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American Journal of Microbiological Research, 2018, Vol. 6, No. 3, 73-78
Available online at http://pubs.sciepub.com/ajmr/6/3/3
©Science and Education Publishing
DOI:10.12691/ajmr-6-3-2
Antimicrobial Activity of Tea Tree oil against Pathogenic
Bacteria and Comparison of Its Effectiveness
with Eucalyptus Oil, Lemongrass Oil
and Conventional Antibiotics
Sinthia Kabir Mumu, M. Mahboob Hossain*
Microbiology Program, Department of Mathematics and Natural Sciences, BRAC University, Mohakhali 66, Dhaka, Bangladesh
*Corresponding author: mmhossain@bracu.ac.bd
Abstract Tea Tree oil (TTO) is known to have antibacterial effects and this study was aimed to determine the
abilities to control pathogenic bacteria and also compared the antimicrobial effectiveness of Eucalyptus oil (ECO),
Lemongrass oil (LGO) and antibiotics those are using for bacterial infection. This study of antimicrobial activity
against ten pathogenic bacteria: Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Proteus
vulgaris, Aeromonas hydrophila, Escherichia coli, Streptococcus pneumoniae, Bacillus subtilis, Klebsiella
pneumonia and Streptococcus agalactiae was done by Broth dilution method and Agar well diffusion method. The
essential oils used in this study were commercially available. The inhibition of bacterial growth after 24 hours
incubation exhibits greater results than 6 hours incubation in most of the cases. After 24 hours incubation, TTO
showed minimum 96.94% against E. coli and maximum 100% inhibition against seven bacteria selected for this
study whereas ECO showed minimum 37.02% against E. coli and maximum 100% inhibition against S. aureus,
P. vulgaris and A. hydrophila and the another essential oil that is LGO exhibited minimum 69.08% against E. coli
and maximum 100% inhibition against five bacteria chosen for this study. The inhibition zones from each extract
were measured and an activity index was calculated from the mean zone sizes. All Essential oils showed some
degree of antimicrobial properties with the highest activity index (1.6) being from TTO against S. agalactiae. At last,
established a comparison between tea tree oil and some broad spectrum antibiotics using well diffusion method. Tea
tree oil exhibited observable zone against all the bacteria contrariwise, among nine antibiotics only two of them
showed noticeable zone of inhibition to all the bacteria tested. According to this study, TTO has demonstrated
remarkable antibacterial activity which was more efficient than ECO and LGO and, moreover, it is expected that
TTO will gradually take place of conventional antibiotics to treat bacterial infection.
Keywords: essential oils, activity index, inhibition percentage, broth dilution, well diffusion
Cite This Article: Sinthia Kabir Mumu, and M. Mahboob Hossain, “Antimicrobial Activity of Tea Tree oil
against Pathogenic Bacteria and Comparison of Its Effectiveness with Eucalyptus Oil, Lemongrass Oil
and Conventional Antibiotics.” American Journal of Microbiological Research, vol. 6, no. 3 (2018): 73-78.
doi: 10.12691/ajmr-6-3-2.
1. Introduction
The world seems to be running out of effective
antibiotics. While any antimicrobial resistance is concerning,
the increasing incidence of antibiotic-resistant Gram-negative
bacteria has become a particular problem as strains
resistant to multiple antibiotics are becoming common and
no new drugs to treat these infections will be available in
the near future [1]. The problem of resistance is also due
to an abuse of antibiotics. Many people will go to a doctor
and demand an antibiotic when they have a cold or a flu,
for which these antibacterial compounds are useless. In
many countries it is possible to buy antibiotics over the
counter. Often, if people are poor, they will not take the
full dose all of that leads to resistance [2]. Nowadays, use
of alternative therapies with mainstream medicine has
gained the momentum. Aromatherapy came into existence
after the scientists deciphered the antiseptic and skin
permeability properties of essential oils [3]. Essential oils
are a rich source of biologically active compounds [4].
