This document describes a study that investigated how exposing E. coli bacteria to various sub-minimum inhibitory concentrations (sub-MICs) of cinnamon essential oil (EO) affects the bacteria's resistance to the cinnamon EO. The study exposed E. coli to concentrations of 0, 2.5, 5, 10, and 20 μl/mL of cinnamon EO for 24 hours over 3 passages. A disk diffusion assay then measured the diameter of inhibition zones to determine resistance. The results showed that E. coli exposed to higher concentrations of cinnamon EO developed greater resistance, as seen by smaller inhibition zone diameters.

1. INTERNATIONAL BACCALAUREATE
EXTENDED ESSAY
BIOLOGY
TOPIC: The effect of various sub-MIC (minimum inhibitory concentration) of Cinnamomum
zeylandicum (cinnamon) essential oils (EO) on the level of resistance to cinnamon EO in non-
pathogenic Escherichia coli
RESEARCH QUESTION: How does 24-hour exposure to various sub-MIC of cinnamon EO (0,
2.5, 5, 10, 20) 𝜇𝑙/𝑚𝐿 affect the level of resistance to cinnamon EO in non-pathogenic
Escherichia coli?
Candidate Name:
Candidate Number:
School name:
School code:
Session: May 2019
Word Count: 4000

2. Table of Contents
INTRODUCTION .......................................................................................................................... 3
1.1 Research question ................................................................................................................ 3
1.2 Hypothesis ............................................................................................................................ 3
1.3 Introduction........................................................................................................................... 3
1.4 Theoretical Basis................................................................................................................... 5
INVESTIGATION.......................................................................................................................... 7
2.1 Variables ............................................................................................................................... 7
2.2 Safety consideration.............................................................................................................. 8
2.3 Materials................................................................................................................................ 8
2.4 Methodology....................................................................................................................... 10
DATA COLLECTION AND PROCESSING .............................................................................. 15
3.1 Processed Data .................................................................................................................... 15
3.2 Sample Calculation............................................................................................................. 15
3.3 Qualitative Data .................................................................................................................. 16
3.4 Graph and Statistical Analysis ............................................................................................ 17
ANALYSIS AND INTERPRETATION ...................................................................................... 18
4.1 Data analysis ....................................................................................................................... 18
4.2 Results................................................................................................................................. 20
CONCLUSION............................................................................................................................. 22
5.1 Evaluation ........................................................................................................................... 23
5.2 Further Investigation........................................................................................................... 24
BIBLIOGRAPHY......................................................................................................................... 25
APPENDIX................................................................................................................................... 26

3. INTRODUCTION
1.1 Research Question
How does 24-hour exposure to various sub-MIC* of cinnamon EO (0, 2.5, 5, 10,
20) 𝜇𝑙/𝑚𝐿 affect the level of resistance* to cinnamon EO in non-pathogenic Escherichia coli?
1.2 Hypothesis
H0: E. coli exposed to all concentrations of cinnamon EO will have similar levels of resistance to
cinnamon EO
H1: E. coli exposed to greater concentrations of cinnamon EO will have a greater level of
resistance to cinnamon EO because there is greater selective pressure
1.3 Introduction
The effectiveness of antibiotics in human populations is decreasing due to increasing
bacterial resistance to antibiotics (Giske). Despite attempts to create new antibiotics, various
causes from overuse to inappropriate prescribing have accelerated the rate at which bacterial
resistance is evolved. Even antibiotics at low concentrations (sub-MIC) can induce a high level
of resistance (Yuen) contrary to previously assumed antibiotic concentration range that would
select for resistant mutants (Drlica and Zhao). The antibiotic resistance crisis is global, and
places a substantial health and economic burden around the world (Ventora). Therefore, there
has been a growing interest in developing new antimicrobial agents from natural compounds to
provide an alternative to antibiotics (Chouhan).
Sub-MIC*: lower than lowest concentrationrequiredtoprevent visible growth ofbacterium
Resistance*: when bacteria mutate when exposedtoantimicrobial substances, which then becomes ineffective tothe bacteria.

