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Molecules 2013, 18, 9334-9351; doi:10.3390/molecules18089334
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
The Potential of Use Basil and Rosemary Essential Oils as
Effective Antibacterial Agents
Monika Sienkiewicz
1,
*, Monika Łysakowska
1
, Marta Pastuszka
2
, Wojciech Bienias
2
and Edward Kowalczyk
3
1
Medical and Sanitary Microbiology Department, Medical University of Lodz, pl. Hallera 1,
Lodz 90-647, Poland; E-Mail: monika.lysakowska@umed.lodz.pl
2
Department of Dermatology, Pediatric Dermatology and Oncology, Medical University of Lodz,
Kniaziewicza 1/5, Lodz 91-347, Poland; E-Mail: dermatologia@umed.lodz.pl (M.P. & W.B.)
3
Pharmacology and Toxicology Department, Medical University of Lodz, Pl. Hallera 1,
Lodz 90-647, Poland; E-Mail: edward.kowalczyk@umed.lodz.pl
* Author to whom correspondence should be addressed; E-Mail: monika.sienkiewicz@umed.lodz.pl;
Tel./Fax: +48-42-639-3342.
Received: 19 June 2013; in revised form: 30 July 2013 / Accepted: 1 August 2013 /
Published: 5 August 2013
Abstract: The considerable therapeutical problems of persistent infections caused by
multidrug-resistant bacterial strains constitute a continuing need to find effective
antimicrobial agents. The aim of this study was to demonstrate the activities of basil (Ocimum
basilicum L.) and rosemary (Rosmarinus officinalis L.) essential oils against multidrug-
resistant clinical strains of Escherichia coli. A detailed analysis was performed of the
resistance of the drug to the strains and their sensitivity to the tested oils. The antibacterial
activity of the oils was tested against standard strain Escherichia coli ATCC 25922 as well
as 60 other clinical strains of Escherichia coli. The clinical strains were obtained from
patients with infections of the respiratory tract, abdominal cavity, urinary tract, skin and
from hospital equipment. The inhibition of microbial growth by both essential oils, presented
as MIC values, were determined by agar dilution. Susceptibility testing to antibiotics was
carried out using disc diffusion. The results showed that both tested essential oils are active
against all of the clinical strains from Escherichia coli including extended-spectrum
β-lactamase positive bacteria, but basil oil possesses a higher ability to inhibit growth.
These studies may hasten the application of essential oils in the treatment and prevention
of emergent resistant strains in nosocomial infections.
OPEN ACCESS
Molecules 2013, 18 9335
Keywords: basil oil; ESBL-positive strains; Escherichia coli; minimal inhibitory
concentration; rosemary oil
1. Introduction
The multidrug-resistant pathogenic strains of Escherichia coli are responsible for opportunistic
infections, including nosocomial ones, which are difficult to treat, especially in immunocompromised
patients. E. coli is responsible for severe cases of urinary tract infection, meningitis in newborns,
digestive system illnesses, and even pneumonia. In recent years, strains of Enterobacteriaceae
producing an extended spectrum β-lactamase have become a concern in the antimicrobial treatment of
persistent infections and control of infection in hospitals [1–5]. The most severe clinical cases are
isolated resistant strains of Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa and
Acinetobacter baumanii. Extended-spectrum β-lactamases are enzymes produced by Gram-negative
bacilli that mediate resistance to penicillin, cephalosporins, and monobactams [6–9]. The widespread
use of antimicrobial drugs, primarily antibiotics, and the transmissibility of resistance determinants
mediated by plasmids, transposons, and gene cassettes in integrons contribute to the spread of resistance.
A worrying development is the fast spread of resistant clones of these bacteria on a global
scale [10–12]. Thanks to growing resistance to drugs commonly used in clinical practice, effective
treatment availability is greatly reduced. This problem of increasing resistance has necessitated the
search for safe and effective factors that may be used to treat persistent bacterial infections.
Experimental research confirms the varied pharmaceutical activities of not only chemical compounds,
but also many plant metabolites such as polysaccharides, flavonoids, coumarins, glycosides, phenolic
acids, saponins and also essential oils. Plant metabolites are a very interesting alternative for synthetic
preparations: many of them have strong antimicrobial activity [13–17]. Their synergy of action with
each other and in combination with antibiotic and chemotherapeutic therapy make them a valued
complement to anti-infective therapy [18–23].
The Ocimum L. (basil) and Rosmarinus L. (rosemary) genera belong to the family Lamiaceae.
Among the plants known for medicinal value, basil and rosemary plants are highly regarded for their
therapeutic potentials. Ocimum basilicum L. and Rosmarinus officinalis L. essential oils offer promise
as biologically active constituents, in that they confer antibacterial, antifungal and antioxidant
properties. Basil and rosemary oils have long been used for treating, among other things, colds, fever,
cough, asthma, sinusitis and rheumatism, as well as accelerating the process of wound healing [24–28].
