Arch. Pharm. Res. DOI 10.1007/s12272-014-0516-0
RESEARCH ARTICLE
Chemical composition and anti-biofilm activity of Thymus sipyleus BOISS. subsp. sipyleus BOISS. var. davisianus RONNIGER essential oil Ozgur Ceylan • Aysel Ugur
Received: 13 June 2014 / Accepted: 4 November 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract In this study, antimicrobial and antibiofilm activities and the chemical composition of Thymus sipyleus BOISS. subsp. sipyleus BOISS. var. davisianus RONNIGER essential oil was evaluated. The essential oil was obtained by hydro-distillation and analyzed by gas chromatography-mass spectrometry. Fourteen compounds were characterized, having as major components thymol (38.31 %) and carvacrol (37.95 %). Minimum inhibitory concentrations (MICs) of oil and the major components were calculated by serial dilution method, and anti-biofilm effects by microplate biofilm assay against five Gram positive (Staphylococcus aureus MU 38, MU 40, MU 46, MU 47, Stahylococcus epidermidis MU 30) and five Gram negative (Pseudomonas aeruginosa MU 187, MU 188, MU 189, Pseudomonas fluorescens MU 180, MU 181) bacteria. It was found that MICs for essential oil, thymol and carvacrol were between 5 and 50 ll/ml, 0.125–0.5 lg/ml and 0.125–05 ll/ml, respectively. The results showed that doses of MIC produced a greater anti-biofilm influence than 0.5, 0.25 and 0.125 MIC. In the presence of essential oil (MIC), the mean biofilm formation value was equal to 67 ± 5.5 % for P. aeruginosa MU 188, and essential oil (MIC) inhibition exceeds 60 % for P. aeruginosa biofilms. The results also showed that carvacrol (MIC) was able to induce an inhibition 72.9 ± 4.1 % for S.aureus (MU 40) biofilm. In addition, thymol (MIC) showed 68.6 ± 5.3 % reduction in biofilm formation of P. fluorescens MU 181. O. Ceylan (&) Apiculture Program, Ula Ali Kocman Vocational School, Mugla Sıtkı Koc¸man University, Ula, Mugla, Turkey e-mail: [email protected] A. Ugur Department of Basic Sciences, Section of Medical Microbiology, Faculty of Dentistry, Gazi University, Emek, Ankara, Turkey
This study demonstrated the antimicrobial and antibiofilm activity of T. sipyleus BOISS. subsp. sipyleus BOISS. var. davisianus RONNIGER essential oil and points out the exceptional efficiency of thymol and carvacrol, which could represent candidates in the treatment of Pseudomonas and Staphylococcus biofilms. Keywords Thymus sipyleus subsp. sipyleus var. davisianus Chemical composition Antibiofilm activity Antimicrobial activity Pseudomonas spp. Staphylococcus spp.
Introduction Biofilms are multicellular matrices which form by the attachment of bacteria by means of physical appendages to a surface followed by coating with extracellular polysaccharides. There are many advantages to an organism forming a biofilm, including protection from antibiotics, disinfectants and dynamic environments (Garrett et al. 2008). In the public health sector, the colonization of medical surfaces, such as catheters and other indwelling devices, by biofilms, plays a decisive role in the problem of healthcare-associated infections (Hammond et al. 2010). Staphylococci are important nosocomial pathogens. Eradication of these micro-organisms is not always successful due to their ability to form biofilms. Experimental evidence has shown that microorganisms in biofilms are less susceptible to conventional treatment (Brown and Gilbert 1993). In addition, S. aureus biofilms are widely found on implanted biomedical devices, accounting (together with Staphylococcus epidermidis) for more than one-half of prosthetic device-associated infections (Fluckiger et al. 2005). Pseudomonas aeruginosa is a species able
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O. Ceylan, A. Ugur
to form biofilms on different abiotic surfaces, including artificial implants, contact lenses, urinary catheters and endotracheal tubes (Davey and O’Toole 2000). On the other hand, as an opportunistic pathogen, P. aeruginosa can cause various infections, which are difficult to treat with conventional antibiotics. Some of the infections, such as periodontitis, prostatitis and infections of patients with cystic fibrosis are considered to be in connection with bacterial attached mode of growth on the biotic surfaces (Lyczak et al. 2002; Hanlon 2007). The safe and traditional folkloric use of plant derived compounds for the prevention and treatment of diseases and infections make wide acceptance of them in the search for alternative antimicrobial agents (Guarrera 2005). Earlier investigations on plants and their active constituents have almost exclusively focused on their effects on planktonic bacteria with little emphasis on the more antimicrobial resistant pathogens and difficult to control biofilm forms (Sandasi et al. 2010). Among the aromatic plants belonging to the family Lamiaceae, the genus Thymus is noteworthy for the numerous species and varieties of wild-growing plants. Many of these species are typical for the Mediterranean area (Consentino et al. 1999). This genus is represented by 39 species and altogether 64 taxa, 24 of which are endemic in Turkey and the East Aegean Islands (Davis 1982; Baser et al. 1995). Several Thymus species are locally known as ‘‘kekik’’ or ‘‘tas kekik’’ and their dried herbal parts are used in herbal tea, condiment and folk medicine (Tumen et al. 1995). It has interesting antibacterial properties (Karaman et al. 2001; Rasooli and Mirmostafa 2002; Rota et al. 2008; Hazzit et al. 2009; Fadli et al. 2012; Kazemi et al. 2012; Ruiz-Navajas et al. 2012; Hussain et al. 2013), which are related to the presence of phenolic compounds: carvacrol, thymol, c-terpinene and p-cymene (Nedorostova et al. 2008; Rota et al. 2008; Hazzit et al. 2009; Sarikurkcu et al. 2010; De Lisi et al. 2011; El Bouzidi et al. 2012; Fadli et al. 2012; Ruiz-Navajas et al. 2012; Hussain et al. 2013). The essential oils of some Thymus spp. are characterized by the presence of high concentration of the isomeric phenolic monoterpenes thymol and/or carvacrol (Baser 1995). In contrast to the numerous studies on antimicrobial activity and chemical composition of Thymus essential oil there are only limited data on the antibiofilm activity. Thus, the aim of the present study was to investigate chemical composition and to assess the in vitro antimicrobial and antibiofilm activities of essential oil obtained from aerial parts of T. sipyleus subsp. sipyleus var. davisianus. To the best of our knowledge, this is the first study to compare the chemical composition and biological activity of T. sipyleus subsp. sipyleus var. davisianus.
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Materials and methods Microorganisms and condition for cultivation Staphylococcus aureus (MU 38, MU 40, MU 46, MU 47), S. epidermidis (MU 30), Pseudomonas aeruginosa (MU 187, MU 188, MU 189) and Pseudomonas fluorescens (MU 180, MU 181) were used as test microorganisms. The strains coded MU (the multi-antibiotic resistant bacteria) used in this study were taken from Mugla University Culture Collection. The antibiotic resistance patterns of the multiresistant bacteria are shown in Table 1. The above-mentioned bacteria were cultured in nutrient broth (NB) (Difco, USA). P. aeruginosa and P. fluorescens strains were incubated at 30 ± 0.1 °C for 18–24 h. Other bacterial strains were incubated at 37 ± 0.1 °C for 24 h. Inocula were prepared by adjusting the turbidity of the medium to match the 0.5 McFarland Standard Dilutions of this suspension in 0.1 % peptone (w/v) solution in sterile water inoculated on NB to check the viability of the preparation. The cultures of microorganisms were maintained in their appropriate agar slants at 4 °C throughout the study and used as stock cultures.