4. EOs are natural compounds that are well-known to have diverse bioactivities including
antimicrobial effects against various bacteria (Chouhan). Because of these properties, EOs have
been used in cosmetics, food additives, and Chinese traditional medicines. However, because of
the strong taste, odor, low water solubility, and low stability (Chouhan), EOs are used in low
concentrations, approximately 3%.
In addition to the antimicrobial effects of EO on various microorganisms, ideally, EO
should not induce antimicrobial resistance to EO in bacteria (Becerril). If resistant traits were to
be induced in bacteria, the EO would be less effective, making it no different from antibiotics.
One study by Raquel Becerril titled Evaluation of Bacterial Resistance to Essential Oils
and Antibiotics After Exposure to Oregano and Cinnamon Essential Oils highlights that only two
of four bacterial strains affected by oregano had resistant strains, while all bacterial strains
affected by cinnamon showed no signs of resistance to cinnamon EO. Therefore, Becerril
concluded that “it is expected that bacteria rarely develop a resistance mechanism for EOs.
Studies on using EO as alternative antimicrobial agents to antibiotics, however, are still
rare. Therefore, it is imperative that further evaluation on bacterial resistance to EOs are
conducted. Thus, I was encouraged to conduct a study on how long-term exposure to various
sub-MIC of cinnamon EO induce resistance in non-pathogenic Escherichia coli. The results of
this experiment can provide an indication to the potential benefits of sub-MIC EOs.
However, it is also important to note that this experiment does not account for the
complex microbial communities of E.coli, including nutrient availability and predation and
competition between many species and genotypes. To take such variables into account would
have been too time-consuming for this research. This experiment, however, should provide an
approximate measure of the effects of sub-MIC EO exposure on E. coli.

5. 1.4 Theoretical Basis
E.coli is a diverse group of gram-negative bacteria frequently used in many fields of
research. Gram negative bacterium have a high pathological importance (Becerril); in fact, 50%
of Enterobacteriaceae represent 50% of clinically significant isolates in humans. Although most
strains of E.coli are safe to humans, some of them can produce severe infections like urinary tract
infections (CDC).
Figure 1: image of E.coli using electron microscopy (Highton)
Composition of EOs
EOs are secondary metabolites formed by plants, in order to protect the plant from biotic
or abiotic stress. The chemical composition of EOs are complex, as there are approximately 20 to

6. 60 various biochemical components in each EO (Laranjo). It is worth noting that only 2 to 3 of
the components are present in high concentrations.
Most EOs consists of terpenes. The major bioactive component of EOs is monoterpenes,
which is synthesized within the cytoplasm of the cell. Some compounds contain monoterpene
hydrocarbon, oxygenated monoterpeens, diterpenes, oxygenated sesquiterpenes, monoterpene
alcohols, sesquiterpene alcohols, aldehydes, phenols, and coumarins (Laranjo).
The compound with the strongest antimicrobial property in essential oils are phenolic
compounds, such as carvacrol, eugenol, and thymol. The hydroxyl component (-OH) of the
phenolic compounds are known to cause inhibitory actions as they can disrupt the cell membrane
and ultimately the membrane structure (Laranjo).
Below is a table of the major component of cinnamon.
Name of EO Major components Structure
Cinnamon Trans-cinnamaldehyde
Table 1: Major component and structure of cinnamon

7. Investigation
2.1 Variables
Independent variable
 The concentration of cinnamon EO (0, 2.5, 5, 10, 20) 𝜇𝑙/𝑚𝐿
o The Swiss legislation on cosmetics, for instance, has fixed maximal
concentrations of 3.0% essential oil, which helps to delimitate cosmetics and
medical products. Hence, the concentrations above are used in this experiment.
 10 𝜇𝑙 of ethanol were used in all solutions to dissolve the oil in LB broth
 10 𝜇𝑙 of ethanol with no EO was used as a control
Dependent variable
 Growth of bacteria measured by inhibition zone diameter (IZD)
o Disk-diffusion test (Kirby-Bauer) was used to measure the diameter of the disk
Constant variable
 LB broth and agar
o The components of LBB and LBA are outlined in the methodology section
 Time of E. coli exposed the EOs
o E. coli was exposed to EO for 24 hours for each treatment group per each trial
 Number of serial passaging
o E. coli was serial passaged three times for each treatment group per trial
 Amount of bacteria serial passaged
o 10 𝜇𝑙 of bacterial culture for each concentration transferred to 4ml of culture
medium