The aim of this work was to determine the antibacterial activity of basil oil from Ocimum basilicum L.
and rosemary oil from Rosmarinus officinalis L. against standard and clinical strains of Escherichia coli
isolated from patients and from hospital equipment.
Molecules 2013, 18 9336
2. Results
2.1. Chemical Composition of the Basil and Rosemary Essential Oils
Chemical analysis showed the presence of forty-eight constituents within basil oil from
Ocimum basilicum L., the main ones being estragole (86.4%), 1,8-cineole (4.9%) and trans-α-bergamotene
(3.0%). The chemical composition of the basil oil is presented in Table 1. The essential oil from
Rosmarinus officinalis L. contains thirty-seven components: the main ones being 1,8 cineole (46.4%),
camphor (11.4%), α-pinene (11.0%), β-pinene (9.2%) and camphene (5.2%). The chemical composition
of the rosemary oil is presented in Table 2.
Table 1. Constituents of basil oil.
Number of
compounds
Compound % RI
1 α-Pinene 0.4 929
2 Camphene 0.1 942
3 Sabinene 0.2 965
4 β-Pinene 0.6 969
5 2,3-Dehydro-1.8-cineole Tr 979
6 Myrcene 0.2 983
7 p-Cymene 0.1 1013
8 1,8-Cineole 4.9 1020
9 Limonene 0.4 1021
10 (E)-β-Ocimene 0.6 1038
11 trans-Linalool oxide (f) Tr 1058
12 Fenchone 0.2 1067
13 cis-Linalool oxide (f) Tr 1073
14 Linalool 1.2 1085
15 endo-Fenchol 0.2 1098
16 Camphor 0.7 1119
17 Menthone 0.1 1134
18 Isomenthone Tr 1143
19 Borneol 0.2 1149
20 Menthol 0.3 1159
21 Terpinen-4-ol 0.1 1163
22 Estragole 86.4 1188
23 Fenchyl acetate 0.3 1209
24 Bornyl acetate 0.3 1269
25 2-Hydroxycineol acetate Tr 1321
26 Eugenol methyl ether 0.5 1373
27 β-Bourbonene Tr 1385
28 β-Elemene 0.3 1389
29 cis-α-Bergamotene Tr 1412
30 β-Caryophyllene 0.1 1419
31 trans-α-Bergamotene 3.0 1435
Molecules 2013, 18 9337
Table 1. Cont.
Number of
compounds
Compound % RI
32 β-Sesquifenchene 0.2 1437
33 (Z)-β-Farnesene tr 1447
34 α-Humulene tr 1452
35 Cadina-1(6),4-diene tr 1459
36 trans-β-Bergamotene 0.2 1479
37 α-Bulnesene 0.1 1499
38 γ-Cdinene 0.5 1506
39 Calamenene tr 1510
40 β-Sesquiphellandrene 0.1 1514
41 p-Methoxycinnamaldehyde 0.5 1525
42 p-Methoxycinnamyl alcohol 0.4 1532
43 Spathulenol 0.1 1565
44 Caryophyllene oxide 0.1 1571
45 Humulene epoxide II 0.1 1595
46 1-epi-Cubenol 0.1 1603
47 T-Cadinol 0.7 1627
48 α-Cadinol tr 1639
RI-Retence Index; tr < 0.05%.
Table 2. Constituents of rosemary oil.
Number of
compounds
Compound % RI
1 Tricyclene 0.2 919
2 α-Thujene 0.1 923
3 α-Pinene 11.0 932
4 Camphene 5.2 944
5 Sabinene 0.1 966
6 β-Pinene 9.2 971
7 Myrcene 1.2 983
8 α-Phellandrene 0.2 997
9 Car-3-ene 0.1 1005
10 α-Terpinolene 0.1 1010
11 p-Cymene 1.3 1017
12 1,8-Cineole 46.4 1027
13 Limonene 1.0 1027
14 γ-Terpinene 1.0 1050
15 trans-Sabinene tr 1054
16 Terpinolene 0.2 1079
17 Linalool 0.5 1087
18 α-Campholenol tr 1096
19 endo-Fenchol tr 1102
20 Camphor 11.4 1124
Molecules 2013, 18 9338
Table 2. Cont.
Number of
compounds
Compound % RI
21 Borneol 3.1 1152
22 Terpinen-4-ol 0.4 1163
23 α-Terpineol 1.8 1175
24 Bornyl acetate 1.0 1269
25 α-Cubebene tr 1349
26 α-Ylangene tr 1372
27 α-Copaene 0.1 1377
28 Longifolene 0.1 1407
29 β-Caryophyllene 3.5 1421
30 α-Humulene 0.4 1452
31 γ-Muurolene 0.1 1471
32 α-Selinene tr 1492
33 α-Muurolene tr 1494
34 γ-Cadinene tr 1506
35 trans-Calamenene tr 1511
36 δ-Cadinene 0.1 1514
37 β-Caryophyllene oxide 0.1 1571
RI-Retention Index; tr < 0.05%.