Table 1 Antibiotic resistance patterns of Staphylococcus sp. and Pseudomonas sp Microorganisms
Resistance patterns
S. aureus MU 38
P, AK, DA, CN, ME, TEC, TE, OX
S. aureus MU 40
P, AK, CN, C, ME, OX, TE
S. aureus MU 46
P, AK, DA, E, CN, TE, OX
S. aureus MU 47
P, AK, DA, CN, OX
S. epidermidis MU 30
P, AK, DA, CN, OX, TEC, TE
P. aeruginosa MU 187
MEZ, PRL, PY, SAM, CFP, CRO, MEM, CN, OFX, C, SXT, AM, B
P. aeruginosa MU 188
MEZ, PRL, PY, SAM, CFP, CRO, C, SXT, AM, B
P. aeruginosa MU 189
MEZ, PRL, PY, SAM, CFP, CRO, OFX, C, SXT, AM, B
P. fluorescens MU 180
MEZ, PRL, PY, SAM, CFP, CRO, MEM, C, SXT, AM, B
P. fluorescens MU 181
PRL, PY, SAM, CFP, CRO, C, AM, B
P penicillin (10 U), AK amikacin (30 lg), DA clindamycin (2 lg), CN gentamicin (10 lg), C chloramphenicol (30 lg), E erythromycin (15 lg), ME methicillin (5 lg), TEC teicoplanin (30 lg), TE tetracycline (30 lg), OX oxacilline (1 lg), MEZ mezlocillin (75 lg), PRL piperacillin (100 lg), PY carbenicillin (100 lg), SAM sulbactam ? ampicillin (10 ? 10 lg), CFP cefaperazone (75 lg), CRO ceftriaxone (30 lg), MEM meropenem (10 lg), OFX ofloxacin (5 lg), SXT trimethoprim ? sulfamethoxazole (1.25 ? 23.75 lg), AM ampicillin (10 lg), B bacitracin (10 U)
Chemical composition and anti-biofilm activity of Thymus
Plant material Aerial parts of T. sipyleus subsp. sipyleus var. davisianus, belonging to the Lamiaceae family, were collected at full flowering stage, in May–July 2010 from Usak province of Turkey. A voucher specimens of plant were taxonomically identified by Ass. Prof. Dr. Mehtap Donmez SAHIN at the Faculty of Education, the University of Usak. These voucher specimens have been deposited at the Herbarium of the Department of Biology, University of Usak, Turkey. Dry aerial materials of plant were subjected to hydrodistillation for 4 h. The oil recovered was stored in darkness at 4 °C. Analysis of the essential oil The chemical composition of the essential oil were analyzed using gas chromatography–mass spectrometry (GC– MS). The GC–MS analyses were carried out using a Shimadzu QP 5050 (Kyoto, Japan) GC–MS system operating in the EI mode at 70 eV, equipped with an Cp-Wax 52 CB column (50 9 0.32 mm; film thickness 1.2 lm). The initial temperature of the column was 60 °C and then rose to 220 °C at a 2 °C/min rate. The carrier gas was helium, the flow rate being 10 psi. Components were identified on the basis of gas chromatographic retention indices, mass spectra from Wiley, NIST98 and Tutor spectral libraries. The relative amounts of each individual component of the essential oil was expressed as the percentage of the peak area relative to total peak area. Kovats retention indices (Kovats 1965) were calculated for each seperate component against n-alkanes standards (C8–C25, Alltech Associates, Deerfield, IL) according to Schomberg and Dielmann (1973), using CP-Wax 52 CB fused silica capillary column. Determination of minimal inhibitory concentrations The minimal inhibitory concentrations (MICs) of essential oil, and its major components (thymol and carvacrol) on planktonic cells were determined in Mueller–Hinton Broth (MHB) using a microtitre broth dilution method as recommended by the Clinical and Laboratory Standards Institute (CLSI 2006). The bacterial suspension was adjusted with sterile saline to a concentration of 5.0 9 105 CFU ml-1. Essential oil, thymol (C99.0 % pure, Aldrich) and carvacrol (C97.0 % pure, Aldrich) was diluted with ethanol (EtOH) at 1:1 ratio, and then with MHB as required. Cell suspensions (200 lL) were inoculated into the wells of 96-well microtitre plates (Nunc F96 MicroWellTM plates; NunclonTM D, Denmark) to get the final concentration ranging from 0.5 to 100 ll/ml for essential oil, 0.0625–1 lg/ml for thymol and 0.0625–1 ll/ml for carvacrol. The wells containing only
MHB and MHB with inoculum were employed as negative and positive controls, respectively. S. aureus and S. epidermidis inoculated microplates were incubated at 37 °C and P. aeruginosa ve P. fluorescens inoculated microplates were incubated at 30 °C for 24 h. The lowest concentration of the tested samples, which did not show any visual growth of tested organisms after macroscopic evaluation, was determined as MIC, which was expressed in ll/ml or lg/ml. Each assay was performed in triplicate for all bacteria. Biofilm inhibition assays The effect of different concentrations of essential oil, thymol and carvacrol (ranging from the MIC to 1/8 MIC) on biofilm forming ability was tested on polystyrene flatbottomed microtitre plates as described by (Meritt et al. 2005). Cultures were grown overnight in 5 ml TSB with 1 % glucose, diluted in growth medium to 5 9 105 CFU ml-1 and 100 ll was dispensed into each well of 96-well polystyrene flat-bottomed microtitre plates in the presence of 100 ll essential oil, thymol and carvacrol (1, 0.5, 0.25 and 0.125 MIC) or 100 ll medium (control). After incubation for 48 h at 37 °C, each well was washed with water to remove planktonic bacteria. The remaining bacteria were subsequently stained with 0.1 % crystal violet solution for 10 min at room temperature. Wells were washed once again to remove the crystal violet solution that had not specifically staining the adherent bacteria. Microplates were inverted and vigorously tap on paper towels to remove any excess liquid then air dried. 200 ll of 95 % ethanol and 33 % glacial acetic acid (Sigma Chemical Co) were poured in Pseudomonas spp. and Staphylococcus spp. wells respectively. Biofilm stains solubilized at room temperature. After shaking and pipetting of the wells, 125 ll of the solution from each well was transferred to a sterile tube and volume reached to 1 ml with distilled water. Finally, the optical density of each well was measured at a wavelength of 550 nm (Thermo Scientific Multiskan FC, Vantaa, Finland). Each strain was tested for biofilm production in duplicate and the assay was repeated three times. Replicate absorbance readings for each concentration tested were averaged and the average of the media control was subtracted. This value was then divided by the mean absorbance of the (cell ? TSB) and multiplied by 100.