8.  Temperature inside the incubator
o Temperature was maintained at 33ºC
 Bacteria species
o Same species of E. coli was used in the experiment
 Bacterial turbidity for antimicrobial susceptibility testing
o Bacterial turbidity was matched to OD (absorption value) of 0.5 at 600nm using a
spectrophotometer (1.6 ∗ 108
𝐶𝐹𝑈/𝑚𝑙). This matches the McFarland standard of
0.5, which is the standard used for antimicrobial susceptibility testing according
to EUCAST.
2.2 Safety Consideration
The experiment strictly adhered to the standards of biosafety level 1.
E.coli is generally considered to be a safe strain of bacteria. Non-pathogenic strains were
used for the experiment. Even then, by mutations, the bacteria may become potentially
dangerous, such as pathogenic. Once a pathogenic bacterium is evolved, pathogenic traits may be
quickly transferred, causing even more danger. To prevent dangerous outcomes, all bacteria were
grown at 33ºC, under 37ºC, which is considered to be the optimal temperature for bacterial
growth. Moreover, gloves and eyewear were worn at all times, work surfaces were sterilized by
spraying ethanol before and after the experiment, and materials used were all autoclaved or
sterilized using a flame. The leftover bacterial suspension in the broth were all autoclaved.
Remaining bacteria on disks were put in a solution of bleach for 24 hours.

9. 2.3 Materials
Bacteria
 E. coli
EO
 Cinnamon bark 100% pure essential oil provided by Satya Inc.
LB broth (LBB) and LB agar (LBA)
 10.0g of tryptone
 10.0g of sodium chloride
 5.0g of yeast extract
 1000mL of distilled water
Ethanol (75%)
Secondary
 Micropipette (0-230 𝜇𝑙)
 Micropipette tips
 21 test tubes
 1 1250mL Erlenmeyer flask
 20 petri dishes
 Vernier caliper
 Forceps
 Filter paper
 Bunsen burner
 2000mL of distilled water
 Inoculation loops

10.  Electronic scale (±0.001)
 Aluminum foils
 Para-films
Appliances
 Autoclave
 Refrigerator
 Laminar Flow Chamber
 Incubator
 Shaker
 Spectrophotometer
2.4 Methodology
 All materials and tools were sterilized in an autoclave.
 During the experiment, all tools were sterilized using a flame when in contact with a new
substance
 The tip of the volumetric pipette was replaced after in contact with a new substance
 The experiment was always conducted inside the laminar flow chamber to prevent any
contamination.
Broth Medium
 Rinsed a 1250mL Erlenmeyer flask with distilled water
 Added 1000mL of distilled water to a 1250mL beaker
 Suspended 10.0g of tryptone, 10.0g of sodium chloride, and 5.0g of yeast extract

11.  to the Erlenmeyer flask
 Substances were all measured using an electric scale
 Solution of LB broth was boiled with a magnetic stirrer to completely dissolve for 10
minutes
 Solution of LB broth was autoclaved at 121ºC for 15 minutes
Agar Medium
 Used the same procedure as the broth medium, but 17.00g of agar was also suspended
before boiling the solution
Preparation of Plates
 The LBA solution was poured into 50 Petri dishes after waiting for it to cool. The
LBA medium was still in liquid form. The plates were undisturbed for approximately
20 minutes for the LBA medium to dry. Afterwards, the lid of the petri dishes was
taped to the dish and placed in the refrigerator at 7ºC upside down to prevent the
formation of moisture.
Preparation of Inoculums
 A colony of E.coli was collected from a stock culture and placed into a test tube of
10mL of LB broth. The solution was stirred thoroughly for 5 minutes. The solution
was then matched to the McFarland standard of 0.5 (1.5 ∗ 108
𝐶𝐹𝑈/𝑚𝐿) by
matching the bacteria turbidity to OD of 0.5 at 600nm of a spectrophotometer. The
inoculum was always used within 15 minutes after matching the solution to the

