Journal of Environmental Treatment Techniques  
2019, Volume 7, Issue 3, Pages: 501-505  
J. Environ. Treat. Tech.  
ISSN: 2309-1185  
Journal web link: http://www.jett.dormaj.com  
Antimicrobial Activity of three Essential Oils  
against Several Human Pathogens  
1
1
2
3
Anesa Dzaferovic , Teofil Gavric , Simin Hagh Nazari , Monika Stojanova , Muamer  
1
4*  
1
Bezdrob , Blazo Lalevic , Saud Hamidovic  
1
The University of Sarajevo, Faculty of agricultural and food sciences, Sarajevo, Bosnia, and Herzegovina  
2
The University of Zanjan, Faculty of agriculture, Zanjan, Iran  
3
University Ss. Cyril and Methodius, Faculty of agricultural sciences and food, Skopje, North Macedonia  
4
The University of Belgrade, Faculty of agriculture, Belgrade - Zemun, Serbia  
Received: 01/04/2019  
Accepted: 16/08/2019  
Published: 29/08/2019  
Abstract  
Essential oils are products derived from various parts of plants. These products have therapeutic, pharmacological and  
antimicrobial properties against human pathogens. In this paper, the impact of clove, spruce and pine essential oils against  
Escherichia coli, Salmonella spp. and Staphylococcus aureus was determined using disc diffusion method. Liquid bacterial  
inoculum was streaked onto Muller-Hinton agar using the sterile swab. Antimicrobial activity was estimated by measurement of  
inhibition zone around the discs previously impregnated by essential oil and placed onto agar. Results showed that the antimicrobial  
activity of essential oils depended on the type of oil and bacterial species. The significantly highest diameter of inhibition zone  
against tested bacteria was recorded using clove essential oil, whilst the lowest diameter was noticed using pine essential oil. Clove  
oil was most effective against Staphylococcus aureus, whilst spruce and pine oil against Salmonella spp. compared to other bacteria.  
Strong positive correlation between the effect of the spruce oil against Escherichia coli and Staphylococcus aureus, spruce and  
with clove oil against Staphylococcus aureus was recorded. The results of this research indicate the possible application of essential  
oil of clove against human pathogens.  
Keywords: antimicrobial activity, essential oil, human pathogens  
growth of human pathogenic bacteria [8]. One of most  
1
Introduction  
efficient methods is the use of plant extracts and essential  
oils containing novel antibacterial properties with potential  
use as food preservatives and alternatives to treat infections  
Essential (volatile) oils are natural extracts obtained  
th  
from plants and have been commonly used since the 16  
century [1]. These plants have medicinal interests and have  
received great attention due to low toxicity and  
pharmacological properties [2]. Besides, ancient reports  
showed that essential oils may be used in control of  
microorganisms, including Gram-positive and Gram-  
negative bacteria [3], as well as for treatment of several  
diseases, such as diabetes, cancer, cardiovascular diseases,  
etc. [4].  
The antimicrobial activity of essential oils has been  
well documented [5]. Unfortunately, some synthetic drugs  
may not be used in control of human pathogens [6].  
Furthermore, multidrug resistance of main human  
pathogens, such as Pseudomonas aeruginosa, Salmonella  
spp., Staphylococcus aureus, Enterococcus sp. and  
Escherichia coli is well described [7].  
[9, 10].  
Several types of research have addressed the  
antimicrobial activity of essential oils produced by conifers,  
such as Picea [11, 12] and Pinus [13]. Odugbemi [14] found  
similar properties using clove (Syzygium aromaticum)  
essential oil.  
Although the impact of essential oil on human  
pathogens have been studied worldwide [15], in Bosnia and  
Herzegovina only a few reports have addressed the influence  
of essential oils on pathogenic bacteria. This paper aimed to  
determinate the antimicrobial activity of spruce (Picea sp.),  
pine (Pinus sp.) and clove (Syzygium aromaticum) essential  
oils.  
