The Effects of Varying Tea Tree Oil Concentrations on Staphylococcus aureus Growth
Yasmin Ghaemmaghami, Nicole Peters, Stevie White
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Staphylococcus aureus has become increasingly resistant to antibiotics. As a result, new methods are being explored to find approaches to fight against antibiotic resistant S. aureus. One approach to fighting against S. aureus is by using essential oils like tea tree oil. The objection[AS1] of this study is to determine the minimum concentration of TTO needed to inhibit S. aureus growth. There could b[AS2]e a statistical difference in the amount of Staphylococcus aureus in the pure nutrient broth versus the Staphylococcus aureus in the nutrient broth combined with the Tea Tree Oil at various concentrations. Fifty test tubes were placed into five different groups based on the TTO concentration used in the experiment: control,3, 1, 0.5, 0.25 (% v/v). A spectrophotometer was used to measure absorbance and percent transmittance of bacteria growth in different tea tree oil concentrations. Absorbance and percent transmittance were used to calculate optical density, which correlated to the amount of bacteria presentThe mean optical density for the control was 0.0105 ± 0.003582 (±SEM), 0.25 M of TTO was 0.0303 ± 0.0058 (±SEM), 0.50 M of TTO was 0.0243 ± 0.004973 (±SEM), 1.0 M of TTO was 0.0247 ± 0.004028 (±SEM), and 3.0 M of TTO was 0.025 ± 0.004624 (±SEM). There was no significant difference (p=0.055, single factor ANOVA) between any of the five groups.If the variation within the four experimental groups had been closer to each other, then there would have been a higher likelihood of there being a statistical difference.
Introduction[AS3]
Staphylococcus aureus, known to cause cutaneous infections,has become increasingly resistant to antibiotics forming dangerous strands like methicillin-resistant S. aureus (MRSA) (Ansari et al. 2012; Leszczyńska et al. 2010). S. aureus is a bacteria commonly found on the skin of the human body (Harris et al. 2002). However, S. aureus can cause health issues like abscesses and superficial lesions if the bacteria enters the underlying skin (Ansari et al. 2012; Harris et al. 2002). This occurs when the skin is breached due to cuts, surgery, or other causes (Ansari et al. 2012; Harris et al. 2002). The threat of antibiotic-resistant S. aureus has led to the exploration of new antibacterial agents(Ansari et al. 2012; Leszczyńska et al. 2010).[AS4]
Currently, antibiotics are being tested to determine how effective they are against antibiotic resistant S. aureus. KatarzynaLeszczyńska from Medical University of Biaystok, Biaystok, Poland assessed “…the antibacterial activity of amoxicillin with clavulanic acid (AMC), tetracycline (T), erythromycin (E) and amikacin (AN)… against different clinical isolates of Staphylococcus aureus in combination with synthetic LL-37” (Leszczyńska et al. 2010). Synthetic LL-37 is the antimicrobial protein cathelicidin LL-37, a natural antimicrobial molecule made by the body that can kill bacteria cells by negatively effecting bacterial cell walls (Leszczyńska et al. 2010). Results suggested that “AMC was strongly potentiated when added in combination with LL-37” (Leszczyńska et al. 2010). Though these antibiotics could provide optimal treatment, the researchers “…did not observe any decrease in the MIC values of T and E, particularly against methicillin-resistant S. aureus…in the presence of LL-37” (Leszczyńska et al. 2010). MIC is Minimal Inhibitory Concentration used to determine the minimal concentration of antibiotics needed to inhibit bacterial growth (Leszczyńska et al. 2010)[AS5]. But, effectiveness of antibiotic drugs are questionable due to antibiotic resistance (Edwards et al. 2004[AS6]). This is only an example of the difficulty of finding antibiotics able to properly inhibit antibiotic-resistant bacteria.
Commercial antiseptics have also been explored as methods to inhibit bacterial growth. Elaine L. Larson and Barbara E. Laughon from the School of Nursing and Division of Infectious Disease of John’s Hopkins University compared four antiseptic products containing gluconate on bacteria growth on the skin (Larson and Laughon 2010). Various participants were given different CHG antimicrobial soaps to test their efficacy on killing bacteria on the skin (Larson and Laughon 2010). The study suggests there was no statistical difference between the CHG products in their inhibition of bacterial growth on the skin, meaning that all the four CHG products were effective in inhibiting general bacteria growth (Larson and Laughon 2010). While certain commercial products are still widely used, the fear is that there could be a day when S. aureus could become resistant to all antibacterial drugs and products (Edwards et al. 2004).