12. McFarland standard. This is to ensure the turbidity of the bacterial suspension is
controlled for all trials.
Evolution of resistance
 A set of 6 cultures were made using 6 test tubes. All 6 test tubes were filled with
4mL of LBB from its stock solution using a volumetric pipette.
 5 test tubes of LBB solutions of varying concentrations of cinnamon EO (0, 2.5, 5,
10, 20) 𝜇𝑙/𝑚𝐿 were made. 10 𝜇𝑙 of ethanol were put in each of the five test tubes.
This is intended to dissolve the EO inside the LBB.
 No cinnamon EO was put in the remaining test tube. 10 𝜇𝑙 of ethanol were put into
the test tube. This was used as a control
 The test tubes were labelled according to their concentrations. Then, 200 𝜇𝐿 of the
inoculum sample were extracted using a volumetric pipette and ejected into each of
the six test tubes.
 Solution from each of the six test tubes were extracted and ejected repeatedly for one
minute using a volumetric pipette.
 All three sets of cultures were incubated at 33ºC with shaking for 24 hours (Figure
2). During that time, 3 serial passages were conducted. (10 𝜇𝐿 of each culture was
transferred for serial passaging.) The serial passage is intended to ensure that
potentially beneficial genes, which in this case are the resistant genes, are not lost.

13. Figure 2: Bacterial cultures in various concentrations of EO on a shaker inside
incubator
Disk-diffusion Method
 After 24 hours, the bacterial suspension’s turbidity of all cultures were adjusted to
OD value of 0.5 at 600nm. All the bacterial suspensions were used within 15 minutes
after the adjustment.
 200 𝜇𝐿 of bacterial cultures of the six varying concentrations and the control were
plated on 12 petri dishes (duplicates). Using an inoculum loop, the bacteria were
spread evenly on the petri dishes.
 Each plate was labelled according to the concentration used.
 Disks of 1𝜇𝐿 of 100% cinnamon EO were placed on all of the petri dishes. Five disks
were placed in each petri dish. The disks were evenly spaced.
Incubator
Test tubes of bacterial cultures
Shaker

14.  All 12 petri dishes were incubated at 33ºC. The results were observed after 24 hours
(Figure 3)
 The diameter of the disk was measured by using a Vernier caliper.
Figure 3: Disk-diffusion method to measure level of resistance of E. coli to cinnamon EO
Petri dish of LBA
Zone of inhibition
EO disk

15. DATA COLLECTION AND PROCESSING
3.1 Processed Data
DV: Diameter (ug/L) (±)
IV:
Cinnamon
EO (ul/L)
0 2.5 5 10 20 Ethanol
Average 21.23 21.79 21.16 21.73 21.6 21.53
𝛿̅* 1.0709 1.2737 0.8776 0.6034 0.8751 0.6320
SEM* 0.3387 0.4028 0.2775 0.2133 0.3094 0.2107
Table 2: average, standard deviation, and standard error measurement of zone of inhibition
𝛿 *: Standard deviation
SEM*: Standard error of the mean
3.2 Sample Calculation
 All Calculation data were processed through Excel
 To find the average of the data, enter “=”, then write “AVERAGE”, then highlight all the
all trials from a treatment group, then press the Enter key
 To find standard deviation of all trials from a treatment group, enter “=”, then write
“STDEV”, then highlight all the values to be evaluated, then press the Enter key

16.  To find the SEM of all trials from a treatment group, enter “=”, then select the
appropriate Standard deviation value, then press “/”, then type SQRT(number of values),”
then press the Enter key
3.3 Qualitative Data
 Streaking pattern of bacterial culture onto petri dishes were inconsistent across all petri
dishes
 The EO initially did not dissolve in the broth medium. After the addition of ethanol and
shaking, the EO thoroughly dissolved in the broth medium.
 Surface of agar plate of bacteria from the broth medium that contained higher
concentrations of EO visibly had greater amounts of EO than bacteria from broth medium
that contained lower concentrations of EO
 The bacterial colonies were sparser on petri dishes containing bacteria from broth
medium containing higher concentration of EO compared to broth medium containing
lower concentration of EO

17. 3.4 Graph and Statistical Analysis
Figure 4: Graph of relation between various concentrations of cinnamon EO and IZD of E. coli
Source of
variation
SS* df* MS* F F crit p-value
Between
groups
3.07 4 0.768 0.753 2.60 0.562
Within
groups
41.9 41 1.02
Total 44.9 45
Table 3: Results of one-way ANOVA test on various cinnamon oil concentration to zone of
inhibition.
SS* Sum of squares
df* Degree of freedom
MS* Mean square
20
21
22
23
0 5 10 15 20 25
ZoneofInhibition(mm)
Cinamon Oil Concentration (ul/L)
Cinnamon Oil Concentration vs Zoneof Inhibition