Results and discussion Chemical analyses of the volatile constituents of the essential oil (percentage content of each compound, elution order, retention times and linear retention indices) are summarized in Table 2. In total, 14 compounds comprising
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O. Ceylan, A. Ugur Table 2 Composition of the essential oil of T. sipyleus subsp. sipyleus. var. davisianus Noa
RTb
Kovats RIc
Compounds name a-pinene
Percentage (%)
1
7.2
909
1.00
2
12.5
1058
myrcene
0.25
3
13.5
1083
a-terpinene
0.58
4
14.9
1118
1, 8-cineole
0.61
5
16.7
1163
c-terpinene
7.28
6
18.2
1200
p-cymene
4.16
7 8
28.2 29.8
1462 1506
1-octen-3-ol Terpineol
0.62 0.83
9
38.9
1783
trans-Caryophyllene
1.83
10
43.7
1947
Borneol
3.83
11
46.2
2036
Bisabolene
2.32
12
61.3
2716
a-bisabolene epoksit
13
68.9
2949
Thymol
38.31
14
70.3
2971
Carvacrol
37.95
0.33
a
Compounds listed in order of elution from a CP-Wax 52 CB column b
Retention time (as minutes)
c
Kovats retention indices calculated for CP-Wax 52 CB in GCMSD
more than 99 % of the total composition were identified in all cases. Thymol (38.31 %) and carvacrol (37.95 %) were the main oxygenated monoterpenes identified from the essential oil of T. sipyleus subsp. sipyleus var. davisianus. The other compounds were c-terpinene (7.28 %), p-cymene (4.16 %), borneol (3.83 %), bisabolene (2.32 %), trans-caryophyllene (1.83 %) and a-pinene (1.0 %). The rest of compounds were identified in trace concentrations. Previous studies on the essential oil of Thymus species have shown that it contains carvacrol and thymol as major components. Hussain et al. (2013) reported thymol
(36.5 %) and carvacrol (9.50 %) in T. linearis essential oil and carvacrol (44.4 %) in T. serpyllum essential oil. El Bouzidi et al. (2012) reported thymol (0.4–12.3 %) and carvacrol (26.0–71.6 %) in T. broussonetii, T. maroccanus and T. satureioides, are endemic Moraccan species. Fadli et al. (2012) reported carvacrol (76.35, 39.77 %) is the major constituent in T. maroccanus and T. broussonetii, respectively. Carvacrol was also reported as a major component by (Jirovetz et al. 2012) on the three thyme species studied. Hazzit et al. (2009) reported four T. pallescens oils have highly similar chemical composition characterized by carvacrol (44.4–57.7 %), p-cymene (10.3–17.3 %) and c-terpinene (10.8–14.2 %) as the major components. Rota et al. (2008) reported thymol (57.7, 68.1 %) and carvacrol (2.8, 3.5 %) in T. vulgaris and T. zygis subsp. gracilis, respectively. The chemical composition of the essential oil from T. guyonii de Noe restricted to Algeria, showed that the main components were carvacrol (55.55 %), thymol (19.51 %) and p-cymene (6.25 %) (Lehbili et al. 2013). The antibacterial activity of the essential oil of T. sipyleus subsp. sipyleus var. davisianus and the phenolic compounds (thymol and carvacrol) was initially evaluated by the microtitre broth dilution method using a multiple antibiotic resistant Staphylococci and Pseudomonas spp. The MICs of the two major compounds and whole essential oil are shown in Table 3. The essential oil of T. sipyleus subsp. sipyleus var. davisianus had MICs in the range of 5–50 ll/ml. The pure major components, thymol and carvacrol, were more active, with MIC (0.125–0.5 lg/ml for thymol and 0.125–05 ll/ml for carvacrol), compared to essential oil. Essential oil inhibited S. aureus (MU 40, MU 46, MU 47) at 5 ll/ml concentration. This result is important in terms of revealing the activity of essential oil against methicilin and oxacilline resistant S. aureus. This value has
Table 3 Minimum inhibitory concentrations (MICs) of T. sipyleus subsp. sipyleus var. davisianus essential oil, thymol, and carvacrol Microorganisms
Minimum inhibitory concentration Essential oil (ll/ml)
Thymol (lg/ml)
Carvacrol (ll/ml)
S. aureus MU 38
25
0.25
0.25
S. aureus MU 40
5
0.25
0.25
S. aureus MU 46
5
0.25
0.25
S. aureus MU 47
5
0.25
0.125
S. epidermidis MU 30
25
0.5
0.5
P. aeruginosa MU 187
50
0.125
0.25
P. aeruginosa MU 188 P. aeruginosa MU 189
25 50
0.125 0.25
0.25 0.5
P. fluorescens MU 180
5
0.125
0.25
P. fluorescens MU 181
5
0.125
0.25
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Chemical composition and anti-biofilm activity of Thymus Table 4 Effects of T. sipyleus subsp. sipyleus var. davisianus essential oil (EO), thymol (T), and carvacrol (C) on biofilm formation expressed as percentage inhibition
Microorganisms
Agent
% Inhibitiona MIC
0.5 MIC
0.25 MIC
0.125 MIC
S. epidermidis MU 30
EO
23.5 ± 3
T
35.7 ± 5.1
11.4 ± 0.91
8.2 ± 1
0.6 ± 0.5
C
53 ± 6
24.3 ± 1.9
8.5 ± 3.5
–
– – –
S. aureus MU 38
MU 40
MU 46
MU 47
EO
28.9 ± 5.4
20.4 ± 3.5
16.4 ± 2.1
T
20.1 ± 2.3
11.4 ± 1.7
4.7 ± 2.9
10.3 ± 1.3
C
69.9 ± 1.9
46.9 ± 1.8
26.6 ± 4.6
5.1 ± 1.2
EO
26.2 ± 3.5
18.4 ± 1.8
11.1 ± 1.3
7.7 ± 1.5
T
46.8 ± 3.1
29.1 ± 1.3
13.2 ± 4.9
–
C EO
72.9 ± 4.1 38.4 ± 4.6
50.7 ± 3.7 19.1 ± 2.5
25.4 ± 3.4 5.9 ± 1.2
13.3 ± 2.1 – –
–
T
42.9 ± 1.7
29.9 ± 1.6
15.4 ± 5.7
C
67.4 ± 1.8
49.1 ± 5.4
23.1 ± 3.2
EO
51.9 ± 9.4
11.2 ± 1.5
–
T
49.2 ± 1.4
35.7 ± 2.7
17.6 ± 1.6
–
C
61.1 ± 1.9
29.8 ± 9
4.1 ± 3.2
–
9.2 ± 3.6 –
P. fluorescens MU 180
MU 181
EO
27.5 ± 2.5
18.2 ± 1.3
6.6 ± 1.2
–
T
47.2 ± 4.7
32.4 ± 2.8
17.3 ± 2.4
–
C
61.4 ± 0.9
39.2 ± 2.6
22.6 ± 0.9
12.5 ± 7.4
EO
65.1 ± 4.9
43.9 ± 5.4
34.3 ± 2
24.9 ± 2.2
T
68.6 ± 5.3
48.9 ± 4.6
23.2 ± 2.6
9.9 ± 2.9
C
45.9 ± 0.5
38.6 ± 3.7
32.3 ± 4.8
12.7 ± 4.1
EO
60.3 ± 9.3
30.9 ± 7.7
26.9 ± 2.7
11.6 ± 1.5
T C
42.7 ± 3.1 54.1 ± 6.5
28.5 ± 2.8 40.5 ± 4.9
12.8 ± 1.2 23.2 ± 3.7
– – –
P. aeruginosa MU 187
MU 188
a
Values are reported as mean ± SD – no inhibition
MU 189
67 ± 5.5
32.9 ± 3.7
14.9 ± 2.2
T
EO
61.7 ± 0.4
43.2 ± 5.6
19.8 ± 3.2
–
C
41.2 ± 4.2
16.3 ± 3.2
6.8 ± 4.8
–
EO
63.7 ± 9.1
42.5 ± 7.4
37.6 ± 4.9
T
54.7 ± 4.1
33.4 ± 2.9
14.4 ± 4.1
–
C
47.7 ± 4.8
30.7 ± 3.2
14.7 ± 4.3
–
been identified as 25 ll/ml for the S. aureus MU 38 (methicilline and oxacilline resistant) and S. epidermidis MU 30 (oxacilline resistant). MIC of thymol were 0.25 lg/ ml for S. aureus strains and 0.5 lg/ml for S. epidermidis. Using carvacrol, a MIC of 0.125 ll/ml was observed for S. aureus MU 47 and 0.5 ll/ml for S. epidermidis MU 30. MIC was determined as 0.25 ll/ml for the other S. aureus strains. These results are consistent with previous studies (Rota et al. 2008; El Bouzidi et al. 2012; Fadli et al. 2012; Hussain et al. 2013) demonstrating that Thymus species’ essential oil rich in thymol and carvacrol possess antibacterial activity on the Staphylococcus spp. Essential oil
14.9 ± 1.5
presented moderate activity against both P. fluorescens (5 ll/ml). On the other hand, essential oil has low activity on the growth of P. aeruginosa which were only inhibited at high concentrations (25 or 50 ll/ml). This antimicrobial effect of essential oil, although at relatively high concentrations, was important because P. aeruginosa strains are multiple antibiotics resistant bacteria (Table 1). This study revealed that thymol showed maximum activity with MIC values ranging from 0.125 to 0.25 mg/ml followed by carvacrol with MIC values ranging from 0.25 to 0.5 ll/ml against all the tested Pseudomonas strains. In this study, thymol and carvacrol exhibited the highest antibacterial
