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The Antimicrobial Activity of Thyme Essential Oil Against Multidrug Resistant Clinical Bacterial Strains

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The aim of this work was to investigate the antimicrobial activity of thyme essential oil against clinical multidrug resistant strains of Staphylococcus, Enterococcus, Escherichia, and Pseudomonas genus. The antibacterial activity of oil was tested against standard strains of bacteria and 120 clinical strains isolated from patients with infections of the oral cavity, abdominal cavity, respiratory and genitourinary tracts, skin, and from the hospital environment. Agar diffusion was used to determine the microbial growth inhibition of bacterial growth at various concentrations of oil from Thymus vulgaris. Susceptibility testing to antibiotics was carried out using disk diffusion. Thyme essential oil strongly inhibited the growth of the clinical strains of bacteria tested. The use of phytopharmaceuticals based on an investigated essential oil from thyme in the prevention and treatment of various human infections may be reasonable.
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The Antimicrobial Activity of Thyme Essential Oil Against
Multidrug Resistant Clinical Bacterial Strains
Monika Sienkiewicz,
1
Monika Łysakowska,
1
Paweł Denys,
2
and Edward Kowalczyk
3
Aim: The aim of this work was to investigate the antimicrobial activity of thyme essential oil against clinical
multidrug resistant strains of Staphylococcus, Enterococcus, Escherichia, and Pseudomonas genus. Materials:
The antibacterial activity of oil was tested against standard strains of bacteria and 120 clinical strains iso-
lated from patients with infections of the oral cavity, abdominal cavity, respiratory and genitourinary tracts,
skin, and from the hospital environment. Methods: Agar diffusion was used to determine the microbial
growth inhibition of bacterial growth at various concentrations of oil from Thymus vulgaris. Susceptibility
testing to antibiotics was carried out using disk diffusion. Results: Thyme essential oil strongly inhibited the
growth of the clinical strains of bacteria tested. Conclusions: The use of phytopharmaceuticals based on an
investigated essential oil from thyme in the prevention and treatment of various human infections may
be reasonable.
Introduction
Opportunistic infections caused by Gram-negative
bacteria, mainly of the Klebsiella sp., Enterobacter sp.,
Serratia sp., Proteus sp., Escherichia sp., and Pseudomonas sp.
present rising problems.
55
There has also been a significant
increase in the number of infections caused by Gram-positive
cocci belonging to Staphylococcus sp. and Enterococcus
sp.
11,20,30
Thus, the search for effective and safe medicines
that could be used to treat particularly persistent bacterial
infections is on. Oils, a diverse group of plant metabolites,
seem to be an interesting solution; in past years, a large
number of essential oils and their constituents have been
investigated for their antimicrobial properties against certain
bacteria, fungi, viruses, and protozoa.
The essential oils of thyme, oregano, mint, cinnamon, cu-
min, salvia, clove, and eucalyptus have been found to pos-
sess the strongest antimicrobial properties.
33
Many of them
appear to have a wide spectrum of antibiotic activity against
microflora that provokes hospital-acquired infections. The
broad and complex activity of essential oils, as well as their
synergy of action, can make them a valued weapon against
multidrug resistant bacterial strains. What is more, there is
no evidence of emergence of resistant bacteria after their
usage, and this is highly promising in the treatment of hu-
man diseases.
25,34,43,49
The aim of the study
The aim of this work was to investigate the antimicrobial
properties of thyme essential oil obtained from thyme (Thy-
mus vulgaris L.) against standard and clinical strains of bac-
teria isolated from patients, clinical staff, and the hospital
environment.
Materials and Methods
Bacterial strains
The standard strains, Staphylococcus aureus ATCC 433000,
Enterococcus faecalis Van B ATCC 51299, En. faecalis, vanco-
mycin VA-sensitive ATCC 29212, Enterococcus faecium, VA-
sensitive ATCC 35667, Enterococcus durans, VA-sensitive
ATCC 6656, Escherichia coli ATCC 25922, and Pseudomonas
aeruginosa ATCC 27853 came from the collection of the
Medical and Sanitary Microbiology Department, Medical
University of Lodz.
The clinical strains of Staphylococcus, Enterococcus, Escher-
ichia, and Pseudomonas genera were collected from different
materials originating from patients and medical staff, as well
as the environment of various wards from two hospitals in
Lodz: internal diseases, surgery, urology, laryngology, and
intensive care unit. Bacterial strains were isolated from ab-
dominal cavity exudates (n=4), bronchial secretions (n=5),
1
Medical and Sanitary Microbiology Department, Medical University of Lodz, Medical University of Lodz, Lodz, Poland.
2
Orthopedic Clinic, Medical University of Lodz, Orthopedic Clinic, Lodz, Poland.
3
Pharmacology and Toxicology Department, Medical University of Lodz, Medical University of Lodz, Lodz, Poland.
MICROBIAL DRUG RESISTANCE
Volume 18, Number 2, 2012
ªMary Ann Liebert, Inc.
DOI: 10.1089/mdr.2011.0080
137
nose (n=9), ear (n=3), pharynx (n=6), hands (n=2), ulcers
(n=14), wounds (n=22), bedsores (n=12), abscesses (n=1),
groin (n=5), bile (n=1), toe (n=1), anus (n=6), sputum
(n=1), blood (n=2), urine (n=14), drains (n=2), hospital
staff (n=2), disinfecting dispensers (n=2), hospital beds
(n=2), and cupboard swabs (n=4).
Bacterial strain identification
S. aureus strains were identified to the genus according to
standard microbiological methods: culturing on Columbia
Agar (bioMerieux) with 5% blood, culturing on Mannitol Salt
Agar (bioMerieux), using Slidex-Staph Kit (bioMerieux), and
determining the ability of bacteria to produce catalase and
coagulase (bioMerieux). Microorganisms were identified to
the species by using API 20 Staph tests (bioMerieux). Bacteria
were incubated at 37C for 24 hours. S. aureus ATCC 29213
strain was used as a control.
Enterococci were identified to the genus according to
standard microbiological methods: culturing on Columbia
Agar (bioMerieux) with 5% sheep blood, culturing on En-
terococcosel Agar (Emapol), determining the presence of D
antigen according to Lancefield (Slidex-Strep Kit, bioMer-
ieux), and determining the ability of bacteria to produce
catalase, pyrrolidonyloarylamidase (Lachema). The micro-
organisms were identified to the species by using API 20
Strep tests (bioMerieux). The bacteria were incubated at 37C
for 24 hours. As a control, En. faecalis Van B ATCC 51299, En.
faecalis ATCC 29212, En. faecium ATCC 35667, and En. durans
ATCC 6656 strains were used.
Es. coli strains were identified according to standard mi-
crobiological methods such as culturing on Columbia Agar
(bioMerieux) with 5% blood and culturing on Mac Conkey
Agar (bioMerieux) and were identified to the species by us-
ing API 20 E tests (bioMerieux). Bacteria were incubated in
37C for 24 hours. As a control, Es. coli ATCC 25922 strain
was used.
P. aeruginosa strains were identified according to standard
microbiological methods including culturing on Agar and
Columbia Agar (bioMerieux) with 5% blood, culturing on
Mac Conkey Agar (bioMerieux), and determining the ability
of the bacteria to produce oxidase (bioMerieux). The micro-
organisms were identified in the species by using API 20 NE
tests (bioMerieux). The bacteria were incubated in 37C for 24
hours. As a control, P. aeruginosa ATCC 27853 strain was used.
Essential oil analysis
Commercial essential oil from thyme—T. vulgaris L. (La-
miaceae) was purchased from the manufacturer (POLLENA-
AROMA) and analyzed by GC-FID-MS in the Institute of
General Food Chemistry, Technical University of Lodz, us-
ing a Trace GC Ultra apparatus (Thermo Electron Corpora-
tion) MS DSQ II detectors and FID-MS splitter (SGE).