2.2. Susceptibility Testing of Clinical Escherichia coli Strains
2.2.1. Susceptibility Testing of Clinical Escherichia coli (ESBL+) Strains
Extended spectrum β-lactamase production for the tested Escherichia coli clinical strains was
detected for strains from the abdominal cavity (n = 4), bronchia (n = 4), wounds (n = 4), urine (n = 4)
and for strains isolated from blood (n = 3) and catheters (n = 3). The results are shown in Figure 1.
The tested strains of Escherichia coli (ESBL+) were generally resistant to β-lactams, aminoglycosides
and quinolones recommended for susceptibility testing. Most of them were resistant to cephalosporins
and β-lactam antibiotics with such inhibitors as clavulanic acid, sulbactam and tazobactam.
2.2.2. Susceptibility Testing of Clinical Escherichia coli (ESBL−) Strains
Escherichia coli ESBL negative strains, characterized by a much lower resistance to β-lactam
antibiotics, were resistant mainly to ampicillin, piperacillin, tikarcillin and also to ticarcillin/clavulanic
acid. Most of them were resistant to aminoglycosides (gentamicin, amikacin) and quinolones
(ciprofloksacin) and tetracycline. The results are shown in Figure 2.
Molecules 2013, 18 9339
Figure 1. The susceptibility testing of clinical Escherichia coli (ESBL+) strains.
The (ESBL+) and (ESBL−) Escherichia coli were found to demonstrate significant resistance to the
reference antibiotics in the susceptibility tests. In our tests, all twenty-two of the tested clinical isolates
of ESBL positive E. coli were resistant to AMC (n = 21, 95%), CZ (n = 10, 45%), CXM (n = 12,
54%), GM (n = 10, 45%), AM (n = 21, 95%), PIP (n = 21, 95%), TIC (n = 21, 95%), TIM (n = 18,
81%), FEP (n = 11, 50%), CIP (n = 14, 64%), AN (n = 12, 54%), NET (n = 13, 59%), C (n = 17, 77%),
TE (n = 21, 95%) and SXT (n = 20, 91%). The thirty-eight tested ESBL negative E. coli strains were
generally resistant to AMC (n = 21, 55%), GM (n = 15, 39%), PIP (n = 23, 60%), TIC (n = 26, 68%),
TIM (n = 13, 34%), CIP (n = 16, 42%), AN (n = 14, 37%), C (n = 15, 39%), TE (n = 34, 89%) and
SXT (n = 26, 68%).
Molecules 2013, 18 9340
Figure 2. The susceptibility testing of clinical Escherichia coli (ESBL−) strains.
2.3. The Susceptibility Escherichia coli Bacterial Strains to Basil Oil
The MIC values for sixty tested E. coli strains were between 8.0 µL/mL to 11.5 µL/mL. The basil
oil showed inhibitory activity against E. coli ATCC 25922 standard strain at 8.0 µL/mL.
2.3.1. The Susceptibility Escherichia coli (ESBL+) Strains to Basil Oil
Most E. coli ESBL+ strains isolated from the abdominal cavity (n = 4) and from the bronchia
(n = 4) were sensitive to basil oil at a concentration range from 8.25 µL/mL to 9.0 µL/mL. For
(ESBL+) clinical strains from wounds (n = 4), MIC values were between 8.5 µL/mL to 9.25 µL/mL.
The growth inhibition concentrations for bacteria isolated from blood were 8.75 µL/mL (n = 1) and
Molecules 2013, 18 9341
9.25 µL/mL (n = 2). Basil oil at a concentration of 8.75 µL/mL inhibited the growth of one of the
tested clinical strains isolated from urine, while 9.0 µL/mL inhibited the growth of three. The MIC
values for strains from catheters were 8.75 µL/mL (n = 2) and 9.25 µL/mL (n = 1). The susceptibility
of Escherichia coli (ESBL+) strains to basil oil is shown in Figure 3.
Figure 3. The susceptibility of Escherichia coli (ESBL+) and (ESBL−) strains to basil oil.
2.3.2. The Susceptibility Escherichia coli (ESBL−) Strains to Basil Oil
The basil oil was active against Escherichia coli (ESBL−) strains at a concentration range from
8.25 µL/mL to 11.5 µL/mL. The most tested strains isolated from the abdominal cavity (n = 4) were
inhibited by concentrations of 11.0–11.5 µL/mL. Clinical strains from the bronchia (n = 5) were
sensitive to basil oil at concentrations of 10.25–11.5 µL/mL. The MIC values for most strains from
wounds (n = 4) were from 10.0 µL/mL to 11.5 µL/mL. Most Escherichia coli (ESBL−) strains isolated
from blood and urine were inhibited by basil oil at concentrations ranging from 9.5 µL/mL to
11.5 µL/mL. The MIC values for bacteria from the catheters (n = 6) were 10.0–11.5 µL/mL.
The susceptibility of Escherichia coli (ESBL−) strains to basil oil is demonstrated in Figure 3.
