J.Sc. Tech Vol.

10(1) 2009
IN VITRO CLOVE OIL ACTIVITY AGAINST PERIODONTOPATHIC BACTERIA
By

Ali, H.S. 1 , Kamal, M. 1 and Mohamed, S.B. 2

1- Dubai Pharmacy College, 2-University of Khartoum

ABSTRACT
Clove oil antimicrobial activity against periodontopathic (ATCC)
bacteria was investigated "in vitro". Bacterial strains tested were: Prevotella
intermedia,
Prevotella
melaninogenica,
Pophyromonas
gingivalis,
Actinobacillus actinomycetemcomitans, Capnocytophaga gingivalis, and
Fusobacterium nucileatum. The minimal inhibitory concentration (MIC) for the
strains tested was determined using the method of broth dilution with the clove
oil in serial concentrations. Results showed MIC of (4 g/ml) for Actinobacillus
actinomycetemcomitans, Capnocytophaga gingivalis; and Fusobacterium
nucleatum were (1 g/ml). Prevotella intermedia and Porphyromonas
gingivalis showed (2 g/ml). But (MIC) for Prevotella melaninogenica was 18
g/ml. Some superinfectant organisms were also tested: Cadida albicans
susceptibility to clove oil was demonstrated at a concentration of (24 g/ml).
The MIC for Pseudomonas aeruginosa, and Staphylococcus aureus was (24
g/ml) but for Escherichia coli was (18 g/ml). All periodontal pathogens and
superinfectants tested were susceptible to the clove oil. The positive results
suggest that the clove oil should be further tested as an adjuvant to periodontal
therapy.
INTRODUCTION
Human periodontal disease has been associated with a complex microbiota. The development of destructive periodontitis seems to be the result of
specific infection [1] . Gram positive coccoid bacteria have been related to
periodontal health, while periodontal disease was associated with Gram
negative rods and spirochetes [ 2 ] . Many authors [ 3] suggest that the presence of
Actinobacillus actinomycetecemilans, Bacteroides gingivlis (Porphyromonas
gingivlis) and Bacteroides intermedius (Prevotella intermedia) is related to
active periodontal disease. Other species like Fusobacterium nucleatum and
Capnocytophaga sp. [ 4 ] were also associated with the periodontics. Carrasco [ 5 ] ,
1

J.Sc. Tech Vol. 10(1) 2009

reported that human periodontitis is initiated and perpetuated by a small group

of bacteria that colonize the subgingival region, mainly Gram-negative,
anaerobic or microaerophilic bacteria. Furthermore, most cases of human
periodontitis are caused by Porphyromonas gingivialis, Bacteroides forsythus
and Actinobacillus actinomycetemcomitans [ 6 ] .
Because periodntitis is an infectious disease, and taking into consideration that some patients do not respond to conventional mechanical therapy,
sometimes antimicrobial agents have been prescribed as adjutants to periodo-ntal
treatment [ 7 ] . However, the emergence of pathogenic bacteria those are resistant
to antibiotics, due to inappropriate systemic usage, has become a serious
clinical problem and has necessitated the search for alternatives.
Clove oil is known for its antibacterial activity, which is due to several
constituents, and will be tested as an alternative to conventional antibiotic
therapy. Slots [ 8 ] pointed out that in order not to contribute to a "coming plague",
dentists should add the knowledge of an infectious disease specialist to their
surgical skills. T.E Rams [ 9 ] suggested that antibiotics should be used only.
MATERIALS AND METHODS
Minimal inhibitory concentrations (MIC) for clove oil [manufactured
locally in United Arab Emirates (UAE)]against the tested strains were determined
using the clove oil in serial concentrations: 0 (negative control), 0.0625, 0.125, 0.5,
1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 g/ml.
Control plates with serial concentrations of ethanolic solution were also
tested. The strains were inoculated by steer apparatus. All tests were performed
in quadruplicate.
All strains were grown in Brain heart Infusion Agar (BHI-Difco) except
for Candida albicans, which was grown in Saboraud agar (Difco), and
incubated at room temperature for 4days.
Pseudomonas aeruginosa, E. coli and S. aureus inoculated in Brain
Heart Infusion agar were incubated aerobically at 370C for 2days. Brain heart
enriched with hemin (1%-Sigma) and mendadione (0.01%-Sigma) was used to
grow strains of: P. intermedia, P. melaninogenica, P. gingivalis, A.
actinomycetemcomitans, C. gingivalis and F. nucleatum. The plates were
incubated anaerobically (Gas-Pak-BBL) at 37C for 7days.
2