Essential oils have been acknowledged for their antimicrobial properties (Carson et al. 2002; BassoleJuliani 2012). Generally, essential oils, like geranium and lavender, attack the cell membrane of bacteria and cause the cell to lyse (Carson et al. 2002). These essential oils have various components responsible for the oils’ medicinal effects (Edwards et al. 2004). One of the most widely researched oils that have the most potential of inhibiting bacterial growth is tea tree oil.
Tea tree oil, extracted from the leaves of Melaleuca alternifolia through steam distillation, has been suggested to have antimicrobial effects (Carson et al. 2002; and Raman and Bloomfield 2012). Tea tree oil and its components work by compromising the cytoplasmic membrane of bacterial cells through various processes; certain methods include inducing the cell to lose essential ions and causing a negative effect on cellular respiration (Carson et al. 2002). Tea tree oil also had the largest zone of inhibition when compared to other oils such as patchouli, lavender, geranium, and CitricidalTM (Edwards et al. 2004). A zone of inhibition shows the potency of how well a substance’s antimicrobial properties have affected the organism’s growth. Since S. aureus has been known to cause many different types of infections, using tea tree oil isas a natural way to treat and prevent these infections, as well as being a natural preservative, is certainly desirable. (Herman et al. 2013).
Studies have reported that S. aureus is susceptible at 0.5% TTO (Hammer et al 1999) or concentrations greater than 2% TTO (Carson et al. 2006) in MIC testing (Minimum Inhibitory Concentration)[AS7]. Therefore in this study concentrations of 3, 1, 0.5, 0.25 (% v/v) are utilized, since these values are above, below, and in between the range of 0.5% and 2%. The objection of this study is to determine the minimum concentration of TTO needed to inhibit S. aureus growth. The implication of this project will hopefully provide better clarification in the amount of TTO needed to inhibit S. aureus growth. There could be a statistical difference in the amount of Staphylococcus aureus in the pure nutrient broth versus the Staphylococcus aureus in the nutrient broth combined with the Tea Tree Oil at various concentrations.
Materials and Methods
Fifty 0.5 mL samples of Staphylococcus. aureusand one 59 mL bottle of De La Cruz Tea Tree Oil were used in the study. The S. aureus was provided to the researchers by Saddleback College Biology Department and the tea tree oil was purchased at Walgreens in Mission Viejo, California. Fifty test tubes were filled with 10 mL of Criterion nutrient broth (lot number 12185).
On October 14, 2015, tea tree oil dilution factors were prepared. Four test tubes filled with 10 mL of sterile deionized water were provided to researchers by the Saddleback College Biology Department. Using aseptic technique, each test tube was pipetted with one of the following amounts of De La Cruz TTO: 300, 100, 50, and 25 microliters. The ratio of the TTO to the 10 mL of DI water correlates to the TTO concentration used in the experiment: 3, 1, 0.5, 0.25 (% v/v). Each test tube was then gently shaken to uniformly mix the DI water and the TTO.
A P-1000 micropipette was used to pipette 500 µL of S. aureus into each test tube that had the previously prepared sterile nutrient broth using aseptic technique. After the transfer of S. aureus each tube was swirled to distribute S. aureus. The tubes were labeled into five groups: control and four experimental groups based on different TTO concentrations.
A Beckman Coulter DU 730 Spectrophotometer was used to measure the absorbance and percent of transmittance at 532 nm. A P-1000 micropipette was used to pipette 1 mL out of each tube into 1.5 mL disposable polystyrene PMMA cuvettes. A cuvette filled with 1 mL of nutrient broth only was used as a blank. The initial absorbance and the percent of transmittance values were recorded.
A P-200 micropipette was used afterwards to aseptically pipette four sets of tubes (n=10) with 0.05 mLs of TTO with the following concentrations: 3, 1, 0.5, 0.25 (% v/v). All test tubes were incubated at 37°C for 48 hours. At 9:20 AM on October 16, 2015 the fifty test tubes were removed from the incubator to measure bacterial growth of each final group.
A P-1000 micropipette was used to pipette 1 mL out of each test tube into 1.5 mL disposable polystyrene PMMA cuvettes. Each set of test tubes (n=10) had a different blank, a 1:1 ratio of TTO concentration used for the particular set and Criterion nutrient broth were placed into the cuvette to be used as the blank.
