Essential Oils in Combination and Their Effect on E. Coli Growth a Thesis Presented to the Faculty of the College of Arts and Sc

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Essential Oils in Combination and Their Effect on E. coli Growth

A thesis presented to the faculty of the College of Arts and Sciences of Ohio University

In partial fulfillment of the requirements for the degree Bachelor’s of Science

This thesis titled Essential Oils in Combination and Their Effect on E. coli Growth

by BRITTNEY E WILLIAMS

has been approved for the Department of Chemistry and Biochemistry and the College of Arts and Sciences by

Marcia Kieliszewski Professor of Chemistry and Biochemistry

Robert Frank Dean, College of Arts and Sciences

Essential Oils in Combination and Their Effect on E. coli Growth WILLIAMS, BRITTNEY E., B.S., May 2018, Chemistry Thesis Advisor: Marcia Kieliszewski

Abstract

The purpose of this thesis is to investigate the effects of four essential oils in combination on the growth of Escherichia coli in liquid and solid culture. This research was undertaken in response to the growth of antibiotic resistant bacteria. Essential oils have historic medicinal properties and it has been deduced by Gupta et. al that “the efficacy of herbals in treatment of diseases for decades suggests that bacteria, fungi and viruses may have a reduced ability to adapt to a plant based antimicrobial regime.” The four oils being studied are pine, orange, oregano, and coriander oils. Each have unique properties related to their chemical structures that impact their antibacterial properties. The goal of this research is to determine if those four essential oils have quantifiable antibacterial properties and if so, are the properties altered when the oils are put in various combinations with different concentrations. It was found that pine and coriander oils in a 2:1 ratio had the lowest final bacterial concentration of 4.38×108 cells/mL.

TABLE OF CONTENTS Page Abstract ...... 3 List of Tables ...... 5 List of Figures ...... 6 Chapter 1: The Medicinal Effects of Essential Oils ...... 7 Justification for oils studied ...... 7 Chapter 2: A Brief History of Antibiotic Resistance...... 11 How the antibiotic resistance epidemic began ...... 11 Explanation of how antibiotic resistance is developed ...... 11 Chapter 3: Experimental Methods ...... 14 Preparation of LB Broth ...... 14 Growing E. coli ...... 14 Measuring the Concentration of E. coli ...... 15 Essential Oil Liquid Culture Test ...... 15 Chapter 4: Results ...... 16 Chapter 5: Discussion and Conclusion ...... 19 Conclusion: ...... 20 References ...... 21

List of Tables

Page

Table 1 Ratios and volumes of oils used in combination test ...... 14

Table 2 OD600 nm values over 5 hours for each oil...... 15

Table 3 OD600 nm values over 4 hours for the combinations of pine with coriander and orange with oregano...... 15

Table 4 The bacterial concentrations in the presence of each individual oil ...... 15

Table 5 The bacterial concentration in the presence of oil combinations ...... 15

List of Figures

Page Figure 1. The dissemination of antibiotics and the uses by humans and animals ...... 14

Figure 2. The absorbance values at 600 nm for E. coli as a function of time in the presence of each individual oil ...... 16

Figure 3. The absorbance values at 600 nm for E. coli as a function of time in the presence of the oil combinations in varying ratios ...... 16

Chapter 1: The Medicinal Effects of Essential Oils

The International Standard Organization defines essential oils as the products obtained from plants “by extraction, effleurage, drag steam vapour, extraction with organic solvents, pressing or by supercritical fluid extraction”.15 These oils can come from various parts of the plant including the leaves, blossoms, peels, wood, roots, bark and resin. The chemical components of these oils give them their healing and fragrant properties. These compounds are predominantly terpenes, alcohols, aldehydes, esters, ethers, and ketones. Although these compounds originate from the secondary metabolism of the plants, essential oils can also be created artificially. However, the synthetic essential oils often lack the healing properties of natural oils. The oils used in this research are the natural-occurring oils that have been distilled using steam distillation.15 Essential oils have been recognized for their antibacterial properties for centuries. They have been used in a wide variety of applications such as cooking oils, medicines, cosmetics, and even aphrodisiacs. The shift to plant oils in industrialized medicine has been rationalized by the “awareness that many diseases have a complex pathophysiology”.21 The focus of research on essential oils as antibacterial agents shifted from a general study of which oils can be used as a medication to finding the mechanism by which these oils inhibit or prevent the growth of bacteria.21

