References
Introduction
Methods
Results
Future Directions
Department of Molecular, Cellular, and Developmental Biology
University of Colorado Boulder, Boulder Colorado
Melody Conde, Lauren Voit, Emily Elmes
Effect of Plicamycin on Salmonella Typhimurium
Abstract
Hypotheses
Acknowledgments
Because there have been previous promising trials using Plicamycin, this experiment only further showcases the effectiveness of the compound. It was hypothesized that even at the max dose for physiological conditions, Plicamycin would be effective in killing bacteria, and our results were consistent enough to accept the hypothesis initially made. In this lab, it was shown to be just as- if not more- effective than ampicillin. Not only is it effective, but Plicamycin kills the bacteria instead of halting bacteria growth. However, it was found that it is most effective between the max dose for physiological conditions at 10 ug/mL to 4 ug/mL.. Some limitations faced during the lab were inconsistent results with our well plates that were visibly off when reviewing some rounds of data. This could have been due to some errors with pipetting techniques. Also, some plates were made and were occasionally misread and data was misconstrued during the 24-48 hour incubation periods, so new well plates had to be made.
A short-term goal for this experiment could be moving trials towards testing in mice. This would show how Plicamycin interacts with larger organisms to potentially predict side effects and to see if it is reasonable to begin future testing on humans.
A long-term goal for this experiment following a successful pre-trial on mice would be entering a Phase 0 study. A Phase 0 study would involve very few participants with no personal benefit to test how the drug acts within the human body at very low doses in hopes to speed up the drug approval process in future phases of the clinical trial.
Conclusions
Antibiotic resistance is something that is more commonplace than the public is aware, and it is occurring at an alarming rate. To preface, no new antibiotics have been discovered since the 1980’s, and yet a single bacteria cell can take no more than 20 minutes to produce progeny cells as they exponentially divide- any one error in replication could produce a mutation. These mutations often serve to increase the fitness of the bacteria, making them resistant to antibiotics intended to treat them. Because of mutations tandem to the lack of new antibiotics, by 2050 more people will die of antibiotic resistance than cancer. Pharmaceutical companies have also garnered most of the funding for research. Pharmaceutical companies are less incentivized financially to invest large amounts of money in their development because their drugs are maximally effective for a short period of time. Providing the population with drugs that manage but don’t treat maladies has allowed pharmacies to flourish under a business model; the continual need for treatment means the continued pressure on the population to pay for these services, pushing funding away from antibiotics and towards pharmacies. This makes it increasingly difficult for scientists interested in antibiotic resistance to allocate funds appropriately to conduct their own research. Antibiotics aren’t quick fixes; they are intended to get to the root of the problem.
In this lab, Salmonellcccccccc. Salmonella
(S. Typhimurium (S.
Typhimurium) will be
tested with
Plicamycin. S.
Typhimurium does
not cause death in
people, but is known to cause symptoms such as vomiting, diarrhea, and fever. Salmonella is an excellent model organism because of its ability to bypass the immune system which can help us understand antibiotic resistance. Salmonella is able to hijack cells through phagocytosis, becoming engulfed inside the cell where they replicate. Salmonella is also able to inject effector proteins through a Type 3 Secretion System (TTSS). The TTSS stimulates actin rearrangement- allowing the Salmonella to gain entry inside the cell once again. By inhibiting Salmonella’s ability to produce proteins, mechanisms such as TTS would no long work, and the cell would eventually die. The compound Plicamycin binds to the minor groove of DNA at GC-rich sites, resulting in inhibition of RNA synthesis. It also inhibits mRNA expression, resulting in a reduction in protein synthesis.
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Algorri , R. (2019, April 8). Foodborne Illness Part 3: How does Salmonella make us Sick? ASM. Retrieved November 18, 2021, from https://asm.org/Articles/2019/April/Foodborne-Illness-Part-3-How-does-Salmonella-make.
Calderone, J. (2015, December 21). Superbugs will soon kill more people than cancer. Business Insider. Retrieved December 2, 2021, from https://www.businessinsider.com/antibiotic-resistance-kill-more-people-than-cancer-2050-2015-6.
Galan, J. E., & Zhou, D. (2000, August 1). Striking a balance: Modulation of the Actin Cytoskeleton by Salmonella. Proceedings of the National Academy of Sciences of the United States of America. Retrieved November 18, 2021, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC34008/.
Jett, J. H. (2015, March 26). How long does it take a cell to divide? . Retrieved November 18, 2021, from https://onlinelibrary.wiley.com/doi/pdf/10.1002/cyto.a.22665.
PubChem. (n.d.). Plicamycin . PubChem.