There has been an increased interest in looking at
antimicrobial properties of extracts from aromatic plants
particularly essential oils. Therefore, it is reasonable to
expect a variety of plant compounds in these oils with
specific as well as general antimicrobial activity and
antibiotic potential [5]. Tea tree oil may have a clinical
application, especially for clearance of methicillin-
resistant Staphylococcus aureus (MRSA) carriage or as a
hand disinfectant to prevent cross-infection with Gram-
positive and Gram-negative epidemic organisms [6]. Tea
tree oil’s antimicrobial terpenes content also makes it
popular for combating acne. Not only does it kill MRSA
74 American Journal of Microbiological Research
or staph infections, but it will also kill Propionibacterium
acnes that live inside hair follicles and can lead to
inflammation and acne. It has the ability to kill parasites
and fungal infections, which is why it’s so popular for use
in fighting toenail fungus, ringworm and athlete’s foot.
Apply undiluted tea tree oil twice daily to affected areas
like nails or feet to relieve symptoms, and possibly completely
heal these unsightly ailments [7]. The antibacterial properties
in eucalyptus essential oil are well established, and
its antiseptic nature makes it appropriate for treating
wounds like burns, sores, cuts, and abrasions [8]. A
diluted lemongrass mixture may assist in facilitating
nutrient assimilation and boosts the functioning of the
digestive system, which is helpful in alleviating bowel
problems and digestive disorders [9]. Lemongrass contains
substances that are used to alleviate muscle pain, reduce
fever, and to stimulate uterus and menstrual flow [10].
2. Materials and Methods
2.1. Strains of Bacteria
The following strains of bacteria were used: Staphylococcus
aureus, Streptococcus pyogenes, Pseudomonas aeruginosa,
Proteus vulgaris, Aeromonas hydrophila, Escherichia coli,
Streptococcus pneumoniae, Bacillus subtilis, Klebsiella
pneumonia and Streptococcus agalactiae. All strains of
bacteria were isolated from clinical sample and maintained in
laboratory fridge through regular subcultures.
2.2.1. Broth Dilution Method
At first, two tubes were prepared where one of them
contain 5 ml of Brain Heart Infusion Broth (BHIB) and
another tube contained mixture of 4ml of BHIB with1ml
of tested oil. Bacterial suspension matched with
McFarland 0.5 and transferred 10 µl per tube prepared
earlier. After mixing the broth and suspension well
incubated the tube at 37°C for 24 hours. After 24 hours
900 µl of saline was taken separately in sets of 6 tubes for
broth and 4 tubes for broth with oil. One hundred
microliters of bacterial suspension from broth were added
to the 1st tube and 100 µl solution was transferred to the
2nd tube and this procedure was repeated till 6th tube.
Similarly, 100 µl of bacterial suspension from broth with
oil was added to the 1st tube and 100 µl solution was
transferred to the 2nd tube and this procedure was
repeated till 4rd tube. Before transferring the solution
every tube was subjected to vortex for uniform mixing. As
the study planned to detect inhibition rate in two different
time interval so the previous step needed to repeat at 6
hours and 24 hours incubation.
2.2.2. Detection of Inhibition Percentage
After specific incubation period 100 µl of the samples
was spread on the agar plate containing nutrient agar from
each diluted both broth and broth with oil tube. One
hundred microliter from original tube containing oil and
broth was spread on agar plate. All the plates were
incubated at 37°C for 24 hours. CFU in Oil mixed with
broth and CFU in broth of each spread plate was counted
and compared. Rate of inhibition in case of every diluted
tube was then calculated and averaged to detect actual
inhibition Percentage.
2.3. Agar Well Diffusion
Fresh subculture plates, incubated for 24 hours, were
placed under the laminar chamber and used to make
standard bacterial suspensions in labeled test tubes. Next,
an autoclaved cotton swab was used to perform lawn
culture for the uniform growth of bacteria. After that, a
cork borer was dipped into the agar to make three holes or
wells in the media. Each well was labelled and
accordingly filled with 50 microlitres of TTO, ECO, LGO
and Cefepime disc as positive control. The plates are
incubated for 24 hours at 37°C, after which clear zones
were formed around the control disc and the extracts
which gave positive results. These inhibition zones were
measured in millimetres using a ruler and recorded. All
antimicrobial tests were repeated twice and the average of
the inhibition zones is noted. An activity index was
calculated from the results to measure the relative efficacy
of the fruit extracts. The following formula was used:
()
Activity Index AI
Zone of Inhibition of EssentialOil
=.