The final absorbance and the percent of transmittance values were recorded in the lab notebook. Optical density was determined with the following formula OD = (2-LOG (%Trans))/abs; optical density was used to quantify the amount of bacteria in solutions. Once calculated, the final optical density of each tube within all five groups was subtracted by its initial optical density. A single factor ANOVA was run to determine if there was a significant statistical difference between the concentrations of each group.
Results
The mean optical density for the control was 0.0105 ± 0.003582 (±SEM), 0.25 M of TTO was 0.0303 ± 0.0058 (±SEM), 0.50 M of TTO was 0.0243 ± 0.004973 (±SEM), 1.0 M of TTO was 0.0247 ± 0.004028 (±SEM), and 3.0 M of TTO was 0.025 ± 0.004624 (±SEM). A single factor ANOVA test was used to determine if there was significant statistical difference between all five groups containing different concentrations of tea tree oil. There was no significant difference (p=0.055, single factor ANOVA) between any of the five groups (figure 1).
Figure 1. Comparison of the mean optical densities between five groups. Error bars are mean ± SEM. There was not a significant statistical difference among any of the five groups (p=0.055, single factor ANOVA).
Discussion
The premise was that there is a[AS8] there could be a statistical difference in the amount of Staphylococcus aureus in the pure nutrient broth versus the Staphylococcus aureus in the nutrient broth combined with the Tea Tree Oil at various concentrations. Since studies have reported that S. aureus is susceptible at 0.5% TTO (Hammer et al. 1999) or concentrations greater than 2% TTO (Carson 2006) in MIC testing (Minimum Inhibitory Concentration), [AS9]concentrations of 3, 1, 0.5, 0.25 (% v/v) were utilized. The reason is 3, 1, 0.5, 0.25 (% v/v) concentrations are above, below, and in between the range of 0.5% (Hammer et al 1999) and 2% (Carson 2006). The objection of this study was to determine the minimum concentration of TTO needed to inhibit S. aureus growth.TTO is a great inhibitor of S. aureusdue to the high numbers of the optical densities. However, Tea Tree Oil did not inhibit S. aureus significantly enough to demonstrate a difference between the control and the various concentrations.
The greatest inhibition was 0.25% TTO concentration. The three other test groups (0.5, 1.0,3.0%) were relatively close to each other according to Figure 1. However, there was no statistical difference between the control with S. aureus and the four experimental groups.
A[AS10] possible reason may have been due to data variation. There was not a lot of variation in the data.If there was even less variation within the data, there may have been a statistical difference.Sample size may have been an issue. Although data suggests that there wasn’t a large variation, more samples in the group may have decreased the variation further.
Literature Cited[AS11]
Ansari, Mohammad Azam, Haris M. Khan, Aijaz A. Khan, Asfia Sultan, and Ameer Azam. (2012). "Characterization of Clinical Strains of MSSA, MRSA and MRSE Isolated from Skin and Soft Tissue Infections and the Antibacterial Activity of ZnO Nanoparticles." World Journal of Microbiology and Biotechnology 28(4): 1605-1613.
Bassole,Imael Henri Nestor and H. Rodolfo Juliani. (2012). “Essential Oils in Combination and Their AntimicrobialProperties.” Molecules17: 3989-4006.
Carson, C F., K A. Hammer, and T V. Riley. "Melaleuca alternifolia (Tea Tree) Oil: Review of Antimicrobial and Other Medicinal Properties." American Society for Microbiology 19, no. 1 (January 2006): 50-62.
Carson, C. F., Mee, B. J., & Riley, T. V. (2002). “Mechanism of Action of Melaleucaalternifolia (Tea Tree) Oil on Staphylococcus aureus Determined by Time-Kill, Lysis, Leakage, and Salt Tolerance Assays and Electron Microscopy.” Antimicrobial Agents and Chemotherapy, 46(6): 1914–1920.
Edwards-Jones, V, R. Buck, S.G. Shawcross, M.M. Dawson, K. Dunn. (2004). “The Effect of Essential Oils on Methicillin-Resistant Staphylococcus aureus Using a Dressing Model.” Burns 30: 772-777.
Hammer, Katherine A., C. F. Carson, and T. V. Riley. "Antimicrobial activity of essential oils and other plant extracts." Journal of applied microbiology 86, no. 6 (1999): 985-990.
