The Effect of active compound Myrcene on Growth of Salmonella Typhimurium
Lili Hirsch, Kevin Ember, Michael Villa
Department of Molecular, Cellular and Developmental Biology
Salmonella Typhi is a bacterium which causes life-threatening Typhoid fever in humans. Typhoid fever is responsible for about 200,000 deaths worldwide annually. There are antibiotics which can treat Typhoid fever, but due to an increasing rate of antibiotic resistance, these drugs are becoming less and less effective against Salmonella Typhi. The need for new antibiotics is always on the rise.
Antibiotic-seekers historically have had great success discovering antibiotics in biological communities such as soils, marine invertebrates, and fungus. Fungi have yielded significant antibiotic resources. According to a 2013 study, coprophilous fungi are elite candidate organisms for antibiotic discovery, they are underexplored and contain significantly underexplored bioactive metabolites. we have tested the active compound Myrcene, derived from Myrocin B, found commonly on coprophilous fungi Stilbella Erythrocephala. When tested in a max dose and dose response curve, it was discovered that three out of five doses of Myrcene had significant inhibitory effects on the growth of Salmonella Typhimurium, indicating that it could have significant potential to act as an antibiotic compound.
Determination of Hits: Values below two standard deviations of the negative control mean prevent the growth of bacteria and are bacteriostatic or bactericidal; these are classified as antibiotic hits, and are of significant interest as a potential drug. Values above two standard deviations of the negative control mean are also valuable as this shows probiotic activity.
The observed results somewhat support the hypothesis, however more work is needed to conclude Myrcene’s effect on Salmonella growth. The main reason why the results aren’t fully reliable is because of inconsistencies with DMSO absorbance. Some DMSO values had an absorbance as high as 0.7 when the expected value is around 0.3-0.4. This creates inconsistencies since the 10mg/mL concentration of Myrcene was a hit in figure one but not figure two. That being said, the average DMSO values for figure one and two, 0.516 and 0.472 respectively which isn’t too far off the expected value for DMSO. This means that although the results aren’t conclusive, they show a inverse correlation with concentration of Myrcene and survival rate of S. Typhimurium. Furthermore, when a sample of Myrcene was tested to be bacteriostatic or bactericidal, it was deduced that Myrcene is bacteriostatic. The reason for this is that when the Salmonella was incubated after being transferred to a new well it had growth, showing that in presence of Myrcene, S. Typhimurium is not able to reproduce, however it is not killed. These results are promising and effective, but future testing on Myrcene is required to confirm our initial hypothesis.
SHORT TERM
LONG TERM
A recognition to Dr. Corrie Detweiler, PhD, the principal investigator of our laboratory. We appreciate her contribution to an underappreciated area of study. We also recognize our funding source: the Howard Hughes Medical Institute; and pay recognition to our department, the CU Boulder Molecular Cellular and Developmental Biology department and its chair, Lee Niswander, PhD. We would also like to thank Dr. Pamela Harvey for her incredible instruction, and our TAs for their project guidance and advice in the lab.
Abstract
Introduction
Methods
Results
Conclusions
Future Directions
Acknowledgements
References
Figure one is showing the absorbance for a dose response curve of Myrcene. Of the 5 doses tested 3 were hits (10mg/mL, 5mg/mL, and 2.5mg/mL). The average Dimethyl sulfoxide (DMSO) absorbance for the dose response curves ran is 0.516 with a standard deviation of 0.098, so a hit is any absorbance value above 0.716 or below 0.32. Figure two is showing the absorbance for all the max dose trials ran and averaged, none of the doses tested were hits with an average DMSO absorbance of 0.472, and a standard deviation of 0.14, so a hit would have to be over 0.752 or under 0.192. A lower absorbance value indicates a well that has less Salmonella growth, and a well with a high absorbance indicates a well that has high amounts of Salmonella growth.
Max Dose Experiment: Investigates if Myrcene inhibits or promotes growth of Salmonella and Ensures all of the Myrcene can dissolve in the patient's blood. Stock solution is used to make a 100mg/mL sample of Myrcene. 10 µL of Myrcene and negative control (DMSO) or positive control (ampicillin) are added, and 90µL of S. Typhimurium is added to all wells in duplicates. The tray will be sealed and incubated for 24 hours at 37°C.