Justification for oils studied

1. Pine Oil Pine oil is a versatile oil due to its large abundance in nature. The Pinus genus is the largest genus of conifers naturally occurring in the northern hemisphere, making them easily attainable for most people.9 People have been using this oil for centuries to solve a host of issues. Pine oil can be used as “industrial and household cleaning products, disinfectants, solvents, fragrances, medicine, and aromatherapy.”13 Pine oil is comprised of 49 terpenic compounds and only 3 nonterpenic compounds. Terpenic compounds are commonly found in plants that give antibacterial properties. Terpenic compounds are those compounds that contain a cyclic structure called terpene. These compounds have a

7 general structure of (C5H8)n, where n is the number of isoprene units that are linked to the structure. In a study conducted in 1971 it was understood that “essential oils containing terpenes and other unsaturated compounds are solely responsible for inhibiting the growth of a number of pathogenic microbes.”12 These properties make pine “one of the most popular plants throughout all civilization” due to its continued uses in therapeutic practice.9 The active compound, turpentine, has been prescribed to treat a large variety of diseases in Indonesia, its main producer, including to treat “rheumatism, sciatica, nephritis, drop, constipation and mercury salivation.”20 Pine oil is used in treatment against gram-positive bacteria such as Staphylococcus aureus which causes skin diseases.20 This is due to the fact that “gram-positive S. aureus was more sensitive to the [essential oils] than gram-negative E. coli”.8 This study by D. Djenane found that oils containing terpenes have the ability to “disintegrate the outer membrane of gram-negative bacteria” which is a potential area of interest for continued study. Pine oil was chosen for this study due to ease of access. When assessing at essential oils with antibacterial properties, it was crucial to find oils that were easily accessible to a wide variety of people. If this oil were to be incorporated into Western medicine, it would need to be something that companies could get in large quantities. Pine oil is highly concentrated in needles, twigs, and buds which is easily obtained.13 It is also a component of certain household cleaners such as Pinesol. This idea was supported by a review article, Essential Oils in Combination and Their Antimicrobial Properties, by Imaël Henri Nestor and H. Rodolfo Juliani, that looked at certain oils in combination. This article described how certain oils in varying combinations could be used as antibacterial agents against the growth of a variety of food-borne pathogens. Pine oil had one of the highest zones of inhibition against gram-negative bacteria.1 Pine was not mixed with the other three oils being tested in this thesis prompting the idea of combining pine oil with other oils to see if it had a synergistic or additive effect. 2. Orange Oil Citrus oils are commonly used in cleaning products, this in part due to the scent, however these scent properties are not the only reason for their use.15 These oils can be

8 used as both antioxidants, flavoring compounds and antimicrobial agents.10 Orange oil, known scientifically as Citrus aurantium, is composed of d-limonene and myrcene. Limonene is a common constituent of citrus oils. Citrus oil antioxidant activity is correlated to presence of both limonene and terpene substances. Limonene is classified as a cyclic terpene and is the most studied component of essential oils with antibacterial properties. Citrus oils are easily extracted from the peels of citrus fruit, the blossoms of the flower or from the juice of the fruit.15 Orange oil can be used to soothe the skin, alleviate colds and strengthen skin tissue in the case of cellulite. These additional uses make it an appealing oil for incorporation into health and beauty products. Orange oil was chosen for this study due to its long understood antibacterial properties. These properties are strong individually and if it was to be combined with other oils with similar properties, I speculated that these properties could be improved. The extract of orange oil is “effective in inhibiting the AcrAB-TolC efflux transporter” present in Gram-negative bacteria.3 This ability qualifies orange oil as a plant synergist meaning that it acts to "increase the activity of other plants used for healing while affecting the body itself.”3 This is the only oil used in this study classified as such. Due to the synergist qualities, this may be an oil that can be used in combination with the other oils in this study to improve its antibacterial properties. 3. Oregano Oil Spices are herbal products that are viewed as potential preservatives for food items. The antibacterial properties of spice “essential oils against different microorganisms have been recognized since antiquity”, which prompted an interest in oils of spices for the current study.5 The same study found that a concentration of 5% oregano oil was most effective at inhibiting the growth of E. coli. The oils used in food to control foodborne pathogens “target the membrane transport activity” of bacteria.5 Origanum or oregano oil is used as a food-flavoring agent because it “possesses a broad spectrum of antimicrobial activity.”17 This is the result of the oil consisting of phenols, carvacrol and thymol which each have antiseptic and antioxidant compounds.14 Oregano oil when combined with other essential oils had a consistent additive effect on the inhibition of bacteria growth.1 Oregano oil is an option to prevent foodborne disease due

