LUCRRI TIINIFICE MEDICIN VETERINAR VOL.

XLII (1), 2009, TIMIOARA

ANTIMICROBIAL POTENTIAL OF SOME LAMIACEAE

ESSENTIAL OILS AGAINST ANIMAL MULTIRESISTANT
BACTERIA
MIHAELA NICULAE, MARINA SPNU, CARMEN DANA ANDRU, F.
BRUDAC, D. CADAR, BIANCA SZAKACS, I. SCURTU, P. BOLF,
C.I. MATE
University of Agricultural Sciences and Veterinary Medicine, Faculty of Veterinary
Medicine, 3-5, Manastur Street, 400372, Cluj-Napoca, Romania
E-mail: niculaemihaela@hotmail.com
Summary
A considerable scientific interest is exhibited nowadays to the antimicrobial
screening of bioactive herbal extracts or constituents, due to their unique and complex
biological potential. The aim of the present study was to investigate the antibacterial
properties of some essential oils derived from several herb species belonging to Lamiaceae
family against animal pathogens well known for their elevate antibiotic resistance level:
Staphylococcus aureus, Salmonella enteritidis, E. coli and Pseudomonas aeruginosa. The
antimicrobial potential was determined performing the disc diffusion assay and also minimal
inhibitory (MIC) and bactericidal (MBC) concentrations were established by broth
microdilution method. The inhibitory ability manifested by the herbal products against the
bacterial strains varied significantly depending fundamentally on herbal source and
concentration, but also on bacterial species. The most resistant bacteria proved to be
Pseudomonas aeruginosa, with MIC and MBC values between 2 and 4% for the most active
herbal products. The results suggest that some of the screened herbal extractions could be
considered for further investigations regarding the therapeutic use in animal bacterial
pathology.
Key words: Lamiaceae essential oils, antimicrobial potential

The increasing occurrence of antimicrobial resistance represents a

worldwide major concern for both human and veterinary medicine (2, 6). Although
several strategies have been proposed to overcome and control this situation, still a
clear solution has not been found due to the antibiotic resistance complexity and
consequences and also considering the side effects of classic antimicrobial drugs.
A powerful trend recommends the herbal preparations to be investigated for their
antimicrobial activity. Therefore, nowadays, there is a growing interest in the
antimicrobial screening of extracts and essential oils from plants in order to
discover new antimicrobial agents. The Lamiaceae is one of the most diverse and
widespread plant families in terms of ethnomedicine and its medicinal value is
manly based on the volatile oils concentration (5). It is well documented that some
plants belonging to this family possess antimicrobial properties (6), however the
reported aspects are not constant.
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The objective of this research was to evaluate the potential of essential oils
derived from Thymus vulgaris, Salvia officinalis, Lavandula officinalis, Mentha
piperita, Rosmarinus officinalis, Ocimum basilicum, Mellisa officinalis and
Origanum vulgare against multi-drug resistant bacteria such as Staphylococcus
aureus, Salmonella enteritidis, E. coli and Pseudomonas aeruginosa which were
isolated from animal clinical cases. The antimicrobial potential was determined
performing the disc diffusion assay and also minimal inhibitory (MIC) and
bactericidal (MBC) concentrations were established by broth microdilution method.
Materials and methods
Plant extracts The present study included essential oils and/ or ethanol
extracts derived from plants belonging to Lamiaceae family: thyme (Thymus
vulgaris), common sage (Salvia officinalis), lavander (Lavandula officinalis),
peppermint (Mentha piperita), rosemary (Rosmarinus officinalis), sweet basil
(Ocimum basilicum), lemon balm (Mellisa officinalis) and oregano (Origanum
vulgare). All of them were purchased from commercial sources and Millipore filter
sterilized prior to antimicrobial assays.
Bacterial strains The set of microorganisms elected for this assay
included standard strains, Staphylococcus aureus ATCC 25923 and several
Staphylococcus aureus, Salmonella enteritidis, E. coli and Pseudomonas
aeruginosa strains isolated from animal clinical cases. These isolates, identified by
standard microbiological methods, were also screened for their susceptibility
towards antimicrobial agents, displaying different levels of antibiotic resistance. In
order to perform the antimicrobial screening, the bacterial isolates were cultured
overnight at 37C on Mueller Hinton agar (Oxoid). Colonies collected from each
twenty-four hours bacterial culture were diluted in sterile saline and the optical
density was adjusted according to the tube 0.5 of McFarland scale to prepare a
standardized inoculum (1.5 x 108cfu/ml).
Antimicrobial assay (Disc Diffusion Assay)
Each strain, prepared as a standardized inoculum, was inoculated on the
surface of Mueller Hinton agar plates (Oxoid). Subsequently, sterile filter paper
discs (Whatman, 6 mm diameter) containing approximately 20L of the herbal
essential oils were applied on and the plates were incubated overnight at 37C. The
study evaluated each herbal sample in triplicate. Discs of Amoxicillin + Clavulanic
Acid (20g +10g, BioMerieux) and enrofloxacin (5g, KRKA) were used as
positive controls, while sterile paper discs without herbal extractions were
considered as negative controls. After the incubation period, the diameters of the
inhibition zone were measured in millimeters and compared to those of the positive
controls.