Dose Response Experiment: Identifies which concentration of Myrcene is most effective: stock solution is diluted 1:2 using 50% DMSO. Five concentrations of Myrcene are made, which when combined with Salmonella give concentrations of 10, 5, 2.5, 1.25, and 0.625 (all mg/mL). 10 µL of a negative (DMSO) and positive control (ampicillin) is under each duplicate. 90 µL of S. Typhimurium is added to all wells. The plate will be sealed and incubated for 24 hours at 37°C to promote Salmonella growth.
Creation of Stock Solution: 1 mL of 50% Dimethyl sulfoxide (DMSO) was added to the sample of concentrated Myrcene, yielding a concentration of 100 mg/mL.
Sources of Reagents used:
-Ampicillin obtained from Research Products International Corp based in Mount Prospect, IL
-Myrcene was obtained from Millipore Sigma, made in Switzerland, and based in St Louis, MO
- Salmonella used this semester was obtained from Corrie Detweiler’s lab, cultured in M9 minimal media and exposed to 10mg/mL Bergamottin (Sigma-Aldrich, St. Louis, MO) dissolved in ethanol
Bacteriostatic/Bactericidal: The max dose plate repeated, then incubated. After first incubation it was transferred to fresh media to test if regrowth occurred, then incubated once more at 37°C for 24 hours. This reveals if regrowth occurred.
Hypothesis
Coprophilous fungi are known to secrete bioactive metabolites from their fruiting bodies as competition mechanisms against other microorganisms for nutrients in their environment. A 2013 Science Direct study analyzed these bioactive metabolites as a reservoir for potential antibiotics and found them to be significantly underexplored and promising for antimicrobial activity. The fungus Stilbella Erythrocephala secretes a substance called Myrcene which we hypothesize to have significant inhibitory effects on the growth of Salmonella Typhimurium when incubated in M9 minimal media.
Antibiotic resistance occurs due to bacteria’s ability to quickly reproduce and obtain spontaneous mutations. Mutations can cause antibiotics to be ineffective against bacteria, leaving infections that are deadlier, harder to treat, and longer in duration. In the golden ages of antibiotic discovery, half of the antibiotics we use today were discovered. Now, those antibiotics have been overused, leaving us with bacteria that are resistant. Since the 1980s, no new classes of antibiotics have been discovered, despite a rising need. By 2050, it is projected that antibiotic resistance will be responsible for more deaths annually than cancer. The need for new antibiotics is higher than ever.
Salmonella Typhimurium is an optimal antibiotic effectiveness due to many favorable characteristics. Salmonella Typhimurium is suitable for lab work because it is cheap and easy to obtain, and models typhoid fever-like symptoms in mice, but only food poisoning in humans. This means that it is much safer for humans to work with, but still models typhoid fever in rodents. Mice and humans are somewhat genetically similar, and findings in an animal study are comparable to what a human would experience with some exceptions. For our compound, we have chosen Myrcene, produced by the Coprophilous Fungus Stilbella Erythrocephala, commonly found growing on rabbit pellets. Stilbella Erythrocephala secretes Myrcene from their fruiting bodies, and it has been shown in initial studies to have antibacterial properties. A 2006 study published in FEMS Microbiology Ecology showed the fungus produces Myrcene in order to eliminate bacterial competition in its range. Myrcene has been shown to use a range of defense mechanisms, depending on the concentration present, such as double nucleotide addition to cross link DNA.
Cross-linking DNA forms a covalent bond instead of the normal hydrogen bond between the two nucleotides, blocking the ability of the cell to translate/transcribe, triggering cell death. Another study in 2006 showed that pure cultures of S. Erythrocephala have been shown to produce antifungal antiamoebins, which attack their target organisms by self assembling into oligomers and forming pores or channels across the target’s cell membrane. A third study published in the Journal of Organic Chemistry in 2020 explains that the entire Myrocin family of fungus has antiproliferative properties that add further evidence to the fungus’ potential to suppress bacterial cell growth. This research leads us to believe that Myrcene is a compound that will show significant antibacterial activity against samples of Salmonella Typhimurium, which is why we have picked it as our lead compound.
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