Operating conditions: apolar capillary column Rtx-1 ms
(Restek), 60 m ·0.25 mm i.d., film thickness 0.25 mm; tem-
perature program, 50C–300Cat4C/minute; SSL injector
temperature 280C; FID temperature 300C; split ratio 1:20;
carrier gas helium at a regular pressure 200 kPa.; FID tem-
perature 260C; carrier gas, helium; 0.5 ml/min; split ratio
1:20. Mass spectra were acquired over the mass range of 30–
400 Da, ionization voltage of 70 eV, and ion source temper-
ature of 200C.
Identification of components was based on the compari-
son 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
1,31
along with the retention indices on 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.
The standard and clinical strains were cultivated in Co-
lumbia agar medium and incubated at 37C for 48 hours in
aerobic conditions. Bacterial suspensions with an optical
density of 0.5 on the Mc Farland scale were prepared and
analyzed with a Bio Merieux densitometer.
The antibacterial properties of the tested oil were investi-
gated by agar dilution. The essential oil was diluted in eth-
anol. This solution was mixed with a nutrient broth to obtain
concentrations from 0.03125 to 2.5 ml/ml and poured into
petri dishes. Inoculum containing 1.5$10
8
CFU (0.1 ml) per
spot was seeded on the surface of the agar with various oil
concentrations, as well as on agar with no oil added (strains
growth control). Minimal inhibitory concentration (MIC)
was determined after 24 hours of incubation at 37C in aer-
obic conditions. The analysis of the antibacterial activity of
the oil was performed thrice independently.
Susceptibility testing
The following antibiotics (Becton Dickinson) were used for
susceptibility testing of S. aureus strains: cefoxitin (FOX;
30 mg) (R £14, 15 £I£17, S 18), erythromycin (E; 15 mg)
(R £13, 14 £I£22, S 23), clindamycin (CC; 2 mg) (R £14,
15 £I£20, S £21), nitrofurantoin (F/M; 300 mg) (R £14,
15 £I£16, S 17) (for isolates from urine), VA (30 mg) (S 15),
teicoplanin (TEC; 30 mg) (R £10, 11 £I£13, S 14), tetracy-
cline (TE; 30 mg) (R £14, 15 £I£18, S 19), chloramphenicol
(C; 30 mg) (R £12, 13 £I£17, S 18), ciprofloxacin (CIP; 5 mg)
(R £15, 16 £I£20, S 21), trimethoprim/sulfamethoxazole
(SXT; 1.25 mg/23.75 mg) (R £10, 11 £I£15, S £16), fusidic acid
(FA; 10 mg) (S 22), linezolid (LZD; 30 mg) (S 21); of En-
terococcus genus:
Ampicillin (AM; 10 mg) (R £16 S 17), C (30 mg) (R £12,
13 £I£17, S 18), CIP (5 mg) (R £15, 16 £I£20, S 21), E
(15 mg) (R £13, 14 £I£22, S £I£23), fosfomycin (FOS; 200 mg)
(R £12, 13 £I£15, S 16) (only for En. faecalis, the isolates
from urine), F/M (300 mg) (R £14, 15 £I£16, S 17) (for iso-
lates from urine), gentamicin (GM; 120 mg) (R £6, 7 £I£9,
S10), LZD (30 mg) (R £20, 21 £I£22, S 23), imipenem
(IPM; 10 mg) (R £13, 14 £I£15, S 16), penicillin (P; 10 mg)
(R £14, S 15), streptomycin (S; 300 mg) (R £6, 7 £I£9, S 10),
synercid (SYN; 4.5 mg/10.5 mg) (R £15, 16 £I£18, S 19) (only
for En. faecium), TE (30 mg) (R £14, 15 £I£18, S 19), TEC
(30 mg) (R £10, 11 £I£13, S 14), VA (30 mg) (R £14,
15 £I£16, S 17), of Es. coli strains: amoxicillin/clavulanic
acid (AMC; 20 mg/10 mg) (R £13, 14 £I£17, S 18), cefalotin
(CF; 30 mg) (R £14, 15 £I£17, S 18), cefazolin (CZ; 30 mg)
(R £14, 15 £I£17, S 18), cefuroxime (CXM; 30 mg) (R £14,
15 £I£17, S 18), GM (10 mg) (R £12, 13 £I£14, S 15), AM
(10 mg) (R £13, 14 £I£16, S 17) (only for the isolates from
urine), norfloxacin (NOR; 10 mg) (R £12, 13 £I£16, S 17) (as
above), F/M (300 mg) (R £14, 15 £I£16, S 17) (as above),
138 SIENKIEWICZ ET AL.
FOX (30 mg) (R £14, 15 £I£17, S 18), cefotaxim (CTX; 30 mg)
(R £14, 15 £I£22, S 23), ceftazidime (CAZ; 30 mg) (R £14,
15 £I£17, S 18), aztreonam (ATM; 30 mg) (R £15, 16 £I£21,
S22), IPM (10 mg) (R £13, 14 £I£15, S 16), CIP (5 mg)
(R £15, 16 £I£20, S 21), netilmicin (NET; 30 mg) (R £12,
13 £I£14, S 15), tobramycin (NN; 10 mg) (R £12, 13 £I£14,
S15), C (30 mg) (R £12, 13 £I£17, S 18), TE (30 mg) (R £14,
15 £I£18, S 19), SXT (1.25 mg/23.75 mg) (R £10, 11 £I£15,
S16) and of P. aeruginosa strains: mezlocillin ( MZ; 75 mg)
(R £17, 18 £I£20, S 21), piperacillin (PIP; 100 mg) (R £17,
18 £I£20, S 21), CAZ (30 mg) (R £14, 15 £I£17, S 18), GM
(10 mg) (R £12, 13 £I£14, S 15), NN (10 mg) (R £12,
13 £I£14, S 15), AMC (20 mg/10 mg) (R £13, 14 £I£17,
S18), piperacillin/tazobactam (TZP; 100 mg/10 mg) (R £17,
18 £I£20, S 21), CTX (30 mg) (R £14, 15 £I£22, S 23), ATM
(30 mg) (R £15, 16 £I£21, S 22), IPM (10 mg) (R £13,
14 £I£15, S 16), meropenem (MEM; 10 mg) (R £13,
14 £I£15, S 16), NET (30 mg) (R £12, 13 £I£14, S 15), CIP
(5 mg) (R £15, 16 £I£20, S 21), SXT (1.25 mg/23.75 mg)
(R £10, 11 £I£15, S 16), C (30 mg) (R £12, 13 £I£17, S 18),
and colistin (CL; 50 mg) (R <15, S >15). Susceptibility testing
was carried out using the disk-diffusion method, on Mueller-
Hinton II Agar. Cultures were incubated at 37C for 16–18
hours, VA for 24 hours. The results were interpreted ac-
cording to Clinical and Laboratory Standard Institute
guidelines.
9
Results
Chemical composition of the tested oil
The composition of the essential oil derived from T. vul-
garis was found to meet the requirements of the Polish
Pharmacopoeia VIII and the European Pharmacopoeia.
18,40
The content of thymol amounts to 38.1% and carvacrol
to 2.3%, as well as other prevailing compounds such as
p-cymene (29.1%), g-terpinene (5.2%), and linalool (3.7%). The
chemical composition of the tested oil is shown in Table 1.
Susceptibility testing
The tested strains of S. aureus were resistant to 50% of
b-lactams and macrolides recommended for susceptibility
testing. Clinical strains of the Enterococcus genus showed
resistance to macrolides in almost 70%, to b-lactams and
aminoglycosides in 60%, and to carbapenems in 40%. The
examined Es. coli strains were resistant to b-lactams in 40%
and to aminoglycosides in 20%. The tested P. aeruginosa
strains were resistant to b-lactams in more than 50%, to
carbapenems in 30%, and to aminoglycosides and mono-
bactams in 20%. The results are shown in Tables 2–5.
The activity of thyme oil against tested bacterial strains
The MIC for S. aureus were between 0.25 and 1.0 ml/ml.