2.4. The Susceptibility Escherichia coli Bacterial Strains to Rosemary Oil
The rosemary oil was less active against the sixty tested Escherichia coli clinical strains obtained
from the diverse clinical materials and the hospital equipment. The MIC values were between
18.0 and 20.0 µL/mL. The standard strain E. coli ATCC 25922 was sensitive to rosemary oil at a
concentration of 18.5 µL/mL.
2.4.1. The Susceptibility of Escherichia coli (ESBL+) Strains to Rosemary Oil
Most Escherichia coli (ESBL+) strains isolated from the abdominal cavity (n = 3) were sensitive to
rosemary oil at concentrations from 18.0 µL/mL to 18.5 µL/mL, and (n = 1) at 19.25 µL/mL
concentration. The MIC values for bacterial strains isolated from the bronchia (n = 4) and wounds
(n = 4) were 18.25–19.0 µL/mL and 18.5–19.25 µL/mL, respectively. E. coli (ESBL+) strains isolated
from blood were sensitive to rosemary oil at concentrations between 18.75 and 19.75 µL/mL. All strains
Molecules 2013, 18 9342
from urine (n = 4) were inhibited in the concentration ranges 18.0 µL/mL to 18.75 µL/mL, while those
from catheters (n = 3) were inhibited from 18.25 µL/mL to 18.75 µL/mL. The results of the tests
detailing the susceptibility of Escherichia coli (ESBL+) strains to rosemary oil are presented in Figure 4.
Figure 4. The susceptibility of Escherichia coli (ESBL+) and (ESBL−) strains to rosemary oil.
2.4.2. The Susceptibility of Escherichia coli (ESBL−) Strains to Rosemary Oil
Rosemary oil was found to have MIC values from 18.5 µL/mL to 19.75 µL/mL for isolates from the
abdominal cavity (n = 6). Growth inhibition was found to occur at concentrations of 19.0–20.0 µL/mL
for clinical bacterial strains obtained from the bronchia (n = 6). Rosemary oil at concentrations ranging
from 18.25 µL/mL to 20.0 µL/mL inhibited the growth of all tested Escherichia coli (ESBL−) strains
isolated from wounds. For the blood (n = 7) and urine (n = 6) isolates, the MIC values were in the
range 18.25-19.75 µL/mL. Finally, similar MIC values were found (18.25–19.75 µL/mL) against
E. coli (ESBL−) strains isolated from catheters (n = 7). The susceptibility of the Escherichia coli
(ESBL−) strains to rosemary oil is shown in Figure 4.
3. Discussion
In our investigation, all clinical strains of Escherichia coli were found to be sensitive to basil
(Ocimum basilicum L.) and rosemary (Rosmarinus officinalis L.) essential oils, irrespective of the
clinical conditions they were obtained under or the pattern of antibiotic resistance they demonstrated,
but basil oil was more active against the tested bacteria. Out of the sixty clinical strains of E. coli,
twenty-two strains were extended-spectrum beta-lactamase positive (ESBL+). Concentrations of basil
oil ranging from 8.25 µL/mL to 9.25 µL/mL were seen to inhibit the growth of eighteen Escherichia coli
(ESBL+) strains. For the extended-spectrum beta-lactamase negative strains, seventeen out of the
thirty-eight were sensitive to basil oil at concentrations between 11.25–11.50 µL/mL. The MIC values
for the other twenty-one Escherichia coli (ESBL−) strains ranged from 8.0 µL/mL to 11.0 µL/mL.
The rosemary oil demonstrated significantly lower activity. No apparent differences in activity of
the essential oil were found against extended-spectrum β-lactamase-positive and negative strains. Out
of the twenty-two clinical strains of E. coli (ESBL+), rosemary oil concentrations ranging from
18.0 µL/mL to 19.0 µL/mL were effective for eighteen of them. Eleven of the Escherichia coli (ESBL−)
Molecules 2013, 18 9343
strains out of the thirty-eight obtained under various conditions were inhibited by 19.75 µL/mL of
rosemary oil. MIC values ranging from 18.25 µL/mL to 19.5 µL/mL for comparable numbers of
strains was obtained.
The results of our tests clearly demonstrate that basil and rosemary essential oils can be widely used
to eliminate clinical strains of Escherichia coli found in different clinical conditions. It is also
significant that extended-spectrum β-lactamase (ESBL)− producing clinical strains of E. coli are
sensitive to these oils. Studies Orhan at al [29], confirm that, essential oils from Foeniculum sp.,
Mentha sp., Ocimum sp., Origanum sp. and Satureja sp. (Lamiaceae family) possess strong antibacterial
activity against extended-spectrum beta-lactamase (ESBL) positive strains of Klebsiella pneumoniae
isolated from food. The obtained MIC values ranged from 32 to 64 μg/ml for a number of strains
resistant to trimetoprime-sulfametoxazole, sulbactam-ampicilin, clavulonate-amoxicilin, ceftriaxon,
cefepime, imipenem, ceftazidime, tobramicine, gentamisine, ofloxacin, and ciprofloxacin. According
our investigations, the Escherichia coli (ESBL+) responsible for human infectious diseases were
significantly more resistant to the basil and rosemary essential oils.