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O. Ceylan, A. Ugur Fig. 1 Effects of essential oil, thymol, and carvacrol on biofilm formation of Staphylococcus spp.
Fig. 2 Effects of essential oil, thymol and carvacrol on biofilm formation of Pseudomonas spp.
effect against all Pseudomonas strains compared with essential oil. Thus, we confirmed the strongest antimicrobial activity of thymol and carvacrol against Pseudomonas sp. which had been previously described by several authors (Sokovic et al. 2010; Soumya et al. 2011). Bassole and Juliani (2012) reported antagonistic effects of the combinations of p-cymene and phenolic monoterpenes (thymol and carvacrol). It was also observed that carvacrol/thymol and carvacrol/myrcene worked antagonistically against S. aureus, B. cereus and E. coli (Gallucci et al. 2009). The different antibacterial activity exhibited by essential oil of
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T. sipyleus subsp. sipyleus var. davisianus when compared to thymol and carvacrol, suggesting that the minor components may contribute to an antagonistic effect on the activity of essential oil. This study also indicates that essential oil of T. sipyleus subsp. sipyleus var. davisianus has lower antimicrobial effects on Gram-negative bacteria than Gram-positive bacteria. This fact has been mentioned in other studies (Maksimovic et al. 2008; El Bouzidi et al. 2012; Fadli et al. 2012; Millezi et al. 2012; Hussain et al. 2013; Lehbili et al. 2013). However, various researchers obtained higher antimicrobial activity for Pseudomonas
Chemical composition and anti-biofilm activity of Thymus
strains with some Thymus essential oils (Adwan et al. 2006; Oussalah et al. 2006; Qaralleh et al. 2009). Essential oils are potential sources of novel anti-biofilm compounds especially against pathogen microorganisms (Nostro et al. 2007; Schillaci et al. 2008; Gursoy et al. 2009; Oral et al. 2010; Sandasi et al. 2010; Sandasi et al. 2011; Soumya et al. 2011). Therefore, the inhibitory effects of T. sipyleus subsp. sipyleus var. davisianus essential oil and major components (thymol and carvacrol) on the development of microbial biofilms were investigated against multiple antibiotic resistant strains of Pseudomonas and Staphylococcus. Our results showed that biofilm formation was significantly inhibited by essential oil, thymol and carvacrol but their effectiveness varied. This activity was dependent on the concentration used to treat the biofilm. Doses of MIC produced a greater influence than doses of 0.5, 0.25 and 0.125 MIC. The activities of essential oil and components tested at several concentrations against the total biomass of S. aureus and S. epidermidis biofilms are shown in Table 4 and Fig. 1. The in vitro activity of the essential oil on biofilm was only slightly lower than on planktonic culture. In the presence of 25 ll/ml essential oil (MIC), the mean biofilm formation values were equal to 23.5 % for S. epidermidis MU 30, and 28.9 % for S. aureus MU 38. At a concentration of 5 ll/ml essential oil, the biofilm formations of MU 40, MU 46 and MU 47 were inhibited to 26.2, 38.4 and 51.9 %, respectively. As the concentration of essential oil decreased, antibiofilm activity showed an obvious decrease in the surface coverage of microplate wells. In our study, carvacrol exhibited strong activity against the S. aureus and S. epidermidis biofilm development. Carvacrol inhibited the biofilm formation of four S. aureus used in the study at various ratios ranging from 61.1 to 72.9 % at MICs. This ratio was determined to be 53 % for S. epidermidis MU 30. The results showed that for S. aureus and S. epidermidis a moderate but not significant reduction in biofilm formation was achieved by treatment with thymol at MICs. The antibiofilm activity of thymol on Staphylococcus biofilm formations was observed lower than carvacrol. In the presence of thymol (MIC), the mean biofilm formation values were equal to 35.7 % for S. epidermidis MU 30 and 49.2 % for S. aureus MU 47. There are no studies in the literature on the anti-biofilm effect of T. sipyleus subsp. sipyleus var. davisianus essential oil, however, there is one study showing the anti-biofilm effect of carvacrol and thymol, the major constituents of T. sipyleus subsp. sipyleus var. davisianus, on S. aureus and S. epidermidis biofilms, at concentrations of 0.031–0.125 %, v/v, for carvacrol and thymol (Nostro et al. 2007). In contrast to Staphylococcus spp., antibiofilm activity of essential oil was more pronounced against Pseudomonas
spp. (Table 4 and Fig. 2). For essential oil, significant antibiofilm inhibition for P. aeruginosa was observed at MIC, inhibiting *65 % biofilm formation (Fig. 2). 5 ll/ml essential oil showed 67 % inhibition against P. aeruginosa MU 188. In the presence of 25 ll/ml essential oil (MIC), the mean biofilm formation values were inhibited to 63.7 % for P. aeruginosa MU 189 and 60.3 % for P. aeruginosa MU 187, respectively. According to our findings, essential oil was more effective on biofilm formation of P. aeruginosa than thymol and carvacrol. In the presence of thymol and carvacrol (MIC), the mean biofilm formations values were equal to 42.7 and 54.1 % for P. aeruginosa MU 187, 61.7 and 41.2 % for P. aeruginosa MU 188, and 54.7 and 47.7 % for P. aeruginosa MU 189, respectively. Treatment with essential oil with MIC gave rise to a 65.1 % inhibition of the P. fluorescens MU 181 biofilm formation. On the other hand, essential oil at concentration of MIC caused at 27.5 % decrease in P. fluorescens MU 180 biofilm formation. Thymol showed the strongest antibiofilm activity against the P. fluorescens MU 181 and it was induced 68.6 % inhibition of the biofilm when used at MIC. In the presence of carvacrol and thymol (MIC), the mean inhibition values were equal to 61.4 and 47.2 % for P. fluorescens MU 180 biofilm formation. Our results pointed out the in vitro potential of T. sipyleus subsp. sipyleus var. davisianus essential oil and major compounds such as thymol and carvacrol as antibiofilm agents and clearly demonstrate the feasibility of using essential oil to prevent biofilm formation. In addition, plant essential oil showed significant antimicrobial activity against Staphylococcus spp. planktonic cells and antibiofilm activity against Pseudomonas spp. biofilms. These results showed that the essential oil of T. sipyleus subsp. sipyleus var. davisianus could be considered as a natural alternative to treat biofilms of medical relevance and be used as an innovative plant-derived antimicrobial agent. Acknowledgments Authors thank Ass. Prof. Dr. M.D. Sahin, Botanist, Usak University, Usak, Turkey, for providing plant taxonomic identification. Conflict of interest
The author declare no conflict of interest.