MIC was 0.25 ml/ml for the standard strain of S. aureus
ATCC 433000 and 6 strains from the clinical material. The
results are shown in Figure 1.
Most Staphylococcus strains were sensitive to oil at a con-
centration of 0.5 ml/ml. Growth inhibition was obtained in 17
out of the 30 clinical bacterial strains. Isolates were taken
from the nose (n=5), ear (n=1), hand (n=2), wound (n=3),
abdominal cavity (n=1), groin (n=1), ulcer swabs (n=3), and
urine (n=1). The results are presented in Figure 2.
The tested clinical strains of S. aureus, resistant to many
antibiotics, were sensitive to the thyme oil at low concen-
trations. S. aureus strain isolated from abscesses was resistant
to 7 out of 11 tested antibiotics (sensitive to VA, TEC, C, and
LZD); the MIC value for the thyme oil was 0.25 ml/ml. The
MIC for highly multidrug resistant bacterial strains from the
hand, wound, ear, foot ulceration, exudates from episiotomy
wounds, stump ulcers, and urine ranged from 0.5 ml/ml to
0.75 ml/ml. Thyme oil at 0.5 ml/ml concentration inhibited the
growth of more than 45% of the resistant strains. Table 2
shows the general characteristics of S. aureus isolates.
The VA resistant standard strain—En. faecalis Van B ATCC
51299 was the most sensitive strain to thyme essential oil
(MIC–0.0625 ml/ml). The MIC was 0.75 ml/ml for VA sensi-
tive strains of En. faecalis ATCC 29212 and En. faecium ATCC
35667 and MIC for the standard strain of En. durans ATCC
6656 was 0.85 ml/ml. The results are shown in Figure 1.
Table 1. Components of the Essential Oil Obtained
from Thyme—Thymus vulgaris L. (Lamiaceae)
No. Compound
Total
oil %
Retention
indices
1a-Thujene 0.6 932
2a-Pinene 1.9 936
3 Camphene 1.2 950
4 Oct-1-en-3-ol 1.0 962
5b-Pinene 0.3 978
6 Myrecene 1.1 987
7p-Cymene 29.1 1,015
8 1.8-Cineole 2.1 1,024
9 Limonene 0.2 1,025
10 g-Terpinene 5.2 1,051
11 p-Cymenene 0.1 1,075
12 Terpinolene 0.1 1,082
13 Linalool 3.7 1,086
14 Camphor 0.5 1,123
15 Borneol 1.9 1,150
16 Terpinen-4-ol 1.3 1,164
17 a-Terpineol 0.3 1,176
18 Thymol methyl ether 1.3 1,215
19 Carvacrol methyl ether 1.0 1,226
20 Borneol acetate 0.3 1,270
21 Thymol 38.1 1,267
22 Carvacrol 2.3 1,278
23 Thymol acetate 0.2 1,329
24 African-1-en 0.1 1,356
25 a-Copaene 0.2 1,379
26 b-Burbonene 0.1 1,386
27 b-Caryophyllene 3.1 1,421
28 Thymohydroquinone 0.1 1,509
29 a-Humulene 0.1 1,455
30 g-Muurolene 0.3 1,474
31 cis-bGuaiene 0.1 1,488
32 Cuparene 0.1 1,498
33 g-Cadinene 0.6 1,507
34 Calamenene B 0.2 1,517
35 dCadinene 0.3 1,520
36 a-Cadinene 0.1 1,534
37 Caryophyllene oxide 0.5 1,578
38 g-Eudesmol 0.1 1,618
39 Eudesm-3-en-7-ol 0.1 1,650
40 Cadalene 0.1 1,659
Bold texts and values indicate the major constituents of thyme oil.
THE ANTIMICROBIAL ACTIVITY OF THYME ESSENTIAL OIL 139
Thyme oil was also very active against clinical strains, the
most active against En. faecium strain being isolated from the
ulcer ( MIC–0.25 ml/ml); it significantly inhibited 16 clinical
strains at a concentration of 0.75 ml/ml and 11 clinical strains
at 0.5 ml/ml. The results are shown in Figure 2.
An MIC value of -0.5 ml/ml was characteristic for all
strains of En. faecalis and En. faecium isolated from urine, and
En. faecalis strains derived from the hospital environment.
The concentration of 0.75 ml/ml inhibited the growth of
En. faecalis clinical strains from wounds and the throat, as
well as of En. faecium strains from blood and from the hos-
pital environment.
It was found that most clinical resistant strains of entero-
cocci were sensitive to the tested oil. The En. faecium strain
isolated from ulcers, resistant to 9 out of 14 antibiotics
(sensitive to LNZ, SYN, TEC, and VA), was sensitive to
thyme essential oil at the lowest concentration of 0.25 ml/ml.
All En. faecium clinical strains isolated from urine, resistant to
8 antibiotics, were also sensitive to the oil of thyme; MIC was
0.5 ml/ml. In all, more than 45% of resistant strains were
sensitive to thyme oil: MIC =(0.75 ml/ml). Table 3 shows the
general characteristics of Enterococcus sp. isolates.
The oil showed bactericidal activity against Es. coli ATCC
25922 standard strain at 0.25 ml/ml (Fig. 1). Figure 2 shows
thatthesameMICvaluewasdeterminedfor13strainsfrom
the clinical material. Thyme oil of this concentration was
effective against clinical strains isolated from the throat
and the majority of strains from the wound swabs. An MIC
of -0.5 ml/ml was obtained for 17 tested clinical strains of
colon bacilli, isolated from the anus, bedsore swabs, and
urine. The clinical strains of Es. coli tested that were highly
resistant to commonly used antibiotics showed sensitivity
to thyme oil.
The multidrug resistant strain isolated from ulcers, re-
sistant to 14 out of 16 antibiotics (sensitive to the ATM,
IPM), was sensitive to thyme oil (MIC =(0.25 ml/ml)). The
same MIC was obtained for two strains from wound and
bedsore swabs, which were sensitive to only 5 out of the 16
tested antibiotics. The growth of nearly 60% of the resistant
strains was inhibited by thyme oil at a concentration of
0.5 ml/ml. Table 4 shows the general features of Es. coli
isolates.
Strains of P. aeruginosa were the most resistant to the
thyme oil. The standard strain—P. aeruginosa ATCC 27853—
was inhibited with 0.5 ml/ml. The results are shown in Figure
1. The number of the clinical strains that were sensitive to the
thyme oil at 1.5, 2.0, and 2.5 ml/ml was similar, and it is
presented in Figure 2. Concentrations of 1.5 and 2.0 ml/ml
Table 2. Characteristics of Staphylococcus aureus Isolates
Susceptibility to antibacterials Total
No.