The antimicrobial properties of essential oils are strictly connected with their chemical composition.
The usefulness of essential oils as effective antimicrobial agents can be evaluated only by analysing
their individual components. Therefore, a thorough GC-FID-MS analysis of the tested basil
and rosemary essential oils was conducted. The composition of the essential oil obtained from
Ocimum basilicum L., is as given in the ISO-11043 standard. The composition of the tested basil oil
corresponded to required standards, according to which the content of estragole must be higher than
75.0%. The composition of the essential oil derived from Rosmarinus officinalis L. was found to meet
the requirements of the European Pharmacopoeia 6 [30] and of the Polish Pharmacopoeia VIII [31] for
the nine main components. The content of β-pinene amounted to 9.2% (required 4.0%–9.0%) and
limonene to 1.0% (required 1.5%–4.0%). Verbenone was not found among the components of the
tested rosemary oil, although EP 6 and the Polish Pharmacopoeia VIII specify its content to be a
maximum of 0.4%. For the tested rosemary essential oil, nine of the thirteen main constituents of the
oil met the requirements given in the ISO-1342 standard: α-pinene, camphene, myrcene, 1,8-cineole,
p-cymene, camphor, bornyl acetate, α-terpineol and borneol.
The highest antibacterial activity is demonstrated by phenolic compounds such as carvacrol, thymol
and eugenol. Another effective group of active compounds are alcohols: terpinen-4-ol, γ-terpineol,
geraniol, cytronellol, menthol and linalol. Many of them are synthesized by plants from the Lamiaceae
family [32]. For instance, the essential oil of Satureja hortensis L. demonstrates high levels of activity.
Mihajilov-Kristev et al. [33], showed that essential oil containing mainly carvacrol (67.0%) and
γ-terpinene (15.3%) is effective against Gram-negative strains, including Escherichia coli, with MIC
values from 0.025 µL/mL to 0.78 µL/mL according to the broth microdilution method. In our study for
basil and rosemary essential oils, we obtained significantly higher MIC values. This high activity
demonstrated by Satureja hortensis L. essential oil is certainly related to the high content of carvacrol,
which is one of the most potent antimicrobial compounds.
The literature reports that basil oil, which contains mainly estragole and linalool, also possesses
antibacterial agents which are effective against a variety of Gram-positive and Gram-negative
bacteria [34]. According to Saković et al. [35], Ocimum basilicum essential oil possesses antibacterial
activity against the human pathogenic bacteria Bacillus subtilis, Enterobacter cloacae, Escherichia coli
Molecules 2013, 18 9344
O157:H7, Micrococcus flavus, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella enteritidis,
S. typhimurium, Staphylococcus epidermidis and S. aureus. According to the authors, basil essential
oil containing 69.3% linalool as a main component possesses antibacterial properties against
Escherichia coli O157:H7 with MIC and MBC values of 6.0 µg/mL when assessed by microdilution.
The multidrug clinical strains of E. coli tested in this study were more resistant to basil essential oil
containing mainly estragole (86.4%).
Results obtained by Sartoratto et al. [36], show that basil oil from Ocimum basilicum containing
mainly linallol 32.6% and eugenol 28.1%, and oil from Ocimum gratissimum containing 93.9%
eugenol, have a broad spectrum of antibacterial activity against reference strains of Gram-positive,
Gram-negative bacteria and Candida albicans. According to the authors, essential oils obtained from
these two genera of Ocimum were active against Escherichia coli CCT0547 standard strain with a MIC
value of >2 mg/mL according to the microplate method. These results also confirm that oils with
active constituents such as eugenol tend to have high antibacterial properties. Our results are slightly
higher than those obtained by Sartoratto et al. The MIC values were in the range from 7.92 mg/mL to
11.04 mg/mL for clinical strains of Escherichia coli which were both positive and negative for
extended-spectrum beta-lactamase activity.
Nakamura et al. [37], demonstrated that the essential oil from Ocimum gratissimum, with eugenol
as its main constituent, possesses antibacterial activity against clinical strains of Escherichia coli
ATCC 25922 with MIC—6 mg/mL. The MICs of this essential oil against a group of other
Gram-negative bacteria comprising Shigella flexneri, Salmonella enteritidis, Klebsiella sp. and
Proteus mirabilis were found to be from 0.3 to 12 mg/mL. The MIC values given in our study were
slightly higher than those given by Nakamura et al. Pereira et al. [10], in their investigations, showed
that oil from Ocimum gratissimum possesses antibacterial activity against clinical Escherichia coli
strains isolated from urinary tract infections. The essential oil was found to be active against
about 70% of tested E. coli clinical isolates. The results of the present study demonstrate that a
chemotype containing mainly 1,8-cineole, eugenol, methyleugenol, thymol, p-cimene, cis-ocimene and
cis-caryophyllene has lower activity.
