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Literature Review Instructions
General Guidelines:
● 5 pages (this does NOT include the cover page or the annotated bibliography).
○ If you go a little over 5 pages, that’s okay.
● Use APA format for your paper. Below are websites to help you write an APA paper. ​Note: you
do not need to have an abstract for your paper.
â—‹ https://owl.english.purdue.edu/owl/resource/560/18/
â—‹ http://www.easybib.com/guides/students/writing-guide/iv-write/a-formatting/apa-paper-f
ormatting/
● Double spaced
● Font size 12
â—‹ Times New Roman
● 1 inch margins
● Number your pages
● Please have a cover page with your name, BSC2011L, your section, TA name, date, and title of
your paper.
â—‹ Make sure to include the scientific names as well as the common names of your
organisms.
■ Make sure to capitalize and italicize the ​Genus​ names and italicize the ​species
names on your cover page and throughout your paper.
■ Ex. Correct: ​Homo sapiens​ or ​H. sapiens​ (after first mention).
Incorrect: Homo sapiens, ​homo sapiens, ​Homo Sapiens, ​Homo Sapiens,
homo sapiens, etc.
● You are basically converting your outline into paragraph form and adding more detail to make
your 5 page paper.
● Remember that paragraphs are generally 5-8 sentences. Do NOT make a paragraph a whole page
long! Break it up.
● If you’re going to include a picture in your literature review, that’s fine. But the pictures must go
on a separate page at the end of the paper and should be labeled as “Figure X. Title.” Any figures
added need to be referred to in the text. Do NOT place the figures within your 5 pages of writing.
○ Ex. You could have a figure called “Figure 1. Angiosperm Life Cycle” and in the text
when you need to refer to the figure you may write “According to the life cycle of the
flower, it goes from being…as in Figure 1.”
● Make sure you cite everything that you paraphrase.
â—‹ Every source that is in your annotated bibliography should be cited at least twice in your
actual paper.
■ In-text citations should (generally) be as follows: (Author’s Last Name, Year).
● If there are 2 authors, cite as: (Last Name 1 and Last Name 2, Year).
● If there are more than 2 authors, cite as (Author’s Last Name ​et al.,​ Year).
● There are NO QUOTES ALLOWED! Everything must be paraphrased!
● If you have taken this class before, you CANNOT resubmit the same paper! You will need to
pick another topic.
● Make sure that you use the right forms of theirs, there’s, its, it’s, etc.
● Make sure that everything is organized and that it flows. Don’t jump around in what you are
talking about.
● Remember, this is not an essay paper. It’s a RESEARCH paper you did on your organisms so
you will not be using the words “I” or “you” anywhere in the paper (aside from the annotations).
Annotated Bibliography:
● APA style
● Make sure sure have a minimum of 10 references, 5 of which MUST be journals. The other 5 can
be more journals and/or books, textbooks, articles.
● NO WEBPAGES ALLOWED!
â—‹ The only exception to this is if you sent the link to your TA for approval beforehand.
● NO ENCYCLOPEDIAS/WIKIPEDIA ALLOWED!
● NO JOURNAL ARTICLES FROM THE FOLLOWING DATABASES ARE ALLOWED:
â—‹ Encyclopedia of Life Sciences via Wiley Interscience
● References must be listed alphabetically by primary author’s last name.
● Don’t number or bullet your references
● If a citation runs on multiple lines make sure the second and third line, etc. are indented.
● Are the annotations done correctly? They should be a paragraph long. You are NOT supposed to
summarize the source (that would be an abstract). In an annotation, you should answer the
following questions:
â—‹ Critique the source/author
â—‹ How is the source relevant to your paper?
â—‹ How are you incorporating the source into your paper?
● Your TA should easily be able to match your references in your in-text citations with your
annotated bibliography.
Running head: ANTIMICROBIAL ACTIVITY
Antimicrobial Activity of Tea Tree (Melaleuca alternifolia) Oil and Cinnamon Oil
(Cinnamomum zeylanicum) on Streptococcus, Lipophilic Bacteria and Listeriosis
Student name
University
Class
Professor
March 16, 2021
1
ANTIMICROBIAL ACTIVITY
2
Antimicrobial Activity of Tea Tree (Melaleuca alternifolia) Oil and Cinnamon Oil
(Cinnamomum zeylanicum) on Streptococcus and Lipophilic Bacteria
Outline
I. Introduction
II. Background information on tea tree oil and cinnamon oil
A. Tea tree oil is distilled from Melaleuca alternifolia a native plant in Australia and
is commercially distributed (Tsao et al., 2010)
B. Essential oils are aromatic and they are obtained from plat material (Chaudhari et
al., 2012)
C. Cinnamon oil is obtained from Cinnamomum zeylanicum or Cinnamomum vervun
tree and it is harvested from either the bark or leaves (El-Shemy, 2018)
D. The main antimicrobial component of cinnamon oil is cinnamaldehyde (Lawrence
& Palombo, 2009)
III. Background information on Streptococcus and lipophilic bacteria
A. Streptococcus is a gram-positive bacteria which is the causal agent of
streptococcosis (Rattanachaikunsopon & Phumkhachorn, 2010)
B. Lipophilic bacteria thrive and multiply in lipids such as personal care products
(Mantil et al., 2015)
C. There are more than 100 species of Streptococcus bacteria (Spellerberg & Brandt,
2015)
D. Most lipophilic bacteria are not pathogenic hence do not pose a serious threat to
human health (Mantil et al., 2015)
IV. Health effects of Streptococcus and lipophilic bacteria
ANTIMICROBIAL ACTIVITY
3
A. Streptococcus iniae causes streptococcosis in fresh and salt water tilapia
(Rattanachaikunsopon & Phumkhachorn, 2010)
B. Streptococcus infections in human cause many inflammations such as skin and
wound inflammation and pneumonia (Spellerberg & Brandt, 2015)
C. Although lipophilic bacteria has no significant health impact, it may affect the
integrity of lipid formulations used in different industries (Mantil et al., 2015)
D. Excessive dependence on antibiotics leads to drug resistance hence the inability to
effectively treat bacteria-caused infections (Rattanachaikunsopon &
Phumkhachorn, 2010)
V. Antimicrobial properties of tea tree oil and cinnamon oil
A. Cinnamon oil has been used successfully to inhibit Streptococcus iniae in tilapia
fish (Rattanachaikunsopon & Phumkhachorn, 2010)
B. Tea tree oil reduces viable spores in Bacillus subtilis by 3 logs when used at 1%
or more concentration (Lawrence & Palombo, 2009)
C. It is hypothesized that tea tree oil antimicrobial activity is through penetration of
bacteria cell membrane, compromising the cytoplasm, and damaging organelle,
leading to cell death (Li et al., 2016)
D. Cinnamon oil, when compared to other common essential oils, has the most
effective antimicrobial activity against Streptococcus (Chaudhari et al., 2012)
VI. Practical use of tea tree oil and cinnamon oil as antimicrobial agents
A. Tea tree oil could be used as a preservative against food-borne bacteria (Shi et al.,
2018)
ANTIMICROBIAL ACTIVITY
B. Cinnamon oil has successfully been used to boost the immunity and metabolism
amongst other productive parameters in broiler chicken (Krauze et al., 2020)
C. Tea tree oil can be used to reduce microbial activity in personal care products
exposed to air and water, hence preserving them (Mantil et al., 2015)
D. Cinnamon oil antimicrobial activity against Streptococcus mutans and
Streptococcus sobrinus presents the potential for use in preventing and treating
dental carries (Choi et al., 2016)
VII.
Conclusion
4
ANTIMICROBIAL ACTIVITY
5
Annotated Bibliography
Rattanachaikunsopon, P., & Phumkhachorn, P. (2010). Potential of cinnamon
(Cinnamomum verum) oil to control Streptococcus iniae infection in tilapia
(Oreochromis niloticus). Fisheries Science, 76(2), 287-293.
https://doi.org/10.1007/s12562-010-0218-6
This study is effective in outlining the antimicrobial effects of cinnamon oil. The main
strength of the article is its analysis of several essential oils including lemongrass oil,
leech lime oil, and turmeric oil. Therefore, in the analysis of the antimicrobial effects of
cinnamon oil, the article justifies the use of the oil as compared to others. This article will
be used in the literature review to outline the antimicrobial activities of essential oils. It
will be used as evidence of the effectiveness of cinnamon in inhibiting Streptococcus
bacteria.
Shi, C., Zhang, X., & Guo, N. (2018). The antimicrobial activities and action-mechanism of
tea tree oil against food-borne bacteria in fresh cucumber juice. Microbial
Pathogenesis, 125, 262-271. https://doi.org/10.1016/j.micpath.2018.09.036
This article presents a thorough analysis of the antimicrobial action of tree tea oil on
bacteria. On the one hand, the authors present a rigorous analysis of the different strains
of L. monocytogenes and E. coli with different concentrations of tree tea oil. These
analysis ensure reliable and valid results. On the other hand, the authors fail to identify
the mode of action or component responsible for the inhibition and hence fail to provide a
conclusive analysis of the oil’s antimicrobial properties. Nevertheless, the article will be
used to outline the potential for using tree tea oil as a preservative in the food industry.
ANTIMICROBIAL ACTIVITY
6
Lawrence, H. A., & Palombo, E. A. (2009). Activity of essential oils against Bacillus subtilis
spores. Journal of Microbiology and Biotechnology, 19(12), 1590-1595.
https://doi.org/10.4014/jmb.0904.04016
Lawrence and Palombo have conducted an effective analysis of essential oils, including
13 such oils and testing their effectiveness in bacteria spore reduction. The laboratory
analysis of 13 oils makes this article’s scope wide and hence more reliable in researching
the spore reduction activity of essential oils. However, the tests were conducted only with
Bacillus subtilis hence the results may not be extended to other species. Regardless of
this limitation, the article is useful in outlining the potential for essential oils in industries
where bacteria spore reduction (antimicrobial activity) is required. It will be used to
report the antimicrobial properties of tea tree oil.
Krauze, M., Abramowicz, K., & Ognik, K. (2020). The effect of addition of probiotic
bacteria (Bacillus subtilis or Enterococcus faecium) or phytobiotic containing
cinnamon oil to drinking water on the health and performance of broiler
chickens. Annals of Animal Science, 20(1), 191-205. https://doi.org/10.2478/aoas2019-0059
This article reports an industry-specific application of essential oils, Bacillus subtilis, and
Enterococcus faecium in boosting the immunity of broiler chicken. The methodology of
this study is credible since the researchers maintained a group for each of the three
additives and a control group (no additives). The study also analyzes different body
components in the chicken to isolate the specific mode of action of the additives. This
article proves the utility of cinnamon oil in boosting the immunity of chicken. It will be
used in the review to provide an industrial application of this essential oil.
ANTIMICROBIAL ACTIVITY
7
Mantil, E., Daly, G., & Avis, T. J. (2015). Effect of tea tree (Melaleuca alternifolia) oil as a
natural antimicrobial agent in lipophilic formulations. Canadian Journal of
Microbiology, 61(1), 82-88. https://doi.org/10.1139/cjm-2014-0667
This article presents a rigorous study of tea tree oil and the potential for use in lipid
formulations. The analysis is thorough because it includes a comparison of antimicrobial
activity in vitro and in lipid-based formulations. The authors also do a great job of
analyzing the essential oil effect on different microorganisms including bacteria and
fungi. The article’s conclusion that tree tea oil could be used in lipid-based personal care
products aligns with the industrial use of the essential oil as an antimicrobial. This article
will be used to outline industry use of essential oils as antimicrobials.
