introduction
Methods
Hypotheses
Future Directions
While our results did not show that elderberry powder had a statistically significant effect on the growth of S. Typhimurium, samples containing elderberry powder consistently showed bacterial growth slightly below that of controls. This suggests that elderberry powder may contain antibacterial compounds in small quantities. Future research into identifying and isolating these compounds could be fruitful, as well as further research into the antibiotic properties of Nordic berries.
Time constraints and lack of access to some compounds hindered a more well-rounded investigation of the potential bioactive properties of the elderberry compound. Future research should not use our results to entirely write off elderberry as a potential antibiotic compound, but rather use these findings as a guide for new angles to take when considering elderberry and other Nordic berries as potentially bioactive.
Department of Molecular, Cellular, and Developmental Biology
University of Colorado Boulder
Abby Ellis, Neko Conner, Tristan Swanson
Sambucus Nigra fails to demonstrate antibiotic activity against Salmonella Typhimurium growth
Abstract
Ashurst, J. V. (n.d.). Salmonella Typhi - StatPearls - NCBI Bookshelf. National Center for Biotechnology Information. Retrieved April 7, 2023, from https://www.ncbi.nlm.nih.gov/books/NBK519002/
CDC. (n.d.). Information for Healthcare Professionals | Typhoid Fever | CDC. Centers for Disease Control and Prevention. Retrieved April 7, 2023, from https://www.cdc.gov/typhoid-fever/health-professional.html#:~:text=An%20estimated%2011%E2%80%9321%20million,fever%20occur%20worldwide%20each%20year.&text=In%20the%20United%20States%2C%20about,diagnosed%20with%20paratyphoid%20fever%20annually
Dan Liu, Xiao-Qin He, Ding-Tao Wu, Hua-Bin Li, Yi-Bin Feng, Liang Zou, and Ren-You Gan. Journal of Agricultural and Food Chemistry, 2022 70 (14), 4202-4220. DOI: 10.1021/acs.jafc.2c00010
Hyun, M.D., (2023, February 7). Antimicrobial Resistance Death Toll Could Catch Up To Cancer By 2050, And Pollution Is Fuelling Its Spread - Health Policy Watch. Health Policy Watch; https://healthpolicy-watch.news/antimicrobial-resistance-deaths-cancer/#:~:text=By%202050%2C%20the%20UN%20estimates,global%20death%20toll%20of%20cancer.
Krawitz, C., Mraheil, M.A., Stein, M. et al. Inhibitory activity of a standardized elderberry liquid extract against clinically-relevant human respiratory bacterial pathogens and influenza A and B viruses. BMC Complement Altern Med 11, 16 (2011). https://doi.org/10.1186/1472-6882-11-16
Liu, D. (2022). Elderberry (Sambucus nigra L.): Bioactive Compounds, Health Functions, and Applications. Journal of Agricultural and Food Chemistry. https://doi.org/10.1021
Results
The Problem
Acknowledgments
Conclusions
This study sought to investigate the antibacterial properties of elderberry berries and flowers on the growth of Salmonella Typhimurium, a gram-negative bacteria with high infectious potential. The results after processing with a spectrophotometer indicate that elderberry powder did not have a statistically significant effect on the growth of S. Typhimurium. Although this may not support the development of elderberry as a new antibiotic, our findings provide valuable insights for future research on the antimicrobial properties of elderberry and other traditional medicines. The continued exploration of new potential antibiotics to combat the rise of antibiotic-resistant bacteria is critical, and studies like this one are key to curtailing this problem.
Null: A solution of elderberry powder and DMSO reduce the growth of Salmonella or have no effect.
Alternative: Increasing the dosage of elderberry powder and DMSO will result in fewer living Salmonella bacteria after treatment and incubation.
A special thanks to the Molecular, Cellular, and Developmental Biology Department at the University of Colorado Boulder and the Howard Hughes Medical institute for offering this lab and generous funding to students irrespective of their major. Thank you to Dr. Corrie Detweiler, the principal investigator of our parent laboratory, for the usage of lab equipment and her support throughout our investigation. Thank you to the Teaching Assistants in Section 12 of this lab, Emma Tilley and Fathima “Zahra” Thatthey, and especially to Cogan Cogan, who oversaw the experiment and offered guidance, advise, tips, and encouragement during each lab. Finally, an enormous thanks to Dr. Pamela Harvey and her teaching assistants, Murphy Brasuel and Vinny Scarato, for all of the time, effort, and guidance they provided throughout the semester in lectures and lab, and for making this class possible.