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Minimum inhibitory concentration (MIC) and Minimum bactericidal

concentration (MBC) (broth dilution method)
The MIC and MBC were determined using the broth microdilution method
(1). Using Mueller Hinton broth, series of twofold dilutions of each essential oil were
performed in sterile 96-well microtiter plates. 100l of each dilution, ranging from
4% to 0.125% (v/v), were mixed with an equal volume of bacterial suspension.
Positive and negative growth controls were prepared. The plates were incubated
for 24h, at 37C, under normal atmospheric conditions. The MIC was defined as
the lowest concentration (highest dilution) of the herbal essential oils that inhibited
the visible growth (no turbidity), when compared to the control. Afterwards, 10l of
each well were transferred to Mueller Hinton agar plates and incubated for 24h, at
37C. The lowest concentration associated with no visible growth of bacteria on the
agar plates was considered the MBC. All dilutions were performed in duplicate.
Results and discussions
Our results, determined using disc diffusion and broth microdilution
methods, are presented as average values in tables 1 and 2.
Table 1
Inhibition zone diameter (d) in mm as established by disc diffusion method
(EO, essential oil; (-), no inhibition) (mean of three tests)
Plant used

Staphylococcus
aureus

Salmonella
enteritidis

E. coli

Pseudomonas
aeruginosa

Thyme

24

14

15

12

Sage

42

30

30

24

Lavander

38

28

28

25

Peppermint

38

27

28

24

Rosemary

36

25

24

10

Sweet basil

28

18

18

Lemon
balm

50

34

34

28

Oregano

35

30

30

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LUCRRI TIINIFICE MEDICIN VETERINAR VOL. XLII (1), 2009, TIMIOARA

Table 2
MIC and MBC (% v/v) for the tested herbal products as established by both
microdilution methods) (mean of two tests)
Plant used

Staphylococcus
aureus

Salmonella
enteritidis

E. coli

Pseudomonas
aeruginosa

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

Thyme

0.125

0.125

0.125

0.125

Sage

0.125

0.125

0.125

0.125

Lavander

Peppermint

Rosemary

Sweet basil

>4

Lemon
balm

0.125

0.125

0.125

0.125

Oregano

>4

This study revealed significant differences in displaying the antimicrobial

potential for the herbal preparations tested. All the herbal species manifested
antibacterial properties, but the level of bacterial growth inhibition induced by plant
materials, as determined by the disc diffusion assay, proved to be dependent
mostly on herbal source and on bacterial strain.
By using disc method we observed that essential oils determined different
inhibition zones on tested bacteria. However, it was established that essential oils
of different Lamiaceae species had antibacterial effects both on Gram-positive
(Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Salmonella
enteritidis and Pseudomonas aeruginosa). Lavandula officinalis, Mentha piperita,
Rosmarinus officinalis and Origanum vulgare displayed similar potential,
whileThymus vulgaris and Ocimum basilicum had the lowest effect on the bacteria
growth. Essential oil of Mellisa officinalis was the most active on the tested strains,
presenting the largest zones of growth inhibition among the sensible strains and
also possessing the lowest MIC and MBCs values.
Comparison of the sensitivity of tested bacteria towards the essential oils
showed that Gram-negative strains, especially Pseudomonas aeruginosa isolates,
were the most resistant. Regarding the strains of Pseudomonas aeruginosa, it was
observed that almost all of the tested substances (except the Ocimum basilicum
and Origanum vulgare essential oils) presented antibacterial activity, characterized
by the presence of zones of growth inhibition with diameters that varied from 10
mm (Rosmarinus officinalis) to 28 mm (Mellisa officinalis). On the other hand, the
most sensitive bacterium was Staphylococcus aureus.
The results obtained in the microdilution assay are well connected with the
above mentioned aspects.
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Most of the essential oils, case of Thymus vulgaris, Salvia officinalis,