Zone of Inhibition of Cefepime
3. Results and Discussion
Essential oils have great medicinal benefits as they
contain the essence of herbs and flowers in concentrated
form. The aroma molecules are very potent organic plant
chemicals that make the surroundings free from disease,
bacteria, virus and fungus. Their versatile character of
antibacterial, antiviral, anti-inflammatory nature along
with immune booster body with hormonal, glandular,
emotional, circulatory, calming effect, memory and
alertness enhancer, is well documented by many scientists
[11]. It’s known to everyone that most antibiotics no
longer work; infections are getting harder to cure. Hence,
it is high time to find alternative to antibiotics from
natural sources.
3.1. Inhibition percentage
Number of colonies
×reciprocal of the dilution factor
CFU = Volume of plated suspension
CFU in oil Dilution
={1- } 100.
CFU in broth Diluti
Inhibition percentag
on
e
×
The results of our investigation showed that in most of
the cases the inhibition percentage of tested essential oils
against selected pathogenic bacteria for this study exhibits
greater results after 24 hours incubation than 6 hours
incubation.
Tea Tree oil shows great promise as an antimicrobial
agent than Eucalyptus oil and Lemongrass oil according to
American Journal of Microbiological Research 75
the results of our investigation. J. May et al., (2000)
researched based on the time-kill approach, determined the
killing rate of tea tree oil against several multidrug-resistant
organisms, including MRSA, glycopeptide-resistant enterococci,
aminoglycoside-resistant klebsiellae, Pseudomonas
aeruginosa and Stenotrophomonas maltophilia, and also
against sensitive microorganisms. A rapid killing time
(less than 60 min) was achieved with both tea tree oils
(standard and chemically cloned) with most isolates, but
MRSA was killed more slowly than other organisms
[6]. In this study, efforts were made to determine the
effectiveness of incubation period on Inhibition percentage.
Figure 1. Inhibition percentage of Essential oils after 6 hours incubation against pathogenic bacteria. Vertical bars indicate Standard deviations from the
mean
Figure 2. Inhibition percentage of Essential oils after 24 hours incubation against pathogenic bacteria. Vertical bars indicate Standard deviations from
the mean
0
20
40
60
80
100
120
Inhibition %
Pathogens
Pathogens vs Inhibition % After 6 Hours Incubation
Tea Tree oil
Eucalyptus oil
Lemongrass oil
0
20
40
60
80
100
120
Inhibition %
Pathogens
Pathogens vs Inhibition % After 24 Hours Incubation
Tea Tree oil
Eucalyptus oil
Lemongrass oil
76 American Journal of Microbiological Research
3.2. Antimicrobial Activity of Essential oils
Tea tree oil proved to be the strongest antimicrobial
agent in this research. In fact, the highest inhibition zones
in our entire investigation, 36.3 mm was that of TTO,
against A. hydrophila, appearing 1.1 times stronger than
the control antibiotic Cefepime. Besides that, TTO showed
significant activity against all the pathogens selected for
this study. Conversely, ECO and LGO didn’t exhibit
satisfactory results; most of the bacteria exhibited very
low degree of sensitivity except against B. subtilis and S.
agalactiae. Fitzpatrick (2010) investigated effectiveness
of Tea tree oil, fresh garlic, an industrial cleaner and
deodorizer Quad 10, and mouthwash Listerine against five
bacteria: B. subtilis, E. coli, M. roseus, S. luteus, and S.
marcescens. TTO showed the most consistent inhibitory
action with all bacteria, except Sarcina luteus, controlled
to at least one centimeter radius when measured from the
edge of the disk solution. Fresh garlic and Listerine had no
effect on controlling the bacteria as they showed no or
little zone of inhibition [12]. In this study, antibacterial
activity of TTO was determined by tested on ten bacteria
which helped to establish a steady comparison with
another essential oils and antibiotics.