Li, W. R., Li, H. L., Shi, Q. S., Sun, T. L., Xie, X. B., Song, B., & Huang, X. M. (2016). The
dynamics and mechanism of the antimicrobial activity of tea tree oil against bacteria
and fungi. Applied Microbiology and Biotechnology, 100(20), 8865-8875.
https://doi.org/10.1007/s00253-016-7692-4
This article is well drafted and the research touches on a unique aspect of tea tree oil
antimicrobial components; specific antimicrobial effects and mechanism. The study thus
researches a topic which had not been effectively tackled previously. The article reports
the possibility that tea tree oil works through compromising cell membrane and leading
to damages to the cytoplasm and organelle. Findings from this article will be used in the
review to present the antimicrobial components of tea tree oil. Specifically, it will be used
to discuss the mechanism of antimicrobial activity for tea tree oil.
Tsao, N., Kuo, C. F., Lei, H. Y., Lu, S. L., & Huang, K. J. (2010). Inhibition of group A
streptococcal infection by Melaleuca alternifolia (tea tree) oil concentrate in the
ANTIMICROBIAL ACTIVITY
8
murine model. Journal of Applied Microbiology, 108(3), 936-944.
https://doi.org/10.1111/j.1365-2672.2009.04487.x
This article effectively tests the effectiveness of tea tree oil on the treatment of group A
streptococcus (GAS) which is relevant because it affects humans and is treated using
antibiotics and surgery. The methodology of the study is well outlined and the authors
used mice to test the effectiveness of the essential oil. The discussion is also relevant
because it connects the findings to utility in human treatment. This article will be used to
provide background information on tea tree oil as well as Streptococcus.
Choi, O., Cho, S. K., Kim, J., Park, C. G., & Kim, J. (2016). Inávitro antibacterial activity
and major bioactive components of Cinnamomum verum essential oils against
cariogenic bacteria, Streptococcus mutans and Streptococcus sobrinus. Asian Pacific
Journal of Tropical Biomedicine, 6(4), 308-314.
https://doi.org/10.1016/j.apjtb.2016.01.007
The evaluation of the use of cinnamon oil against Streptococcus mutans and
Streptococcus sobrinus is well outlined in this article with a thorough analysis of current
literature on the topic. The authors extracted numerous essential oils before focusing on
Cinnamomum verum as the most potent, hence presenting an informed and credible
approach to the study. This article will be used for analysis of the potential for essential
oils in industry use as well as outlining background of cinnamon oil and Streptococcus. It
will be referenced as evidence for the potential use in preventing dental carries as well as
providing information on background.
Chaudhari, L. K., Jawale, B. A., Sharma, S., Sharma, H., Kumar, C. D., & Kulkarni, P. A.
(2012). Antimicrobial activity of commercially available essential oils against
ANTIMICROBIAL ACTIVITY
9
Streptococcus mutans. J Contemp Dent Pract, 13(1), 71-74. https://doi.org/10.5005/jpjournals-10024-1098
This article presents credible study of the antimicrobial activity of essential oils.
Although the article is well developed and research is conducted using credible
approaches, its main limitation is the analysis of activity in vitro which may not
necessarily be transferable to practical use. Nevertheless, results from this study could be
used for further research. This study will be used to detail background information on
Streptococcus as well as the antimicrobial activity of cinnamon oil.
El-Shemy, H. (Ed.). (2018). Potential of essential oils. BoD–Books on Demand.
This book presents an extensive analysis of essential oils and their utility and potential.
The importance of this book is that it presents chapters written by different authors and
hence it presents a wide scope of studies on essential oils. As an edited book, it compiles
different aspects of essential oils and cinnamon and tea tree oils are some of them. This
book will be used generally for background information on these essential oils as well as
specific information on their utility. It will be cited in the background sections of the
paper to provide a background on essential oils and bacteria.
ANTIMICROBIAL ACTIVITY
10
References
Chaudhari, L. K., Jawale, B. A., Sharma, S., Sharma, H., Kumar, C. D., & Kulkarni, P. A.
(2012). Antimicrobial activity of commercially available essential oils against
Streptococcus mutans. J Contemp Dent Pract, 13(1), 71-74. https://doi.org/10.5005/jpjournals-10024-1098
Choi, O., Cho, S. K., Kim, J., Park, C. G., & Kim, J. (2016). Inávitro antibacterial activity and
major bioactive components of Cinnamomum verum essential oils against cariogenic
bacteria, Streptococcus mutans and Streptococcus sobrinus. Asian Pacific Journal of
Tropical Biomedicine, 6(4), 308-314. https://doi.org/10.1016/j.apjtb.2016.01.007
El-Shemy, H. (Ed.). (2018). Potential of essential oils. BoD–Books on Demand.
Krauze, M., Abramowicz, K., & Ognik, K. (2020). The effect of addition of probiotic bacteria
(Bacillus subtilis or Enterococcus faecium) or phytobiotic containing cinnamon oil to
drinking water on the health and performance of broiler chickens. Annals of Animal
Science, 20(1), 191-205. https://doi.org/10.2478/aoas-2019-0059
Lawrence, H. A., & Palombo, E. A. (2009). Activity of essential oils against Bacillus subtilis
spores. Journal of Microbiology and Biotechnology, 19(12), 1590-1595.
https://doi.org/10.4014/jmb.0904.04016
Li, W. R., Li, H. L., Shi, Q. S., Sun, T. L., Xie, X. B., Song, B., & Huang, X. M. (2016). The
dynamics and mechanism of the antimicrobial activity of tea tree oil against bacteria and
fungi. Applied Microbiology and Biotechnology, 100(20), 8865-8875.
https://doi.org/10.1007/s00253-016-7692-4
ANTIMICROBIAL ACTIVITY
11
Mantil, E., Daly, G., & Avis, T. J. (2015). Effect of tea tree (Melaleuca alternifolia) oil as a
natural antimicrobial agent in lipophilic formulations. Canadian Journal of
Microbiology, 61(1), 82-88. https://doi.org/10.1139/cjm-2014-0667
Rattanachaikunsopon, P., & Phumkhachorn, P. (2010). Potential of cinnamon (Cinnamomum
verum) oil to control Streptococcus iniae infection in tilapia (Oreochromis
niloticus). Fisheries Science, 76(2), 287-293. https://doi.org/10.1007/s12562-010-0218-6
Shi, C., Zhang, X., & Guo, N. (2018). The antimicrobial activities and action-mechanism of tea
tree oil against food-borne bacteria in fresh cucumber juice. Microbial Pathogenesis, 125,
262-271. https://doi.org/10.1016/j.micpath.2018.09.036
Spellerberg, B., & Brandt, C. (2015). Streptococcus. Manual of Clinical Microbiology, 383-402.
https://doi.org/10.1128/9781555817381.ch22
Tsao, N., Kuo, C. F., Lei, H. Y., Lu, S. L., & Huang, K. J. (2010). Inhibition of group A
streptococcal infection by Melaleuca alternifolia (tea tree) oil concentrate in the murine
model. Journal of Applied Microbiology, 108(3), 936-944. https://doi.org/10.1111/j.13652672.2009.04487.x
https://books.google.co.ke/books?id=Dm-QDwAAQBAJ&pg=PA32&dq=ElShemy,+H.+(Ed.).+(2018).+Potential+of+essential+oils.+BoD%E2%80%93Books+on+Demand
.&hl=en&sa=X&ved=2ahUKEwjH9_eA9a3vAhUGT8AKHWsVCkoQ6AEwAHoECAUQAg#v
=onepage&q=ElShemy%2C%20H.%20(Ed.).%20(2018).%20Potential%20of%20essential%20oils.%20BoD%E2
%80%93Books%20on%20Demand.&f=false
Spreading Disease – It’s Contagious! Using a Model & Simulations to Understand How
Antibiotics Work
Author(s): EVA M. OGENS and RICHARD LANGHEIM
Source: The American Biology Teacher , Vol. 78, No. 7 (SEPTEMBER 2016), pp. 568-574
Published by: University of California Press on behalf of the National Association of
Biology Teachers
Stable URL: https://www.jstor.org/stable/10.2307/26411105
REFERENCES
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University of California Press and National Association of Biology Teachers are collaborating
with JSTOR to digitize, preserve and extend access to The American Biology Teacher
This content downloaded from
131.94.16.10 on Tue, 09 Feb 2021 03:09:18 UTC
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Spreading Disease – It’s
Contagious! Using a Model &
Simulations to Understand How
Antibiotics Work
INQUIRY &
INVESTIGATION
•
EVA M. OGENS, RICHARD LANGHEIM
ABSTRACT
We describe how to enable students to learn about the transmission of disease,
resistant bacteria, and the importance of taking a “full course” of antibiotics by
developing models and simulations to represent the growth and demise of
bacteria. By doing these activities, students experience a model of the effects of
antibiotics on the population of disease-causing bacteria during an infection.
Students learn about the spread of infection through game playing and then,
using a simulation, investigate how different variables, such as skipping a day
of medication, affect the persistence of the disease. A key concept is that almost
every naturally occurring population of bacteria that causes disease has a
component that is resistant to antibiotics. Therefore, through graphing data and
computer models, students can visually understand why it is important to take
a complete course of antibiotics to kill all the bacteria and decrease the
likelihood of bacteria becoming resistant, which can be harmful to human
health. In this hands-on, inquiry-based activity that is seamlessly integrated
with technology, the teacher becomes the facilitator of learning while the
student is an active, engaged partner.
Key Words: Antibiotics; resistance; models; simulations; bacteria.
“Can you come over and hang out?” Katie texted Elena, her friend
from Ms. Collins’s sixth-grade class, on a Saturday afternoon. “I can’t,”
Elena replied. “I just got back from the doctor’s office. I got sick last
night with a high fever and a bad sore throat.
The doctor said I have strep throat and now I
am on antibiotics.”
“That’s too bad,” Katie wrote. “Well, I
wonder how you got that. Will you be back
in school on Monday?” “Yes, since I will have
been taking the medicine for more than
24 hours,” texted Elena. “But I don’t know
why I have to keep taking the medicine for
10 days! My stomach is getting upset already.”
Does this sound like a scenario that could take place in a typical
classroom? How can we teach students so that they understand how
diseases are transmitted and why they need to take their medication
for the entire length of time it is prescribed? Conversely, we need to
convey the importance of not taking antibiotics for colds or other
viruses, since antibiotics only kill bacteria. Here, we present a way
to teach students about the transmission of disease and the importance of taking a “full course” of antibiotics. Using technology, students develop models to represent the growth and demise of bacteria.
Background
The Next Generation Science Standards (NGSS) were developed by
the National Research Council (NRC), the National Science Teachers Association, and the American Association for the Advancement
of Science (AAAS) and managed by Achieve, Inc. In the standards,
the expectation is that students will develop the behaviors and
skills practiced by scientists when they investigate and build models and theories about the natural world, and emulate engineers in
designing and building models and systems. For example, “Practice
2” is “Developing and Using Models.” Students are expected to use
models to “describe, test, and predict more abstract phenomena
and design systems” (NGSS Lead States, 2013).
Both scientists and engineers construct and use models as helpful
tools for representing ideas and explanations. These tools include diagrams, drawings, physical replicas, mathematical
representations, analogies, and computer simulations (NSTA, 2014). According to the NGSS,
“Although models do not correspond exactly to
the real world, they bring certain features into
focus while obscuring others. All models contain
approximations and assumptions that limit the
range of validity and predictive power, so it is
important for students to recognize their limitations” (NSTA, 2014).