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RESEARCH ARTICLE
Chemical composition and anti-biofilm activity of Thymus sipyleus BOISS. subsp. sipyleus BOISS. var. davisianus RONNIGER essential oil Ozgur Ceylan • Aysel Ugur
Received: 13 June 2014 / Accepted: 4 November 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract In this study, antimicrobial and antibiofilm activities and the chemical composition of Thymus sipyleus BOISS. subsp. sipyleus BOISS. var. davisianus RONNIGER essential oil was evaluated. The essential oil was obtained by hydro-distillation and analyzed by gas chromatography-mass spectrometry. Fourteen compounds were characterized, having as major components thymol (38.31 %) and carvacrol (37.95 %). Minimum inhibitory concentrations (MICs) of oil and the major components were calculated by serial dilution method, and anti-biofilm effects by microplate biofilm assay against five Gram positive (Staphylococcus aureus MU 38, MU 40, MU 46, MU 47, Stahylococcus epidermidis MU 30) and five Gram negative (Pseudomonas aeruginosa MU 187, MU 188, MU 189, Pseudomonas fluorescens MU 180, MU 181) bacteria. It was found that MICs for essential oil, thymol and carvacrol were between 5 and 50 ll/ml, 0.125–0.5 lg/ml and 0.125–05 ll/ml, respectively. The results showed that doses of MIC produced a greater anti-biofilm influence than 0.5, 0.25 and 0.125 MIC. In the presence of essential oil (MIC), the mean biofilm formation value was equal to 67 ± 5.5 % for P. aeruginosa MU 188, and essential oil (MIC) inhibition exceeds 60 % for P. aeruginosa biofilms. The results also showed that carvacrol (MIC) was able to induce an inhibition 72.9 ± 4.1 % for S.aureus (MU 40) biofilm. In addition, thymol (MIC) showed 68.6 ± 5.3 % reduction in biofilm formation of P. fluorescens MU 181. O. Ceylan (&) Apiculture Program, Ula Ali Kocman Vocational School, Mugla Sıtkı Koc¸man University, Ula, Mugla, Turkey e-mail: [email protected] A. Ugur Department of Basic Sciences, Section of Medical Microbiology, Faculty of Dentistry, Gazi University, Emek, Ankara, Turkey
This study demonstrated the antimicrobial and antibiofilm activity of T. sipyleus BOISS. subsp. sipyleus BOISS. var. davisianus RONNIGER essential oil and points out the exceptional efficiency of thymol and carvacrol, which could represent candidates in the treatment of Pseudomonas and Staphylococcus biofilms. Keywords Thymus sipyleus subsp. sipyleus var. davisianus Chemical composition Antibiofilm activity Antimicrobial activity Pseudomonas spp. Staphylococcus spp.
Introduction Biofilms are multicellular matrices which form by the attachment of bacteria by means of physical appendages to a surface followed by coating with extracellular polysaccharides. There are many advantages to an organism forming a biofilm, including protection from antibiotics, disinfectants and dynamic environments (Garrett et al. 2008). In the public health sector, the colonization of medical surfaces, such as catheters and other indwelling devices, by biofilms, plays a decisive role in the problem of healthcare-associated infections (Hammond et al. 2010). Staphylococci are important nosocomial pathogens. Eradication of these micro-organisms is not always successful due to their ability to form biofilms. Experimental evidence has shown that microorganisms in biofilms are less susceptible to conventional treatment (Brown and Gilbert 1993). In addition, S. aureus biofilms are widely found on implanted biomedical devices, accounting (together with Staphylococcus epidermidis) for more than one-half of prosthetic device-associated infections (Fluckiger et al. 2005). Pseudomonas aeruginosa is a species able
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to form biofilms on different abiotic surfaces, including artificial implants, contact lenses, urinary catheters and endotracheal tubes (Davey and O’Toole 2000). On the other hand, as an opportunistic pathogen, P. aeruginosa can cause various infections, which are difficult to treat with conventional antibiotics. Some of the infections, such as periodontitis, prostatitis and infections of patients with cystic fibrosis are considered to be in connection with bacterial attached mode of growth on the biotic surfaces (Lyczak et al. 2002; Hanlon 2007). The safe and traditional folkloric use of plant derived compounds for the prevention and treatment of diseases and infections make wide acceptance of them in the search for alternative antimicrobial agents (Guarrera 2005). Earlier investigations on plants and their active constituents have almost exclusively focused on their effects on planktonic bacteria with little emphasis on the more antimicrobial resistant pathogens and difficult to control biofilm forms (Sandasi et al. 2010). Among the aromatic plants belonging to the family Lamiaceae, the genus Thymus is noteworthy for the numerous species and varieties of wild-growing plants. Many of these species are typical for the Mediterranean area (Consentino et al. 1999). This genus is represented by 39 species and altogether 64 taxa, 24 of which are endemic in Turkey and the East Aegean Islands (Davis 1982; Baser et al. 1995). Several Thymus species are locally known as ‘‘kekik’’ or ‘‘tas kekik’’ and their dried herbal parts are used in herbal tea, condiment and folk medicine (Tumen et al. 1995). It has interesting antibacterial properties (Karaman et al. 2001; Rasooli and Mirmostafa 2002; Rota et al. 2008; Hazzit et al. 2009; Fadli et al. 2012; Kazemi et al. 2012; Ruiz-Navajas et al. 2012; Hussain et al. 2013), which are related to the presence of phenolic compounds: carvacrol, thymol, c-terpinene and p-cymene (Nedorostova et al. 2008; Rota et al. 2008; Hazzit et al. 2009; Sarikurkcu et al. 2010; De Lisi et al. 2011; El Bouzidi et al. 2012; Fadli et al. 2012; Ruiz-Navajas et al. 2012; Hussain et al. 2013). The essential oils of some Thymus spp. are characterized by the presence of high concentration of the isomeric phenolic monoterpenes thymol and/or carvacrol (Baser 1995). In contrast to the numerous studies on antimicrobial activity and chemical composition of Thymus essential oil there are only limited data on the antibiofilm activity. Thus, the aim of the present study was to investigate chemical composition and to assess the in vitro antimicrobial and antibiofilm activities of essential oil obtained from aerial parts of T. sipyleus subsp. sipyleus var. davisianus. To the best of our knowledge, this is the first study to compare the chemical composition and biological activity of T. sipyleus subsp. sipyleus var. davisianus.