J.Sc. Tech Vol. 10(1) 2009

RESULTS
The Minimum Inhibitory Concentration (MIC) was determined as the
lowest concentration of the clove oil, which inhibited the growth of the tested
microorganisms.
The clove oil showed antimicrobial activity against all tested strains.
(Table 1) presents the Minimal Inhibitory concentration obtained for each strain
tested. All control plates, including those with different clove oil concentrations
and the negative controls presented regular bacterial growth.
Clove Oil against Candida albicans:

Clove Oil against Staphylococcus aureus:

Table (1): Minimum Inhibitory Concentration (MIC) of clove oil obtained for each strain tested
Tests in quadruplicates.
Table (1): Minimum Inhibitory Concentration (MIC) of *clove oil obtained for each strain tested
Tests in quadruplicates
Microorganism

(MIC* -

Actinobacillus actinomycetemcomitans
Capnocytophaga gingivalis
Fusobacterium nucleatum
Porphyromonas gingivalis
Prevotella intermedia
Prevotella melaninogenica
Staphylococcus aureus
Pseudomonas aeruginosa
Candida albicans
Escherichia coli

4
1
1
2
2
18
24
24
24
18

g/ml)

J.Sc. Tech Vol. 10(1) 2009

DISCUSSION
The antimicrobial activity of clove oil has been studied by several
authors [10] , and have investigated its activity towards oral pathogens.
Besides showing antimicrobial activity against periodotopathic bacteria,
the clove oil did not demonstrate selection of superinfectant organisms.
The verification of the antimicrobial action of the clove oil is not
surprising. The primary function of clove oil in the clove paste in Aphthous
Ulcer treatment act as a biocide, being active against invasive bacteria, fungi
and even invading larvae [11] . The spectrum of activity is fairly broad, with action
against Gram positive and Gram negative rods and cocci, yeast and fungi [12] .
The present study has shown clove oil antimicrobial activity against the
following periodontal pathogens: A. actinomycetemcomitans, P. intermedia, P.
melaninogenica, P. gingivalis, C. gingivaiis and F. nucleatum. Antimicrobial
activity against Candida albicans, Pseudomonas aeruginosa, Escherichia coli
and Staphylococcus aureus was also demonastrated in this study, confirming
previous results [13] .
Different results were achieved by Nieva et al., [14] that reported
antimicrobial activity against Staphylococcus aureus, but no action against
Pseudomonas aesuginosa and Escherichia coli. A possible explanation for
these diverse results is the fact that clove oil composition is variable depending
on the region and season that it is collected [15] . Consequently, the active
compounds may not be present in sufficient quantities or quality.
One of the limitations to clove oil use is the variability in composition
and action as a consequence of variation in the flora of the region where it is
produced. However, according to Bankova et al. [16] , antimicrobial action is
expected to be always present because of its vital importance as an
antimicrobial agent in the bees wax paste, independently of the region where
the clove oil is produced. In a recent study, Sforcin et al. [17 ] did not find
a seasonal effect on the antimicrobial activity of the Brazilian clove oil in its
early collection period.
Clove oil mechanism of antimicrobial action, though not completely
understood, seems to be complex and may vary according to its composition.
4

J.Sc. Tech Vol. 10(1) 2009

The compounds known to have antimicrobial action are mainly the flavonoids
and cinamic acids [18] .
Regarding susceptibility of the tested microorganisms, it seems that
they were more susceptible to clove oil than to some antibiotics. Carrasco et al.
[5]
Showed the MIC for Doxycycline, Tetracycline, Metronidazole, Ofloxaxin,
and Amoxicillin were (8, 4, 8, 9, and 4g/ml respectively) against P. gingivalis,
P. intermedia and F. nucleatum in vitro. When their MIC results are compared
to ours, it is observed that frequently used antibiotics had greater MIC than
clove oil against P. gingivalis, P. inteamedia and F. nucleatum. These
periodontal pathogens may be more susceptible to the antibiotics shown above.
Previous study on the susceptibility of A. actinomycecomitans to
selected antimicrobial agents indicated that MIC 90g/ml to penicillin varied
from 1.0 to 6.25g/ml, to amoxicillin from 1.0 to 2.0g/ml, to tetracycline from
0.5 to 8.0g/ml, to doxycycline from 1.o to 3,1g/ml, and to metronidazole
from 12.5 to 32g/ml [19] . In the present study, the MIC of this pathogen to
Clove Oil was 2g/ml.
Susceptibility tests of P. gingivalis have shown that MIC90 to penicillin
varied from 0.016 to 0.29g/ml, amoxicillin from 0.023 to < 1.0g/ml,
metronidazole from 0.023 to 2.1g/ml [ 20 ] . Interestingly, in present study MIC to
clove oil was (0.5g/ml).
In 1990, Rams et al. [ 6 ] noted that some strains of S. aureus isolated
from the periodontal pocket were resistant to tetracycline, penicillin,
metronidazole and erythromycin. Additionally, when the antimicrobial activity
of 18antibiotics was tested against Enterobacteriaceae and pseudomonadaceae,
only ciprofloxacin was able to eliminate these microorganisms from the
periodontal pocket [ 21] .
Our results showed that clove oil presented "in vitro" antimicrobial
activity, not only against some periodontophatic bacteria (F. nucleatum, P.
gingivalis, P. intermedia, P. melaninogenica, A. actinomycetemcomitans S.
aureus, P. aeruginosa, E. coli and Candida albicans).
The antimicrobial action observed for the clove oil suggests its usage as
an adjuvant to periodontal therapy. A step further should be given to verify if
a dose sufficient to kill the target microorganisms can be reached within the
5