By participating in the activities described, students experience
a model of the effects of antibiotics on the population of diseasecausing bacteria during an infection. In this case, the action of
Using technology,
students develop
models to represent
the growth and
demise of bacteria.
The American Biology Teacher, Vol. 78, No 7, pages. 568–574, ISSN 0002-7685, electronic ISSN 1938-4211. © 2016 National Association of Biology Teachers. All rights
reserved. Please direct all requests for permission to photocopy or reproduce article content through the University of California Press’s Reprints and Permissions web page,
www.ucpress.edu/journals.php?p=reprints. DOI: 10.1525/abt.2016.78.7.568.
568
THE AMERICAN BIOLOGY TEACHER
VOLUME. 78, NO. 7, SEPTEMBER 2016
This content downloaded from
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antibiotics on bacteria is simplified and, as with any model, there
are limitations, but students can get a good idea of how antibiotics
work. Students learn about the spread of infection through game
playing and then, using a simulation, investigate how different variables, such as skipping a day of medication, affect the persistence
of the disease.
A key concept is that almost every naturally occurring population of bacteria that causes disease has a component that is resistant
to antibiotics. Therefore, through graphing data and computer
models, students can visually understand why it is important to
take a complete course of antibiotics to kill all the bacteria and
decrease the likelihood of bacteria becoming resistant, which can
be harmful to human health. Students also learn that some bacteria
are “stronger” than others and more resistant to drugs.
Prior to the NGSS, the National Science Education Standards
(NRC, 1996) and state standards across the country expected students to learn that scientists often create models to explain scientific phenomena. The NRC’s Framework describes a vision of what
it means to be proficient in science; it rests on a view of science
as both a body of knowledge and an evidence-based, model- and
theory-building enterprise that continually extends, refines, and
revises knowledge (NRC, 2012). The National Science Education
Standards state that many students in middle and high school “view
models as physical copies of reality and not as conceptual representations. Teachers should help students understand that models are
developed and tested by comparing the model with observations
of reality” (NRC, 1996, p. 127). The NGSS have been adopted by
17 states and the District of Columbia so far (Heitin, 2014). Helping students develop a strong foundation in their understanding of
models can only improve their future understanding of science and
mathematical concepts. Unifying concepts in the standards – such
as systems, order, and organization as well as models, evidence,
and explanation – provide students with powerful ideas to help
them understand the natural world (NRC, 1996, p. 115).
Introductory Activity
We begin by having students play an engaging game that encourages
them to think about how diseases spread. “The Infection Game,”
developed by Rob Quaden, Alan Ticotsky, and Debra Lyneis
(2009), is described in the Creative Learning Exchange newsletter.
This game simulates the generic structure of the spread of a contagious activity, or infection, and once students understand how it
works, they will be able to understand the spread of a rumor, a
fad, or a computer virus (Quaden et al., 2009, p. 1). It is best played
with 35 players, so classes can be combined. The rules for the students are as follows:
Table 1. Data.
Number of New
Zeros
Total Number of
Zeros
Start
1
1
1
1
2
2
2
4
3
4
8
4
8
16
Round
For example, if you have a “1” and the other student has a “0,” you
will both get 1 × 0 = 0 as the new number on your next line. In the
next round, find another student and tell each other your number,
multiply them, and record the new product on the next line.
5. Continue to do this until the teacher ends the game (Quaden
et al., 2009, p. 1).
After the teacher explains the rules, students can play for about
four minutes. The game works like this. The spread of a disease is
represented by the multiplication of numbers. Each student is given
the number “1” except for one student, who, unknowingly, is given
the number “0.” The interactions represent the exchange of germs.
Each student approaches another student in class and shares his or
her number with the other student. Each student then multiplies
his or her own number by the number of the other student to
arrive at his or her new number for the next round. Since everyone
in class has been given a worksheet with “1” as their number except
for one student who, unknowingly, has been given a “0,” the results
are an increasing number of “0s” in the class. By the end of about
four minutes, most, if not all, students will have a “0” as their final
product.
After the game, the teacher should ask who had the first entry of
“0” in the beginning of the game. (It should only be one person.) Then
ask how many had “0” in the second round, and so on. The teacher’s
class record should look like Table 1 (Quaden et al., 2009, p. 3).
After all the students have interacted, the class should create a
graph with a spreadsheet and plot the number of new “0s” and
the total number of “0s” for each round of the game (see Figure 1).
The graph shows one curve on the bottom that represents the
new infections each round and another curve above that is “s”
shaped for the total number of infected students. Students, either
in small groups or as a whole class, can engage in building a table
of results and a graph to see the pattern of the spread of the disease.
The creation and interpretation of graphs such as this are common
expectations of middle school students.
1. You will receive a sheet to track the results of the game.
2. You will be given a secret number, which will already be
filled in on your record sheet.
3. Secrecy and accuracy are very important!
4. You will play the game for several rounds. In the first round,
find any other student and quietly tell each other your numbers. Then, on your own, secretly multiply your two numbers together and record the product on the next line of
your sheet. This will be your new number for the next
round.
Using a Computer Simulation
A computer simulation is a “computer program that recreates an
abstract, real-world system. Computer simulations are used in science to explore concepts that are difficult or impossible to observe
in the real world. In this case, a computer simulation can conduct
many trials in a fraction of the time it would take to do it manually.
Thus, the next step, which helps further students’ understanding
of why things change, is to teach them about “stocks” and “flows.” A
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569
Hands-On Simulation
of a Bacterial Infection
The next activity is from the Science Education for Public Understanding Program
(SEPUP, 2010). The introduction to the
activity explains that when harmful bacteria
reproduce too quickly, the body’s immune
system cannot control the growing population of bacteria, which leads to an infection.
Antibiotics help by killing off the bacteria;
however, some bacteria are more resistant to
the antibiotic and will not be killed as
quickly, thus continuing to grow and multiply. By completing the “full course” of antibiotics, the patient helps ensure that the
bacteria are killed off and the infection cured.
We started by asking the students, by a
show of hands, how many had ever had an
Figure 1. Students “infected” over time (Quaden et al., 2009, p. 6).
ear infection or strep throat or had taken
antibiotics. Predictably, they all raised their
hands. We explained that by playing a
game using chips and dice, which simulates
the bacteria and taking the medication, they
would see how antibiotics help cure an
infection.
The activity materials include 50 disks
(poker chips can be used) in three colors,
15 orange, 15 blue, and 20 green. The
orange disks represent the most resistant
bacteria, the blue represent resistant bacteria, and the green represent the least resistant bacteria. Students (working in pairs)
begin with 20 chips (13 green, 6 blue,
and 1 orange) and roll a die to determine
if they “took” the antibiotic or if they “forgot” to take it. Students work in pairs and
each pair has a die. Students roll the dice,
and after each roll, they follow the correFigure 2. Stock-and-flow diagram (Quaden et al., 2009, p. 11).
sponding directions: rolling a “1,” “3,” “5,”
or “6” indicates taking the medication;
rolling a “2” or a “4” means that a dose was
stock can be viewed as any accumulation and a flow as the increasing or forgotten. Because the colors represent antibiotic resistance, studecreasing of the stock. Many people think of a stock as a bathtub. In dents remove the green first, then blue, and then orange. In every
this image the person’s body (the bathtub) fills up with bacteria. The case, however, if there is a chip of any color left, another chip of
inflow to the stock is like the faucet that fills the tub, while the outflow that color is added to simulate that the bacteria are still reproducis like the drain that empties it. We did this with sixth-graders; we first ing. Students keep track of the number of each type of bacteria
explained what a stock is by simply demonstrating how water flows until all are “killed” by the antibiotic.
from one cup to another, representing a stock of water and the flow.
Students record their data on a table, tracking how many disks
Once this is understood, and students are at a more sophisticated of each color are left after each roll. Finally, students graph their
level, students and the teacher build a stock-and-flow diagram to rep- data so that they have a visual representation, drawing the demise
resent the behavior of the system (Figure 2).
of the bacteria with green, blue, and orange markers. They can also
When this is built and run with software such as STELLA use Microsoft Excel to graph the data.
(http://www.iseesystems.com/), the students can watch the graphs
There is no set number of rounds in this game, because it depends
being built and run the model with different values to see the same on how many times the players roll a “2” or a “4,” which means they
behavior develop over time. Through this dramatizing exercise, stu- forgot to take the medication on time, and therefore the bacteria keep
dents can see how quickly germs can spread.
multiplying. It can be as few as eight rounds or many more. The
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Table 2. Chart for recording number of harmful bacteria in your body
(Science Education for Public Understanding Program, 2010, C-269).
Round
Number
Initial
Least resistant
Bacteria
Resistant
Bacteria
Extremely
Resistant
Bacteria
Total
13
6
1
20
1
2
3
4
5
6
7
8
9
10
teacher can choose 10 rounds to simulate taking antibiotics for ten
days. If the “infection” is not cleared up by then, this would be a great
opportunity to discuss why sometimes people have to take an additional course of medication or even switch to a more powerful antibiotic. The data can be charted as shown in Table 2. An example of
game play is recorded in Table 3.
Using this data and creating a graph is an excellent way to review
math graphing skills. Being able to interpret or construct graphical
representations is a crucial skill for every student, whether or not they
want to pursue science- or math-related careers (Ozgun-Koca, 2001).
The Common Core Standards require that students use mathematics
to model real-world problems, to communicate mathematically, to
solve problems, and to use and interpret graphs and organize and
describe data. This is also an opportunity to discuss antibiotic resistance. The first antibiotic (penicillin) was discovered by Sir Alexander
Fleming in 1929. By the 1930s and ’40s, other antibiotics were developed and used to treat urinary tract infections, pneumonia, and other
conditions (Todar, 2012, p. 1). Very soon after, in the late 1940s,
resistance to antibiotics was noted. Evidence also began to accumulate
that bacteria could pass genes for drug resistance between strains and
even between species (Todar, 2012, p. 2).
Antibiotic-resistant bacteria constitute a growing public health
crisis because infections from resistant bacteria are increasingly difficult and expensive to treat (Sustainable Table, 2015). Resistant
bacterial strains have become more prevalent. More than 90 strains
of Staphylococcus aureus, a common cause of hospital staph infections, are now resistant to penicillin (SEPUP, 2010).
Antibiotic resistance is one of the most pressing public
health issues facing the world today. The Centers for Disease Control and Prevention (CDC) estimates that each
year at least two million illnesses and 23,000 deaths are
caused by antibiotic-resistant bacteria in the United States
alone. Antibiotic resistance limits our ability to quickly
and reliably treat bacterial infections, and the rise of resistance
could hamper our ability to perform a range of modern medical
procedures from joint replacements to organ transplants, the
safety of which depends on our
ability to treat bacterial infections
that can arise as post-surgical complications. Antibiotic-resistant bacteria also pose economic threats.