Lavandula officinalis, Mentha piperita, Rosmarinus officinalis and Mellisa officinalis,
showed strong antimicrobial activity (both bacteriostatic and bactericidal effect)
against Staphylococcus aureus, Salmonella enteritidis and E. coli, at
concentrations that ranged from 0.125 to 2% (v/v). Mellisa officinalis essential oils
showed significant bactericidal effects against the bacterial strains tested, with
minimal bactericidal concentration 0.125% (v/v). Salvia officinalis essential oils also
displayed an antimicrobial activity, however, less efficient comparing to Mellisa
officinalis essential oils as MIC and MBC against the Pseudomonas aeruginosa
strains were 4% (v/v).
The use of plant extracts with known antimicrobial properties can be of
great significance in therapeutic treatments. In the last few years, a number of
studies have been conducted in different countries to prove such efficiency (2, 3,
6). Many plants have been used because of their antimicrobial traits, which are due
to compounds synthesized in the secondary metabolism of the plant. Several
studies indicate that the essential oils of the herbal belonging to Lamiaceae family
possess biological activity against several bacteria and yeast (2, 3, 5, 6). According
to them, there are differences in the activity of an essential oil to another or even of
their subproducts, due to the structure activity relationship. This discrepancy in
exerting the antibacterial potential may be caused by a variation that concerns the
chemical composition as this one is determined at a greater extent by the
distillation and extraction technique or by the different geographical chemotype.
This happens, probably because the essential oils are composed by,
several substances that can interact jointly in a synergic/potential form, giving them
strong antimicrobial activity (1, 2, 3). Other factor that should also be studied and
considered is related to the diffusion ability of the tested product in the culture
medium, which can favor in some situations, the diameter of the zones of growth
inhibition.
Many of the oil components may possess the ability to break or to
penetrate the lipid structure present in the Gram-negatives bacteria wall.
Due to its remarkable antibiotic-resistance, P. aeruginosa represents a
problem of increasing significance in veterinary medicine. According to our results,
susceptibility of Pseudomonas aeruginosa to most essential oils was relatively
poor, but we observed that the essentials oils obtained from Mellisa officinalis
seemed to manifest bacteriostatic and bactericidal activity against these bacteria.
Conclusions
All the screened Lamiaceae species showed antibacterial potential, but
with significant differences concerning the level of bacterial growth inhibition. The
antimicrobial activity seemed to be dependent mostly on herbal source and also on
bacterial genera. Mellisa officinalis demonstrated the strongest antimicrobial
efficacy against all bacterial strains. Also, Salvia officinalis, Rosmarinus officinalis
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and Lavandula officinalis essential oils possessed promising antibacterial

properties against animal multiresistant bacterial pathogens.
The results of this study showed that most of the essential oils presented
similar antimicrobial activity when challenged to a particular bacterial strain,
diverging discreetly in relation to the size of the zones of growth inhibition, as well
as to the MIC and MBC values.
These data suggest the potential application of some herbal preparations
alone or in combination with antibiotics for the treatment and prevention of
pathologies associated with multiresistant bacteria. Further investigations regarding
the in vitro and in vivo toxicity should be conducted in order to develop such
products.
References
1. Carson, C.F., Cookson, B.D., Riley, T.V., Susceptibility of methicillinresistant Staphylococcus aureus to the essential oils of Melaleuca alternifolia,
Journal of Antimicrobial Chemotherapy, 1995, 35, 421-424.
2. Delamare, A.P.L., Pistorello, I.T.M., Artico, Liane, Serafini, L.A.,
Echeverrigaray, S., Antibacterial activity of the essential oils of Salvia
officinalis L. and Salvia triloba L. cultivated in South Brazil, Food Chemistry,
2007, 100, 603-608.
3. Faleiro, M.L., Miguel, M.G., Ladeiro, F., Venancio, F., Tavares, R., Brito,
J.C., Figueiredo, A.C., Barroso, J.G., Pedro, L.G., Antimicrobial activity of
essential oils isolated from Portuguese endemic species of Thymus, Lett
Appl Microbiol., 2003, 36, 1, 35-40.
4. Pasqua R. di Feo, V. de Villani F., Mauriello G., In vitro antimicrobial
activity of essential oils from Mediterranean Apiaceae, Verbenaceae and
Lamiaceae against foodborne pathogens and spoilage bacteria, Annals of
Microbiology, 2005, 55, 2, 139-143.
5. Sarac, N., Ugur, A., Antimicrobial activities and usage in folkloric medicine of
some Lamiaceae species growing in Mugla, Turkey. EurAsia J BioSci, 2007,
1, 4, 28-34.

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