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Department of Molecular, Cellular, and Developmental Biology

University of Colorado Boulder

Sedique Pitsuwan-Meier, Bailey Weibel and Ketzia Ojeda

The Effects of Picea Abies Resin on Salmonella Typhimurium

Abstract

Introduction

Results

Reference

Future Directions

Hypothesis

Methods

Conclusion

Acknowledgements

Figure 1: The effects of different concentrations of Picea Abies and Picea Mariana products on Salmonella Typhimurium growth as seen through absorbances of S. Typhimurium at 620 nm in comparison with two standard deviations of the mean absorbances of Salmonella in the presence of 70% ethanol. The bar through the graph represents the positive hit line of two standard deviations above the mean absorbance of 70% ethanol, which was the negative control. Bars represent average absorbance values of each concentration. Error bars represent the standard deviation of 0.0052156.

1. Centers for Disease Control and Prevention (U.S.), National Center for Emerging Zoonotic and Infectious Diseases (U.S.), Division of Healthcare Quality Promotion, Antibiotic Resistance Coordination and Strategy Unit (2019), Antibiotic resistance threats in the United States. https://stacks.cdc.gov/view/cdc/82532

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2. Jokinen, J. J., & Sipponen, A. (2016). Refined Spruce Resin to Treat Chronic Wounds: Rebirth of an Old Folkloristic Therapy. Advances in wound care, 5(5), 198–207. https://doi.org/10.1089/wound.2013.0492

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2. Sipponen, A., & Laitinen, K. (2011). Antimicrobial properties of natural coniferous rosin in the European Pharmacopoeia challenge test. APMIS, 119, 720-724. https://doi.org/10.1111/j.1600-0463.2011.02791.x

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2. Sipponen, P., Sipponen, A., Lohi, J., Soini, M., Tapanainen, R., & Jokinen, J. J. (2012). Natural coniferous resin lacquer in treatment of toenail onychomycosis: an observational study. Mycoses, 56(3), 289–296. https://doi.org/10.1111/myc.12019

Every year there are more than 2.8 million antibiotic-resistant bacterial infections in the US with more than 35,000 deaths annually [1]. Due to the rapid growth in resistant bacterial strains, mortal infections could one day again critically affect public health. Exacerbating the issue of antibiotic resistance, no new class of antibiotics has been found since the 1980s. The lack of development is due in part to today’s lack of profitability for research in antibiotics. Current profit motives notwithstanding, the future demand for a novel safe and efficacious drug will predictably rise as lethal cases of bacterial infections increase due antibiotic resistance. It is critical that science rises up to meet the challenge before infections become a death sentence.

While antibiotic research generally investigates isolated compounds, the group’s curiosity in traditional medicines motivated a look into the viable sources of antibiotics in folk remedies.

Though somewhat limited in scope, the group came upon a series of papers that researched the antifungal and antibacterial characteristics of Picea Abies (Norwegian Spruce) resin used in traditional Scandinavian salves. First, the group noted some evidence of antifungal properties of Norwegian Spruce resin in topical treatment of onychomycosis [4]. Upon additional research, there appeared to be support for some antibacterial applications of 10% concentration of Picea Abies resin in salves treating chronic wounds [2, 3]. Given the interesting evidence, the viability of Picea Abies as a source for antibiotics warrants additional investigation.

(a.) Picea abies (Norwegian Spruce) (b.) Picea Abies resin/sap

Source: Reference photo from Refined Spruce Resin to Treat Chronic Wounds: Rebirth of an Old Folkloristic Therapy

The model organism the group will test Norwegian spruce resin against is Salmonella Typhimurium, a bacteria that causes food poisoning in humans and typhoid-fever-like symptoms in mice. Importantly, in vivo testing resin and using Salmonella as the model organism at this point in development lowers possible outside errors, returns data faster, and the process is relatively inexpensive. Overall, differing effects on humans and mice by Salmonella Typhimurium will help to determine the effectiveness and potential of the substances for further development as a clinical candidate.

Considering prior research showing antibacterial and antifungal properties in Picea Abies resin, it can inhibit the growth of Salmonella Typhimurium and therefore will act as an effective antibiotic.