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Materials and methods Microorganisms and condition for cultivation Staphylococcus aureus (MU 38, MU 40, MU 46, MU 47), S. epidermidis (MU 30), Pseudomonas aeruginosa (MU 187, MU 188, MU 189) and Pseudomonas fluorescens (MU 180, MU 181) were used as test microorganisms. The strains coded MU (the multi-antibiotic resistant bacteria) used in this study were taken from Mugla University Culture Collection. The antibiotic resistance patterns of the multiresistant bacteria are shown in Table 1. The above-mentioned bacteria were cultured in nutrient broth (NB) (Difco, USA). P. aeruginosa and P. fluorescens strains were incubated at 30 ± 0.1 °C for 18–24 h. Other bacterial strains were incubated at 37 ± 0.1 °C for 24 h. Inocula were prepared by adjusting the turbidity of the medium to match the 0.5 McFarland Standard Dilutions of this suspension in 0.1 % peptone (w/v) solution in sterile water inoculated on NB to check the viability of the preparation. The cultures of microorganisms were maintained in their appropriate agar slants at 4 °C throughout the study and used as stock cultures.
Table 1 Antibiotic resistance patterns of Staphylococcus sp. and Pseudomonas sp Microorganisms
Resistance patterns
S. aureus MU 38
P, AK, DA, CN, ME, TEC, TE, OX
S. aureus MU 40
P, AK, CN, C, ME, OX, TE
S. aureus MU 46
P, AK, DA, E, CN, TE, OX
S. aureus MU 47
P, AK, DA, CN, OX
S. epidermidis MU 30
P, AK, DA, CN, OX, TEC, TE
P. aeruginosa MU 187
MEZ, PRL, PY, SAM, CFP, CRO, MEM, CN, OFX, C, SXT, AM, B
P. aeruginosa MU 188
MEZ, PRL, PY, SAM, CFP, CRO, C, SXT, AM, B
P. aeruginosa MU 189
MEZ, PRL, PY, SAM, CFP, CRO, OFX, C, SXT, AM, B
P. fluorescens MU 180
MEZ, PRL, PY, SAM, CFP, CRO, MEM, C, SXT, AM, B
P. fluorescens MU 181
PRL, PY, SAM, CFP, CRO, C, AM, B
P penicillin (10 U), AK amikacin (30 lg), DA clindamycin (2 lg), CN gentamicin (10 lg), C chloramphenicol (30 lg), E erythromycin (15 lg), ME methicillin (5 lg), TEC teicoplanin (30 lg), TE tetracycline (30 lg), OX oxacilline (1 lg), MEZ mezlocillin (75 lg), PRL piperacillin (100 lg), PY carbenicillin (100 lg), SAM sulbactam ? ampicillin (10 ? 10 lg), CFP cefaperazone (75 lg), CRO ceftriaxone (30 lg), MEM meropenem (10 lg), OFX ofloxacin (5 lg), SXT trimethoprim ? sulfamethoxazole (1.25 ? 23.75 lg), AM ampicillin (10 lg), B bacitracin (10 U)
Chemical composition and anti-biofilm activity of Thymus
Plant material Aerial parts of T. sipyleus subsp. sipyleus var. davisianus, belonging to the Lamiaceae family, were collected at full flowering stage, in May–July 2010 from Usak province of Turkey. A voucher specimens of plant were taxonomically identified by Ass. Prof. Dr. Mehtap Donmez SAHIN at the Faculty of Education, the University of Usak. These voucher specimens have been deposited at the Herbarium of the Department of Biology, University of Usak, Turkey. Dry aerial materials of plant were subjected to hydrodistillation for 4 h. The oil recovered was stored in darkness at 4 °C. Analysis of the essential oil The chemical composition of the essential oil were analyzed using gas chromatography–mass spectrometry (GC– MS). The GC–MS analyses were carried out using a Shimadzu QP 5050 (Kyoto, Japan) GC–MS system operating in the EI mode at 70 eV, equipped with an Cp-Wax 52 CB column (50 9 0.32 mm; film thickness 1.2 lm). The initial temperature of the column was 60 °C and then rose to 220 °C at a 2 °C/min rate. The carrier gas was helium, the flow rate being 10 psi. Components were identified on the basis of gas chromatographic retention indices, mass spectra from Wiley, NIST98 and Tutor spectral libraries. The relative amounts of each individual component of the essential oil was expressed as the percentage of the peak area relative to total peak area. Kovats retention indices (Kovats 1965) were calculated for each seperate component against n-alkanes standards (C8–C25, Alltech Associates, Deerfield, IL) according to Schomberg and Dielmann (1973), using CP-Wax 52 CB fused silica capillary column. Determination of minimal inhibitory concentrations The minimal inhibitory concentrations (MICs) of essential oil, and its major components (thymol and carvacrol) on planktonic cells were determined in Mueller–Hinton Broth (MHB) using a microtitre broth dilution method as recommended by the Clinical and Laboratory Standards Institute (CLSI 2006). The bacterial suspension was adjusted with sterile saline to a concentration of 5.0 9 105 CFU ml-1. Essential oil, thymol (C99.0 % pure, Aldrich) and carvacrol (C97.0 % pure, Aldrich) was diluted with ethanol (EtOH) at 1:1 ratio, and then with MHB as required. Cell suspensions (200 lL) were inoculated into the wells of 96-well microtitre plates (Nunc F96 MicroWellTM plates; NunclonTM D, Denmark) to get the final concentration ranging from 0.5 to 100 ll/ml for essential oil, 0.0625–1 lg/ml for thymol and 0.0625–1 ll/ml for carvacrol. The wells containing only
MHB and MHB with inoculum were employed as negative and positive controls, respectively. S. aureus and S. epidermidis inoculated microplates were incubated at 37 °C and P. aeruginosa ve P. fluorescens inoculated microplates were incubated at 30 °C for 24 h. The lowest concentration of the tested samples, which did not show any visual growth of tested organisms after macroscopic evaluation, was determined as MIC, which was expressed in ll/ml or lg/ml. Each assay was performed in triplicate for all bacteria. Biofilm inhibition assays The effect of different concentrations of essential oil, thymol and carvacrol (ranging from the MIC to 1/8 MIC) on biofilm forming ability was tested on polystyrene flatbottomed microtitre plates as described by (Meritt et al. 2005). Cultures were grown overnight in 5 ml TSB with 1 % glucose, diluted in growth medium to 5 9 105 CFU ml-1 and 100 ll was dispensed into each well of 96-well polystyrene flat-bottomed microtitre plates in the presence of 100 ll essential oil, thymol and carvacrol (1, 0.5, 0.25 and 0.125 MIC) or 100 ll medium (control). After incubation for 48 h at 37 °C, each well was washed with water to remove planktonic bacteria. The remaining bacteria were subsequently stained with 0.1 % crystal violet solution for 10 min at room temperature. Wells were washed once again to remove the crystal violet solution that had not specifically staining the adherent bacteria. Microplates were inverted and vigorously tap on paper towels to remove any excess liquid then air dried. 200 ll of 95 % ethanol and 33 % glacial acetic acid (Sigma Chemical Co) were poured in Pseudomonas spp. and Staphylococcus spp. wells respectively. Biofilm stains solubilized at room temperature. After shaking and pipetting of the wells, 125 ll of the solution from each well was transferred to a sterile tube and volume reached to 1 ml with distilled water. Finally, the optical density of each well was measured at a wavelength of 550 nm (Thermo Scientific Multiskan FC, Vantaa, Finland). Each strain was tested for biofilm production in duplicate and the assay was repeated three times. Replicate absorbance readings for each concentration tested were averaged and the average of the media control was subtracted. This value was then divided by the mean absorbance of the (cell ? TSB) and multiplied by 100.