The CDC reports that antibioticresistant infections account for at
least $20 billion in excess direct
health care costs and up to $35
billion in lost productivity due to
hospitalizations and sick days each
year. (“Fact Sheet,” 2015)
Recently, the Washington Post (March
27, 2015) reported that antibiotic resistance
is a mounting problem that causes an estimated 2 million illnesses and 23,000 deaths
every year in the United States. There are
several factors contributing to the increase
in antibiotic-resistant bacteria, in addition
to the natural resilience of the bacteria in developing resistance to
the antibiotics. One important factor is the overuse and misuse of
antibiotics. The antibiotic use rate was 0.85 prescriptions per capita
in 2009 for the entire United States, which means almost one prescription for every person in the country (“Antimicrobial prescription data,” 2010). In 1998, 80 million prescriptions for human
antibiotic use were filled (Todar, 2012, p. 1). Inappropriate uses of
antibiotics in both medicine and agriculture drive up the use rate
and the development of resistance; agricultural practices account
for over 60% of antibiotic use in the United States. The result is that
once-powerful drugs are losing their ability to kill emerging “superbug” strains of disease-causing bacteria.
Random mutations in microbes and the ability of bacteria to
exchange genetic material have long been hypothesized to foster
the evolution of resistant strains. However, a recent study in the
journal Science Advances found that preventing antibiotic resistance
may be much more difficult than believed, because the microbes
long ago evolved the ability to fight toxins, including antibiotics.
Scientists studied the people called Yanomami – hunter-gatherers
in the remote mountains of the Amazon jungle of Venezuela – in
2009. These people had never been exposed to Western medicine
or diets. Yet the scientists found that the “Yanomami’s gut bacteria have already evolved a diverse array of antibiotic-resistance
genes . . . even though these mountain people had never ingested
antibiotics or animals raised with drugs” (Gibbons, 2015). This
new study suggests that resistance genes have been around in
the human microbiome (the trillions of bacteria that live in and
on the body) for thousands of years – long before antibiotic drugs
were invented. The team of scientists speculated that “antibiotics
are not just a human invention. Bacteria evolved strategies to kill
each other long before we were around – they were the first
inventors of antibiotics. And to defend against this, they developed resistance mechanisms” (Gibbons, 2015).
ANTIBIOTICS & RESISTANT BACTERIA
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571
Table 3. Example of playing the game. In this case, the person “forgot” to take the antibiotics twice,
so the antibiotics worked in 12 days instead of 10.
Number of Harmful Bacteria in Your Body
Round
Number
Roll of Die
Meaning
Initial
Least
Resistant
Bacteria
Resistant
Bacteria
Extremely
Resistant
Bacteria
Total
Action Taken
13
6
1
20
Removed 5 green, added
1 of green, blue, and orange
1
3
Took
antibiotics
9
7
2
18
Removed 5 green, added
1 of green, blue, and orange
2
2
Forgot to
take
antibiotics
10
8
3
21
Added 1 of each color
3
1
Took
antibiotics
6
9
4
19
Removed 5 green, added
1 of each color
4
3
Took
antibiotics
2
10
5
17
Removed 5 green, added
1 of each color
5
5
Took
antibiotics
0
8
6
14
Removed 5 (2 green, 3 blue)
added 1 blue and 1 orange
6
6
Took
antibiotics
0
4
7
11
Removed 5 blue, put back 1
blue and added 1 orange
7
4
Forgot to
take
antibiotics
0
5
8
Added 1 blue and 1 orange
8
1
Took
antibiotics
0
0
9
Removed 5 blue, added 1
orange
9
3
Took
antibiotics
0
0
5
Removed 4 orange
10
4
Forgot to
take
antibiotics
0
0
6
Added one orange
11
6
Took
antibiotics
0
0
2
Removed 4 orange
12
5
Took
antibiotics
0
0
0
Removed remaining 2 orange
Antibiotic Discovered That Is Resistant
to Resistance!
A study published recently in Nature (Ling et al., 2015) revealed a
new antibiotic called “teixobactin,” which might keep working for
more than 30 years because of its unique method of action. It
may be resistant to resistance (Feltman, 2015).
Lossee Ling from NovoBiotic pharmaceuticals and Tanja
Schneider at the University of Bonn showed that teixobactin works by withholding two molecules – Lipid II,
which bacteria need to make the thick walls around their
cells, and Lipid III, which stops their exiting walls from
572
breaking down. When teixobactin is around, bacterial
walls come crumbling down, and don’t get rebuilt. The
drug also sticks to parts of Lipid Ii and Lipid III that
are constant across different species of bacteria. It’s likely
that these parts can’t be altered without disastrous consequences, making it harder for bacteria to avoid teixobactin’s double punch. This might explain why it’s so hard
to evolve resistance to the drug. (Yong, 2015)
Stopping Antibiotics Prematurely
Many people stop taking an antibiotic prescription after a few days,
when they feel better, although not all the bacteria have been killed.
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This contributes to the more resistant bacteria reproducing, increasing
their population, and increasing the chances that future infections will
not respond to the typical course of antibiotics. By playing this game,
students will visually see, by graphing, how the population spikes
when a dose is “missed.”
Another valuable tool to develop problem-solving skills is the
use of simulations. Simulations enable users to play and replay a
problem, while changing the values of one variable at a time and
then observing the behavior of the system. One simulation that we
have developed presents students with the problem of explaining
the impact of taking a portion of a prescribed medicine, of sporadically
taking a prescription, or of taking the entire prescription (“full course”)
as prescribed. The simulation generates a table of values and a graphic
representation for each run that engages students in explaining the
contrasting patterns of behavior of the system.
One of the advantages of simulations built with an application like
STELLA is that the organization of the system is clearly presented in a
language that expresses accumulations (i.e., stocks) as rectangles, the
movements (i.e., flows) of a substance (e.g., a disease) as pipes, and
feedback loops as lines with arrows. With this simple vocabulary,
extraordinarily complex systems can be depicted and tested by
running the simulation repeatedly.
Our students also learned that by using computer simulations,
much time is saved in doing experiments and that scientists can
repeat the scenarios many times to investigate the results. In the
game play described above, the stock of healthy people flowed to
the stock of infected people. The flow at the outset was gradual,
since only one student began with a “0” to indicate that they were
ill, and then increased as more and more students spread the illness
to others. Finally, when most students had been infected, the number
of new infections tapered off because many of the interactions were
between already ill students. When students have an opportunity to
see the system in the iconic representation of stocks and flows or
through the generated graphs, they gain valuable conceptual tools that
can be applied to any problem that involves change.
A significant network of schools has been integrating systems
thinking and the use of dynamic modeling into the curriculum for
the past two decades. Information about these efforts is available
from the Creative Learning Exchange and the Waters Foundation.
Conclusion
Student feedback included the following comments:
• “I learned that even if you take antibiotics, the bacteria keep
multiplying. That is why you must take antibiotics for an
extended period of time.”
• “I liked this project because it helped me understand how an
infection works; if you don’t treat it the bacteria get stronger.”
• “I learned that bacteria grow in a certain way. I thought it was
cool and a lot of fun on learning about bacteria.”
• “I liked the activity. Something I learned is [that] the bacteria
still grow when you are getting better.”
• “It showed me how to take care of myself better.”
These student-centered, inherently collaborative learning activities are effective pedagogical techniques that meet the call for the
development of communication, collaboration, critical-thinking, and
problem-solving skills identified by the Partnership for 21st Century
Learning. As an aspect of critical thinking, students are expected to
analyze how parts of a system interact to produce outcomes. In addition, one of the interdisciplinary themes articulated by the Partnership
is that of health literacy. Student expectations include, but are not limited to, being able to use available information to make appropriate
health-related decisions, and to understand national and international
public health and safety issues.
The entire concept of scientific literacy includes the ability to
make decisions based on evidence and the interpretation of information. The NGSS expect students to understand how models
are used by scientists and engineers to explain phenomena and
solve problems. The NGSS also “aim to prepare students to be better decision makers about scientific and technical issues and to
apply science to their daily lives” (Achieve, Inc., 2013). What better
way to apply science than to a common experience – taking antibiotics? In this hands-on, inquiry-based activity that is seamlessly
integrated with technology, the teacher becomes the facilitator of
learning while the student is an active, engaged partner.
References
Achieve, Inc. (2013). Final Next Generation Science Standard released.
[Press release.] Available at http://www.nextgenscience.org/final-nextgeneration-science-standards-released.
“Antimicrobial prescription data reveal wide geographic variability in
antimicrobial use in the United States, 2009” (2010, October 22).
Conference session abstract, retrieved from http://idsa.confex.com/
idsa/2010/webprogram/Paper3571.html.
Common Core State Standards Initiative (2015). Standards for mathematical
practice. Available at http://www.corestandards.org/Math/Practice/.
“Fact Sheet: President’s 2016 budget proposes historic investment to
combat antibiotic-resistant bacteria to protect public health” (2015).
[Press release.] Retrieved from http://www.whitehouse.gov/the-pressoffice/2015/01/27/fact-sheet-president-s-2016-budget-proposeshistoric-investment-combat-a.
Feltman, R. (2015, January 7). New class of antibiotic found in dirt could
prove resistant to resistance. Washington Post. Available at http://www.
washingtonpost.com/news/speaking-of-science/wp/2015/01/07/newclass-of-antibiotic-found-in-dirt-could-prove-resistant-to-resistance/.
Gibbons, A. (2015, April 17). Resistance to antibiotics found in isolated
Amazonian tribe. Science. Available at http://news.sciencemag.org/
biology/2015/04/resistance-antibiotics-found-isolated-amazoniantribe.
Heitin, L. (2014, August 15) Next Generation Science Standards: which
states adopted and when? Education Week. Available at http://blogs.
edweek.org/edweek/curriculum/2014/08/next_generation_science_
standa.html.
Ling, L.L., Schneider, T., Peoples, A.J., Spoering, A.L., Engels, I., Conlon, B.P.
et al. (2015). A new antibiotic kills pathogens without detectable
resistance. Nature, 517, 455–459.
National Research Council (1996). National Science Education Standards.
Washington, DC: National Academies Press.
National Research Council (2012). A Framework for K–12 Science
Education: Practices, Crosscutting Concepts, and Core Ideas.
Washington, DC: National Academies Press.
New Jersey Core Content Curriculum Standards (2009). Science standards
learning progressions. Available at http://www.state.nj.us/education/
aps/cccs/science/frameworks/2009progressions.pdf.
ANTIBIOTICS & RESISTANT BACTERIA
THE AMERICAN BIOLOGY TEACHER
This content downloaded from
131.94.16.10 on Tue, 09 Feb 2021 03:09:18 UTC
All use subject to https://about.jstor.org/terms
573
NGSS Lead States (2013). Next Generation Science Standards: For States, By
States. Washington, DC: National Academies Press.
NSTA (2014). [NGSS] Science and Engineering Practices: developing and
using models. Available at http://ngss.nsta.org/Practices.aspx?id=2.
Ozgun-Koca, S.A. (2001). The graphing skills of students in mathematics and
science education. ERIC Digest. ERIC Identifier: ED464804. Available at
http://www.ericdigests.org/2003-1/graphing.htm on April 20, 2015.
Partnership for 21st Century Learning (n.d.). Framework for 21st century
learning. Available at http://www.p21.org/index.php?option=com_
content&task=view&id=254&Itemid=120.
Quaden, R., Ticotsky, A. & Lyneis, D. (2009). The infection game. The Creative
Learning Exchange, 18(1). [Used with permission.] Available at http://
static.clexchange.org/ftp/newsletter/CLEx18.1.pdf.
SEPUP (Science Education for Public Understanding Program) (2010). Issues
& Life Science [Full Course]. Berkeley, CA: University of California,
Lawrence Hall of Science. Available at http://sepuplhs.org/about.html.
Sustainable Table (2015). Antibiotics. New York, NY: Grace Communications
Foundation. Available at http://www.sustainabletable.org/issues/
antibiotics.