Carson et al. (2006), Carson and Riley (1995), Cox et al.
(2001) Tea tree oil has shown inhibitory effects on bacteria
with E. coli [13,14,17]. Similarly, Lee et al., 2013 found
that TTO presented dose-dependent inhibitory effects against
the growth of P. acnes and S. aureus, while the inhibitory
effects against P. acnes were stronger than those against
S. aureus [16]. .In this experiment antimicrobial action
of TTO was tested on other bacteria and moreover, this
experiment also determined how TTO compared to other
essential oils and conventional antibiotics thought to control
bacteria.
3.3. Tea Tree Oil vs. Conventional Antibiotic
Aggarwal, (2006) in his research stated that the use of
herbal medicine is becoming popular due to toxicity and side
effects of antibiotics. This has led to sudden increase in the
number of herbal drug manufacturing [15]. Another
important section of this study was to establish comparative
analysis of antimicrobial efficacy between TTO and
conventional antibiotics against selected pathogenic
organism. The purpose of this section was to find out a
replacement of antibiotics which has nearly similar ability
to control bacterial growth as antibiotics has several side
effects and it was done by agar diffusion assay. Nine
antibiotics and TTO involved in this segment. TTO
appeared 30.6 mm ZOI against A. hydrophila which is
greater than eight antibiotics out of tested nine antibiotics.
Undoubtedly TTO proved itself as a great substitution of
conventional antibiotics as TTO showed remarkable activity
against all the pathogenic bacteria selected for this study.
On the other hand, all the bacteria used in this study were
resistant to only two antibiotics (Cefepime and Cefuroxime
Sodium) out of nine antibiotics. The claimed statement
presented by constructed table of average zone of
inhibition in response to TTO and conventional antibiotic
discs against selected bacteria for this study (Table 2).
Figure 3. Antibacterial effect of Essential oils Against K. pneumonia
Figure 4. ZOI of Tea Tree Oils and Antibiotics against A. hydrophila
Table 1. Antimicrobial Activity for Essential Oils
Bacteria
Mean ZOI*
for Cef* (mm)
Mean ZOI for
TTO (mm)
AI* for TTO
(mm)
Mean ZOI for
ECO (mm)
AI for ECO
(mm)
Mean ZOI for
LGO (mm)
AI for LGO
(mm)
S. aureus
27.3
16.7
0.6
12.3
0.4
11.7
0.4
S. pyogenes
34.3
20.7
0.6
13.3
0.3
15.0
0.4
P. aeruginosa
18.7
22.7
1.2
11.7
0.6
15.3
0.8
P. vulgaris
32.0
20.0
0.6
12.0
0.3
22.7
0.7
A. hydrophila
33.0
36.3
1.1
22.3
0.6
17.7
0.5
E. coli
32.3
24.3
0.7
0.0
0.0
0.0
0.0
S. pneumoniae
34.7
19.7
0.5
13.7
0.3
14.7
0.4
B. subtilis
21.0
22.7
1.0
18.3
0.8
24.3
1.1
K. pneumoniae
25.0
19.3
0.7
9.3
0.3
0.0
0.0
S. agalactiae
10.3
17.3
1.6
14.0
1.3
16.3
1.5
*ZOI = Zone Of Inhibition, *Cef = Cefepime, *AI = Activity Index
American Journal of Microbiological Research 77
Table 2. Average zone of inhibition in response to TTO and conventional antibiotic
Bacteria
Name of antibiotics
Zone of Inhibition (mm)
Rifampicin
Cefepime
Cephalexin
Erythromycine
Amoxycillin
Sulphamethoazole
Doxycycline
Cefuroxime Sodium
Clindamycin
Tea Tree Oil
S. aureus 32.6 25.3 33.3 28.6 35.3 24.3 30.6 34.3 29.3 18.6
S. pyogenes 0 34.2 0 0 0 0 0 14.6 0 17.3
P. aeruginosa 0 31.6 0 0 0 0 0 16.3 0 18.6
P. vulgaris 17.6 35.3 19.3 14.3 0 21.6 27.6 28.3 0 21.3
A. hydrophila 8.6 31.3 19.6 0 0 24.3 21.3 23.6 0 30.6
E. Coli 37.6 30.3 37.6 0 38.6 22.3 34.3 35.3 24.6 15.6
S. pneumoniae 25.6 17.6 28.6 26.3 40.3 27.3 10.3 35.3 23.3 17.6
B. Subtilis 0 24.3 11.3 0 0 9.6 11.6 14.6 0 14.3
K. pneumoniae 26.6 10.3 35.6 30.3 36.6 38.3 35.3 16.6 28.3 16.6
S. agalactiae
20.3 18.3 24.3 24.3 32.3 29.6 16.6 31.3 24.3 16.3
3.4. Limitations
Wilkinson and Cavanagh (2005), and Carson et al.,
(2006) showed that TTO presented better antibacterial
activity toward anaerobic bacteria than aerobic bacteria.