In few last decades, the resistance of antimicrobial  
drugs led to the development of techniques for the  
production of novel compounds, which may inhibit the  
2 Material and methods  
Flower buds of clove were obtained from local  
distributer and used for extraction. The essential oil of clove  
Corresponding author: Blazo Lalevic, University of Belgrade, Faculty of agriculture, Department of environmental microbiology,  
Nemanjina 6, 11080 Belgrade-Zemun, Serbia, Email: blazol@agrif.bg.ac.rs.  
501  
Journal of Environmental Treatment Techniques  
2019, Volume 7, Issue 3, Pages: 501-505  
was produced using distillation technique by Clevenger  
apparatus. Picea sp. and Pinus sp. essential oils were  
obtained from a local distributor. In this research, two  
bacteria from family Enterobacteriaceae (Escherichia coli,  
and Salmonella spp.) and one member of family  
Staphylococcaceae (Staphylococcus aureus) were used.  
Pure cultures of bacteria originated from the collection of  
Department of microbiology at Faculty of agriculture and  
food sciences (Sarajevo, Bosnia, and Herzegovina).  
Antimicrobial activity of essential oils was determined  
using paper-disc diffusion method [16]. Pure bacterial  
cultures were grown on Muller-Hinton agar plates at 37°C  
for 24h. Colonies of bacteria were transferred into sterile  
normal saline. Bacterial inoculum was streaked onto Muller-  
Hinton agar using the sterile swab. Six sterile filter discs  
previously, the activity of clove oil against S. aureus can be  
characterized as good, which is significantly different  
(p<0.05) from spruce and pine oil, whilst both of them have  
average antibacterial activity.  
Table 2: The diameter of inhibition zone (mm) of clove,  
spruce and pine essential oils against Staphylococcus  
aureus  
Clove  
Spruce  
Pine  
Essential oil  
Diameter of inhibition zone (mm)  
Disc 1  
Disc 2  
Disk 3  
Disk 4  
Disk 5  
Disk 6  
30  
28  
26  
27  
31  
26  
20  
14  
15  
18  
17  
15  
15  
16  
15  
14  
20  
16  
(
diameter 6mm, Whatman paper No. 3) were previously  
±
SD  
± SD  
± SD  
16.0 ± 2.10b  
Average  
impregnated by essential oil (10 μl/disc) and placed onto  
Muller-Hinton agar. Incubation was performed in an  
incubator at 37°C for 16-24h. Antimicrobial activity was  
estimated by measurement of the inhibition zone around the  
discs. All experiments were carried out in triplicate. The  
occurrence of an inhibition zone > 18 mm was characterized  
as good antimicrobial activity, 16-18 mm as an average  
activity, 14-16 as moderate activity, and 10-14 mm as low  
activity. Presented data are statistically processed with  
ANOVA test using SPSS 20 package program.  
2
8.0 ± 2.10a 16.5 ± 2.26b  
Values marked with different letters have a statistically  
significant difference between the examined essential oils (p<0.05)  
a,  
b
–
As can be seen from Table 3, highest antimicrobial  
activity against Salmonella spp. was obtained using clove oil  
(diameter of inhibition zone was 27.7 mm), whilst another  
essential oil has a lower diameter of inhibition zone.  
However, the antibacterial activity of all tested oils can be  
classified as good. According to that, a statistically  
significant difference (p<0.05) between the effect of all the  
examined essential oils against Salmonella spp. was  
determined.  
3
Results and discussion  
Our results showed that antimicrobial activity of  
essential oils depended on the type of oil and bacterial  
species (Table 1, 2 and 3). From Table 1 it is obvious that  
clove essential oil has the highest zone of inhibition against  
Escherichia coli compared to other essential oils (average  
Table 3: The diameter of inhibition zone (mm) of clove,  
spruce and pine essential oils against Salmonella spp.  