Staphylococcus aureus
strain/clinical material
MIC of thyme
essential oil ll/ml FOX E CC F/M VA TEC TE C CIP SXT FA LZD R I S
1. Swab/nose 0.25 R S R S S R S I S S S 3 1 7
2. Swab/nose 0.5 S S S S S R S S S S S 1 — 10
3. Swab/nose 0.75 R S R S S R S I R R S 5 1 5
4. Swab/nose 0.5 R S R S S R S I I R S 4 2 5
5. Swab/nose 0.5 R S R S S R S I S R S 4 1 6
6. Swab/nose 0.25 S S S S S S S S R R S 2 9
7. Swab/nose 0.5 S S S S S R S S R S S 2 9
8. Swab/nose 0.5 S S S S S S S S I S S — 1 10
9. Swab/ear 0.5 R R R S S R S R R R S 7 — 4
10. Swab/hend 0.5 S S S S S R S S S S S 1 — 10
11. Swab/hend 0.5 R R R S S R S R R R S 7 — 4
12. Swab/wound 0.75 R S R S S R S I R R R 6 1 4
13. Swab/wound 0.75 R S R S S R S I R S S 4 1 6
14. Swab/wound 0.5 S S S S S R S R S S S 2 — 9
15. Swab/wound 0.5 I R R S S R R S S S S 4 1 6
16. Swab/wound 0.5 S S S S S R S S S S S 1 — 10
17. Swab/wound 1.0 R R R S S R S R S R S 6 — 5
18. Exudation/abdominal cavity 0.25 R R R S S R S R S S S 5 6
19. Exudation/abdominal cavity 0.5 I R R S S R S R S S S 4 1 6
20. Exudation/abdominal cavity 0.25 S R R S S R S R S S S 4 7
21. Swab/ulceration 0.5 R R R S S R S R I R S 6 1 4
22. Swab/ulceration 0.5 S R R S S R S R S S S 4 — 7
23. Swab/ulceration 0.75 R R R S S S R R R R S 7 4
24. Swab/ulceration 0.5 S S S S S S S S S S S — — 11
25. Swab/groin 0.5 S S S S S S S S S S S — — 11
26. Swab/groin 1.0 I R R S S R R I S S S 4 2 5
27. Swab/abscess 0.25 R R R S S R S R R R S 7 4
28. Urine 0.5 I R R S S S R S R S S R 5 1 6
29. Urine 0.25 R S R S S S R S I R S S 4 1 7
30. Swab/drain 0.75 S S S S S R S S S S S 1 — 10
FOX, cefoxitin; E, erythromycin; CC, clindamycin; F/M, nitrofurantoin; VA, vancomycin; TEC, teicoplanin; TE, tetracycline; C,
chloramphenicol; CIP, ciprofloxacin; SXT, trimethoprim/sulfamethoxazole; FA, fusidic acid; LZD, linezolid; R, resistant; I, intermediate
susceptible strain; S, susceptible strain; MIC, minimal inhibitory concentration.
140 SIENKIEWICZ ET AL.
inhibited the growth of strains mainly isolated from wounds
and bedsores. MIC values of -2.0 and 2.5 ml/ml were ob-
tained for blue pus bacilli isolated from ulcers. The highest
concentration of thyme oil, 2.5 ml/ml, was effective against
the bacteria from bronchial secretions and from the anus
swabs. It was found that the most resistant micro-organisms
were isolated from bronchial secretions. Two of them were
found to be resistant to all 16 antibiotics, and the third was
sensitive to only 6 of them. The MIC obtained for thyme oil
against these strains ranged from 2.0 to 2.5 ml/ml.
Higher oil activity (MIC-1.5 ml/ml) was obtained against
the P. aeruginosa strain isolated from the flank, which showed
resistance to 12 antibiotics (sensitive to AMC, TZP, IPM, and
NET). At the same time, these strains were sensitive in 40%
of thyme oil used at a concentration of 1.5 ml/ml.
Table 5 shows the general characteristics of P. aeruginosa
isolates.
Control media containing alcohol (at concentrations used
in the dilutions) did not inhibit the growth of bacterial
strains.
Discussion
A number of chemotypes were identified in red thyme,
T. vulgaris; the most important are the thymol (65% thymol,
5%–10% carvacrol) and carvacrol chemotypes (85% carvacrol,
1%–5% thymol).
42
According to the requirements of the Polish
Pharmacopeia VIII and European Pharmacopeia, the oil
should contain thymol (36%–55%) and carvacrol (1%–4%).
18,40
In our study, the oil showed antimicrobial activity against
standard and clinical strains of S. aureus, En. faecalis,
En. faecium, En. durans, Es. coli, and P. aeruginosa. The ob-
tained results are in accordance with the literature, and show
that thyme oil has strong antimicrobial properties against all
tested strains. The activity is due to the high content of
phenolic compounds with antibacterial properties, such as
thymol and carvacrol, which constitute more than 40% of the
ingredients of the oil.
33
In our tests, most of the clinical strains of S. aureus were
sensitive to thyme oil at a concentration of 0.5 ml/ml (17
strains), therefore relatively low compared with the high
concentrations of antibiotics usually required. These strains
came from diverse materials and hospital wards (e.g., swabs
from wounds, swabs from nose, and urine). As far as sus-
ceptibility to antibiotics was concerned, many isolates were
resistant to TE (n=25, 83.3%), CC (n=20, 66.6%), FOX (n=14,
46.6%), E (n=13, 43.3%), and CIP (n=12, 40%). However, all
the strains were susceptible to VA, and TEC and only two
(from wound and urine) were resistant to LZD. Strains with
MIC at 1 ml/ml were isolated from swabs (groin and wound),
wards (nephrology and ICU), and remained sensitive to
several antibiotics (VA, TEC, C, SXT, and LNZ). There were
Table 3. Characteristics of Enterococcus sp. Isolates
Susceptibility to antibacterials Total
No.
Enterococcus spp.
Strain/clinical material
MIC of thyme
essential
oil ll/ml AM C CIP E FOS F/M GM IPM LNZ P S SYN TE TEC VA R I S
1. En. faecalis/urine 0.5 S S R S S S S S S S S I S S 1 1 12
2. En. faecalis/urine 0.5 R S R R S S R R S R R R S S 8 6
3. En. faecalis/urine 0.5 S S I I S S S S S S S R S S 1 2 11
4. En. faecalis/urine 0.5 S R R R S S R S S R R R S S 7 — 7
5. En. faecalis/urine 0.5 S R R R S S R S S S R — R S S 6 — 8
6. En. faecalis/swab/wound 0.75 S S I S S S S S S S S S 1 11
7. En. faecalis/swab/wound 0.75 S R R R — S R S S S R — R S S 6 — 7
8. En. faecalis/swab/wound 0.75 S R R R — R S S S R — R S S 6 — 6
9. En. faecalis/swab/ulceration 0.75 S R S R S S S S I R S S 3 1 8
10. En. faecalis/bile 0.75 S S S S S S S S S S S R S S 1 — 13
11. En. faecalis/swab/bedsore 1.25 S S I S S S S S S R S S 1 1 10
12. En. faecalis/swab/pharynx 0.75 S S S I S S S S S R S S 1 1 10
13. E. faecalis/swab/pharynx 0.75 S R I R R S S S R R S S 5 1 6
14. En. faecalis/hospital staff 0.75 S S S R S S S S R R S S 3 9
15. En. faecalis/drain 0.75 S S R S R S S S S R S S 3 9
16. En. faecalis/cupboard 0.5 S S R S R S S S S R S S 3 9
17. En. faecalis/disinfecting dispencer 0.5 S S I S S S S S S R S S 1 1 10
18. En. faecalis/disinfecting dispencer 0.5 S S R S R S S S S R S S 3 9
19. En. faecium/urine 0.5 R S R R S R R S R R S R S S 8 6
20. En. faecium/urine 0.5 R S R R R R R S R R S S S S 8 6
21. En. faecium/urine 0.5 R S R R I R R S R R S R S S 8 1 5
22. En. faecium/blood 0.75 R S R R S R S S R S R S S 6 7
23. En. faecium/blood 0.75 R S R R S R S R R S R S S 7 6
24. En. faecium/swab/ulceration 0.25 R I R R R R R S R R S R S S 9 1 4
25. En. faecium/hospital staff 0.75 R S R R S R S R R S R S S 7 6
26. En. faecium/hospital bed 0.75 R S R R R R S R R S S S S 7 6
27. E. faecium/hospital bed 0.75 S S S S S S S S S S S S S — — 13
28. En. faecium/cupboard 0.75 R S R R R S S S S R S S 5 7
29. En. faecium/cupboard 0.75 R S R R S R R S R R S R S S 8 6
30. En. durans/cupboard 1.0 R S R R S R R S R R S R S S 8 6
AM, ampicillin; FOS, fosfomycin; GM, gentamicin; IPM, imipenem; LZD, linezolid; P, penicillin; S, streptomycin; SYN, synercid.
THE ANTIMICROBIAL ACTIVITY OF THYME ESSENTIAL OIL 141
Table 4. Characteristics of Escherichia coli Isolates
Susceptibility to antibacterials Total
No.