Todar, K. (2012). Todar’s Online Textbook of Bacteriology. Available at
http://textbookofbacteriology.net/resantimicrobial.html.
Yong, E. (2015, January 7). A new antibiotic that resists resistance.
Available at http://phenomena.nationalgeographic.com/2015/01/07/
antibiotic-resistance-teixobactin/.
EVA M. OGENS is an Associate Professor of Math/Science Methods at
Ramapo College of New Jersey, Mahwah, NJ 07430; e-mail:
eogens@ramapo.edu. RICHARD LANGHEIM is an Associate Professor of
Education (Retired) at Ramapo College of New Jersey; e-mail:
richlangheim@gmail.com.
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Microbial Pathogenesis 125 (2018) 262–271
Contents lists available at ScienceDirect
Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath
The antimicrobial activities and action-mechanism of tea tree oil against
food-borne bacteria in fresh cucumber juice
T
Ce Shi, Xiaowei Zhang, Na Guo∗
Department of Food Quality and Safety, College of Food Science and Engineering, Jilin University, 130062, Changchun, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords:
Foodborne pathogens
Tea tree oil
Antimicrobial mechanism
Food system
Food preservative
Background: Foodborne diseases caused by foodborne pathogens have increasingly become a worldwide public
health concern. Due to potential harmful effects of synthetic chemicals, there is a pressure for adoption of natural
alternatives to obtain microbial safety of food. Tea tree oil (TTO) exhibited a wide range of pharmacological
actions attribute to the broad spectrum activities. However, to the best of our knowledge, no systematic research
on the mode of antibacterial actions of TTO against Listeria monocytogenes (L. monocytogenes) and Escherichia coli
(E. coli) in vitro models have been conducted so far.
Results: The present investigation reported on the antimicrobial activities of TTO and examined its possible
antimicrobial mode of action against L. monocytogenes and E. coli. Results showed that the susceptibility of L.
monocytogenes were excellent with the lower minimal inhibitory concentration (MIC) values and larger inhibition zones. TTO changed the integrity of the membrane, as evidenced by the release of 260 nm absorbing intracellular materials and the alteration of membrane potential. The results of flow cytometry showed that TTO
caused bacterial membrane permeabilization in a dose-dependent manner. The remarkable cellular morphological changes in bacteria caused by TTO were observed using the scanning electron microscope, indicating cell
damage. In addition, antimicrobial preserving properties of TTO were evaluated by time-kill assay after its
incorporation in cucumber juice, the results showed TTO successfully inhibited L. monocytogenes and E. coli
development, at room temperature and in refrigerator (25 °C and 4 °C) respectively, demonstrating it had good
preservative activities in food system.
Conclusions: These findings suggested that TTO exhibited good antimicrobial effect against food-borne pathogens and could be potentially used in food industries as a food preservative.
1. Introduction
Food spoilage is a major problem that restricts the storage and
transportation of food and also seriously impacts food safety. Food
products contaminated with pathogens can not only lead to the reduction the quality and quantity of food products [1], but also generating illness and diseases [2], which have increasingly become a
public health concern in developing as well as developed countries.
Listeria monocytogenes (L. monocytogenes) is recognized as one of the
most important foodborne pathogens of concern for the food industries
worldwide. Foodborne listeriosis is a relatively rare but serious disease
with high fatality rates (30%) compared with other foodborne microbial pathogens [3]. L. monocytogenes is an ubiquitous microorganism
and it had previously been isolated from foods of poultry, seafood and
animal origin, mainly meat and milk [4]. Furthermore, the incidence of
foodborne outbreaks caused by contaminated fresh fruit and vegetables
∗
has also been found to increase in recent years such as cabbage, cucumbers, parsley and watercress, especially fresh produce and ready-toeat food products [5,6]. Escherichia coli (E. coli) pathogenic strains,
which have been found in a variety of foodstuffs including milk, yogurt,
salad vegetables, fruits, fruit juices and meat products, have been associated with foodborne illnesses [7]. In addition to different food
processing for reducing or eliminating spoilage or pathogenic microorganisms in food, chemical synthetic preservatives are extensively
used in food industry to extend food shelf-life or enhance food safety
[8]. Nevertheless, it is considered that chemical synthetic preservatives
have many carcinogenic and teratogenic attributes as well as residual
toxicity [9]. Therefore, it resulted in increasing search for safe and
natural antimicrobial agents, particularly those from natural plants and
fruits to be used in food conservation systems.
Tea tree oil (TTO), an essential oil extracted from the leaves of
Melaleuca alternifolia, is a complex mixture of volatile oil, terpinen-4-ol,
Corresponding author.
E-mail addresses: jlnaguo@126.com, abas1980@163.com (N. Guo).
https://doi.org/10.1016/j.micpath.2018.09.036
Received 9 November 2017; Received in revised form 19 September 2018; Accepted 24 September 2018
Available online 25 September 2018
0882-4010/ © 2018 Elsevier Ltd. All rights reserved.
Microbial Pathogenesis 125 (2018) 262–271
C. Shi et al.
γ-terpinene, α-terpinen, terpinolene and α-terpineol are the major
components of TTO [10]. Updated data showed that TTO has a wide
range of pharmacological actions due to the broad spectrum activities,
including antibacterial, antifungal, antiviral, antiprotozoal activities
and immune effects [11]. Therefore, TTO has widely been used as an
antimicrobial and anti-inflammatory agent and applied to traditional
medicine in Australian and more recently worldwide [12]. However, to
the best of our knowledge, no systematic research on the mode of antimicrobial action of TTO against Listeria monocytogenes (L. monocytogenes) and Escherichia coli (E. coli) in vitro models has been conducted so far. In present study, it is the first time to study on
antimicrobial effects of TTO, exploring its antimicrobial activities and
mode of action against L. monocytogenes and E. coli strains in a food
system, which could provide scientific data for TTO to be an alternative
natural food preservative.
respectively. TTO was prepared in MH broth to obtain subinhibitory
concentrations by serial dilutions in 96-well microtiter plates. 100 μl of
the bacterial suspension and 100 μl of TTO dilutions of different subinhibitory concentrations were added into individual wells of 96-well
microtiter plates. Wells without TTO treatment served as negative
growth controls. Then the plates were incubated at 37 °C for 16–24 h.
The MICs were defined as the lowest concentration of antibiotic that
produced complete inhibition of visible growth. All tests were performed in triplicate.
2.2.2. Agar disk diffusion tests
The antimicrobial activities of TTO against L. monocytogenes and E.
coli were measured by agar disk diffusion method according to previous
studies [15,16]. In Brief, overnight cultures of bacteria were adjusted to
1 × 107 CFU ml−1 in MH broth respectively. A 100 μl aliquot of the
bacteria suspension was spread on the MH agar plates uniformly by
confluent swabbing of the surface. Sterile paper disks (6 mm in diameter) impregnated with 10 μl the diluted TTO (0.25 mg ml−1,
0.5 mg ml−1, 1 mg ml−1 and 2 mg ml−1) aliquots were placed on the
surfaces of seeded agar plates. Paper disks treated with PBS served as
controls. The plates were incubated at 37 °C for 24 h. After incubation,
bacteria inhibitions were visually evaluated as the diameter of the
zones of inhibition (ZOI) surrounding the disks (disk diameter included)
with a caliper. The antimicrobial activity of plant essential oils (EOs)
can be classified into three levels: inhibition zone ≤ 12 mm, weak activity; 12 mm < inhibition zone < 20 mm, moderate activity; inhibition zone ≥ 20 mm, strong activity [17]. 2. Materials and methods 2.1. Chemical reagents and bacterial strains TTO was obtained from Nanjing Chemlin Chemical Industry Co., Ltd. (Nanjing, China). The purity of TTO exceeds 98%, and Terpinen-4ol (38.1%), γ-terpinene (22.2%), α-terpinen (9.1%), terpinolene (3.5%), 1, 8-cineole (6.3%) and α-terpineol (4.6%) are the main components of components of our TTO sample. The Mueller-Hinton (MH) broth and the Tryptic Soy Broth (TSB) were purchased from Qingdao Hope BioTechnology Co., Ltd. (Qingdao, China). Rhodamine 123 and Propidium iodide (PI) were purchased from Sigma-Aldrich Chemicals Company (St. Louis, USA). The fresh cucumbers were purchasd from the local market (Changchun, China). In this study, L. monocytogenes ATCC 19115 (L. monocytogenes) and E. coli ATCC 25922 (E. coli) was obtained from the China Medical Culture Collection Center. Thirteen food-borne isolates of L. monocytogenes strains and thirteen food-borne isolates of E. coli strains were obtained from Jilin Entry-Exit Inspection and Quarantine Bureau (Table 1). All microorganisms were cultured in TSB at 37 °C for 24 h with shaking. 2.3. Antimicrobial mechanisms 2.3.1. Integrity of cell membrane The cell membrane integrity of L. monocytogenes and E. coli was examined respectively by measuring the release of cell constituents into cell suspension according to the methods described by previous research with slight modifications [18,19]. The bacterial cells cultured in TSB overnight were collected by centrifugation for 15 min at 5, 000 × g, and then washed three times and resuspended in phosphate buffer saline (PBS, pH 7.4). The final cell suspension was adjusted to an absorbance at 420 nm of 0.7. Then 100 ml of cell suspension was incubated at 37 °C with shaking for 1 h, 2 h, 4 h, 6 h and 8 h in the presence of TTO at three different concentrations (1/2 × MIC, 1 × MIC and 2 × MIC). Control flasks without TTO treatment were tested similarly. Then 10 ml of samples were collected and centrifuged at 11, 000 × g for 10 min. After that the supernatants were collected, and the absorption at 260 nm was measured using an UV spectrophotometer. Results were expressed in terms of optical density of 260-nm absorbing materials in each interval. 