Clove  
Spruce  
Pine  
Essential oil  
Diameter of inhibition zone (mm)  
26.7 mm). Antibacterial activity of spruce essential oils can  
Disc 1  
Disc 2  
Disk 3  
Disk 4  
Disk 5  
Disk 6  
30  
27  
25  
29  
28  
27  
20  
28  
26  
20  
19  
20  
18  
19  
19  
19  
19  
18  
be classified as average, whilst pine oil has moderate  
antibacterial activity. Based on the statistical analysis, can  
be seen that the effect of clove oil against Escherichia coli  
has a statistically significant difference (p<0.05) compared  
to the determined effects of spruce and pine oil. There is no  
statistically significant difference between the effect of  
spruce and pine oil.  
±
SD  
± SD  
22.2 ± 3.82b  
± SD  
18.7 ± 0.52c  
Average  
7.7 ± 1.75a  
2
a, b,  
c
–
Values marked with different letters have a statistically  
Table 1: The diameter of inhibition zone (mm) of clove,  
significant difference between the examined essential oils (p<0.05)  
spruce and pine essential oils against Escherichia coli  
Clove  
Spruce  
Pine  
Obviously, there is no statistically significant  
difference in the effect of the clove oil against three  
determined pathogenic bacteria (Table 4). Nevertheless,  
there is a statistically significant difference (p<0.05) in the  
effect of spruce and pine oil against Salmonella spp.,  
compared to that against Escherichia coli and  
Staphylococcus aureus. Results presented in Table 5 showed  
that there is a strong positive correlation (r= +.896*)  
between the effect of the spruce oil against Escherichia coli  
and Staphylococcus aureus. This oil has a strong positive  
correlation (r= +.847*) with clove oil against  
Staphylococcus aureus, too. On the other hand, the effect of  
spruce oil against Staphylococcus aureus has a strong  
positive correlation (r= +.860*) with the effect of clove oil  
against Salmonella spp.  
Essential oil  
Diameter of inhibition zone (mm)  
Disc 1  
26  
25  
29  
25  
27  
28  
21  
15  
14  
18  
15  
13  
17  
12  
14  
13  
15  
14  
Disc 2  
Disk 3  
Disk 4  
Disk 5  
Disk 6  
±
SD  
± SD  
± SD  
Average  
6.7 ± 1.63a  
16.0 ± 2.96b  
14.2 ± 1.72b  
2
a,  
b
–
Values marked with different letters have a statistically  
significant difference between the examined essential oils (p<0.05)  
Similar results were obtained for selected oils and  
antimicrobial activity against Staphylococcus aureus (Table  
). The more pronounced activity of all tested essential oils  
against S. aureus compared to E. coli was noticed. As  
Several previous researchers suggest that eugenol is a  
major component of clove essential oil [17]. Eugenol  
belongs to the phenolic compounds. These compounds have  
2
502  
Journal of Environmental Treatment Techniques  
2019, Volume 7, Issue 3, Pages: 501-505  
antiseptic, antimicrobial and antioxidant properties [18].  
Hamad et al. [19] detected β-caryophyllene as a constituent  
of clove essential oil, which also posses antibacterial  
influence [20]. According to Hamad et al. [19], clove  
essential oil showed strong antimicrobial activity against S.  
aureus, which is confirmed in this research. On the other  
hand, inhibition of E. coli growth was not detected in the  
same research, which is in contrast with our results. Ayoola  
et al. [18] found that the antimicrobial activity of clove oil  
was most expressed against S. aureus.  
Radulescu et al. [21]. Several studies have addressed the  
presence of antibacterial compounds in spruce essential oil,  
such as p-cymene [22], limonene [23], camphene [24], etc.  
Nevertheless, the efficiency of spruce essential oils against  
human pathogens was not reported in several kinds of  
research. Canilac and Mourey [25] found that coliform  
bacteria were more resistant in the presence of essential oil  
of Picea excelsa. On the other hand, Tanase et al. [26]  
reported that spruce extract may be effective against E. coli.  