Escherichia coli
strain/clinical material
MIC of thyme
essential oil ll/ml AMC CF CZ CXM GM AM NOR F/M FOX CTX CAZ ATM IPM CIP NET NN C TE SXT R I S
1. Swab/pharynx 0.25 R R R R S S S S S S S S S S I R 5 1 10
2. Swab/pharynx 0.25 R R R R S S S S I S S S S S I R 5 2 9
3. Swab/nose 0.5 S S S S S S S S R S S S R S R S 3 13
4. Secretion/bronchical 0.25 R I I S S S R S S S S S S I S S 2 3 11
5. Secretion/bronchical 0.5 R R I S S S R S S S S S S I S S 3 2 11
6. Swab/groin 0.5 R R R R S — S S S S S S S S R R R 6 9
7. Swab/groin 0.25 R I S S S S S S S S S S S S I S 1 2 13
8. Exudation/abdominal
cavity
0.5 RRS S S—R R R S S S SRRRR97
9. Swab/anus 0.25 S S S S S S S S S S S S S S S S — — 16
10. Swab/anus 0.5 R I I I S S S S S S S S S S I S 1 4 11
11. Swab/anus 0.5 I R S S S S S S S S S S S S R R 3 1 12
12. Swab/anus 0.5 S S S I S S S S S S S S S I R S 1 2 13
13. Swab/bedsore 0.5 I R R R R S I R S S R R R R R R 11 2 3
14. Swab/bedsore 0.5 S I S I S S S I S S S S S S S S — 3 13
15. Swab/bedsore 0.5 R R S S S S S S S S S S S S R S 3 13
16. Swab/bedsore 0.5 R R R R S R R R S S R R S S R R 11 — 5
17. Swab/bedsore 0.25 R S S S S S S S S S S S S S R S 2 14
18. Swab/ulceration 0.5 R R R R R R R R S S R R R R R R 14 — 2
19. Swab/ulceration 0.25 R R R S S — — R S S S S S S S S R R 6 10
20. Swab/wound 0.25 S S S S S S S S S S S S S S R S 1 15
21. Swab/wound 0.5 R R S R S — S S R S S S S S S R R 6 10
22. Swab/wound 0.5 R R S R S — S S S S S S S S R R R 6 10
23. Swab/wound 0.25 I I S S S S S S R S S S R S R R 4 2 10
24. Swab/wound 0.25 R S S S S S S S S S S S S S I S 1 1 14
25. Swab/wound 0.25 R R R R R S R S S S R R R S R R 11 — 5
26. Swab/wound 0.25 R R R R R S S S R S R R R S R R 11 5
27. Urine 0.5 I R S S S R S S R S S S S S S S S R S 4 1 14
28. Urine 0.5 R R R S S R S S R S S S S S S S R R S 7 — 12
29. Urine 0.25 R R R S S R R S S S S S S R S S S R R 8 11
30. Urine 0.5 R R R I S R I S R S S S S I S S I R I 6 5 8
AMC, amoxicillin/clavulanic acid; CF, cefalotin; CZ, cefazolin; CXM, cefuroxime; NOR, norfloxacin; CTX, cefotaxim; CAZ, ceftazidime; ATM, aztreonam; NET, netilmicin; NN, tobramycin.
142
Table 5. Characteristics of Pseudomonas aeruginosa Isolates
Susceptibility to antibacterials Total
No.
Pseudomonasaeruginosa
strain/clinical material
MIC of thyme
essential oil ll/ml MZ PIP CAZ GM NN AMC TZP CTX ATM IPM MEM NET CIP SXT C CL R I S
1. Swab/ear 1.0 S S S R S R S S S S S S S S S S 2 — 14
2. Swab/ear 1.0 S S S I S I S S S S S S S S S S 2 14
3. Swab/pharynx 2.5 R S S S S R S R S S S S I R R S 5 1 10
4. Swab/pharynx 2.0 R S S S S R S R S R S S S R R R 7 9
5. Swab/groin 1.5 R R R R R I S R R S R S R R R R 12 1 3
6. Swab/toe 1.5 S S S S S S S R R S R S R R R R 7 — 9
7. Swab/anus 2.5 S S S S S S S R S S S S S R R R 4 — 12
8. Swab/anus 2.5 S S S R R S S R S S S S S R R R 6 — 10
9. Sputum 1.0 S S S S S R S S S S S S S S R I 2 1 13
10. Secretion/bronchical 2.0 R R S I S R S R R R R S R R R S 10 1 5
11. Secretion/bronchical 2.5 R R R R R R R R R R R R R R R R 16
12. Secretion/bronchical 2.5 R R R R R R R R R R R R R R R R 16
13. Swab/bedsore 1.5 R R R S R R S I R S R I I R R R 10 3 3
14. Swab/bedsore 1.5 R R S S S R S R S S S R R R R S 8 8
15. Swab/bedsore 1.5 R S S S S R S R S S S S S R R S 5 11
16. Swab/bedsore 2.0 R S S S S R R R S S S S S R R R 7 — 9
17. swab/bedsore 2.0 R R S S S R R R S S S R R R R S 9 — 7
18. Swab/bedsore 2.0 R S S S S R S R S S S S S R R R 6 10
19. Swab/wound 1.5 S R S S R R S S S S R S S R R R 7 — 9
20. Swab/wound 1.5 R R S S I R S S S S R S S R R S 6 1 9
21. Swab/wound 2.0 S R S S I R S S S S R S S R R S 5 1 10
22. Swab/wound 2.0 R S S S S R S R S S S S S R R R 6 10
23. Swab/wound 1.5 R S S S S R S I S S S S S R R S 4 1 11
24. Swab/wound 1.5 R S S S S R R R S S R S S R R R 8 — 8
25. Swab/ulceration 2.5 S S S S R R S R S S R S S R R S 6 — 10
26. Swab/ulceration 2.5 R S S S S R R R S S R S S R R R 8 — 8
27. swab/ulceration 2.5 R S S S S R S R S S S S S R R R 6 10
28. Swab/ulceration 2.0 R S S S S R S I S S S S S R R S 4 1 11
29. Swab/ulceration 2.0 R R S S I R S S S S R S S R R S 6 1 9
30. Swab/ulceration 2.0 R R S S S R R R S S S R R R R S 9 — 7
MZ, mezlocillin; PIP, piperacillin; TZP, piperacillin/tazobactam; MEM, meropenem.
143
five isolates for which the MIC was 0.75 ml/ml, isolated from
diverse materials, and with different susceptibility to tested
drugs.
The increasing prevalence of methicillin resistance among
S. aureus strains is an increasing problem, one that has re-
newed interest in treating S. aureus infections with CC.
However, widespread use of MLS
B
antibiotics has led to an
increase of resistance to them.
15
Although Prabhu et al. re-
ports that 28.42% of S. aureus strains are resistant to TE, our
results showed much greater resistance to TE as well as
CC.
41
Resistant strains were a significant problem in our
study -66.6% of the strains were resistant at the same time to
at least three antibiotics. However, they were sensitive to
glycopeptides and generally to LZD and chloramfenicol.
Only 10% of the strains that were susceptible/had interme-
diate susceptibility to all drugs.
Most of the resistant isolates occurred in the ICU, and the
major sites of isolation were swabs (wounds, nose, or ul-
ceration), which is in accord with the literature.
41,57
For-
tunately, despite the fact that there were many multidrug
resistant strains, all of them showed susceptibility to ‘‘last
line’’ antibiotics.
In accordance with the literature, T. vulgaris L. oil was
shown to inhibit the growth of S. aureus strains isolated from
respiratory infections. The tested strains of S. aureus, sensi-
tive to 0.0125 ml/ml of oil, were resistant to oxacylin, GM,
and NN, and many of them, to NOR.
19
Oil obtained from
Thymus fontanesii Boiss. Et Reut. containing carvacrol at a
FIG. 1. Standard strains susceptibility to thyme essential oil. MIC, minimal inhibitory concentration.