They used mass spectrophotometry to separate two major
components, terpinen-4-ol and 1,8-cineole, were used to
evaluate skin toxicity by a single topical application
[13,18]. On the other hand, in our study, the components
were not separated through mass spectrophotometry as
they did in their research and also they had applied TTO
directly on erythema and edema instead of pathogenic
bacteria which was not done in this study. Hammer et al.,
(2006) concluded that it may be used externally in its diluted
form by the majority of individuals without adverse effect
(provided oxidation is avoided) [19]. Topical application of
high concentrated TTO can cause adverse reactions like
skin irritation, allergic contact dermatitis, systemic contact
dermatitis, erythema multiform like reactions, and systemic
hypersensitivity reactions [19, 20]. We didn’t provide any
constructed information about appropriate dose which will
treat infection without any kind of adverse effect.
Therefore, this study could have been better if we could
separate components through spectrophotometry and applied
essential oils directly with ascertained dose on bacterial
infection.
3.5. Further Scope
As the antimicrobial efficacy of the tested essential oil
have been established, further research is required keeping
the limitations in mind. In this study, we have used
commercial essential oils since preparing fresh oil wasn’t
feasible in our country. This results could be compared with
freshly prepared essential oils. Analysis by spectrophotometry
could help isolate and identify the major components
contributing to the antimicrobial properties of the essential
oils. In our investigation we tested essential oils on
selected pathogenic bacteria those are responsible for
bacterial infection. These essential oils would be more
acceptable for treatment if the tested oils were applied
directly on infection area with safe doses instead of
individual bacteria.
4. Conclusion
Slowly, science is catching up in explaining why tea
tree oil is such an effective antimicrobial agent. In this
study, it’s been proven that Tea Tree oil has noticeable
antimicrobial activity against bacteria which are responsible
for bacterial infection in compared to Eucalyptus oil,
Lemongrass oil and conventional antibiotics. In the final
analysis, the potential of Tea Tree Oil to be used as natural
antimicrobial agent is recommendable as antimicrobial
activity against Staphylococcus aureus, Streptococcus
pyogenes, Pseudomonas aeruginosa, Proteus vulgaris,
Aeromonas hydrophila, Escherichia coli, Streptococcus
pneumoniae, Bacillus subtilis, Klebsiella pneumonia and
Streptococcus agalactiae were demonstrated. The use
antibiotics would never be in the first place. Tea tree oil
itself as fully effective against all the bacteria used in this
study. The development of Tea Tree oil would be a great
alternative to conventional antibiotics against bacterial
infections.
References
[1] Schneider, E.K., Ortega, F.R., Velkov, T., Li, J. Antibiotic–non-
antibiotic combinations for combating extremely drug- resistant
Gram-negative ‘superbugs’. Essays In Biochemistry, 2017; 61 (1):
115.
[2] Moskvitch, K. (2015, January 28). How to Solve the Problem of
Antibiotic Resistance. Retrieved November 14, 2017, from
https://www.scientificamerican.com/article/how-to-solve-the-
problem-of-antibiotic-resistance/ .