Results and discussion Chemical analyses of the volatile constituents of the essential oil (percentage content of each compound, elution order, retention times and linear retention indices) are summarized in Table 2. In total, 14 compounds comprising
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O. Ceylan, A. Ugur Table 2 Composition of the essential oil of T. sipyleus subsp. sipyleus. var. davisianus Noa
RTb
Kovats RIc
Compounds name a-pinene
Percentage (%)
1
7.2
909
1.00
2
12.5
1058
myrcene
0.25
3
13.5
1083
a-terpinene
0.58
4
14.9
1118
1, 8-cineole
0.61
5
16.7
1163
c-terpinene
7.28
6
18.2
1200
p-cymene
4.16
7 8
28.2 29.8
1462 1506
1-octen-3-ol Terpineol
0.62 0.83
9
38.9
1783
trans-Caryophyllene
1.83
10
43.7
1947
Borneol
3.83
11
46.2
2036
Bisabolene
2.32
12
61.3
2716
a-bisabolene epoksit
13
68.9
2949
Thymol
38.31
14
70.3
2971
Carvacrol
37.95
0.33
a
Compounds listed in order of elution from a CP-Wax 52 CB column b
Retention time (as minutes)
c
Kovats retention indices calculated for CP-Wax 52 CB in GCMSD
more than 99 % of the total composition were identified in all cases. Thymol (38.31 %) and carvacrol (37.95 %) were the main oxygenated monoterpenes identified from the essential oil of T. sipyleus subsp. sipyleus var. davisianus. The other compounds were c-terpinene (7.28 %), p-cymene (4.16 %), borneol (3.83 %), bisabolene (2.32 %), trans-caryophyllene (1.83 %) and a-pinene (1.0 %). The rest of compounds were identified in trace concentrations. Previous studies on the essential oil of Thymus species have shown that it contains carvacrol and thymol as major components. Hussain et al. (2013) reported thymol
(36.5 %) and carvacrol (9.50 %) in T. linearis essential oil and carvacrol (44.4 %) in T. serpyllum essential oil. El Bouzidi et al. (2012) reported thymol (0.4–12.3 %) and carvacrol (26.0–71.6 %) in T. broussonetii, T. maroccanus and T. satureioides, are endemic Moraccan species. Fadli et al. (2012) reported carvacrol (76.35, 39.77 %) is the major constituent in T. maroccanus and T. broussonetii, respectively. Carvacrol was also reported as a major component by (Jirovetz et al. 2012) on the three thyme species studied. Hazzit et al. (2009) reported four T. pallescens oils have highly similar chemical composition characterized by carvacrol (44.4–57.7 %), p-cymene (10.3–17.3 %) and c-terpinene (10.8–14.2 %) as the major components. Rota et al. (2008) reported thymol (57.7, 68.1 %) and carvacrol (2.8, 3.5 %) in T. vulgaris and T. zygis subsp. gracilis, respectively. The chemical composition of the essential oil from T. guyonii de Noe restricted to Algeria, showed that the main components were carvacrol (55.55 %), thymol (19.51 %) and p-cymene (6.25 %) (Lehbili et al. 2013). The antibacterial activity of the essential oil of T. sipyleus subsp. sipyleus var. davisianus and the phenolic compounds (thymol and carvacrol) was initially evaluated by the microtitre broth dilution method using a multiple antibiotic resistant Staphylococci and Pseudomonas spp. The MICs of the two major compounds and whole essential oil are shown in Table 3. The essential oil of T. sipyleus subsp. sipyleus var. davisianus had MICs in the range of 5–50 ll/ml. The pure major components, thymol and carvacrol, were more active, with MIC (0.125–0.5 lg/ml for thymol and 0.125–05 ll/ml for carvacrol), compared to essential oil. Essential oil inhibited S. aureus (MU 40, MU 46, MU 47) at 5 ll/ml concentration. This result is important in terms of revealing the activity of essential oil against methicilin and oxacilline resistant S. aureus. This value has
Table 3 Minimum inhibitory concentrations (MICs) of T. sipyleus subsp. sipyleus var. davisianus essential oil, thymol, and carvacrol Microorganisms
Minimum inhibitory concentration Essential oil (ll/ml)
Thymol (lg/ml)
Carvacrol (ll/ml)
S. aureus MU 38
25
0.25
0.25
S. aureus MU 40
5
0.25
0.25
S. aureus MU 46
5
0.25
0.25
S. aureus MU 47
5
0.25
0.125
S. epidermidis MU 30
25
0.5
0.5
P. aeruginosa MU 187
50
0.125
0.25
P. aeruginosa MU 188 P. aeruginosa MU 189
25 50
0.125 0.25
0.25 0.5
P. fluorescens MU 180
5
0.125
0.25
P. fluorescens MU 181
5
0.125
0.25
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Chemical composition and anti-biofilm activity of Thymus Table 4 Effects of T. sipyleus subsp. sipyleus var. davisianus essential oil (EO), thymol (T), and carvacrol (C) on biofilm formation expressed as percentage inhibition
Microorganisms
Agent
% Inhibitiona MIC
0.5 MIC
0.25 MIC
0.125 MIC
S. epidermidis MU 30
EO
23.5 ± 3
T
35.7 ± 5.1
11.4 ± 0.91
8.2 ± 1
0.6 ± 0.5
C
53 ± 6
24.3 ± 1.9
8.5 ± 3.5
–
– – –
S. aureus MU 38
MU 40
MU 46
MU 47
EO
28.9 ± 5.4
20.4 ± 3.5
16.4 ± 2.1
T
20.1 ± 2.3
11.4 ± 1.7
4.7 ± 2.9
10.3 ± 1.3
C
69.9 ± 1.9
46.9 ± 1.8
26.6 ± 4.6
5.1 ± 1.2
EO
26.2 ± 3.5
18.4 ± 1.8
11.1 ± 1.3
7.7 ± 1.5
T
46.8 ± 3.1
29.1 ± 1.3
13.2 ± 4.9
–
C EO
72.9 ± 4.1 38.4 ± 4.6
50.7 ± 3.7 19.1 ± 2.5
25.4 ± 3.4 5.9 ± 1.2
13.3 ± 2.1 – –
–
T
42.9 ± 1.7
29.9 ± 1.6
15.4 ± 5.7
C
67.4 ± 1.8
49.1 ± 5.4
23.1 ± 3.2
EO
51.9 ± 9.4
11.2 ± 1.5
–
T
49.2 ± 1.4
35.7 ± 2.7
17.6 ± 1.6
–
C
61.1 ± 1.9
29.8 ± 9
4.1 ± 3.2
–
9.2 ± 3.6 –
P. fluorescens MU 180
MU 181
EO
27.5 ± 2.5
18.2 ± 1.3
6.6 ± 1.2
–
T
47.2 ± 4.7
32.4 ± 2.8
17.3 ± 2.4
–
C
61.4 ± 0.9
39.2 ± 2.6
22.6 ± 0.9
12.5 ± 7.4
EO
65.1 ± 4.9
43.9 ± 5.4
34.3 ± 2
24.9 ± 2.2
T
68.6 ± 5.3
48.9 ± 4.6
23.2 ± 2.6
9.9 ± 2.9
C
45.9 ± 0.5
38.6 ± 3.7
32.3 ± 4.8
12.7 ± 4.1
EO
60.3 ± 9.3
30.9 ± 7.7
26.9 ± 2.7
11.6 ± 1.5
T C
42.7 ± 3.1 54.1 ± 6.5