FIG. 2. Clinical strains of Staphylococcus aureus, Enterococcus sp., Escherichia coli, and Pseudomonas aeruginosa susceptibility to
thyme essential oil.
144 SIENKIEWICZ ET AL.
concentration of 0.3 ml/ml inhibited the growth of the stan-
dard and clinical strains of S. aureus isolated from clinical
materials.
4
The oil was derived from the carvacrol chemo-
type of Thymus ciliatus (Desf.) Benth. ssp. eu-ciliatus Maire.
Thyme oil was also found to inhibit the growth of pristina-
micin-sensitive S. aureus strains isolated from respiratory
diseases with MIC 0.8 ml/ml; our results were similar.
7
Re-
search on the antimicrobial properties of thyme oils obtained
from different chemotypes of Thymus spinulosus Ten. con-
firms the relationship between inhibitory activity and the
thymol and carvacrol content. These oils contained myrecene,
limonene, and g-terpinene as the dominant components and
low concentration of phenolic compounds. Their antimicrobial
activity was less effective, with MIC values ranging from 2.25
to 9.0 ml/ml against the standard strain of S. aureus ATCC
25923 and also against En. faecalis ATCC 29212.
14
Many clinical strains of Enterococcus spp. were sensitive to
thyme oil at concentrations of 0.5 (n=11) and 0.75 ml/ml
(n=16), but En. faecalis isolate was inhibited at 1.25 ml/ml.
These strains came from diverse materials and hospital
wards. The En. faecium strains were, in general, more resis-
tant to antibiotics than En. faecalis strains. Fortunately, no
isolates were resistant to last-line drugs: glycopeptides, SYN,
and LZD. However, resistance to TE (n=25, 83.3%), E (n=19,
63.3%), CIP (n=20, 66.6%), GM, and S (n=17, 56.6%) was
common. Strains inhibited by 1.25 or 0.75 ml/ml of the es-
sential oil were isolated from different hospital wards and
presented diverse susceptibility to antibiotics.
In the last two decades, the importance of En. faecium as a
nosocomial pathogen has increased throughout the world
due to the greater ability of this species to acquire resistance
to drugs than En. faecalis. Very often, resistance among en-
terococci results from the presence of a putative pathoge-
nicity island. What is interesting is that an increase in
AM-resistant En. faecium usually precedes increasing rates of
VA-resistant strains.
10,54
In our study, there were no isolates
showing resistance to VA, but 10 of 11 En. faecium strains
were resistant to AM. Regarding TE, although the entero-
cocci resistance rate decreased over time, it remains high in
some centers (57%).
10
In our study, the resistance to TE was
even higher (83.3%), especially among En. faecalis strains. In
addition, erythromycin may be ineffective in therapy. Re-
sistance worldwide is very high, which is in accord with our
results.
6,46
Overall, rates of high-level resistance to ami-
noglycosides were higher than those observed recently in
Brazil and in the United States.
23,45
Due to the high content of active phenols and p-cymene,
the oil of T. vulgaris L. showed a very strong activity against
standard strains of Enterococcus genus, with MIC values
ranging from 0.0625 to 0.85 ml/ml in our studies. MIC values
obtained for the clinical strains cultured in the presence of
the oil were also significantly lower (0.25–1.25 ml/ml). Men-
tion is made within the literature of the inhibiting proper-
ties of thymol from Thymus sp. species on the adhesion of
S. aureus and Es. coli clinical strains to epithelial cells of
genitourinary system, which may be an alternative to syn-
thetic drugs in the prevention of urinary tract infections.
12
We have shown that clinical strains of Es. coli were sensi-
tive to thyme oil at concentrations of 0.25 ml/ml (13 strains)
and 0.5 ml/ml (17 strains). They were more sensitive to oils
than P. aeruginosa, S. aureus, and Enterococcus spp. strains. As
far as susceptibility to antibiotics was concerned, many iso-
lates were resistant to AMP and TE (n=21, 70%), CF (n=19,
63.3%), SXT (n=15, 50%), and CZ (n=12, 40%). However, all
the isolates were sensitive to IPM, and most of them were
susceptible to ATM, CAZ, and NET. Strains inhibited by
0.5 ml/ml of essential oil were isolated from diverse materi-
als, in different hospital wards, and, generally, were more
resistant to antibiotics than strains inhibited by 0.25 ml/ml.
There was an increase observed in antimicrobial resistance
in Es. coli between 2002 and 2009. It was suggested that the
observed trends regarding resistance to third-generation
cephalosporins, a more than fivefold overall growth in re-
sistance (from 0.6% to 3.4%), may be the result of the addi-
tion of resistance traits to strains that were already
resistant.
22
It may be explained by the spread of multidrug-
resistant plasmids that also contain genes for the production
of extended-spectrum beta-lactamase (ESBL).
2,35
Resistant
strains were a significant problem in our study. The most
resistant strains came from wounds (n=13) and were resis-
tant at the same time to even 11 or 14 of the tested drugs.
Contrary, there were two isolates (n=6.6%) entirely suscep-
tible to antibiotics and seven that were resistant to one or two
of them (n=23.3%).
Studies on the antimicrobial properties of the essential oil
obtained from T. fontanesii Boiss. Et Reut. containing carva-
crol explain its very strong activity against clinical strains of
Es. coli, with an MIC value of -0.35 ml/ml.
4
Similar MIC
values were obtained for the carvacrol chemotype of T. ci-
liatus (Desf.) Benth. ssp. eu-ciliatus Maire eu. against clinical
strains of colon bacilli isolated from the respiratory system.
7
In our tests, clinical strains of Es. coli were sensitive to thyme
oil at concentrations of 0.25 and 0.5 ml/ml.
P. aeruginosa strains were sensitive to thyme oil at con-
centrations of 1.0–2.5 ml/ml. Many strains required 2.0–
2.5 ml/ml of the essential oil to be inhibited; therefore, they
were the most resistant when compared with other tested
bacterial species. At the same time, some isolates obtained
from bronchial secretions were resistant to all tested antibi-
otics (n=2). Besides, the great majority of strains were re-
sistant to C (n=28, 93.3%), STX (n=27, 90%), AMC (n=25,
83.3%), PIP and MZ (n=21, 70%), and CTX (n=20, 66.6%).
Fortunately, most isolates were susceptible to CAZ (n=4),
GM (n=5), IPM, and NET (n=5). Eight strains with an MIC
of 2.5 ml/ml were isolated from diverse materials, and their
resistance to antibiotics was similar to isolates more sus-
ceptible to the essential oil.
A lot of published studies on antibiotic use and resistance
have reported increasing resistance among P. aeruginosa
strains,
21
some show a decrease in CIP and CAZ resistance
among P. aeruginosa isolates as a result of decreased use of
those antibiotics.
37
There is also a global problem with
MBLs P. aeruginosa strains, which are very often resistant to
other important groups of antibiotics tested, including third-
generation cephalosporins, aminoglycosides, and quino-
lones.
8,13
In our study, there were some isolates resistant not
only to IPM but also to all the other antibiotics tested, which
could cause problems with therapy. In this situation, the only
therapeutic option may be polymyxins, which should not be
used as monotherapy.
53
The most resistant to the tested oil were clinical strains of
P. aeruginosa, where inhibition of growth fell in the range of
1.5–2.5 ml/ml. Similar MIC values were obtained using the
disk-diffusion method for oil obtained from Thymus persicus
THE ANTIMICROBIAL ACTIVITY OF THYME ESSENTIAL OIL 145
L. (thymol -10%, carvacrol -25%) and Thymus eriocalyx
(Ronniger) Jalas (thymol -66%).
24
The action of T. spinulosus
Ten. essential oil, which has a much lower content of active
phenolic compounds (thymol) than that of the oil derived
from T. vulgaris L., was much weaker against blue pus bacilli.
The obtained MIC values were within the limits of 4.5–
9.0 ml/ml, which was in accord with the literature.