Antibiotic resistance has been an exponentially growing threat to global public health since the stagnation of antibiotic class discovery in the early 2000s, and it has been largely ignored by pharmaceutical companies and research and development firms at the detriment of those affected. In 2019, antibiotic resistant bacteria were responsible for nearly 1.3 million deaths globally, and by 2050, antimicrobial resistance is expected to be responsible for 10 million deaths worldwide, matching the annual global deaths from all cancers combined (Hyun M.D., 2023). These figures represent a 770% increase in just half a lifetime. This research project aims to assess the efficiency and legitimacy of the antimicrobial properties in traditional medicines, particularly of elderberry powder.
Multiple studies have been conducted concerning the antimicrobial properties of elderberry, and the compound has been proven to exhibit an array of bioactive ingredients, such as phenolic compounds, that have the potential to inhibit the growth of pathogenic bacteria (Liu et al., 2022) (Krawitz, C. et al., 2011). Despite these discoveries, there is limited research about antimicrobial properties in elderberries and other sources of traditional medicine and antioxidants; for instance, there has not been any research conducted observing the behavior of elderberry power in the presence of a pathogenic bacteria, such as S. Typhimurium, but there are strong implications that it could be bacteriostatic.
Salmonella Typhi is a gram-negative bacteria and human pathogen that has burdened communities in the global south for decades; it is best known for causing typhoid fever, which remains a concern in global public health, which an estimated 11-21 million cases occurring worldwide each year (CDC, n.d.). Unlike S. Typhi, which poses significant risk to human researchers, S. Typhimurium is a safer model organism for this experiment. It has a broader host range and has a nearly identical structure to the serovar of S. Typhi, as depicted in the figure. In rodents, for example, S. Typhimurium causes symptoms resembling typhoid-fever, whereas it simply causes gastroenteritis in humans. Additionally, because there is a wide range of well-established, peer-reviewed protocols and procedures utilizing S. Typhimurium in the development of new antibiotics, and the bacterium is particularly easy to culture and contain with minimal risk of harm to those conducting the experiment (Ashurst, n.d.). S. Typhimurium is also highly infectious and therefore an efficient model of host-pathogen interactions, in both animal and human cells.
This research seeks to observe the effect of elderberry powder on S. Typhimurium, which will serve as an analog for pathogenic bacteria that have been developing antibiotic resistant strains, and assess the legitimacy and extent of its antimicrobial potential. This research could, therefore, serve as a basis for the development of a new antibiotic that utilizes elderberry as well as determine which properties in elderberry powder were effective against a pathogenic bacteria, and could be present in other sources of traditional medicine.
The results of this study were gathered by a spectrophotometer, which measures the bacterial concentration via proxy by measuring the absorbance of light in the sample. A compilation of 48 negative controls (DMSO) was used to determine statistical significance. Results greater than 2 standard deviations above or below the mean of the negative controls were considered statistically significant.
The max dose experiment returned values that all fell within the the range of two standard deviations of the mean absorbance of the negative control.
The dose response curve returned values with slightly more variation from the mean of the negative control, but still fell within the range of being statistically insignificant.
Starting with an elderberry extract which was powdered elderberry flower (20%) and berry (80%), a compound manufactured by Nature’s Way Brands LLC in Green Bay, WI, a stock was made with a concentration of 100 ug of elderberry powder per 1 mL of 50% concentration DMSO solvent. Two experiments were performed with this stock: a maximum dose experiment and a dose-response experiment. Both experiments evaluated the growth of S. Typhimurium using absorbance spectrophotometry on a 96-well microtiter plate, which correlates linearly with bacterium count. Both a negative and positive control were included in the experiments, with DMSO acting as the negative control and ampicillin as the positive. Results are considered statistically significant if they fall outside of 2 standard deviations from the mean of the negative control.
Maximum Dose Experiment
Dose Response Curve