[3] Essential oils used in aromatherapy: A systemic review. (2015,
July 10). Retrieved November 14, 2017, from
http://www.sciencedirect.com/science/article/pii/S2221169115001
033?via%3Dihub.
[4] Milhau G, Valentin A, Benoit F, Mallie M, Bastide J, Pelissier Y,
Bessiere J. In vitro antimicrobial activity of eight essential oils. J
Essent Oil Res. 1997; 9: 329-333.
78 American Journal of Microbiological Research
[5] Darokar MP, Mathur A, Dwivedi S, Bhalla R, Khanuja SPS,
Kumar S. Detection of antibacterial activity in the floral petals of
some higher plants. Curr Sci. 1998; 75: 187.
[6] May, J. (2000). Time-kill studies of tea tree oils on clinical
isolates. Journal of Antimicrobial Chemotherapy, 45(5),
pp.639-643.
[7] 13 Uses For Tea Tree Oil That Will Change Your Life. (2017,
April 05). Retrieved November 14, 2017, from
http://www.naturallivingideas.com/tea-tree-oil-uses/.
[8] Benefits Of Eucalyptus Oil. (n.d.). Retrieved November 14, 2017,
from
http://www.lisasfragrancensuch.com/apps/blog/show/43944925-
benefits-of-eucalyptus-oil.
[9] Herbal Oil: Eucalyptus Oil Benefits and Uses. (n.d.). Retrieved
November 14, 2017, from
https://articles.mercola.com/herbal-oils/eucalyptus-oil.aspx.
[10] Boldt, E. (2017, August 25). What is Lemongrass Essential Oil
Good For? Retrieved November 14, 2017, from
https://draxe.com/lemongrass-essential-oil/.
[11] Ali B., Al-Wabel N. A., Shams S., Ahamad A., Khan S. A.,
Anwar F. Essential oils used in aromatherapy: a systemic review.
Asian Pacific Journal of Tropical Biomedicine. 2015; 5(8):
601-611.
[12] Fitzpatrick, M. (February 17, 2010). Antimicrobial action of tea
tree oil (Melaleuca alternifolia) on five common bacteria.
[13] Carson, C.F., Hammer, A., Riley, T.V. (2006). Melaleuca
alternifolia (Tea Tree) Oil: a Review of Antimicrobial and Other
Medicinal Properties. Clinical Microbiology Reviews, Jan. 2006,
p. 50-62.
[14] Carson, C.F. and Riley, T.V. (1995). Antimicrobial activity of the
major components of the essential oil of Melaleuca alternifolia.
Journal of applied bacteriology. 78 (3), 264-269.
[15] B.B. Aggarwal, H. Ichikawa, P. Garodia, et al. From traditional
Ayurvedic medicine to modern medicine: identification of
therapeutic targets for suppression of inflammation and cancer.
Expert Opin Ther Targets, 10 (2006), pp. 87-118.
[16] Lee, C., Chen, L., Chen, L., Chang, T., Huang, C., Huang, M. and
Wang, C. (2013). Correlations of the components of tea tree oil
with its antibacterial effects and skin irritation. Journal of Food
and Drug Analysis, 21(2), pp.169-176.
[17] Cox, S., Mann, C., Markham, J., Bell, H., Gustafson, J.,
Warmington, J. and Wyllie, S. (2001). The mode of antimicrobial
action of the essential oil of Melaleuca alternifolia (tea tree oil).
Journal of Applied Microbiology, 88(1), pp.170-175.
[18] Wilkinson, JM, Cavanagh HM. Antibacterial activity of essential
oils from Australian native plants. Phytother Res 2005; 19: 643-6.
[19] Hammer, KA; Carson, CF; Riley, TV; Nielsen, JB (May 2006).
"A review of the toxicity of Melaleuca alternifolia (tea tree) oil."
Food and Chemical Toxicology. 44 (5): 616-25.
[20] Pazyar, N; Yaghoobi, R; Bagherani, N; Kazerouni, A (July 2013).
"A review of applications of tea tree oil in dermatology".
International Journal of Dermatology. 52 (7): 784-90.