2.2. Antimicrobial activities 2.2.1. Antimicrobial susceptibility testing To determine the minimal inhibitory concentrations (MICs) of TTO for the above-mentioned L. monocytogenes strains and E. coli strains, antimicrobial susceptibility testing was carried out on the basis of the Clinical and Laboratory Standards Institute guidelines (CLSI 2009) and Ce et al. (2016) [13,14]. Briefly, L. monocytogenes strains and E. coli strains cultured overnight (37 °C, shaking) were diluted with MH broth to a final concentration of 105 colony-forming units (CFU) ml−1 Table 1 In vitro TTO against L. monocytogenes strains and E. coli strains. Strains L. L. L. L. L. L. L. L. L. L. L. L. L. L. monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes JL-10110 JL-10111 JL-10112 JL-10113 JL-10114 JL-10115 JL-10116 JL-10117 JL-10118 JL-10119 JL-10120 JL-10121 JL-10122 ATCC 19115 MIC (range) of TTO (mg ml−1) Strains 1 (1) 1 (1–2) 1(1) 2 (1–2) 1 (1) 2 (2) 1 (1) 1 (1) 1 (1–2) 2 (2) 1 (1–2) 1 (1–2) 2 (1–2) 1 (1) Escherichia Escherichia Escherichia Escherichia Escherichia Escherichia Escherichia Escherichia Escherichia Escherichia Escherichia Escherichia Escherichia Escherichia 263 MIC (range) of TTO (mg ml−1) coli coli coli coli coli coli coli coli coli coli coli coli coli coli JL-10001 JL-10002 JL-10003 JL-10004 JL-10005 JL-10006 JL-10007 JL-10008 JL-10009 JL-10010 JL-10011 JL-10012 JL-10013 ATCC 25922 2 2 2 2 2 4 2 2 2 2 2 4 2 2 (1–2) (1–2) (2) (1–2) (2) (2–4) (1–2) (2) (1–2) (2) (2) (2–4) (2) (2) Microbial Pathogenesis 125 (2018) 262–271 C. Shi et al. 2.3.2. Membrane potential To analyze the effects of TTO on the metabolic activity changes of bacteria, the membrane potential (MP) of L. monocytogenes and E. coli was measured respectively on the basis of the Rhodamine fluorescence method as described by previous studies with slight modifications [20,21]. Bacterial cells were incubated in TSB medium at 37 °C overnight. TTO was added to the cell solutions (approximately 1 × 107 CFU ml−1) at a final concentration at 1 × MIC. Then the mixtures were incubated for 15 min, 30 min, 45 min, 60 min, 75 min and 90 min respectively. Control flasks without TTO treatment were tested similarly. Rhodamine 123 was added to PBS to aquire a 1 mg ml−1 stock solution. The suspensions were washed twice with PBS and then Rhodamine 123 was added to the suspensions at a final concentration of 0.2 μg ml−1 from the stock solution. Then the samples were completely washed and resuspended in PBS after standing in dark for 30 min. The cell suspensions were placed in the spectrofluorometer (Bio-Tek, USA). Rhodamine123 fluorescence was excited at 480 nm and emission wavelength was collected at 530 nm. The data was expressed by mean fluorescence intensity (MFI). All the tests were conducted in triplicate. incubation at 37 °C and the number of CFU ml−1 was recorded. Control samples (no TTO treatment) were incubated under the same conditions. A bactericidal effect was defined as a ≥3 log10 CFU ml−1 decrease after 24 h of incubation, compared to the density of the initial inoculum [24]. 2.3.3. Membrane damages assessment The membrane damages of L. monocytogenes and E. coli were assessed respectively by PI staining. Logarithmic phase cells of the two strains (∼107 CFU ml−1) were firstly treated with TTO at final different concentrations of 1/2 × MIC, 1 × MIC and 2 × MIC for 3 h. Control groups without TTO treatment was tested similarly. Then the bacteria were harvested by centrifugation at 8, 000 × g for 10 min. PI was added into each group at a suitable concentration. After incubation for 30 min in the dark, membrane permeability of the cells stained by PI was analyzed using a flow cytometry method (FCM) with the excitation of the light at the wavelength of 488 nm. %inhibition = 100 - (CFUsample/CFUblank) × 100 (1) %inhibition = 100 - (CFUsample/CFUblank) × 100 - Tinhibition (2) Tinhibition = 100 - (CFUTgrowth/CFUT0growth) × 100 (3) 2.4.2. In situ antimirobial assays Food preserving property was evaluated with the methods on the basis of previous studies with slight modifications and the results were expressed as percentage inhibition [25]. The sterile cucumber juice was inoculated with L. monocytogenes and E. coli grown overnight at 37 °C respectively which were adjusted to 1 × 106 CFU ml−1. The samples mixed with different concentrations of TTO ranging from 0.25 to 2 mg ml−1 thoroughly. Experimental microplates were divided into two groups: one group was kept at room temperature (25 °C) and the other group was kept at 4 °C. Both groups contained equal amounts of TTO. The inhibition percentages of the samples at 25 °C (1) and 4 °C (2, 3) were calculated by the reduction of bacterial colonies numbers using the following equations: Where CFUsample and CFUblank is the CFU of the antimicrobial samples and the blank sample incubation at 25 °C respectively. CFUTgrowth and CFUT0growth presented the growth of strains at 4 °C after and before incubation respectively. 2.5. Statistical analysis All the experiments in the present study were conducted in triplicate, and average values with standard deviation (S.D.) were revealed, all values are reported as mean ± standard deviation. The one-way analysis of variance (ANOVA), followed by Tukey's test was used for all statistical analyses to determine significant differences. A value of P < 0.05 denoted the presence of a statistically significant difference. 2.3.4. SEM analysis Morphology changes of L. monocytogenes and E. coli exposed to TTO at different concentrations were observed using a SEM (SHitachi S3400N, Japan) according to previous methods [22,23]. Logarithmic phase bacteria were allowed to adhere to polylysine-coated coverslips for approximately 12 h and exposed to TTO at 1/2 × MIC, 1 × MIC and 2 × MIC for 3 h. Then cells were washed with PBS buffer after incubation and fixed in 2.5% glutaraldehyde at 4 °C for about 12 h. After this, the cells were dehydrated using sequential exposure per ethanol concentrations ranging from 30% to 100% and the ethanol was replaced by tertiary butyl alcohol at last. The resulting samples were placed on a silicon wafer and subjected to vacuum freeze-drying (Hitachi ES-2030, Japan). Finally, the dried samples were mounted on aluminum stubs with sticky double-side conductive metal tape and gold-coated by ion sputtering (Hitachi E-1010, Japan) for about 2 min. The morphology of the bacterial cells was observed on a SEM (Hitachi S-3400N, Japan). The bacterial cells without TTO treatment served as the control groups and they were similarly processed. 3. Results and discussion 3.1. MICs and ZOI determination In this assay, the antimicrobial activities of TTO against L. monocytogenes strains and E. coli strains were assessed by MIC values. As shown in Table 1, TTO exerted potent inhibitory effects against L. monocytogenes and E. coli strains with the MIC values ranging for 1–2 mg ml−1 (0.1%–0.2% approximately, v/v) and 2–4 mg ml−1 (0.2%–0.4% approximately, v/v) respectively, and the MIC value of L. monocytogenes ATCC 19115 and E. coli ATCC 25922 was 1 mg ml−1 (0.1% approximately, v/v) and 2 mg ml−1 (0.2% approximately, v/v) respectively. In addition, the ZOI values of TTO against L. monocytogenes strains and E. coli strains were presented in Table 2. The ZOI values for L. monocytogenes ATCC 19115 were in the range of 2.4. Challenge antimicrobial tests in fresh cucumber juice 2.4.1. Time-kill curves tests The fresh cucumbers used in this assays was purchased from the local market (Changchun, China), washed, peeled and squeezed to obtain cucumber juice. And then the cucumber juice was autoclaved at 110 °C for 10 min to inactivate the naturally existing bacterial population. The bactericidal kinetics of TTO were studied by inoculating cucumber juice containing 1/2 × MIC, 1 × MIC and 2 × MIC of TTO with an initial inoculum of 1 × 105 CFU ml−1. Briefly, the samples were cultured in cucumber juice with or without TTO treatment at 37 °C. At each predetermined time point (0 h, 3 h, 6 h, 9 h, 12 h and 24 h), a 100 μl sample was removed from each test suspension, serially diluted in sterile saline (0.85% w/v NaCl) and plated on TSB agar plates to allow for growth. A viable count was performed after overnight Table 2 ZOI values of TTO against tested bacteria strains. TTO Control 0.25 mg ml−1 0.5 mg ml−1 1 mg ml−1 2 mg ml−1 ZOI values (mm) L. monocytogenes ATCC 19115 E. coli ATCC 25922 6.4 ± 0.80 13.6 ± 0.42 18.5 ± 0.44 26.8 ± 0.80 34.4 ± 1.20 6.3 ± 0.60 10.8 ± 0.40 14.6 ± 0.62 22.5 ± 0.80 28.4 ± 1.00 The results are expressed as mean ± S.D. 264 Microbial Pathogenesis 125 (2018) 262–271 C. Shi et al. Fig. 1. Effects of TTO exposed to different concentrations on the UV absorption at 260 nm of L. monocytogenes ATCC 19115 (a) and E. coli ATCC 25922 at different time points. Error bars are ± SD of the means. revealed an increasing release of 260-nm absorbing materials with respect to exposure time (Fig. 1). However, no changes in the OD of control group cells of L. monocytogenes and E. coli were observed. The results indicated that after exposure to TTO at different final concentrations to the bacteria, the cell constituents’ release of both the strains increased significantly with the increasing final concentrations of TTO. For L. monocytogenes, compared to the control group, the concentration the cell constituents (OD260nm) in suspensions treated with 1/2 × MIC TTO increased from 0.021 at 0 h to 0.888 at 8 h. While they increased from 0.021 at 0 h to 1.298 when treatment at 1 × MIC. Furthermore, after treatment with 2 × MIC TTO, the absorbance value at OD260nm increased significantly from 0.021 at 0 h to 1.681 at 8 h. For E. coli, the concentration the cell constituents (OD260nm) exposed to 1/ 2 × MIC and 1 × MIC TTO grew from 0.03 at 0 h to 0.761 and 2.298 at 8 h respectively. After exposure to 2 × MIC TTO, the absorbance value at OD260nm increased significantly from 0.03 for 0 h to 2.685. These 13.6–34.3 mm. And they were in the range of 12.8–28.4 mm for E. coli ATCC 25922. The data indicated that TTO was a potentially effective antimicrobial agent against L. monocytogenes strains and E. coli strains. Previous studies have suggested the MIC values of TTO against some foodborne pathogenic bacteria and clinical isolates of bacteria. It had reported that the MICs of TTO was 0.25% for both E. coli AG100 and Staphylococcus aureus NCTC 8325, and the MIC value for Candida albicans KEM H5 was 0.125% [26]. In addition, Renata et al. have revealed that the MIC values were 0.28% or 0.31% for E. coli and 0.56% or 0.62% for Staphylococcus aureus respectively [27]. 3.2. Measurement of release of 260-nm absorbing cellular materials The optical density (OD260nm) of the culture filtrates of L. monocytogenes and E. coli cells exposed to TTO at different concentrations 265 Microbial Pathogenesis 125 (2018) 262–271 C. Shi et al. MP is measured as the difference in electric potential between the interior and the exterior of a biological cell, and MP alterations of bacteria can affect cell metabolic activity, eventually leading to cell death. A MP of normal bacteria is generated by differences in the concentrations of ions on opposite sides of the cell membrane. Any treatments depolarize the cell membrane are deemed to reduce the volume of MP [20]. In this study, measurements of MFI of Rhodamine 123 in exponentially growing cells revealed a sharp decrease after the addition of TTO. The results demonstrated that TTO caused membrane hyperpolarization of bacteria cells, as evidenced by a decrease in fluorescence (Fig. 2). Consistent with our results, a recent investigation showed that 3-p-trans-Coumaroyl-2-hydroxyquinic acid, a novel phenolic compound from pine needles of Cedrus deodara could destroy the cell membrane of Staphylococcus aureus, causing membrane hyperpolarization [29]. A previous study of this phenomenon have demonstrated that pH or K+ change could be the primary cause which led to the hyperpolarization occurs [30]. results clearly indicated the confirmation of leakage of 260 nm absorbing materials from the bacterial cells treated with TTO. The irreversible damage to the cytoplasmic membranes might occur, bacterial cell membrane integrity may also have been compromised, which led to the losses of cell constituents such as protein and some essential molecules and consequently lead to cell death. The integrity of the cytoplasmic membrane