18. Figure 5: Graph of relation between no treatment and ethanol (control) and zone of inhibition of
E. coli
Comparison t-value
No treatment and 10uL of Ethanol 2.262
Table 4: statistical analysis between no treatment group and 10uL ethanol group using t-test
ANALYSIS AND INTERPRETATION
4.1 Data Analysis
According to the data presented in table 2, no statistically significant change was
observable in the diameter of the zone of inhibition as the concentration of EO increased. The
average zone of inhibition of the bacteria were 21.23, 21.79, 21.16, 21.73, and 21.60 mm for 0,
15
16
17
18
19
20
21
22
23
0 Ethanol
ZoneofInhibition(mm) Null Treatmentand Ethanol to Zone of Inhibition

19. 2.5, 5, 10, 20μL/L respectively. The greatest difference amongst the groups in IZD was between
5 and 10 μL/L, which was only 0.57mm.
The error bars in the graph represent standard error measurements of the five average
IZDs (Figure 4). It is important to note that majority of the error bars from the five different
treatments overlap. 2.5 μL/L group had the largest SEM of 0.4028mm, which overlaps with the
error bars of all four remaining groups. 10 μL/L group had the smallest SEM of 0.21333mm,
which still overlaps with 0, 2.5and 20 μL/L groups. Because the error bars overlap, the
differences amongst the IZDs across all five groups are likely to be statistically insignificant.
In addition, the one-way ANOVA test was conducted using the Stateplus feature in
Microsoft excel to analyze the statistical significance of the data from table 2. An ANOVA test is
appropriate for this experiment as there are over 3 different treatment group in one independent
variable.
The results of the ANOVA test showed a p-value of 0.5621 at the 45 degree of freedom
(table 3). The value is far above 0.05, which is generally agreed to be the maximum value for a
relationship to not be due to chance. Additionally, the F-critical value is much higher than the F-
value: The F-value of the test was 0.7525, while the F critical value is 2.6000. This indicates that
the relationship between the various treatment groups of cinnamon EO concentrations is not
statistically significant.
The ANOVA test in addition to the standard error measurements of the error bars shows
that the null hypothesis (Various concentrations of cinnamon EO exposed to E. coli will have no
effect on the level of resistance to cinnamon EO measured by the diameter of the zone of
inhibition) cannot be rejected. However, the ANOVA test assumes that each group has the same

20. variance, which is not true for the data in this experiment. Because the data does not conform to
this assumption, the results from the ANOVA test may be incorrect.
Finally, a t-test was conducted between bacteria exposed to broth of no EO and broth of
10 μL of ethanol using the “Data Analysis” feature on Microsoft excel to ensure the ethanol had
minimal effect on the experiment. The t-value was 2.262 while the critical t-value is 2.101 at a
significance level of 0.05 and 18 degree of freedom (Table 4). Because the t-value is smaller than
the t-critical value, there is no significant difference between the two groups. Therefore, 10 μL,
which was used to dissolve the EO in the broth, did not significantly affect the results of the
experiment.
4.2 Results
This essay intended to measure the effect of varying concentrations of cinnamon EO on the IZD
of E. coli. In response to the research question, “How does 24-hour exposure to various sub-MIC
of cinnamon EO (0, 2.5, 5, 10, 20) 𝜇𝑙/𝑚𝐿 affect the level of resistance to cinnamon EO in non-
pathogenic E. coli?,” the null hypothesis stated that “E. coli exposed to all concentrations of
cinnamon EO will have similar levels of resistance (IZD) to cinnamon EO” while the alternative
hypothesis stated that “E. coli exposed to greater concentrations of cinnamon EO will have a
greater level of resistance value (IZD) to cinnamon EO because greater concentration of EO has
greater selective pressure and thus greater fitness value for resistant genes.” The data collected,
however, appeared to show no significant difference amongst the various concentrations of
cinnamon EO as not only did most of the error bars overlap, but the ANOVA single-factor test
showed a high p-value of 0.56. Thus, any differences that may have occurred amongst the
various treatment groups are more likely to be due to chance rather than differences in EO