In our tests, basil oil obtained from Ocimum basilicum, containing mainly estragole (86.4%),
inhibited the growth all strains isolated from various clinical materials. Among them were bacteria
isolated from urine, which were also extended-spectrum beta-lactamase positive. Our studies confirm
that antibacterial activity is possessed by not only basil oil chemotypes with linalool or eugenol as their
main components, but also that of Ocimum basilicum, containing mainly estragole. Our research
showed that basil essential oil was significantly more effective against all clinical isolates than
rosemary essential oil. Similar results were obtained by Hammer et al [38], who studied the antimicrobial
activity of basil and rosemary essential oils against Acinetobacter baumanii, Aeromonas veronii
biogroup sobria, Candida albicans, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae,
Pseudomonas aeruginosa, Salmonella enterica subsp. enterica serotype typhimurium, Serratia marcescens
and Staphylococcus aureus, using an agar dilution method. The authors confirmed that basil (Ocimum
basilicum) oil is more active against Escherichia coli than rosemary (Rosmarinus officinalis). The
MIC values were 0.5 and 1.0% (v/v), respectively.
According to Lopez et al. [39], the oils from Ocimum basilicum and Rosmarinus officinalis have an
antibacterial potential against the Gram-positive bacteria Staphylococcus aureus, Enterococcus faecalis
Molecules 2013, 18 9345
and Listeria monocytogenes and against Gram-negative bacteria Escherichia coli, Yersinia enterocolitica,
Salmonella choleraesuis and Pseudomonas aeruginosa as foodborne bacterial strains. The authors
present a detailed analysis of the tested oils and their ability to inhibit the growth of bacteria. Their
basil and rosemary essential oils were of a similar composition to the essential oils in our
investigations. The main component of the basil essential oil was estragole—82% ± 1.2%, while the
rosemary essential oil contains 1.8-cineole—48% ± 9.1%, camphor—17% ± 4.0, β-pinene—4.8% ± 0.9%
and β-caryophyllene—6.8% ± 3.7%. The authors confirm that the basil oil is more effective at
inhibiting the growth of Escherichia coli strains.
Probuseenivasan et al [27], confirmed that rosemary essential oil strongly inhibits Escherichia coli
ATCC 25922. Although the basil oil was also seen to demonstrate low activity against the tested
bacteria, no data was given about the constituents of the essential oils. The minimal inhibitory
concentration for rosemary oil against E. coli was >6.4 mg/mL. The MIC values obtained by the
present study were higher and ranged from 16.02 mg/mL to 17.35 mg/mL against E. coli clinical
strains. Fabio et al [40], report that rosemary oil has an antibacterial effect on a number of
microorganisms responsible for respiratory infections, isolated from clinical specimens, among which
were antibiotic-sensitive and antibiotic-resistant strains such as Streptococcus pyogenes, S. agalactiae,
S. pneumoniae and Klebsiella pneumoniae, Staphylococcus aureus and Stenotrophomonas maltophilia.
In our tests, rosemary oil was also found to demonstrate antibacterial activity against Escherichia coli
strains with different patterns of resistance, including extended-spectrum β-lactamase positive strains
isolated from various clinical materials. The rosemary essential oil used in the present study obtained
from Rosmarinus officinalis contains mainly 1,8-cineole (46.4%), camphor (11.4%) and α-pinene
(11.0%). The composition of the rosemary essential oil used by Jiang et al. [41], was similar to that
used by us: mainly 1,8-cineole (26.54%) and α-pinene (20.14%). The authors show that the tested oil
possesses antibacterial activity against Gram-positive bacteria (Staphylococcus epidermidis,
Staphylococcus aureus and Bacillus subtilis), Gram-negative bacteria (Proteus vulgaris, Pseudomonas
aeruginosa and Escherichia coli) and fungi (Candida albicans and Aspergillus niger). Bendeddouche
et al [42], showed that of essential oil from Rosmarinus tournefortii De Noé growing wild in the
occidental region of Algeria possesses antimicrobial activity also against Gram-negative (Escherichia
coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) pathogenic bacteria.
The main constituents of the tested essential oil were camphor (37.6%), 1,8-cineole (10.0%),
p-cymene-7-ol (7.8%) and borneol (5.4%).
A number of studies show that essential oils and their constituents possess useful properties
concerning human health. Many of them may be applied in anticancer therapy, cardiovascular and
nervous system disorders to reduce the level of cholesterol, to regulate the glucose level or to stimulate
hormone production [43]. They also might have great value in preventing and treating infectious
diseases. Essential oils not only have bactericidal activity but also can inhibit multidrug bacterial strain
formation. Their multiple antibacterial, antifungal, antiviral and also anti-inflammatory and antioxidant
effects, have made them valuable agents in human treatment and for the prevention of pathological
changes. In addition, essential oils have a number of beneficial properties as natural preservatives in
cosmetics, toiletries, drugs and food products [44–46]. Considering the huge increase in the number of
multidrug resistant bacterial strains in health care facilities, essential oils may prove to be effective
natural antimicrobial agents.