Monoterpenes compounds in Pinus sp. essential oil are  
known to have antimicrobial properties [27]. Antimicrobial  
activity of various pine essential oils is well described [13].  
Our study showed that the highest antimicrobial  
activity of spruce oil was detected against Salmonella spp.  
These results are in accordance with observations of  
Table 4: Statistical analysis of average values from the effects of the examined essential oils against three different pathogens  
Clove  
Spruce  
Pine  
Essential oil  
nn  
±
SD  
± SD  
16.0 ± 2.96  
16.5 ± 2.26  
± SD  
14.2 ± 1.72  
16.0 ± 2.10  
a
a
a
a
a
a
a
Escherichia coli  
Staphylococcus aureus  
66  
66  
66  
26.7 ± 1.63  
28.0 ± 2.10  
27.7 ± 1.75  
b
b
Salmonella spp.  
22.2 ± 3.82  
18.7 ± 0.52  
a, b  
–
Values for the essential oils marked with different letters have a statistically significant difference between the examined pathogens (p<0.05)  
Table 5: Correlation between the effects of examined essential oils against three different pathogens  
Clove  
Salmonella  
spp.  
Spruce  
Salmonella  
spp.  
Prine  
Salmonella  
spp.  
Clove Spruce  
E. coli E. coli  
Pine  
Clove  
Spruce  
Pine  
E. coli S. aureus S. aureus S. aureus  
Pearson Correlation  
Sig. (2-tailed)  
N
1
(.578)  
.230  
.237  
.651  
(.350)  
.496  
(.325)  
.529  
.175  
.740  
(.676)  
.140  
6
.011  
.984  
6
(.158)  
.765  
6
Clove  
E.coli  
6
6
6
6
6
Pearson Correlation  
Sig. (2-tailed)  
N
1
.548  
.260  
.482  
.333  
.896*  
.016  
(.354)  
.492  
.847*  
.033  
6
(.336)  
.515  
6
(.261)  
.617  
6
Spruce  
E.coli  
6
6
6
6
Pearson Correlation  
Sig. (2-tailed)  
N
1
.554  
.254  
.746  
.089  
.166  
.753  
.486  
.328  
6
(.583)  
.224  
6
(.600)  
.208  
6
Pine  
E. coli  
6
6
6
Pearson Correlation  
Sig. (2-tailed)  
N
1
.549  
.259  
.636  
.174  
.599  
.209  
6
(.375)  
.464  
6
.000  
1.000  
6
Clove  
S. aureus  
6
6
Pearson Correlation  
Sig. (2-tailed)  
N
1
(.127)  
811  
.860*  
.028  
6
(.685)  
.134  
6
(.343)  
.506  
6
Spruce  
S. aureus  
6
Pearson Correlation  
Sig. (2-tailed)  
N
1
(.054)  
.918  
6
(.250)  
.633  
6
.185  
.726  
6
Pine  
S. aureus  
Pearson Correlation  
1
(.648)  
.164  
6
(.369)  
.472  
6
Clove  
Salmonel Sig. (2-tailed)  
la spp.  
N
Pearson Correlation  
1
.440  
.383  
6
Spruce  
Salmonel Sig. (2-tailed)  
la spp.  
N
Pearson Correlation  
1
Prine  
Salmonel Sig. (2-tailed)  
la spp.  
N
*
Correlation is significant at the 0.05 level (2-tailed).  
503  
Journal of Environmental Treatment Techniques  
2019, Volume 7, Issue 3, Pages: 501-505  
In our research, the various antibacterial activity of pine  
oil was noticed, which corroborate with previous findings  
[9] Astani, A., et al., Comparative study on the antiviral activity of  
selected monoterpenes derived from essential oils.  