is a critical factor to bacteria growth. The cytoplasmic cell membrane is a structural component, which may become damaged and functionally invalid when bacterial suspensions are exposed to antimicrobial agents. If bacterial membrane became compromised, release of cytoplasmic constituents of the cell can be monitored at the absorbance of 260 nm using ultraviolet (UV) spectrophotometer, small ions such as K+ tend to leach out first, and then followed by large molecules such as nucleic acids, proteins and other materials. The release of these intracellular components with strong UV absorption at 260 nm is an indication of membrane damage [18,28]. Therefore, analyzing the leakage of cell constituents can therefore give further insight to the mechanism of antibacterial action. Current study revealed that TTO could act on the cytoplasmic membrane, affecting the integrity of membrane. Consequently, nucleic acids, ions and proteins were released through the malfunction membrane as reflected by the significant higher OD260nm in respond to the addition of TTO. 3.4. Membrane damages assay In present study, the effects of TTO on the membrane permeability of L. monocytogenes and E. coli cells were measured with PI-based FCM, and more red fluorescence was observed in bacteria after exposure to TTO. As shown in Fig. 3, FCM with PI staining observed more bacteria with red fluorescence after TTO treatment as the concentration increasing than that of the controls (in the area of R2). For L. monocytogenes (Fig. 3A), in control group, only a small proportion was stained with PI, the positive rate of dead bacterial cells was 0.04% (in the area of R2). However, after treatment with 1/2 × MIC TTO and 1 × MIC TTO, FCM showed that the positive rate of dead bacterial cells stained by PI increased to 73.93% and 80.24% respectively (in the area of R2). In addition, most of the population was stained with PI after exposure to TTO at 2 × MIC, the proportion of membrane damaged cells increased to 96.32% (in the area of R2). Similarly, for E. coli, the positive rate of dead bacterial cells stained by PI increased rapidly with the concentrations of TTO increasing (Fig. 3B). In control group, the positive rate of dead bacterial cells was 0.44% (in the area of R2). After treatment with TTO at 1/2 × MIC, 1 × MIC and 2 × MIC, the positive rate of dead bacterial cells stained by PI increased to 77.33%, 84.92% and 95.94% respectively (in the area of R2). PI exhibit substantially increased fluorescences on binding to intracellular nucleic acids. And the dye normally bear two positive 3.3. MP determination MP was selected as another aspect to illustrate the antimicrobial mechanism as it plays an important role in bacterial physiology. MP alteration is an early indication of injury in bacteria, and can be evaluated by measuring the fluorescent intensity. The result of bacterial membrane potential was showed in Fig. 2. The magnitude of MP was expressed by MFI values (AU) of Rhodamine 123. After exposure to TTO at 1 × MIC, a rapidly decrease were observed in both L. monocytogenes and E. coli with the MFI values. As shown in Fig. 2, in the control groups (without TTO treatment), the MFI value was 766 and 784 for L. monocytogenes and E. coli respectively, while after treatment with TTO for 30 min, the MFI value decreased to 545 and 556 respectively. And when exposure to TTO for 60 min, the MFI value continued to drop to 477 and 435 respectively. The MFI values decreased to 336 and 294 when treatment with TTO for 90 min respectively. The results revealed that TTO treatment decreased the MFI values of L. monocytogenes and E. coli in a time-dependent manner. Fig. 2. Membrane potential of L. monocytogenes ATCC 19115 and E. coli ATCC 25922 treated with TTO at 1 × MIC. Values are expressed as mean ± SD of three different experiments performed in triplicate. Error bars indicate the SD. *p < 0.05 and **p < 0.01 were considered significant versus the control groups. 266 Microbial Pathogenesis 125 (2018) 262–271 C. Shi et al. Fig. 3. Membrane permeability of L. monocytogenes ATCC 19115 (A) and E. coli ATCC 25922 (B) was measured using FCM with PI staining after treatment with TTO at different concentrations for 3 h respectively. (a) Control (b) Treatment with TTO at 1/2 × MIC (c) Treatment with TTO at 1 × MIC (d) Treatment with TTO at 2 × MIC. 267 Microbial Pathogenesis 125 (2018) 262–271 C. Shi et al. charges and are excluded from cells with intact cell membranes of both bacteria and eukaryotes, while they stain nucleic acids in cells with damaged membranes. It is generally used as an indicator of cell membrane permeability [31]. All the cells are bounded by the cytoplasmic membrane, which allows the cell to communicate selectively with its immediate environment. Passive and active transport systems across the cytoplasmic membrane exist and generate an electrochemical gradient. The presence of both an intact polarised cytoplasmic membrane and active transport systems are essential for a fully functional healthy cell [32]. In this assay, TTO induced severe damage in the bacterial membranes of L. monocytogenes and E. coli at 3 h of contact: up to 96.32% and 95.94% of the population was stained with PI, which strongly indicated that losses in viability may be associate with membrane damage. 3.5. SEM observation Electron microscopy observations are powerful tools to better understand the impact of a stressor on bacterial cells [33]. The physical and morphological alterations in L. monocytogenes and E. coli cell wall structures were observed by SEM analysis. The results showed the destructive effects of TTO on bacteria directly. As shown in Fig. 4, the surfaces of the TTO-treated bacteria cells underwent obvious morphological changes compared with the cells treated without TTO. In control groups, the cells showed the distinctive characteristics of normal rod shaped, intact, regular and smooth cell membrane surface. However, when the cells exposed to TTO at different concentrations, remarkable morphological changes were observed. The cells treated with TTO at 1/ 2 × MIC became pitted, deformed and irregular, and when they exposed to TTO at 1 × MIC, the cells became more wrinkled, shriveled and irregular. Furthermore, treatment with TTO at 2 × MIC, the cells showed a series of severe alterations including swelling even broken, surface roughening, disruption and adhesive to each other. These damage may give rise to the leaching out of nutrient and genetic materials. In conclusion, larger populations of sunken and malformed cells were observed in the microphotograph of the two strains. Furthermore, the changes became more severe with the increasing of treatment concentration of TTO. In addition, the observations were in agreement with the results of the membrane damages assay and FCM assay, and indicated that TTO may have severe effects on the cell wall and cytoplasmic membrane. Previously such morphological alterations have been observed for various of organisms when exposed to different essential oils [34,35]. The literature suggested that the active components of the essential oil might bind to the cell surface and then penetrate to the target sites possibly the plasma membrane and membrane-bound enzymes, leading to the disruption of cell wall structures. And other reports have revealed that the changes in membrane fluidity usually occur due to membrane lipid composition alterations and are thought to be a compensatory mechanism to counter the lipid disordering effects of the treatment agent [36]. We investigated the mechanism by measurement of release of 260-nm absorbing cellular materials, the MP changes, membrane damages assay, and SEM were also carried out to verify the mechanism. These results indicated that TTO enable to decrease the content of cellular materials and decline the MFI values, by permeating and disrupting cell membranes. However, TTO is composed of a variety of active components, so it seems unlikely that there is only one mechanism of action or that only one component is responsible for the antimicrobial action. Therefore, further work is required to understand fully the mechanisms involved in order to justify the real applications of TTO in food practices as a natural antimicrobial agent. Fig. 4. SEM observation of L. monocytogenes ATCC 19115 (A) and E. coli ATCC 25922 (B) exposed to TTO at different concentrations for 3 h respectively. (a) Control (b) Treatment with TTO at 1/2 × MIC (c) Treatment with TTO at 1 × MIC (d) Treatment with TTO at 2 × MIC. how quickly an agent acts on an organism. In vitro time-kill studies, one of the most commonly used experimental models to assess antibacterial activity, efficiently characterize the rate, extent, and timing of bacterial killing and regrowth [37]. The assay results are shown in Fig. 5, with the bactericidal effect of different concentrations of TTO reported as the log10 reduction in viable bacterial cells in cucumber juice compared to the initial inoculum. Treatment of L. monocytogenes with TTO at two times the MIC reduced the viability by 6-log10 over approximately 12 h, whereas treatment with TTO at the MIC resulted in a 2.7-log10 reduction over 24 h. However, treatment with TTO at one-half the MIC did not result in reduced viability (Fig. 5A). Similarly, TTO at one-half the MIC had no effect on the viability of E. coli. In contrast, treatment with TTO at two times the MIC and the MIC effected reductions of 6-log10 and 2.5-log10 over 24 h (Fig. 5B). These time-kill results suggested that 2 × MIC of TTO has bactericidal activity. At each time point in food system, TTO at 1/2 × MIC against bacteria demonstrated the smallest or even no reduction in the number of viable cells, followed by 1 × MIC, which demonstrated a greater reduction, and 2 × MIC, which demonstrated the greatest reduction in the number of viable cells. 