J.Sc. Tech Vol. 10(1) 2009

subgingival environment, without causing major local or systemic adverse

effects.
REFERENCES
1-

2-

3-

4-

5678910-

1112-

Ambrose, U.; Middleton, K.; Seal, D. (1991). In

vitro studies of water activity and

bacterial growth inhibition of sucrose-polyethylene glycol-400-hydrogen
peroxide and xylose-polyethylene glycol-400-hydrogen peroxide past used
to treat infected wounds. Antim. Agents and Chemoth., 35: [1799-1803].
Benjilali, B.; Tantaoui-Elaraki, A.; Ayadi, A.; Ihlal, M. (1984). Method to study
antimicrobial effects of essential oils: application to the antifungal activity
of six Moroccan essences. J. Food Protec., 47: [748-752].
Briozzo, J.; Nuez, L.; Chirife, J.; Herszage, L.; D'Aquino, M. (1988). Antimicrobial
activity of clove oil dispersed in a concentrated sugar solution. J. Appl.
Bacteriol., 66: [69-77].
Chirife, J.; Herszage, L.; Joseph, A.; Kohn, E.S. (1983). In vitro study of bacterial
growth inhibition in concentrated sugar solutions: microbiolog-ical basis for
the use of sugar in the treatment of infected wound. Antim. Agents
Chemoth., 23: [766-773].
Carrasco Rev. Arg. Microbiol., 24: [32-39], 1992.
Rams et al., (1987). Antibacterial properties of plant essential oils. Int. J.
Food Microbiol. 5: [165-180].
Mahoud, L.E. (1994). Antifungal action and antiaflatoxigenic properties of
some essential oil constituents. Lett. Appl. Microbiol., 19: [110-113].
Morris, J.A.; Khettry, A.; Seitz, E.W. (1979). Antimicrobial activity of aroma
chemicals and essential oils. J. Amer. Oil Chemists' Soc., 56: [595-603].
T. E Rams (1991). The Lancet, 338: [571-572].
Glenn N Wagner, Thomas D Singer, R (2003). Scott McKinley Aquaculture
Research Vol. 34(13): [1139-1146], Nov 2003.
Alison C Holloway, Joel L Keene, David G Noakes, Richard D Moccia (2004).
Aquaculture Research Vol. 35(11): [1025-1030], Sep 2004.
S.A. Burt, R.D. (2003). Reinders Letters in Applied Microbiology Vol. 36(3): [162167], Mar 2003.

J.Sc. Tech Vol. 10(1) 2009

13- P. J. Frosch, J.D. Johansen, T. Menn, C. Pirker, S.C. Rastogi, K.E. (2002).
Andersen, M. Bruze, A. Goossens, J. P. Lepoittevin, I. R. White Vol. 47(5):
[279-287], Nov 2002.
14- T.E Rams (1999). Journal of Applied Microbiology Vol. 86(6): [985-990],
Jun 1999.
15- N. Chami, S. Bennis, F. Chami, A. Aboussekhra, A. Remmal (2005). Oral
Microbiology and Immunology Vol. 20(2): [106-111], Apr 2005.
16- Bankova et al (2003). Microbiology Vol. 58(2): [158-158], Feb 2003.
17- Sforcin et al (1999). Letters in Applied Microbiology Vol. 29(5): [334-336],
Nov 1999.
18- S. E. Walsh, J.-Y. Maillard, A.D. Russell, C.E. Catrenich, D.L. Charbonneau, R.G.
Bartolo (2003). Journal of Applied Microbiology Vol. 94(2): [240-247], Feb 2003.
19- L. R. G Fava, W. P Saunders (1999). International Endodontic Journal Vol. 32(4):
[257-282], Jul 1999.
20- M Battino, M. S Ferreiro, D Fattorini, P Bullon (2002). Journal of Clinical
Periodontology Vol. 29(5): [462-467], May 2002.
21- Saun-Joo Lee Yoon PhD RN, Claydell H. Horne PhD RN (2001). Journal of
Advanced Nursing Vol. 33(1): [51-59], Jan 2001.