Due to the lack of funding and research behind antibiotics, antibiotic resistance is on the rise with 2.8 million bacterial infections and more than 35,000 deaths per year in the US. There have been no new classes of antibiotics since the 80’s which raises worry. Antibiotic resistance occurs due to random mutations in the bacteria that develop over time and it is a growing problem due to the misuse and overuse of antibiotics. In this lab, Salmonella Typhimurium is used as a model organism in this lab because it is safe and easy to grow in the lab, more affordable, mimics typhoid fever in mice and survives in macrophages. Picea Abies was chosen due to its history of having antifungal and antibacterial properties. Drug screens were performed to discover a new antibiotic using Picea Abies (Norwegian spruce) resin sample from Finland. Throughout the experiment, drug screens were performed using max dose and dose response curves to determine if the bacteria grew or not in the presence of our compound. The data showed that the bacteria did grow when in contact with Picea Abies which would not make a good antibiotic.

• Spruce resin, Picea Abies (Norwegian Spruce) from Karelia, Finland and Picea Mariana (Canadian Spruce) from Rosevear, Alberta, Canada.
• Picea Abies resin is a complex mixture of compounds ranging from water soluble sugars to hydrophobic lipids.

• To separate out the water soluble and insoluble compounds in Picea Abies resin, process the resin by boiling the raw resin in water.
• Ideally, boiling resin allows for extraction of water soluble and insoluble compounds in separate aqueous and organic layers

• Ethanol extraction of Picea Abies and Picea Mariana sap taffy, water/ juice, and raw.

• Conduct four serial dilutions of the max dose
• Each dilute will be a 1:2 dilution from the previous tube
• Fill 20 wells with S. Typhimurium solution
• Add in two duplicate wells for the dilution series and add in the negative and positive controls (EtOH and ampicillin respectively)
• Now place the plate in an incubator for 24 hours at 37°C

Figure 2: Results of the Mueller-Hinton agar well diffusion tests of Picea Abies (Raw), Picea Abies (sap taffy), and Picea Mariana (Sap taffy) as well as positive and negative controls (ampicillin and 70% ethanol. The plates show that the solutions have no effect on the growth of salmonella.

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While we initially hypothesized due to research showing antibacterial and antifungal properties of Picea Abies resin that the resin would inhibit the growth of Salmonella Typhimurium, this is not shown by our trials in the form of gel plates or drug dilution absorbances. However, our project is still significant for understanding of bacterial population growth as we did observe some growth of the bacterial population.It is important to note that the terms sap taffy, sap water/juice, and raw refer to the extraction process used. Sap taffy refers to the solid product of the boiling process, sap water refers to the liquid product of the boiling process, and raw refers to the resin that did not undergo the boiling process.

Both Picea Abies or Picea Mariana show some positive effect on the growth of Salmonella Typhimurium in certain concentrations and forms, as their absorbance values fall above two standard deviations of 70% ethanol, the negative control’s mean. These hits are from Picea Abies sap juice at concentrations of 100%, 50%, 25%, and 12.5%, Picea Abies raw sap at 100% concentration, Picea Mariana sap taffy at 50% concentration, and Picea Mariana sap water at 25% concentration. Furthermore in the Mueller-Hinton agar well diffusion tests do not show a decrease in salmonella growth and instead show no effect to very small amounts of increased growth in comparison with our negative control of 70% ethanol.

The possible limitations of this project include the amount of substances tested created limitations of time to do as many trials of each substance as we would have liked. This created possible limitations in the accuracy of our data. The use of 70% ethanol as a negative control may have also skewed results due to the fact that the ethanol may have killed some of the bacteria. We did not use a lower ethanol concentration due to us doing ethanol extraction and wanting to get as much of the substance as possible.

1. For future experiments, it would be important to know the extraction process for the resin sample. In common parlance, sap and resin are often used interchangeably. By knowing whether the resin source provides sap (has high sugar concentration) or resin (has higher concentrations of complex organic compounds), a more appropriate comparison between the results of this lab and the research sources could be determined.
2. Since the group had multiple statistically significant hits, future testing could increase the number of Mueller-Hinton agar well diffusion tests to include all hits.
3. For long-term studies, it may be interesting to test other spruce species for possible antibiotic compounds.
4. Since 70% ethanol is a highly antimicrobial solution, future research could look into using lower ethanol concentrations for the extraction of resin.

• The resulting data would be analysed by an OD620 spectrophotometer for the absorbance value of the wells. Statistical significant values are absorbances with +/- 2 standard deviations from the mean of the negative control absorbance value.

This project would not have been accomplished without the support, guidance and teaching from Dr. Pamela Harvey throughout the semester and endorsement from Dr. Lee Niswander. We would like to thank Dr. Corrie Detweiler for advising and facilitating lab work. We would also like to give credit to our teaching assistants, Kiana Roberts and Zahra Thathey, for guiding us through the process and helping us in the lab for countless hours. Lastly, we would like to thank the Howard Hughes Medical Institute for sponsoring the Discovery lab.

Dilution Series

Dose Response Plate