Harris, Llinos G., S J. Foster, and Robert G. Richards. (2002). "An Introduction toStaphylococcus aureus, and Techniques for Identifying and Quantifying S. aureus Adhesins in Relation to Adhesion to Biomaterials: Review." European Cells and Materials 4: 39-60.
Herman, Anna, Andrzej Przemysław, Herman Beata, Wanda Domagalska, Andrzej Młynarczyk. (2013)."Essential Oils and Herbal Extracts as Antimicrobial Agents in Cosmetic Emulsion." Indian Journal of Microbiology 53(2): 232-237.
Laughon, Barbara E. and Elaine L Larson. (1987). "Comparison of Four Antiseptic Products Containing Chlorhexidine Gluconate." American Society for Microbiology 31(10): 1572 1574.
Leszczyńska, Katarzyna, Andrzej Namiot, Paul A. Janmey, and Robert Bucki. (2010)."Modulation of Exogenous Antibiotic Activity by Host Cathelicidin LL-37." Apmis 118(11): 830-836.
Raman, A., U. Weir, and S.F. Bloomfield. (1995). “Antimicrobial Effects of Tea-Tree Oil and its Major Components on Staphylococcus aureus, Staph. epidermidis and Propionibacterium acnes.” Letters in Applied Microbiology 21(4): 242-245.
Review Form
Department of Biological Sciences
Saddleback College, Mission Viejo, CA 92692
Author (s):_Yasmin Ghaemmaghami, Nicole Peters, Stevie White
Title: The Effects of Varying Tea Tree Oil Concentrations on Staphylococcus aureus Growth
Summary
Summarize the paper succinctly and dispassionately. Do not criticize here, just show that you understood the paper.
This project analyzed the effects of tea tree oil as an antibiotic. Previous experiments looked at various concentrations of tea tree oil, this one looked at another specific concentration of tea tree oil. Careful analysis with a spectrophotometer was used to indicate different concentrations of the tea tree oil. Overall, the analysis showed no significant difference between the amounts of tea tree oil used and their effects on staphylococcus aureus growth. This experiment sought to look for the minimum amount of tea tree oil necessary to inhibit bacterial growth.
General Comments
Generally explain the paper’s strengths and weaknesses and whether they are serious, or important to our current state of knowledge.
This paper is extremely knowledgeable and solid. Some things I would change were indicated in the comments provided on the paper, however, many weaknesses were not found. Personally, I would limit the amount of references used in the introduction. However, overall, the paper was done extremely well and not many changes other than spelling errors and formatting changes should be done.
Technical Criticism
Review technical issues, organization and clarity. Provide a table of typographical errors, grammatical errors, and minor textual problems. It's not the reviewer's job to copy Edit the paper, mark the manuscript.
This paper was a final versionThis paper was a rough draft
- There is an error in your abstract. “The objection of this study” should be “The objective.”
- Second error comes from word choice. Reword your statements so they are more firm.
- There are a lot of references in your introduction. While it’s good to have sources for your statements, you do not need nearly that many references for your statements.
- Also, your introduction is pretty long. I’d advise condensing it down a bit, maybe get rid of just one paragraph.
- There are some formatting issues in your discussion session.
- I changed around some wording in a couple of your sentences.
- Like I said, overall this essay is very strong. Other than a few wording, spelling, and formatting errors, it looks pretty solid
Recommendation
This paper should be published as is
This paper should be published with revision
This paper should not be published
Signature: Patrick Stumps Date: 12/5/2015
Print your name: Patrick Stumps
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[AS1]Objective
[AS2]Reword
[AS3]Your intro is solid, educated, and well rounded. It seems like you guys really know what you’re talking about. However, I would suggest condensing it down a bit.
[AS4]Is the reference in this sentence really necessary?
[AS5]Unnecessary reference?
[AS6]Again, this reference is also unnecessary. You should use more of your own commentary. Not every sentence needs some sort of reference.
[AS7]Again, this reference is also unnecessary. You should use more of your own commentary. Not every sentence needs some sort of reference.
[AS8]The data suggested that
[AS9]You explained what MIC was earlier.
[AS10]Formatting for past two paragraphs is incorrect; delete the space in-between two paragraphs
[AS11]Overall, this project is solid. You guys did a great job. I didn’t edit much, simply because it didn’t seem like there was too much to edit.