Molecules 2013, 18 9346
4. Experimental
4.1. Bacterial Strains
The standard strain, E. coli ATCC 25922, was obtained from the collection of the Medical and
Sanitary Microbiology Department, Medical University of Lodz. The clinical strains of Escherichia
coli were collected in 2011 and 2012 from a range of clinical materials recovered from patients and
from the hospital equipment in various wards from one of the Medical University hospitals in Lodz:
internal medicine, surgery, urology and the intensive care unit. The tested bacterial strains were
isolated from the abdominal cavity (n = 10), bronchia (n = 10), wounds (n = 10), blood (n = 10), urine
(n = 10) and from catheters (n = 10).
4.2. Bacterial Strain Identification
E. coli strains were cultured on Columbia Agar (bioMerieux, Craponne, France) and on Mac
Conkey Agar (bioMerieux). They were identified to the species by using API 20 E tests (bioMerieux).
The bacteria were incubated at 37 °C for 24 h.
4.3. Essential Oil Analysis
Commercial essential oils from basil—Ocimum basilicum L. and rosemary—Rosmarinus officinalis L.
were purchased from the manufacturer (POLLENA-AROMA, Warsaw, Poland) and analyzed by
GC-FID-MS in the Institute of General Food Chemistry, Lodz University of Technology, using a Trace
GC Ultra apparatus (Thermo Fisher Scientific Inc., Waltham, MA, USA) MS DSQ II detectors and FID-MS
splitter (SGE). Operating conditions: apolar capillary column Rtx-1ms (Restek Corporation, Bellefonte,
PA, USA), 60 m × 0.25 mm i.d., film thickness 0.25 µm; temperature program, 50–300 °C at 4 °C/min;
SSL injector temperature 280 °C; FID temperature 300 °C; split ratio 1:20; carrier gas helium at regular
pressure 200 kPa.; FID temperature 260 °C; carrier gas, helium; 0.5 mL/min; split ratio 1:20. Mass
spectra were acquired over the mass range 30–400 Da, ionization voltage 70 eV; ion source temperature
200 °C. The analysis of the constituents of the oils were performed two times independently.
Identification of components was based on the comparison of their MS spectra with those of the
laboratory-made MS library, commercial libraries (NIST 98.1, Wiley Registry of Mass Spectral Data,
8th Ed. and MassFinder 3.1) and with literature data [47,48] along with the retention indices on the
apolar column (Rtx-1, MassFinder 3.1) associated with a series of alkanes with linear interpolation
(C
8
-C
26
). A quantitative analysis, expressed as percentages of each component, was carried out by
peak area normalization measurements without correction factors.
4.4. Antibacterial Tests
The standard and clinical strains were cultivated in Columbia Agar medium and incubated at 37 °C
for 48 h in aerobic conditions. The microbial suspension was standardized to a cell density of
1–2·× 10
8
cells/mL, equal to an optical density of 0.5 on the Mc Farland scale, by a bioMerieux
densitometer. The agar dilution method was employed for the screening of antimicrobial activities of
the essential oils [32,38,49–51]. The tested essential oils were diluted in 96% ethanol PURE (POCH,
Molecules 2013, 18 9347
Gliwice, Poland) yielding a concentration of 97% v/v of oils. Although the tested essential oils dissolve
well in ethanol, only minimum amounts were used, as it can inhibit the growth of the tested bacteria.
This solution was mixed with a culture medium to obtain concentrations from 7.25 µL/mL to
11.75 µL/mL for basil oil and 17.75 µL/mL to 20.25 µL/mL for rosemary oil and poured into sterile
Petri dishes. An inoculum containing 1–2 × 10
8
cells/mL (0.1 mL) per spot was seeded upon the
surface of the agar with various oil concentrations, as well as on agar with no oil added (acting as a
control for strain growth). The Minimal Inhibitory Concentration, MIC, was determined after 24 h of
incubation at 37 °C under aerobic conditions. The MIC was considered the lowest concentration of the
sample at which no visible growth was observed. The analysis of the antibacterial activity of the oil
was performed three times independently. Control media containing only alcohol at concentrations
used in the dilutions of tested essential oils did not inhibit the growth of bacterial strains.