3.6. Time-kill curves The bactericidal activity of TTO against L. monocytogenes and E. coli was determined using a time-kill assay respectively, which determines 268 Microbial Pathogenesis 125 (2018) 262–271 C. Shi et al. Fig. 5. Time-kill curves for TTO with different concentrations against L. monocytogenes ATCC 19115 (a) and E. coli ATCC 25922 (b). Values are expressed as mean ± SD of three different experiments performed in triplicate. Error bars indicate the SD. 3.7. In situ antimicrobial activity in cucumber juice respectively. Table 3 and Table 4 showed that the antimicrobial activities of TTO against both of the two strains were better at 4 °C. Under refrigerated conditions, the inhibition percentage was stabilized for all the concentrations during different periods of storage, with the range of 87.8%–100%. With the highest tested concentrations (2 mg ml−1), Regarding the receivable antibacterial activity of TTO against all the tested bacteria, the antibacterial activity of TTO against L. monocytogenes and E. coli was tested under 25 °C and 4 °C in cucumber juice Table 3 The antimicrobial activity of TTO in cucumber juice against L. monocytogenes ATCC 19115. Concentrations of TTO (mg ml−1) Temperature (°C) Percentage of inhibition of L. monocytogenes ATCC 19115 0h 0.25 0.5 1 2 25 4 25 4 25 4 25 4 0 0 0 0 0 0 0 0 ± ± ± ± ± ± ± ± 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 The results are expressed as mean ± S.D. 269 12 h 24 h 36 h 48 h 0 ± 0.00 91.5 ± 0.50 45.5 ± 0.50 92.2 ± 0.40 95.5 ± 0.50 100 ± 0.00 100 ± 0.00 100 ± 0.00 0 ± 0.00 88.9 ± 0.70 44.2 ± 0.80 92.1 ± 0.70 94.8 ± 0.40 100 ± 0.00 100 ± 0.00 100 ± 0.00 0 ± 0.00 88.6 ± 0.40 36.4 ± 0.80 90.6 ± 0.40 93.6 ± 0.40 100 ± 0.00 100 ± 0.00 100 ± 0.00 0 ± 0.00 87.8 ± 0.60 34.2 ± 0.60 90.5 ± 0.50 93.8 ± 0.40 100 ± 0.00 100 ± 0.00 100 ± 0.00 Microbial Pathogenesis 125 (2018) 262–271 C. Shi et al. Table 4 The antimicrobial activity of TTO in cucumber juice against E. coli ATCC 25922. Concentrations of TTO (mg ml−1) Temperature (°C) Percentage of inhibition of E. coli ATCC 25922 0h 0.25 0.5 1 2 25 4 25 4 25 4 25 4 0 0 0 0 0 0 0 0 ± ± ± ± ± ± ± ± 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 12 h 24 h 36 h 48 h 0 ± 0.00 93.5 ± 0.50 66.7 ± 0.40 95.5 ± 0.50 72.5 ± 0.50 100 ± 0.00 100 ± 0.00 100 ± 0.00 0 ± 0.00 89.9 ± 0.30 64.5 ± 0.50 94.9 ± 0.60 68.4 ± 0.60 100 ± 0.00 100 ± 0.00 100 ± 0.00 0 ± 0.00 90.6 ± 0.80 64.4 ± 0.40 91.2 ± 0.60 66.5 ± 0.50 100 ± 0.00 100 ± 0.00 100 ± 0.00 0 ± 0.00 90.8 ± 0.40 62.2 ± 0.80 90.8 ± 0.80 65.6 ± 0.40 100 ± 0.00 100 ± 0.00 100 ± 0.00 Conflicts of interest 100% inhibition was achieved in 12 h regardless of the incubation temperature. No inhibition was found at the lowest concentrations (0.25 mg ml−1) at room temperature. The effect was also dose dependent, decreasing at lower doses with period of storage. However, from the results of Tables 3 and 4, TTO showed the highest inhibition zones at 12 h, but after then, the diameters of the inhibition zone of TTO decreased with the incubation time delayed. As we all known, TTO presents poor solubility and high volatility [38]. In previous study, it was found that composite essential oils showed the highest inhibition zone, then the diameters of the inhibition zone of composite essential oils were decreased with the incubation time delayed. Especially, composite essential oils produced no observable inhibition zones against tested bacteria at 9 day [39]. Therefore, the good antibacterial activity of TTO can not last for long time and it could be speculated that the results are relevant to the high volatility. Overall, the antimicrobial activity of TTO provides a strong evidence that it is a green and efficient way in the control of foodborne bacteria. In order to control food spoilage, the application of low amounts of naturally occurring antimicrobial agents as food ingredients should be investigates deeply. The present work provides a basis for developing effective naturally promising antimicrobial agents to extend the shelf-life of food and provides strong evidence that TTO might be efficient in the control of L. monocytogenes and E. coli strains in vegetable juice. The authors declare that there was no conflict of interest. Acknowledgements Financial support for this work came from the following sources: the National Nature Science Foundation of China (No. 31772082) and Natural Science Foundation of Jilin Province (No. 20180101249JC). References [1] K.M. Soliman, R.I. Badeaa, Effect of oil extracted from some medicinal plants on different mycotoxigenic fungi, Food Chem. Toxicol. 40 (2002) 1669–1675. [2] C. Jacob, L. Mathiasen, D. Powell, Designing effective messages for microbial food safety hazards, Food Contr. 21 (2010) 1–6. [3] J. Ponniah, T. Robin, M.S. Paie, S. Radu, F.M. Ghazali, C.Y. Kqueen, M. Nishibuchi, Y. Nakaguchi, P.K. Malakar, Listeria monocytogenes in raw salad vegetables sold at retail level in Malaysia, Food Contr. 21 (2010) 774–778. [4] A. Schuchat, B. Swaminathan, C.V. Broome, Epidemiology of human listeriosis, Clin. Microbiol. Rev. 4 (1991) 169–183. [5] J.A. Odumeru, S.J. Mitchell, D.M. Alves, J.A. Lynch, J.A. Yee, S.L. Wang, et al., Assessment of the microbiological quality of ready-to-use vegetables for health-care food services, J. Food Protect. 60 (1997) 954–960. [6] A. Mukherjee, D. Speh, A.T. Jones, K.M. Buesing, F. Diez-Gonzalez, Longitudinal microbiological survey of fresh produce grown by farmers in the upper Midwest, J. Food Protect. 69 (2006) 1928–1936. [7] J.L. Kornacki, E.H. Marth, Foodborne illness caused by Escherichia coli: a review, J. Food Protect. 45 (1982) 1051–1067. [8] R. Gyawali, S.A. Ibrahim, Natural products as antimicrobial agents, Food Contr. 46 (2014) 412–429. [9] L.X. Hou, Y.H. Shi, P. Zhai, G.W. Le, Inhibition of foodborne pathogens by Hf-1, a novel antibacterial peptide from the larvae of the housefly (Musca domestica) in medium and orange juice, Food Contr. 18 (2007) 1350–1357. [10] J.T. Callander, P.J. James, Insecticidal and repellent effects of tea tree (Melaleuca alternifolia) oil against Lucilia cuprina, Vet. Parasitol. 184 (2012) 271–278. [11] M. Li, L. Zhu, B. Liu, L. Du, X. Jia, L. Han, Y. Jin, Tea tree oil nanoemulsions for inhalation therapies of bacterial and fungal pneumonia, Colloids Surfaces B Biointerfaces 141 (2016) 408–416. [12] C.F. Carson, K.A. Hammer, T.V. Riley, Melaleuca alternifolia (tea tree) oil: a review of antimicrobial and other medical properties, Clin. Microbiol. Rev. 19 (2006) 50–62. [13] Clinical and Laboratory Standards Institute (CLSI), Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, eighth ed., CLSI. Approved Standard M7-A8, Wayne, PA, USA, 2009. [14] S. Ce, Z. Xiaowei, Z. Xingchen, M. Rizeng, L. Zonghui, C. Xiangrong, G. Na, Synergistic interactions of nisin in combination with cinnamaldehyde against Staphylococcus aureus in pasteurized milk, Food Contr. 71 (2017) 10–16. [15] F. Lv, H. Liang, Q. Yuan, C. Li, In vitro antimicrobial effects and mechanism of action of selected plant essential oil combinations against four food-related microorganisms, Food Res. Int. 44 (2011) 3057–3064. [16] G. Na, Z. Yu-Ping, C. Qi, G. Qing-Yan, J. Jiao, W. Wei, Yuan-Gang Zu, F. Yu-Jie, The preservative potential of Amomum tsaoko essential oil against E. coil, its antibacterial property and mode of action, Food Contr. 75 (2017) 236–245. [17] M.C. Rota, J.J. Carraminana, J. Burillo, A. Herrera, In vitro antimicrobial activity of essential oils from aromatic plants against selected foodborne pathogens, J. Food Protect. 67 (2004) 1252–1256. [18] C.Z. Chen, S.L. Cooper, Interactions between dendrimer biocides and bacterial membranes, Biomaterials 23 (2002) 3359–3368. [19] W. Du, C. Sun, Z. Liang, Y. Han, J. Yu, Antibacterial activity of hypocrellin A against Staphylococcus aureus, World J. Microbiol. Biotechnol. 28 (2012) 3151–3157. [20] J. Zhang, K.P. Ye, X. Zhang, D.D. Pan, Y.Y. Sun, J.X. Cao, Antibacterial activity and mechanism of action of black pepper essential oil on meat-borne Escherichia coli, Front. Microbiol. 7 (2016) 2094. 4. Conclusions In summarize, based on the present research, TTO exhibited good antibacterial activities against the two food-borne pathogenic bacteria using in vitro model. TTO treatment caused the physical and morphological alterations in the cell wall and membrane of L. monocytognes and E. coli. The results demonstrated that the major probable mechanism of action of TTO towards L. monocytognes and E. coli appears to be alteration of permeability and integrity of bacterial cell membranes, and then leading to leakage of intracellular materials, such as electrolytes, ATP, proteins, and DNA materials. The ability of TTO to disrupt the morphology of the two bacteria was clearly shown in SEM observation. In addition, TTO successfully preserved cucumber juice by inhibiting the growth of L. monocytogenes and E. coli during the product storage period. These changes led to disorder, decomposition, and death eventually, which were corresponded to a simultaneous reduction in the number of viable L. monocytognes and E. coli. According to present results, it should lead to effective application of TTO as natural antimicrobial food preservative to control foodborne pathogens in food industries. However, due to heterogeneous compositions of TTO, it seems likely that there is not only one mode of action or only one component is responsible for the antibacterial mechanisms, and how it works in food systems is not clear. As a result, further research should be carried out in future research before its real application in food industry, such as the interactions with other food additives or other ingredients in order to verify TTO applications as a natural food preservative. 270 Microbial Pathogenesis 125 (2018) 262–271 C. Shi et al. [30] C. Bot, C. Prodan, Probing the membrane potential of living cells by dielectric spectroscopy, Eur. Biophys. J. 38 (2009) 1049–1059. [31] J. Novo, N.G. Perlmutter, R.H. Hunt, H.M. Shapiro, Multiparameter flow cytometric analysis of antibiotic effects on membrane potential, membrane permeability, and bacterial counts of Staphylococcus aureus and Micrococcus luteus, Antimicrob. Agents Chemother. 44 (2000) 827–834. [32] A. Reis, T.L. da Silva, C.A. Kent, M. Kosseva, J.C. Roseiro, C.J. Hewitt, Monitoring population dynamics of the thermophilic Bacillus licheniformis CCMI 1034 in batch and continuous cultures using multi-parameter flow cytometry, J. Biotechnol. 115 (2005) 199–210. [33] I.M. Helander, E.L. Nurmiaho-Lassila, R. Ahvenainen, J. Rhoades, S. Roller, Chitosan disrupts the barrier properties of the outer membrane of gramnegative bacteria, Int. J. Food Microbiol. 71 (2001) 235–244. [34] S.Y. Zhu, Y. Yang, H.D. Yu, Y. Ying, G.L. Zou, Chemical composition and antimicrobial activity of the essential oils of Chrysanthemum indicum, J. Ethnopharmacol. 96 (2005) 151–158. [35] V.K. Bajpai, S.M. Al-Reza, U.K. Choi, J.H. Lee, S.C. Kang, Chemical composition, antibacterial and antioxidant activities of leaf essential oil and extracts of Metasequioa glyptostroboides Miki ex Hu, Food Chem. Toxicol. 47 (2009) 1876–1883. [36] J. Sikkema, J.A.M. De Bont, B. Poolman, Mechanism of membrane toxicity on hydrocarbons, Microbiol. Rev. 59 (1995) 201–222. [37] S.E. Cheah, J. Li, R.L. Nation, J.B. Bulitta, Novel rate-area-shape modeling approach to quantify bacterial killing and regrowth for in vitro static time-kill studies, Antimicrob. Agents Chemother. 59 (2015) 381–388. [38] M.E. Souza, L.Q.S. Lopes, P.C. Bonez, et al., Melaleuca alternifolia nanoparticles against Candida species biofilms, Microb. Pathog. 104 (2017) 125–132. [39] Jing Hu, Yudi Zhang, Zuobing Xiao, et al., Preparation and properties of cinnamonthyme-ginger composite essential oil nanocapsules, Ind. Crop. Prod. 122 (2018) 85–92. [21] J. Comas, J. Vives-Rego, Assessement of the effects of gramicidin, formaldehyde, and surfactants on Escherichia coli by flow cytometry using nucleic acid and membrane potential dyes, Cytometry 29 (1997) 58–64. [22] S.X. Shen, T.H. Zhang, Y. Yuan, S.Y. Lin, J.Y. Xu, H.Q. Ye, Effects of cinnamaldehyde on Escherichia coli and Staphylococcus aureus membrane, Food Contr. 47 (2015) 196–202. [23] L.H. Wang, M.S. Wang, X.A. Zeng, Z.W. Liu, Temperature mediated variations in cellular membrane fatty acid composition of Staphylococcus aureus in resistance to pulsed electric fields, Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1858 (2016) 1791–1800. [24] K.L. LaPlante, In vitro activity of lysostaphin, mupirocin, and tea tree oil against clinical methicillin-resistant Staphylococcus aureus, Diagn. Microbiol. Infect. Dis. 57 (2007) 413–418. [25] D. Stojković, J. Zivković, M. Soković, J. Glamočlija, I.C.F.R. Ferreira, T. Janković, Z. Maksimović, Antibacterial activity of Veronica Montana L. extract and of protocatechuic acid incor... Purchase answer to see full attachment

  
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