Phytotherapy Research, 2010. 24: p. 673-679.  
[28,29]. Ait Mimoune et al. [30] reported that S. aureus was  
[
10] Safaei-Ghomi, J., et al., Antimicrobial and antifungal properties  
of the essential oil and methanol extracts of Eucalyptus  
largiflorens and Eucalyptus intertexta. Pharmacognosy  
Magazine, 2010. 6: p. 172-175.  
one of the most sensitive bacteria against pine essential oil,  
which is confirmed in this study.  
4
Conclusion  
[11] Krauze-Baranowska, M., et al., Antifungal activity of the  
essential oils from some species of the genus Pinus. Zeitschrift  
fur Naturforschung C, 2002. 57(5-6): p. 478-482.  
The results of the present research have shown that  
essential oils of clove, spruce and pine inhibit the growth of  
Escherichia coli, Staphylococcus aureus, and Salmonella  
spp. Clove oil was most effective against Staphylococcus  
aureus, whilst spruce and pine oil against Salmonella spp.  
compared to other bacteria. In all treatments, clove oil  
showed the highest inhibition of bacterial growth compared  
to other oils. Thus, the results of current research indicate the  
possible application of essential oil of clove in medicinal  
protocol against human pathogens.  
[
12] Yang, S.A., et al., Radical scavenging activity of the essential  
oil of Silver Fir (Abies alba). Journal of Clinical Biochemistry  
and Nutrition, 2009. 44: p. 253-259.  
[
13] Apetrei, C.L., et al., Composition, and antioxidant and  
antimicrobial activities of the essential oils of a full-grown  
Pinus cembra L. tree from the Calimani Mountains (Romania).  
Journal of Serbian Chemical Society, 2013. 78(1): p. 27-37.  
14] Odugbemi, T.O., Outlines and pictures of medicinal plants  
from Nigeria, University of Lagos Press, Lagos, Nigeria. 2006.  
p. 91.  
[
[
15] Sartoratto, A., et al., Composition and antimicrobial activity of  
essential oils from aromatic plants used in Brazil. Brazilian  
Journal of Microbiology, 2004. 35: p. 275-280.  
Ethical issue  
Authors are aware of, and comply with, best practice in  
publication ethics specifically with regard to authorship  
[16] Mighri, H., et al., Antimicrobial and antioxidant activities of  
Artemisia herba-alba essential oil cultivated in Tunisian arid  
zone. Comptes Rendus Chimie, 2010. 13: p. 380-386.  
(
avoidance of guest authorship), dual submission,  
manipulation of figures, competing interests and compliance  
with policies on research ethics. Authors adhere to  
publication requirements that submitted work is original and  
has not been published elsewhere in any language.  
[
17] Ayoola G. A., et al., Chemical analysis and antimicrobial  
activity of the essential oil of Syzigium aromaticum (clove).  
African Journal of Microbiology Research, 2008. 2(13): p. 162-  
1
66.  
[
18] Pelczar, M.J., et al., Control of microorganisms, the control of  
microorganisms by physical agents. In: Microbiology. New  
York: McGraw-Hill International, 1998. p. 469-509.  
19] Hamad, A., et al., Chemical constituents and antimicrobial  
activities of essential oils of Syzigium polyanthum and Syzigium  
aromaticum. Rasayan Journal of Chemistry, 2017. 10(2): p.  
564-569.  
Competing interests  
The authors declare that there is no conflict of interest  
that would prejudice the impartiality of this scientific work.  
[
Authors’ contribution  
All authors of this study have a complete contribution  
for data collection, data analyses and manuscript writing.  
[20] Dorman, H.J.D., et al., Antimicrobial agents from plants:  
antibacterial activity of plant volatile oils. Journal of Applied  
Microbiology, 2000. 88: p. 308-316.  
[
[
[
21] Radulescu, V., et al., Chemical composition and antimicrobial  
activity of essential oil from shoots spruce (Picea abies L).  