9 to its ability to inhibit bacteria growth and its lack of effect on taste. Since oregano oil is common and widely used, I added it to this study. The extensive applications of this oil as a potential food preservative make it a potential choice for fighting antibiotic resistant foodborne pathogens. 4. Coriander Oil The choice for coriander oil in the current study was due to having access to a coriander plant through Dr. Kieliszewski. Her garden in Michigan provided the seeds to distill the coriander oil using steam distillation. Although the distillation process was futile, it prompted further research into the uses of coriander oil. Coriander, better known as cilantro, is a common food seasoning in Spanish, Latin, Mexican and Indian cuisines. When the leaves are consumed, they are found to lower low-density lipoproteins and increase the high-density lipoprotein levels. Coriander contains anti-inflammatory properties and antiseptic properties.15 These properties are similar to the other four oils being studied. The antiseptic properties are of greatest interest for the eventual use of these oils as antibacterial agents. Like oregano, coriander is a spice that is used typically in food preparation. The antiseptic properties may have applications in food preservation.

Chapter 2: A Brief History of Antibiotic Resistance

The major motivation of this research is to use natural essential oils to fight antibiotic resistance. Antibiotic resistance as described in the introduction has been affecting the world since the creation of antibiotics.5 To better understand the significance of the present study, it is necessary to understand how the antibiotic-resistance epidemic began and the other methods suggested as a remedy.

How the antibiotic resistance epidemic began

Since the creation of the first widely use antimicrobial drug, bacteria and microbes have developed resistance to these drugs. The first effective antimicrobial was introduced in 1937 and was faced with mechanisms of resistance in the 1930s.5 Penicillin was met with a similar resistant strain capable of “inactivating the drug” when it was being researched. A similar case occurred when streptomycin, used to treat tuberculosis, was met with a mutant strain of Mycobacterium tuberculosis which was resistant to the antibiotic concentrations of the drug. The idea of “superbugs” was coined in the twentieth century as microbes “with enhanced morbidity and mortality due to multiple mutations” evolved a resistance to the antibiotics used in their treatment.5

Explanation of how antibiotic resistance is developed

The oldest cause for antibiotic resistance is simply the random mutations of “genes encoding the enzymes” which has “given rise to modified catalysts with increasingly extended spectra of resistance.”5 As these microbes replicate and are mutated through DNA replication, alterations made to the genetic makeup that give rise to resistance against certain antibiotics. As a result of the principle of natural selection, these bacteria have developed genes that allow for survival in the presence of these antibiotics in previously considered therapeutic concentrations. An example of this is the plasmid-encoded beta-lactamase which have acquired resistance and mutations through environmental strains of Kluyvera.5 Some bacteria develop an intrinsic resistance due to “the existence of genes in bacterial genomes that could generate a resistance phenotype.5