44
Other
species of thyme such as Thymus zygis L., T. serpyllum L.,
T. kotschyanus Boiss. & HoH., T. persisus L., and T. longicaulis
C. Presl also demonstrate antibacterial activity due to the
active phenol content.
7,14
The literature reports not only the antimicrobial properties
of thyme and oregano essential oils, but also their antioxi-
dant properties as demonstrated by chemotypes from Thy-
mus munybyanus De Noe, T. pallescens De Noe, T. numidicus
Poiret, T. guyonii De Noe and oregano: Origanum glandulo-
sum Desf., and O. floribundum Munby.
29
Studies on the bio-
logical properties of savory oil (Satureja hortensis L.),
containing phenols as predominant compounds, showed
antioxidant and antimicrobial properties against standard
strains of Gram-positive and Gram-negative bacteria.
27
Due to the bactericidal and fungicidal properties of es-
sential oils, their use in pharmaceuticals and food are more
and more widespread as alternatives to synthetic chemical
products. Essential oils or some of their components are used
in perfume products, in sanitary products, in dentistry, in
agriculture, as food preservers and additives, and as natural
remedies.
28,39
Essential oils are effective in anticancer ther-
apy, cardiovascular, and nervous system disorders; they
lower cholesterol levels, decrease and regulate glucose level,
and have antioxidative properties.
17
Now, investigations on
the mechanism of action of essential oils and their compo-
nents are carried out both in vitro and in vivo on ani-
mals.
5,38,47,48
Analytical monographs have been published
(National Pharmacopeia, European Pharmacopoeia, ISO,
WHO, Council of Europe) to ensure the necessary informa-
tion about essential oils: their source, concentration of active
components, and therapeutic doses. The cytotoxic capacity of
essential oils based on their pro-oxidant activity can make
them excellent antiseptic and antimicrobial agents. A big
advantage of essential oils is the fact that they are usually
devoid of long-term genotoxic risks. Moreover, some of them
show a very clear antimutagenic activity that could well be
linked to an anticarcinogenic activity. Recent studies have
demonstrated that the pro-oxidant activity of essential oils or
some of their constituents, as also that of some polyphenols,
is very efficient in reducing local tumor volume or tumor cell
proliferation by apoptotic and necrotic effects.
32,36,50,51,56
However, essentials oils may be toxic not only against
bacteria, fungi, or viruses but also can have an adverse effect
on the human body when overdosed. In eukaryotic cells,
essential oils can provoke depolarization of the mitochon-
drial membranes by decreasing the membrane potential, af-
fect ionic Ca
2+
cycling and other ionic channels, and reduce
the pH gradient.
52
Some essential oils contain photoactive
molecules such as furocoumarins. The essential oils posses-
sing phototoxic activity are obtained from some plant fami-
lies, such as Apiaceae Rutaceae, Polygonaceae, and
Hypericaeae. This may cause damage to cellular macromol-
ecules and, in some cases, the formation of covalent adducts
to DNA, proteins, and cellular membrane.
16
Some essential
oils or rather some of their constituents may be considered
secondary carcinogens after metabolic activation.
26
For ex-
ample, psoralen, a photosensitizing molecule found in some
essential oils, for instance from Citrus bergamia, can induce
skin cancer after formation of covalent DNA adducts under
ultraviolet A or solar light.
3
In addition, pulegone, a com-
ponent of essential oils from many mint species, can induce
carcinogenesis through metabolism generating the glutathi-
one depletory p-cresol.
58
The application of essential oils in the treatment of many
human diseases, particularly infectious diseases caused by
multidrug resistant bacterial strains, may be an interesting
alternative for synthetic drugs that also show side effects.
Essential oils used in combination with antibiotics might
prevent antibiotic-resistant strain formation. Due to the
therapeutic problems associated with particularly resistant
strains, essentials oils can be useful in fighting diseases
caused by nosocomial pathogens.
Conclusions
Thyme oil obtained from T. vulgaris L.:
1. Shows very strong activity against standard and clinical
strains belonging to: Staphylococcus sp., Enterococcus sp.,
Escherichia sp., and Pseudomonas sp. genus.
2. It shows lower efficacy against clinical strains of P.
aeruginosa.
3. It is active against clinical strains resistant to most tes-
ted antibiotics.
Acknowledgments
The authors wish to thank Danuta Kalemba for thyme oil
analysis. The research reported in this article was supported
by grant no 503/5015-02/503-01 and has not been submitted
elsewhere.
Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Monika Sienkiewicz, Ph.D.
Medical and Sanitary Microbiology Department
Medical University of Lodz
Plac Hallera 1
90-647 Lodz
Poland
E-mail: monika.sienkiewicz@umed.lodz.pl
148 SIENKIEWICZ ET AL.
... The results revealed the strong antimicrobial activity of the tested thyme oil against multidrug resistant microbes previously mentioned. The authors would relate the bioactivity of thyme oil to its main volatile ingredients pcymene and thymol recording 29.10 and 38.10%, respectively [34]. The GC-MS analysis of the Egyptian Thymus vulgaris oil in this current study revealed its enrichment with eucalyptol (1,8 cineole) by (24.3%), in addition to thymol (17.4%). ...
... The GC-MS analysis of the Egyptian Thymus vulgaris oil in this current study revealed its enrichment with eucalyptol (1,8 cineole) by (24.3%), in addition to thymol (17.4%). Eucalyptol itself possessed potent antimicrobial activity as reported in previous works [34,35]. The volatile oil of Mentha virdis enriched with mainly carvone "monoterpene ketone" 42.50% followed by eucalyptol "monoterpene oxide" 17.40% then finally dihydrocarveol "monoterpene alcohol" 13.00% as in Figure 2c and Table 4 where these data matched with the previous literature by Mkaddem et al., 2022 [36] upon which an analysis of the oil obtained from Mentha virdis collected from Tunisia. ...
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The purpose of this study was to demonstrate the antimicrobial effects of natural essential oils (EO) and determine their preservative action. Eight natural essential oils were tested against Staphylococcus aureus, Escherichia coli, and Candida albicans representing gram positive, gram negative, and fungi, respectively. The plant materials were used in this study viz. Thymus vulgaris—thyme (TV), Mentha virdis (MV), Mentha longifolia (ML), Rosmarinus officinalis—rosemary (RO), Lavandula dentata—lavender (LD), Origanum majorana—oregano (OM), which belong to the Lamiaceae family. The other two plants were Cymbopogon citratus—lemon grass (family Poaceae) (CC), and Eucalyptus globulus (family Myrtaceae) (EG). Employing the disc diffusion susceptibility test, minimum inhibitory and minimum bactericidal concentrations were estimated for each oil, followed by the addition of oils to pasteurized apple juice after microbial induction. The results revealed that thyme oil showed the maximum zone of inhibition against all tested microbes enriched with monoterpenes class viz. eucalyptol (24.3%), thymol (17.4%), and γ-terpinene (15.2%). All other tested oils exhibited a concentration-dependent inhibition of growth and their MIC ranged from 0.1 to 100 µL/mL. The recorded minimum bactericidal concentration values were apparently double the minimum inhibitory concentration. The EO of Mentha virdis followed by Mentha longifolia showed maximum antimicrobial activity against the tested organisms in pasteurized apple juice. A gas chromatography–mass spectroscopy (GC–MS) analysis of lemon grass, thyme, and Mentha virdis essential oils showed their enrichment with monoterpenes class recording 97.10, 97.04, and 97.61%, respectively.
... A less frequent one is Klebsiella pneumoniae [5]. Several studies have proven the antibacterial efficacy against E. coli of EO extracted from: Thymus vulgaris L., thyme (Lamiaceae) [6][7][8], other plants in the genus Thymus [9][10][11][12], Origanum vulgare L., oregano (Lamiaceae) [13,14], other plants in the genus Origanum [15], Eucalyptus globulus Labill., eucalyptus (Myrtaceae) [16], other plants in the genus Eucalyptus [17], Melaleuca alternifolia (Maiden and Betche) Cheel, tea tree (Myrtaceae) [18,19], Syzygium aromaticum (L.) Merrill and Perry Clove, clove (Myrtaceae) [20]. ...