As a group, we would like to thank Dr. Pamela Harvey for assisting us as a class not only during lecture hours but also during laboratory sessions. With her pure dedication to teach us laboratory skills and techniques, we were able to test our compound with ease in each laboratory session. We would also like to thank Biological Sciences Initiative and Howard Hughes Medical Institute for funding the drug discovery lab as well as Dr. Corrie Detwiler. Finally, we would like to acknowledge CU Boulder Molecular, Cellular, and Developmental Biology at the University of Colorado Boulder for this lab to be continued.
Create Stock Solution:
-Max dose must be 10uM for physiological conditions
-Received 1mg of Plicamycin derived from Streptomyces Plicatus, purchased from Research Projects International Corp. Mount Prospect, Illinois
-Mix 1mg of Plicamycin with 10mL of 20% DMSO to create our stock solution of 1mg/10mL
Max/Min Doses:
-Aim is to find the highest therapeutic dose with the lowest toxicity
-Original dilutions were made in centrifuge tubes starting with stock solution, then jumping to 5ug/mL, 2.5ug/mL, 1.25ug/mL, and 0.625ug/mL.
-Dilutions further examined to find the minimum dose. Dilutions were 9ug/mL, 8ug/mL, 7ug/mL, 6ug/mL, 5ug/mL, 4ug/mL.
-Dilutions are then placed into a well plate in triplicates mixed with 90uL of Salmonella with 2% DMSO and 5 mg/mL AMP in the last two columns.
-Incubate at 37°C for 24 hours to mimic physiological conditions
Bacteriostatic/Bactericidal:
-Aim is to discover whether Plicamycin is bacteriostatic or bactericidal.
-In a triplicate, distribute 90uL of Salmonella, followed by the first column adding 10uL of stock solution, second column of 10uL DMSO, and third column of 10uL AMP.
-Incubate at 37°C for 24 hours to mimic physiological conditions
DMSO: 0 cm
Plicamycin
Avg Radius: .73 cm
Ampicillin
Avg Radius: .6 cm
Figure 2: The image above illustrates the effects of Ampicillin, Plicamycin and DMSO on Salmonella. Measurements were taken the from center of agar holes; clearing of bacteria around holes indicates bacteria death.
Figure 1: This graph illustrates a 1:2 dilution series of Plicamycin with 10 ug/mL being the stock concentration. On the X axis is the compounds that we tested with Salmonella Typhimurium and on the Y axis is the absorbances for each.
Discussion
Plicamycin was chosen as the choice of compound because how it provided promising data from prior experiments that were performed with it. Data from the previous semesters had shown that Plicamycin was effective in killing bacteria such as Salmonella. We then wanted to further explore Plicamycin by testing at what doses it was most effective in killing the bacteria. This led us to making several dilution series testing the absorbance rates allowing us to see what would effectively kill the bacteria. A bactericidal/bactericidal test was done, confirming that when S. Typhimurium absorbs the Plicamycin, the bacteria actually die and their growth isn’t halted. Looking at labs outside of the University of Colorado, Plicamycin had been successful in acting as an antineoplastic antibiotic, but still was found to be toxic in lingering amounts of certain effective doses. With this in mind, we would have liked to narrow down our effective doses with further experimentation but were unable to due to lack of time. More than once, plates we had made were incorrectly read and the data was not accurate and thus could not be used. This then led us to using future lab periods remaking well plates.
Salmonella Typhimurium is a bacterium that can cause infections in both humans and animals. In humans, S. Typhimurium does not cause death, but does induce diarrhea, vomiting, fever, and abdominal pain. Antibiotics may be used to combat the presence of harmful bacteria, but due to the increasing presence of antibiotic resistance, many are being rendered useless. Because of this, the number of deaths due to antibiotic resistance will be greater than those of cancer by 2050. The compound Plicamycin (Mithramycin) has been found effective in inhibiting RNA synthesis, which is the compound being tested against S. Typhimurium. Using a stock solution, a max dose at physiological conditions was created tandem to several dilutions testing the minimum dose of effectiveness. After, a bactericidal/bacteriostatic test was done to see if the bacteria died off or if growth was halted. Through thorough experimentation, Plicamycin was found to be extremely effective in killing bacteria growth. Our results have shown that the compound Plicamycin acts as a bactericidal antibiotic, completely killing off the population of bacteria instead of halting the growth. Future experiments for this lab would include moving to pre-clinical trials involving testing in mice. If the results with Plicamycin are promising, further experiments could move towards using people to see the full potential of the interaction between the compound and bacteria.
Knowing that Plicamycin was a hit in previous semesters and that it is an antibiotic, we hypothesized that the Salmonella Typhimurium in the well plates will be killed. We\ know that the max dose in physiological conditions of a human would be 10 uM, so we hypothesized that the max dose will be effective dose in killing the Salmonella. We also hypothesized that if we diluted the maximum dose, that our minimum doses would also work as well.