Genetic amplification has been labeled a common route for this enhanced resistance notably for resistance against sulfonamides and trimethoprim.2 A study was done on a mutant E. coli gene, Keio, and it was found to contain 140 isolates that were hypersensitive to a range of antibiotics.5 These hypersensitive genes are the ones that are known to generate a resistant phenotype when the wild-type is overexpressed. Some of these resistances, however, can be man-made. Resistomes are generated when bacterial strains resistant to antibiotics are separated by plating on media containing antibiotics.5 This is the proposed expression by Gerard D. Wright for the collection of antibiotic resistance genes and their precursors. He and his colleagues “screened a collection of morphologically distinct spore-forming actinomycetes” for over 20 antibiotics. Most strains were resistant to an average of 7.5 antibiotics meaning they were naturally multidrug resistant. 6 In conjunction with this study, Dantas took a complementary approach to suggest that soil bacteria can be screened for biochemical processes that either degrade or stop antibiotics.4 Most strains identified grew on common antimicrobials and this research showed that there are new mechanisms of resistance through more than just catabolic pathways.5 The study by Dantas revealed the full extent of degradation genes in the environment. However, the environment and natural mutations are not the main cause in these antibiotic resistant bacteria. A large cause of this influx of resistance is the role of human activities. Since the first commercial uses of antibiotics in the 1940s, these antibiotics have been released into the environment providing the ability for these resistant strains to thrive. The use of antibiotics directly in humans is not the main problem as it constitutes less than half of all applications of commercially produced antibiotics.5 The following figure shows how the use of antibiotics coming in direct contact with food animals and humans contributes to the dissemination of antibiotics.

Figure 1. The dissemination of antibiotics and the uses of antibiotics by humans and animals.7

It can be seen in the figure that although antibiotics come into direct contact with four components of the figure, the effects of these drugs resonate all the way to aquaculture. Studies performed on wastewater treatment plants have shown they are “rich reservoirs of resistant organisms” and these genes are frequently carried and transmitted through plasmids providing sources of resistance determinants.19 Although genetic variation is a downstream cause for the spread of antibiotic resistance, the transfer of these genes typically originates from a human cause. There are, however, other ways for genetic transmission of genes that occurs naturally.

Chapter 3: Experimental Methods

Preparation of LB Broth

To grow a liquid culture, lysogeny (LB) broth was prepared according to the following procedure. Three hundred milliliters of deionized, MilliQ water was measured into a flask and to this, 5g tryptone, 2.5g yeast extract and 2.5g of sodium chloride are added. The contents were mixed on a stir plate with a metal stir bar. The solution was brought to a final volume of 500 mL by adding deionized, MilliQ water. The flask was stoppered with a foam stopper and aluminum foil was used to cover the stopper. The flask was autoclaved on liquid cycle at 121°C for 20 minutes. This procedure was amended to make the LB agar plates to use to grow streaked plates of E. coli. To create agar, 7.5 g bacterioagar was added to the flask prior to autoclaving. The mixture was autoclaved on liquid cycle at 121°C for 20 minutes. Once removed from the autoclave, the broth was moved into an isotemp to cool to 50°C. The media was poured into culture dishes as follows: a. Remove lid to a dish and remove the foil cover to the flask. b. Pour just enough LB agar into the dish to completely cover the bottom. c. Use a flame to sterilize the opening of the flask. d. Repeat to fill all dishes. The plates were stored in the plastic they originally came in and placed into the fridge.

Growing E. coli

The E. coli was grown on both LB agar plates and in an LB liquid culture. To grow the bacteria on a plate, the original culture of Top 10 E. coli was removed from the -80°C freezer and allowed to thaw to room temperature. A wire hoop was used to streak the plate with the bacteria. The hoop is sterilized in a flame prior to using. The plate was streaked in the pattern shown in Figure 2. The streaked plate was placed into an incubator set at 37°C and allowed to grow overnight for 16 hours. The plated culture was used to create the liquid culture. 10 mL of L broth was pipetted into an autoclaved culture tube. An autoclaved toothpick is used to select a

14 single colony from the plate to inoculate the broth. The toothpick is placed into the tube and the tube was capped and placed into a shaker at 37°C and 250 rpm overnight for 16 hours.

Measuring the Concentration of E. coli

To measure the concentration of E. coli in a liquid culture, a spectrometer was used to measure the absorbance of the bacteria at 600 nm. This value was recorded once after the initial growing time and again each hour during the essential oil test.

