References
The rise of antibiotic resistance in bacteria responsible for human infectious disease has underscored the need for the discovery of new antibiotic drugs. Nigella sativa essential oil has been observed to inhibit bacterial growth in both gram-positive and gram-negative bacteria. Two biologically active compounds in the essential oil, thymoquinone and longifolene, may be responsible for this antimicrobial activity. To test this, we exposed Salmonella Typhimurium bacteria to thymoquinone and longifolene at realistic medical doses and measured the bacterial growth. By comparing these to positive and negative controls–water and ampicillin–we determined whether thymoquinone and longifolene caused statistically significant change to the growth of S. Typhimurium.
Introduction
Methods
Results
Further Study
Among many courses of action which could be taken, we believe the following represent the most important areas in which further study should be done on thymoquinone and longifolene.
Department of Molecular, Cellular, and Developmental Biology
University of Colorado Boulder
Peter K, Jamie O, Ian D, Oscar R.
Antimicrobial activities of major compounds in Nigella sativa extract on Salmonella Typhimurium
Abstract
Hypothesis
Conclusions
Although it has been shown that both thymoquinone and longifolene greatly inhibit growth in S. aureus and, to a lesser extent, in E. Coli (Bourgou, 2010), our experiments yielded inconclusive results for the effectiveness of thymoquinone and longifolene as antimicrobial agents.
While some tests, such as the maximum dose experiments and the solid media cultures indicated that thymoquinone had no effect on the growth of S. Typhimurium, the dose response testing and preliminary results suggested that thymoquinone may inhibit its growth even more than ampicillin. However, further examination of thymoquinone’s effects on S. Typhimurium failed to provide results due to human error. Since some results were promising, further study of thymoquinone is advisable.
Longifolene was only tested at a 1M concentration in the solid media culture but successfully inhibited some level of bacterial growth. This compound must studied further at concentrations more similar to what may be found in the human body. However, previous studies have not shown longifolene to inhibit bacterial growth in E. coli, another gram- negative bacteria, so future research should feature gram-positive bacteria for testing longifolene as a potential antibiotic.
Throughout this experiment and the antibiotic discovery lab as a whole, we experienced challenges in accurate data collection and the quality of standard solutions and bacteria cultures. Improving experimental design and reducing human error would be an important step in increasing the efficacy of future experimental data.
Antibiotic development is an increasingly important area of research, since existing antibiotics are becoming obsolete due to the rise of antibiotic resistant bacteria. Meanwhile, pharmaceutical companies lack the financial incentive to fund development of new antibiotics because antibiotics are curative in nature. As a result, almost no significant advancements have been made in the discovery of novel antibiotics since the 1980s, while numerous bacteria have become multi-drug resistant (Ventola, 2015). Without intervention, death by bacterial infection is projected to be the leading cause of medical death by 2050 (Murray, 2022). Among the most impactful of these bacterial infections is Salmonella Typhimurium, with 1.35 million US infections per year (CDC, 2022).
Salmonella Typhimurium are gram-negative proteobacteria that cause gastroenteritis in humans and are closely related to S. Typhi, which causes deadly typhoid fever. S. Typhimurium is safe to work with, a frequent cause of illness, and readily available for research. Moreover, some strains of S. Typhimurium have gained resistance to the antibiotics including ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline, highlighting the need for novel antibiotic solutions for this specific bacteria.
Vascular plants maintain environments favorable to parasitic infection. Available nutrients, stable temperature and osmotic conditions, and a lack of adaptive immunity all permit the growth of parasitic microorganisms. To counteract these disadvantages, plants produce phytochemicals–naturally occurring plant compounds–such as phenols and terpenes which have known antimicrobial effects (A. Mahizan, 2019). Thymoquinone is a monoterpene found in Nigella sativa (commonly known as black cumin), the essential oil of which has been found to be antimicrobial against Escherichia coli and Staphylococcus aureus. When tested in isolation, thymoquinone was also found to inhibit growth of both of these bacteria (Bourgou, 2010).
In the majority of our testing, thymoquinone at 10µM concentration failed to inhibit Salmonella Typhimurium growth with an average average absorbance of approximately 0.73 at 620nm which was slightly below the negative control of 0.75 as shown in figure 1. To be significant, the bacterial growth would need to be below absorbance 0.67 as seen as the red line on figure 1.
However, when testing the response to varying doses of thymoquinone (figure 2), much greater growth inhibition was observed at 10µM. At this concentration, the culture had an absorbance of 0.040, which is far below the threshold for a “hit” of 0.24 (shown in red on figure 2) in this experiment. Nonetheless, the antimicrobial effect greatly dropped off at lower doses and this effect was not replicated.