Revista de Chimie Bucharest, 2011. 62(1): p. 69-74.  
22] Burt, S., Essential oils: Their antibacterial properties and  
potential applications in foods - a review. International Journal  
of Food Microbiology, 2004. 94(3): p. 223-253.  
23] Aggarwal K.K., et al., Antimicrobial activity profiles of the two  
enantiomers of limonene and carvone isolated from the oils of  
Mentha spicata and Anethum sowa. Flavour and Fragrance  
Journal, 2002. 17: 59-63.  
References  
[
1] Man, A., et al., Antimicrobial activity of six essential oils against  
a group of human pathogens: a comparative study. Pathogens,  
2
019. 8(1): p. 15.  
[
2] Auddy, B., et al., Screening of antioxidant activity of three  
Indian medicinal plants, traditionally used for the management  
of neurodegenerative diseases. Journal of Ethnopharmacology,  
2
003. 84: p. 131-138.  
[
[
[
3] Galli, A., et al., Attività antimicrobica in vitro di oli essenziali  
ed estratti di spezie di uso alimentare. Industrie Alimentari,  
[
[
[
[
24] Husnu, K.C.B., et al., Handbook of essential oils. Science,  
technology and applications, CRC Press Taylor and Francis  
Group, 2010. p. 133.  
25] Canillac N., et al., Antibacterial activity of the essential oil of  
Picea excelsa on Listeria, Staphylococcus aureus and coliform  
bacteria. Food Microbiology, 2001. 18: p. 261-268.  
26] Tanase, C., et al., Antibacterial activity of spruce barke (Picea  
abies L.) extract against Eschericia coli. Acta Biologica  
Marisiensis, 2018. 1(1): p. 5-9.  
27] Fekih, N., et al., Chemical composition and antibacterial  
activity of Pinus halepensis Miller growing in West Northern  
of Algeria. Asian Pacific Journal of Tropical Disease, 2014.  
1
985. 24: p. 463-466.  
4] Ali, B., et al., Essential oils used in aromatherapy: a systemic  
review. Asian Pacific Journal of Tropical Biomedicine, 2015.  
5
(8): p. 601-611.  
5] Politeo, O., et al., Chemical composition and antimicrobial  
activity of the essential oil of endemic Dalmatian black pine  
(
8
Pinus nigra ssp. dalmatica). Chemistry and Biodiversity, 2011.  
(3): p. 540-547.  
[
6] Swamy, M.K., et al., Antimicrobial properties of plant essential  
oils against human pathogens and their mode of action: an  
updated review. Evidence-Based Complementary and  
Alternative Medicine, 2016. Article ID 3012462.  
4
(2): p. 97-103.  
[7] Chouhan, S., et al., Antimicrobial activity of some essential oils-  
present status and future perspectives. Medicines, 2017. 4: p. 1-  
[
28] Hmamouchi, M., et al., Chemical composition properties of  
essential oils of five Moroccan Pinaceae. Journal of Essential  
Oil Research, 2001. 13(4): p. 298-302.  
2
3.  
[
8] Rudramurthy, G.R., et al., Nanoparticles: alternatives against  
drug-resistant pathogenic microbes. Molecules, 2016. 21(7): p.  
E836.  
[
29] Lee, J.H., et al., Comparison of chemical compositions and  
antimicrobial activities of essential oils from three conifer trees;  
Pinus densiflora, Cryptomeria japonica, and Chamaecyparis  
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Journal of Environmental Treatment Techniques  
2019, Volume 7, Issue 3, Pages: 501-505  
obtuse. Journal of Microbiology and Biotechnology, 2009.  
1
9(4): p. 391-396.  
[
30] Ait Mimoune, N., et al., Chemical composition and  
antimicrobial activity of the essential oils of Pinus pinaster.  
Journal of Coastal Life Medicine, 2013. 1(1): p. 55-59.  
505