21. concentration. Therefore, the experiment does not support the alternate hypothesis and fails to
reject the null hypothesis.
Theoretical studies showed similar results. According to Becerril’s study, when multiple
gram-negative bacteria were exposed to cinnamon essential oil for 50 passages, no increase in
resistance to cinnamon EO was detected (Becerril). Becerelli’s study used the minimum
inhibitory concentration (MIC) to measure the amount of resistance the bacteria had, and for
cinnamon EO, the MIC value remained at 400mg/L for three of the four bacteria tested. It is
worth noting that Becerril’s study exposed EO to gram-negative bacteria for a longer period of
time and more number of serial passages than this extend essay, yet still showed no growth in
bacterial resistance to cinnamon EO (Becerril).
There are numerous possible factors that explain the failure to induce resistance to
cinnamon EO in E. coli when exposed to various low concentrations of cinnamon EOs. EOs
contain many more chemical compounds that have antimicrobial properties (Chouhan), which
makes it difficult for bacteria to become resistant. This is because in order for bacteria to become
resistant, they would need to undergo many more mutations unlike antibiotics, which has a lot
fewer antimicrobial compounds (Chouhan).
Moreover, EOs are known to have the capability to reverse mechanism of resistance in
gram-negative bacteria. One way bacteria become resistant to antimicrobials like antibiotics is by
the overproduction of protein pumps at the bacterial membrane that pumps the antimicrobial out
faster than it can diffusion into the bacteria, rendering the antimicrobial ineffective. Bacteria are
able to efflux a wide range of synthetic drugs not present in the natural ecosystem. As plant
extracts are not synthetic, bacteria cannot as easily resist EOs (Polly).

22. Quorum sensing is another component that is responsible for stress resistance based on
the signaling of molecules. Numerous EOs are known to have the ability to inhibit quorum
sensing. There are many other possible reasons E. coli did not become resistant to the cinnamon
EOs, such as beta-lactamase inhibition (Polly).
The factor that caused the lack of resistance in E. coli to cinnamon EO cannot be
identified in this experiment because of lack of resources to observe the experiment at a
molecular level. Nevertheless, it is likely that at least one of the factors mentioned contributed to
the lack of resistance in E.coli to cinnamon EO.
CONCLUSION
The results from this study suggest that various low concentration of cinnamon EO does not
increase bacterial resistance to cinnamon EO in E. coli. It was noted by Yuen et al. that sub-MIC
concentration of antibiotics can induce high levels of resistance to antibiotics in bacteria (Yuen).
Given that low concentrations of antibiotics are often used for several applications such as
agriculture (Chang), bacteria in the environment may become resistant posing danger to people.
In fact, up to 90% of antibiotics given to livestock are excreted in urine and stool, then widely
dispersed through fertilizer, groundwater and surface runoff (Ventola). Therefore, plant extracts
like cinnamon EOs should be considered as potential alternative as antimicrobial agents to
antibiotics. Though the efficacy of EOs may be lower than antibiotics, EOs are safer to use with
regards to development of resistant traits in bacteria.

23. 5.1 Evaluation
Random Error:
Due to time constraints, the bacteria could only be exposed to cinnamon EO for 24 hours.
Had the bacteria been exposed to EO for a longer period of time, the bacteria may have acquired
enough mutations to become resistant to EO. In the future when there is more time, exposing
bacteria to EO for 120 hours could induce resistance to cinnamon EO in E. coli, at least to a
certain degree.
Systematic Error:
Only non-pathogenic strains of bacteria were used in the experiment. According to a
study conducted by Martinez and Banquero, antimicrobial resistance and virulence genes can be
linked in the same replicon. By only taking non-pathogenic bacteria into account, I exempted the
pathogenic bacteria in the environment. Due to inadequate appliances as well as IB safety
guidelines, experiments with pathogenic E. coli could not be conducted. However, if possible,
experiments with pathogenic bacteria should be conducted at biosafety level 2.
When transferring the bacteria from the broth medium to the agar plates for testing
resistance, the bacteria were plated on the plate with the broth containing the essential oil the
bacteria had been growing in. Thus, the bacteria in the broth medium of lower concentration of
EO may have had an unfair advantage compared to the bacteria in the broth medium of higher
concentration of EO. To improve upon the issue, centrifuging the bacteria and suspended the
pellet in broth medium containing no essential oil for a more accurate reading.