On the Mueller-Hinton Agar plates neither drug inhibited bacterial growth to the same level as Ampicillin which prevented the growth of S. Typhimurium for a radius of 10mm around the well (image 3). However, longifolene did inhibit bacterial growth to a radius of 4mm around the well which was greater than both the negative control of water and the thymoquinone.
Thymoquinone and Longifolene may inhibit the growth of Salmonella Typhimurium as an antibiotic agent.
A. Mahizan, N, et. al. (2019). Terpene Derivatives as a Potential Agent against Antimicrobial Resistance (AMR) Pathogens. Molecules, 24(14), 2631. https://doi.org/10.3390/molecules24142631
CDC (2019). Antibiotic Resistance Threats in the United States. Atlanta, GA: U.S. Department of Health and Human Services.
Jørgensen, M. G., Raaphorst, R. van, & Veening, J.-W. (2013). Noise and Stochasticity in Gene Expression: A Pathogenic Fate Determinant. Methods in Microbiology, 40, 157-175.
https://doi.org/10.1016/B978-0-12-417029-2.00006-6
J. Murray, C., et al. (2022). Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. The Lancet, 399(10325), 629-655. https://doi.org/10.1016/S0140-6736(21)02724-0
National Center for Biotechnology Information (2022). PubChem Compound Summary for CID 10281, Thymoquinone. Retrieved November 4, 2022 from https://pubchem.ncbi.nlm.nih.gov/compound/Thymoquinone
S. Bourgou, A. Pichette, B. Marzouk, J. Legault (2010). Bioactivities of black cumin essential oil and its main terpenes from Tunisia. South African Journal of Botany, 76(2),
210-216. https://doi.org/10.1016/j.sajb.2009.10.009
Ventola C. L. (2015). The antibiotic resistance crisis: part 1: causes and threats. P & T : a peer-reviewed journal for formulary management, 40(4), 277–283. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4378521/
Vinod N, Noh HB, Oh S, Ji S, Park HJ, et al. (2017). A Salmonella typhimurium ghost vaccine induces cytokine expression in vitro and immune responses in vivo and protects rats against homologous and heterologous challenges. PLOS ONE 12(9): e0185488. https://doi.org/10.1371/journal.pone.0185488
Image 1. Thymoquinone is a monoterpene produced by Nigella sativa, the essential oil of which has been shown to be antimicrobial against bacteria (National Center for Biotechnology Information, 2022).
Thymoquinone (Fisher Scientific, Hampton NH) was diluted in water. Pure DI water was our negative control and ampicillin (50µg/mL) was our positive control.
The Salmonella Typhimurium was tested against the maximum possible dose of each compound in the human bloodstream–10µM. 90µL of S. Typhimurium at 105 bacteria per mL in liquid M9 media (Detweiler Lab, Boulder CO) was mixed with 10µL of each compound or control yielding 10µM. After incubation for 24 hours at 37℃, we measured bacterial growth via spectrophotometry at 620 nm. Absorbances ± 2 standard deviations from the mean of the negative control were considered “hits”ーpotential antibiotics or growth factors.
Thymoquinone was diluted in 1:2 serial dilutions to concentrations from 100µM to 1025nM. In duplicate on a 96-well plate, 10µL of each compound was added to 90µL of S. Typhimurium in liquid culture (yielding a test concentration of 10µM) and measured for level of bacterial growth against water and ampicillin as positive and negative controls.
Both thymoquinone and longifolene at 1M concentrations were tested on Mueller-Hinton Agar plates. First four separate 7.5mm wells were cut into the agar. Next, 3 µL of highly concentrated S. Typhimurium was pipetted onto the plate and spread around evenly. Then, 50 µL of each compound was added to a well in the plate was then put into the incubator to grow. The radius of inhibited bacterial growth surround the wells was measured and compared to ampicillin and water.
Compound Preparation
Maximum Dose Testing
Dose Response Curve
Image 2. Mueller-Hinton Agar plate before incubation.
Image 3. Mueller-Hinton Agar plate after incubation.
Solid Media Culture
Figure 1. Growth of Salmonella Typhimurium exposed to 10µM thymoquinone was measured in absorbance at 620nm against controls.
Ampicillin
Longifolene
Thymoquinone
Water
Figure 2. Growth of Salmonella Typhimurium exposed to thymoquinone at varying concentrations measured in absorbance at 620nm.
We would like to thank Dr. Pamela Harvey for guiding us through the background and design for this experiment. She has encouraged scientific exploration through education of young scientists as well as our teaching assistants, Josh Fandel, Madelyn MaClaughlin, and Logan Faberowski. We would also like to extend our gratitude to Dr. Corrie Detweiler for supplying the groundwork for our research. In addition, we want to recognize the MCDB department at University of Colorado, Boulder and the chair of the MCDB department Lee Niswander. We would also like to thank the Howard Hughes Medical Institute for their support.
Acknowledgments