4.5. Susceptibility Testing
The following antibiotics (Becton Dickinson) were used for susceptibility testing of Escherichia coli
strains (R—resistance; I—intermediate susceptibility; S—susceptibility): AM—ampicillin (10 µg)
(R ≤ 13, 14 ≤ I ≤ 16, S ≥ 17), AMC—amoxicillin/clavulanic acid (20 µg/10 µg) (R ≤ 13, 14 ≤ I ≤ 17,
S ≥ 18), CF—cefalotin (30 µg) (R ≤ 14, 15 ≤ I ≤ 17, S ≥ 18), CZ—cefazoline (30 µg) (R ≤ 14, 15 ≤ I ≤ 17,
S ≥ 18), CXM—cefuroxime (30µg) (R ≤ 14, 15 ≤ I ≤ 17, S ≥ 18), GM—gentamicin (10 µg) (R ≤ 12,
13 ≤ I ≤ 14, S ≥ 15), TE—tetracycline (30 µg) (R ≤ 14, 15 ≤ I ≤ 18, S ≥ 19), NOR—norfloxacin
(10 µg) (R ≤ 12, 13 ≤ I ≤ 16, S ≥ 17) (only for the isolates from urine), FTN—nitrofurantoin (300 µg)
(R ≤ 14, 15 ≤ I ≤ 16, S ≥ 17) (as above), FOS—fosfomycin (200 µg) (R ≤ 12, 13 ≤ I ≤ 15, S ≥ 16)
(as above), STX—trimethoprim/sulfamethoxazole (1.25 µg/23.75 µg) (R ≤ 10, 11 ≤ I ≤ 15, S ≥ 16),
PIP—piperacillin (100 µg) (R ≤ 17, 18 ≤ I ≤ 20, S ≥ 21), TIC—tikarcillin (75 µg) (R ≤ 14, 15 ≤ I ≤ 19,
S ≥ 20), TZP—piperacyllin/tazobaktam (100/10 µg) (R ≤ 17, 18 ≤ I ≤ 20, S ≥ 21), TIM—
ticarcillin/clavulanic acid (75 µg/10 µg) (R < 16, S > 16), FOX—cefoxitin (30 µg) (R ≤ 14, 15 ≤ I ≤ 17,
S ≥ 18), CTX—cefotaxim (30 µg) (R ≤ 14, 15 ≤ I ≤ 22, S ≥ 23), CAZ—ceftazidime (30 µg) (R ≤ 14,
15 ≤ I ≤ 17, S ≥ 18), FEP—cefepim (30 µg) (R ≤ 14, 15 ≤ I ≤ 17, S ≥ 18), ATM—aztreonam (30 µg)
(R ≤ 15, 16 ≤ I ≤ 21, S ≥ 22), IMP—imipenem (10 µg) (R ≤ 13, 14 ≤ I ≤ 15, S ≥ 16), MEM—meropenem
(10 µg) (R ≤ 13, 14 ≤ I ≤ 15, S ≥ 16), ETP—ertapenem 10 µg) (R ≤ 15, 16 ≤ I ≤ 18, S ≥ 19), DOR—
doripenem (10 µg) (R ≤ 19, 20 ≤ I ≤ 23, S ≥ 24), CIP—ciprofloxacin (5 µg) (R ≤ 15, 16 ≤ I ≤ 20, S ≥ 21),
AN—amikacin (30 µg) (R ≤ 14, 15 ≤ I ≤ 16, S ≥ 17), NET—netilmicin (30 µg) (R ≤ 12, 13 ≤ I ≤ 14,
S ≥ 15), TOB—tobramycin (10 µg) (R ≤ 12, 13 ≤ I ≤ 14, S ≥ 15), C—chloramphenicol (30 µg)
(R ≤ 12, 13 ≤ I ≤ 17, S ≥ 18), TGC—tigecyclin (15 µg) (R ≤ 14, 15 ≤ I ≤ 18, S ≥ 19).
Susceptibility testing was carried out using the disc-diffusion method, on Mueller-Hinton II Agar
(bioMerieux). Cultures were incubated at 37 °C for 16–18 h. The results were interpreted according to
EUCAST guidelines [52].
The double-disk synergy test and combination disk method were used to determine ESBL
production. The sensitivity of the DDST can be improved by reducing the distance between the disks
of cephalosporins and clavulanate. The disk approximation method was performed on a Muller-Hinton
agar plate inoculated with the clinical bacterial strain, by placing disks containing CAZ—ceftazidime
(30 µg), CTX—cefotaxime (30 µg) and ATM—aztreonam (30 µg) 20 mm (edge to edge) from a disk
Molecules 2013, 18 9348
of AMC—amoxicillin/clavulanic acid (20/10 µg). Following incubation for 16–18 h at 37 °C, any
enhancement of the zone of inhibition between a cephalosporin and monobactam-aztreonam disk from
the amoxicillin/clavulanic acid disk, was indicative of the presence of an ESBL. E. coli ATCC 25922
was used as a positive control [11,53].
5. Conclusions
(1) The results of these experiments indicate the potential use of basil and rosemary essential
oils against resistant Escherichia coli clinical strains, and also against extended-spectrum
β-lactamase positive bacteria.
(2) The tested basil oil was more active against all Escherichia coli clinical strains.
(3) The action of essential oils against bacteria exhibiting different mechanisms of resistance may
be useful, not only in treating but also preventing the spread of resistant strains.
Acknowledgements
The authors wish to thank Danuta Kalemba from the Institute of General Food Chemistry,
Lodz University of Technology for essential oil analysis. The research reported in this manuscript was
supported by grant no 503/5-020-03/503-01 and has not been submitted elsewhere.
Conflict of Interest
The authors declare no conflict of interest.
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