Essential Oil Liquid Culture Test

To test the antibacterial properties of each oil, the E. coli was grown in the presence of the oil for 5 hours after the initial 16-hour growth period. Each hour the absorbance at 600 nm was recorded to detect any changes in the growth of the E. coli. For the first test, 30 µL of the oil being tested is pipetted into a culture tube with grown E. coli. For the combination test, oils were added to the tubes in the following ratios:

Table 1. The ratios and volumes of oils used in the combination of oils test. Ratio Volume of Each Oil 1:1 15 µL of each 1:2 10 µL and and 20 µL 2:1 20 µL and 10 µL

Chapter 4: Results

Each absorbance value is used to calculate the cell concentration per milliliter of inoculated broth. Calculation 1 shows how the absorbance at 600 nm is proportional to the bacterial cell concentration. Coriander oil correlated to a decrease in bacteria absorbance while oregano and orange oils showed the least inhibitory properties with absorbances values that were higher than that of the control. These values are shown in Table 4 and plotted in Fig. 2. The coriander oil was combined with the pine oil because these oils had the two lowest final absorbance values during the initial test. Similarly, orange and oregano oils had the highest absorbance values and were combined to test their inhibitory properties. Pine and coriander oil in a 2:1 addition ratio correlated to the greatest decrease in absorbance after 4 hours of growing. Each of the pine and coriander combinations had a final bacteria concentration lower than the control. Orange and oregano in a 2:1 ratio had a final concentration most similar to that of the control. Growing the cells in the presence of a 1:1 ratio of oregano and orange oil created the highest initial increase in concentration although orange and oregano in a 1:2 ratio corresponded to the highest bacteria concentration after 3 hours with 1.72x109 cells/mL.

Table 2. OD600 nm values over 5 hours for each oil.

Time (hr) Orange Pine Oregano Coriander Control 0 1.743 1.743 1.743 1.743 1.743 1 2.07 1.754 2.128 1.678 1.771 2 2.202 1.983 2.424 1.724 1.762 3 2.183 2.03 2.19 1.867 1.745 4 2.227 2.021 2.313 1.844 1.822 5 2.115 1.964 2.196 1.745 1.787

Table 3. OD600 nm values over 4 hours for the combinations of pine with coriander and orange with oregano. OrO OrO OrO Time PC 1:1 PC 1:2 PC 2:1 Control 1:1 1:2 2:1 0 1.007 1.007 1.007 1.007 1.007 1.007 1.007 1 1.319 1.386 1.217 1.69 1.602 1.609 1.257 2 1.182 1.382 1.148 1.892 1.972 1.633 1.522 3 0.841 1.282 1.105 1.916 2.156 1.729 1.697 4 1.195 1.243 0.547 1.951 1.985 1.655 1.683

Table 4. The bacterial concentrations in the presence of each individual oil. Time (hr) Orange Pine Oregano Coriander Control 0 1.39E+09 1.39E+09 1.39E+09 1.39E+09 1.39E+09 1 1.66E+09 1.40E+09 1.70E+09 1.34E+09 1.42E+09 2 1.76E+09 1.59E+09 1.94E+09 1.38E+09 1.41E+09 3 1.75E+09 1.62E+09 1.75E+09 1.49E+09 1.40E+09 4 1.78E+09 1.62E+09 1.85E+09 1.48E+09 1.46E+09 5 1.69E+09 1.57E+09 1.76E+09 1.40E+09 1.43E+09

Table 5. The bacterial concentration in the presence of oil combinations. Time PC 1:1 PC 1:2 PC 2:1 OrO 1:1 OrO 1:2 OrO 2:1 Control 0 8.06E+08 8.06E+08 8.06E+08 8.06E+08 8.06E+08 8.06E+08 8.06E+08 1 1.06E+09 1.11E+09 9.74E+08 1.35E+09 1.28E+09 1.29E+09 1.01E+09 2 9.46E+08 1.11E+09 9.18E+08 1.51E+09 1.58E+09 1.31E+09 1.22E+09 3 6.73E+08 1.03E+09 8.84E+08 1.53E+09 1.72E+09 1.38E+09 1.36E+09 4 9.56E+08 9.94E+08 4.38E+08 1.56E+09 1.59E+09 1.32E+09 1.35E+09

Individual Oil Growth Curve 3 Orange 2 Pine 1 Oregano OD600 nm 0 Coriander 0 2 4 6 Control Time (h)

Figure 2. The absorbance values at 600 nm for E. coli as a function of time in the presence of each individual oil.