24. Lastly, in this experiment, I was unable to investigate the cause of the lack of resistance
to cinnamon EO in bacteria or if any mutations did occur. Whole genome sequencing would help
identify where potential mutations or structural rearrangements may have occurred. Dye
sequencing, pyrosequencing, or SMRT sequencing are three technologies that can conduct whole
genome sequencing. They will allow me to further analyze the cause of how bacteria are
acquiring high levels of resistance to cinnamon EO.
Strengths:
Despite the numerous limitations in the experiment, there were some strengths that
increased the validity of the results. Due to time issues, although only 3 serial passages were
done for each treatment group per trial, they help rare genes, in this case mutated resistant genes,
survive. Without serial passaging, there would be too many bacteria with non-resistant genes for
bacteria with resistant genes to grow, even with selective pressure.
5.2 Further Investigation
 This experiment only investigated the relationship using one bacterium and cinnamon
EO. However, there are many more EOs, such as garlic and cloves, that could be
investigated. Relationship between more bacteria and EOs should be investigated for a
stronger establishment of the effect sub-MIC EOs have on the bacteria.
 Investigate the effect time that bacteria are exposed to EO on the bacteria’s level of
resistance to EO. It is possible that if the bacteria were exposed to EO for a longer period
of time resistant strains grow.

25. BIBLIOGRAPHY
Becerril, Raquel, Cristina Nerín, and Rafael Gómez-Lus. "Evaluation of bacterial resistance to
essential oils and antibiotics after exposure to oregano and cinnamon essential
oils." Foodborne pathogens and disease 9.8 (2012): 699-705.
Bengtsson-Palme, Johan, and DG Joakim Larsson. "Concentrations of antibiotics predicted to
select for resistant bacteria: Proposed limits for environmental regulation." Environment
International 86 (2016): 140-149.
Chang, Qiuzhi, et al. "Antibiotics in agriculture and the risk to human health: how worried
should we be?." Evolutionary applications 8.3 (2015): 240-247.
Chouhan, Sonam, Kanika Sharma, and Sanjay Guleria. "Antimicrobial activity of some essential
oils—present status and future perspectives." Medicines 4.3 (2017): 58.
“E.coli (Escherichia Coli).” Centers for Disease Control and Prevention, Centers for Disease
Control and Prevention, 26 Feb. 2018, www.cdc.gov/ecoli/general/index.html.
Highton, Melissa. An Image of E.coli Using Early Electron Microscopy. University of
Edinburgh, 31 July 2016.
Laranjo, Marta, et al. "Use of essential oils in food preservation." (2017).
Martínez, José L., and Fernando Baquero. "Interactions among strategies associated with
bacterial infection: pathogenicity, epidemicity, and antibiotic resistance." Clinical
microbiology reviews 15.4 (2002): 647-679.
Ventola, C. Lee. "The antibiotic resistance crisis: part 1: causes and threats." Pharmacy and
Therapeutics 40.4 (2015): 277.
Wistrand-Yuen, Erik, et al. "Evolution of high-level resistance during low-level antibiotic
exposure." Nature communications9.1 (2018): 1599.
Yap, Polly Soo Xi, et al. "Essential oils, a new horizon in combating bacterial antibiotic
resistance." The open microbiology journal 8 (2014): 6.
Zhao, Xilin, and Karl Drlica. "Restricting the selection of antibiotic-resistant mutants: a general
strategy derived from fluoroquinolone studies." Clinical Infectious
Diseases33.Supplement_3 (2001): S147-S156.

26. APPENDIX
Inhibition Zone Diameter (mm)
Concentration
of Cinnamon
EO (𝜇𝐿/𝑚𝑙)
0 2.5 5 10 20 Ethanol
1 22.2 22.65 20.3 21.3 21.5 21.15
2 19.85 21.4 20.45 22.5 22.2 23.65
3 20.5 23.6 20.9 24.2 24.2 21.3
4 21.4 21.4 21.4 27.0 25.0 21.3
5 20.6 23.2 22.3 21.7 22.1 22.0
6 21.4 20.7 22.3 21.5 21.3 21.5
7 20.5 21.4 20.3 22.2 25.1 21.0
8 20.5 20.5 20.1 22.1 22.1 23.0
9 23.3 19.9 22.2 22.2 22.2 21.0
10 22.3 23.1 21.3 23.2 22.0 21.5
Table 5: Raw data of effect of various concentrations of cinnamon EO on IZD