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Background : In view of the high recurrence rate of urinary tract infections and the increasing number of germs resistant to multiple antibiotics, the aim of the present study was to evaluate the antibacterial properties of clove, oregano, thyme, eucalyptus, tea tree essential oils (EOs) against 32 isolates of Escherichia coli and 28 isolates of Klebsiella pneumoniae from patients with urinary tract infections (UTI). Methods : The agar disk diffusion method was used to assess the susceptibility of these isolates to essential oils and the minimal inhibitory concentration (MIC), and the minimal bactericidal concentration (MBC) were determined. Results : Our results suggest that volatile phenols (such as carvacrol in oregano EO, thymol in thyme EO, and eugenol in clove EO) are more efficacious as antibacterial than non-aromatic compounds (such as eucalyptol in eucalyptus EO and terpinene derivatives in tea tree EO). Conclusion : The oregano EO, followed by thyme appear to have the highest efficacy against Escherichia coli and Klebsiella pneumoniae isolates investigated.
... There was no growth inhibition in the disc impregnated with DMSO, an indication that it has no activity against R. solanacearum. (Sienkiewicz et al., 2012). Using disc diffusion test to study the antibacterial and antifungal activity of eugenol, Pavesi et al. (2018) reported inhibition zones of 12.1, 4.2 and 3.2 mm for Candida albicans, E. coli and Staphylococcus aureus, respectively. ...
Bacterial wilt disease caused by Ralstonia solanacearum impacts negatively on potato production. This study evaluated the effects of thymol and eugenol encapsulated in chitosan nanoparticles (TCNP and ECNP) on the virulence genes (PhcA, XpsR and HrpG) of R. solanacearum and their in vivo efficacy against bacterial wilt. Gene expression levels of virulence genes in the presence of TCNP (5.6 and 11.3 µg mL⁻¹) and ECNP (11.3 and 22.5 µg mL⁻¹) together with those of plant defense genes (chitinase and β-l,3-glucanase) were determined through RT-qPCR. All the virulence genes were downregulated when exposed to both TCNP and ECNP while glucanase and chitinase genes increased and peaked after 18 hours post inoculation. The lowest disease severity index (10.3%) was recorded with plants treated with 90 µg mL⁻¹ ECNP. The results of this study show that both TCNP and ECNP have a potential to be used as bacterial wilt bactericides.
... Almost a decade ago Sienkiewicz et al. explored the antimicrobial activity of thyme essential oil against clinical multidrug resistant Staphylococcus, Enterococcus, Escherichia, and Pseudomonas genera, that were isolated in the hospital setting from infections of the oral cavity, abdominal cavity, respiratory tract, genitourinary tract, and skin. Using an agar diffusion method, the essential oil of thyme was shown to strongly inhibit the growth of the tested clinical strains, suggesting that it may be reasonable to investigate it as a phytopharmaceutical for treatment and prevention of bacterial infections caused by both Gram-positive and Gram-negative bacteria [97]. The results were reproducible with a panel of 30 E. coli strains isolated from patients with various conditions, where thyme essential oil was active against all the strains and was more potent than other oils [98]. ...
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A perennial wild shrub from the Lamiaceae family and native to the Mediterranean region, thyme is considered an important wild edible plant studied for centuries for its unique importance in the food, pharmaceutical, and cosmetic industry. Thyme is loaded with phytonutrients, minerals and vitamins. It is pungent in taste, yet rich in moisture, proteins, crude fiber, minerals and vitamins. Its chemical composition may vary with geographical location but is mainly composed of flavonoids and antioxidants. Previous studies have illustrated the therapeutic effects of thyme and its essential oils, especially thymol and carvacrol, against various diseases. This is attributed to its multi-pharmacological properties that include, but are not limited to, antioxidant, anti-inflammatory, and antineoplastic actions. Moreover, thyme has long been known for its antiviral, antibacterial, antifungal, and antiseptic activities, in addition to remarkable disruption of microbial biofilms. In the COVID-19 era, some thyme constituents were investigated for their potential in viral binding. As such, thyme presents a wide range of functional possibilities in food, drugs, and other fields and prominent interest as a nutraceutical. The aims of the current review are to present botanical and nutritive values of this herb, elaborate its major constituents, and review available literature on its dietetic and biological activities.
... The large inhibition zones (>25 mm) and low MIC values (0.1 mg/mL) recorded here related to thyme EO confirm the findings of previous authors [31]. Its strong antimicrobial effect is attributed to its high phenolic compound content, including thymol and carvacrol, which constitute > 40% of this oil [32]. Kačániová et al. have previously shown that cinnamon EO was very effective against B. subtilis, where the MIC value was 0.10 mg/mL, similarl to our results in the cases of all the tested bacteria [33]. ...
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Pitted keratolysis (PK) is a bacterial skin infection mostly affecting the pressure-bearing areas of the soles, causing unpleasant symptoms. Antibiotics are used for therapy, but the emergence of antiobiotic resistance, makes the application of novel topical therapeutic agents necessary. The antibacterial effects of 12 EOs were compared in the first part of this study against the three known aetiological agents of PK (Kytococcus sedentarius, Dermatophilus congolensis and Bacillus thuringiensis). The results of the minimal inhibitory concentration, minimal bactericidal concentration and spore-formation inhibition tests revealed that lemongrass was the most effective EO against all three bacterium species and was therefore chosen for further analysis. Seventeen compounds were identified with solid-phase microextraction followed by gas chromatography–mass spectrometry (HS-SPME/GC-MS) analysis while thin-layer chromatography combined with direct bioautography (TLC-BD) was used to detect the presence of antibacterially active compounds. Citral showed a characteristic spot at the Rf value of 0.47, while the HS-SPME/GC-MS analysis of an unknown spot with strong antibacterial activity revealed the presence of α-terpineol, γ-cadinene and calamenene. Of these, α-terpineol was confirmed to possess an antimicrobial effect on all three bacterium species associated with PK. Our study supports the hypothesis that, based on their spectrum, EO-based formulations have potent antibacterial effects against PK and warrant further investigation as topical therapeutics.
... Several works describe even broadly known EO, like, Syzygium aromaticum L. [35], Mentha piperita L. [36], Origanum vulgare L. [37,38], Cinnamomum cassia Persl. [35], Rosmarinus officinalis L. [38], Cymbopogon citratus DC. [37] and Thymus vulgaris L. EO [39] as large spectrum antimicrobial agents. In 2019, Malik reported that EO obtained from Thymus vulgaris L., Origanum vulgare L., Syzygium aromaticum L., Ocimum basilicum L., Myristica fragrans Houtt. ...
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Adhesion is an important starting event in the pathogenesis of bacterial infection because the microorganisms must first adhere to host tissue in order to multiply and create a colony or colonies before specific symptoms allow the disease process to be detected. This is particularly true in the case of female urogenital infections, including urinary tract infections, bacterial vaginosis and vaginitis. Thymol is a component of thyme essential oil, which has been reported to possess interesting antimicrobial effects on various microorganisms; however, its ability to interact with the adhesion of bacteria (an important determinant of bacterial virulence) has not been investigated. The aim of this study was to assess whether thymol interferes with the adhesion of Escherichia coli and Staphylococcus aureus to human vaginal epithelial cells. The adhesiveness of three strains of E. coli to vaginal cells was significantly reduced at thymol concentrations ranging from 1/2 MIC to 1/32 MIC, and in three strains of S. aureus at concentrations ranging from 1/2 MIC to 1/16 MIC. Sub-MICs down to 1/8 MIC also significantly reduced the hemagglutination of E. coli, which is correlated with fimbriation and thus provides a clue relating to the interference of thymol, a phenolic structure with an hydroxyl group, on the physicochemical characteristics of the outer surface of bacteria. This is of interest for the strategy of protecting against vaginitis or vaginosis using drugs other than antibiotics.