Essential Oils in Combination Growth Curves 2.5

2 PC 1:1

1.5 PC 1:2 PC 2:1 1 OrO 1:1 OD600 nm OrO 1:2 0.5 OrO 2:1 0 Control 0 1 2 3 4 5 Time (h)

Figure 3. The absorbance values at 600 nm for E. coli as a function of time in the presence of the oil combinations in varying ratios.

The concentration of E. coli can be determined using the following equation:

�� /�� = �� × × (1) 8 where OD600nm is 1, 8×10 is the number of cells per milliliter.

Chapter 5: Discussion and Conclusion

The results of this research showed that pine and coriander oils mixed in a 2:1 ratio had the highest inhibitory potential. This value is an area of interest for potential future research. In the future, pine could be mixed with other oils in varying ratios to see if pine is a synergistic oil. The same could be done with coriander to see which of the two was the cause of the decrease in bacterial concentration. Others have found that orange oil had high inhibitory properties however this research does not correlate to those findings.3 This may be due to the use of E. coli instead of the bacteria previously studied. When combined with oregano, the total bacteria concentration was greater than that of the control which again disputes the claim that orange oil is a plant synergist.3 Future research can be done to determine which plant oils work best in combination with orange oil. Similarly, pine oil did not work well in a liquid culture alone. Others found that pine had one of the highest zones of inhibition against gram-negative bacteria however this research was not done in a liquid culture.1 In the future, the pine oil could be tested on a plate culture both alone and in combination with other oils. Other oils that could be tested include tea tree, lemon, lavender and peppermint. These oils could be used in both liquid and plated cultures to compare inhibitory properties and see if growing E. coli in the presence of the oil in a liquid culture or on a plate works better. The antibacterial properties of essential oils are currently a major topic of discussion due to the antibiotic resistant bacteria epidemic. The bacteria of interest are typically food-borne pathogens. Essential oils were historically used in food preservation, but the optimal concentrations were unknown.15 Some of these essential oils can be used in combination with EDTA, ethylenediamine tetracetic acid, to potentially inhibit E. coli growth.18 Researchers noticed that “the cell membrane becomes unstable and consequently susceptible to hydrophobic agents” due to the activity of a chelating agent.16 This advancement provided a potential mechanism for how essential oils could be used as antibiotics if coupled with a chelating agent. The addition of essential oils to a chelating agent such as EDTA “increased the inhibition dramatically” from that observed with oregano essential oil.18 This capability is possible with any essential oil due to the similar chemical structure of the oils.12 It was shown that “permeabilizing agents are

19 defined as chemical agents that increase the permeability of the outer membrane of the Gram-negative bacteria.”18 The EDTA allowed for the oregano oil to permeate the membrane to kill the bacteria. Although at this point the exact mechanism was unknown, essential oils were beginning to be investigated as a potential additive to food to prevent bacteria growth. The active components of essential oils have been thought to contain the antibacterial properties. A 2011 study, “Chemical composition and antimicrobial effects of essential oils of Eucalyptus globulus, Myrtus communis and Satureja hortensis against Escherichia coli O157:H7 and Staphylococcus aureus in minced beef” by D. Djenane, concentrated on how the active chemical compound in each of these oils interacted with the bacteria found in meat. This study found that by using essential oils as a preservative in minced meat, bacteria growth was prevented, and the taste was not affected. Essential oils can potentially be used in the future as a food preservative that does not alter the taste or nutritional content. Further studies may be done to test this theory.

Conclusion:

This thesis looked at the antibacterial effects of four essential oils on the growth of E. coli in a liquid culture. Pine and coriander in a 2:1 ratio was found to have the lowest bacteria concentration in the study. Future research could be done to test the efficacy on a plated culture and different ratios of oils. Other research may be performed on other oils to find the optimal combination of oils for the inhibition of E. coli growth.

References

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