28.5 ± 2.8 40.5 ± 4.9
12.8 ± 1.2 23.2 ± 3.7
– – –
P. aeruginosa MU 187
MU 188
a
Values are reported as mean ± SD – no inhibition
MU 189
67 ± 5.5
32.9 ± 3.7
14.9 ± 2.2
T
EO
61.7 ± 0.4
43.2 ± 5.6
19.8 ± 3.2
–
C
41.2 ± 4.2
16.3 ± 3.2
6.8 ± 4.8
–
EO
63.7 ± 9.1
42.5 ± 7.4
37.6 ± 4.9
T
54.7 ± 4.1
33.4 ± 2.9
14.4 ± 4.1
–
C
47.7 ± 4.8
30.7 ± 3.2
14.7 ± 4.3
–
been identified as 25 ll/ml for the S. aureus MU 38 (methicilline and oxacilline resistant) and S. epidermidis MU 30 (oxacilline resistant). MIC of thymol were 0.25 lg/ ml for S. aureus strains and 0.5 lg/ml for S. epidermidis. Using carvacrol, a MIC of 0.125 ll/ml was observed for S. aureus MU 47 and 0.5 ll/ml for S. epidermidis MU 30. MIC was determined as 0.25 ll/ml for the other S. aureus strains. These results are consistent with previous studies (Rota et al. 2008; El Bouzidi et al. 2012; Fadli et al. 2012; Hussain et al. 2013) demonstrating that Thymus species’ essential oil rich in thymol and carvacrol possess antibacterial activity on the Staphylococcus spp. Essential oil
14.9 ± 1.5
presented moderate activity against both P. fluorescens (5 ll/ml). On the other hand, essential oil has low activity on the growth of P. aeruginosa which were only inhibited at high concentrations (25 or 50 ll/ml). This antimicrobial effect of essential oil, although at relatively high concentrations, was important because P. aeruginosa strains are multiple antibiotics resistant bacteria (Table 1). This study revealed that thymol showed maximum activity with MIC values ranging from 0.125 to 0.25 mg/ml followed by carvacrol with MIC values ranging from 0.25 to 0.5 ll/ml against all the tested Pseudomonas strains. In this study, thymol and carvacrol exhibited the highest antibacterial
123
O. Ceylan, A. Ugur Fig. 1 Effects of essential oil, thymol, and carvacrol on biofilm formation of Staphylococcus spp.
Fig. 2 Effects of essential oil, thymol and carvacrol on biofilm formation of Pseudomonas spp.
effect against all Pseudomonas strains compared with essential oil. Thus, we confirmed the strongest antimicrobial activity of thymol and carvacrol against Pseudomonas sp. which had been previously described by several authors (Sokovic et al. 2010; Soumya et al. 2011). Bassole and Juliani (2012) reported antagonistic effects of the combinations of p-cymene and phenolic monoterpenes (thymol and carvacrol). It was also observed that carvacrol/thymol and carvacrol/myrcene worked antagonistically against S. aureus, B. cereus and E. coli (Gallucci et al. 2009). The different antibacterial activity exhibited by essential oil of
123
T. sipyleus subsp. sipyleus var. davisianus when compared to thymol and carvacrol, suggesting that the minor components may contribute to an antagonistic effect on the activity of essential oil. This study also indicates that essential oil of T. sipyleus subsp. sipyleus var. davisianus has lower antimicrobial effects on Gram-negative bacteria than Gram-positive bacteria. This fact has been mentioned in other studies (Maksimovic et al. 2008; El Bouzidi et al. 2012; Fadli et al. 2012; Millezi et al. 2012; Hussain et al. 2013; Lehbili et al. 2013). However, various researchers obtained higher antimicrobial activity for Pseudomonas
Chemical composition and anti-biofilm activity of Thymus
strains with some Thymus essential oils (Adwan et al. 2006; Oussalah et al. 2006; Qaralleh et al. 2009). Essential oils are potential sources of novel anti-biofilm compounds especially against pathogen microorganisms (Nostro et al. 2007; Schillaci et al. 2008; Gursoy et al. 2009; Oral et al. 2010; Sandasi et al. 2010; Sandasi et al. 2011; Soumya et al. 2011). Therefore, the inhibitory effects of T. sipyleus subsp. sipyleus var. davisianus essential oil and major components (thymol and carvacrol) on the development of microbial biofilms were investigated against multiple antibiotic resistant strains of Pseudomonas and Staphylococcus. Our results showed that biofilm formation was significantly inhibited by essential oil, thymol and carvacrol but their effectiveness varied. This activity was dependent on the concentration used to treat the biofilm. Doses of MIC produced a greater influence than doses of 0.5, 0.25 and 0.125 MIC. The activities of essential oil and components tested at several concentrations against the total biomass of S. aureus and S. epidermidis biofilms are shown in Table 4 and Fig. 1. The in vitro activity of the essential oil on biofilm was only slightly lower than on planktonic culture. In the presence of 25 ll/ml essential oil (MIC), the mean biofilm formation values were equal to 23.5 % for S. epidermidis MU 30, and 28.9 % for S. aureus MU 38. At a concentration of 5 ll/ml essential oil, the biofilm formations of MU 40, MU 46 and MU 47 were inhibited to 26.2, 38.4 and 51.9 %, respectively. As the concentration of essential oil decreased, antibiofilm activity showed an obvious decrease in the surface coverage of microplate wells. In our study, carvacrol exhibited strong activity against the S. aureus and S. epidermidis biofilm development. Carvacrol inhibited the biofilm formation of four S. aureus used in the study at various ratios ranging from 61.1 to 72.9 % at MICs. This ratio was determined to be 53 % for S. epidermidis MU 30. The results showed that for S. aureus and S. epidermidis a moderate but not significant reduction in biofilm formation was achieved by treatment with thymol at MICs. The antibiofilm activity of thymol on Staphylococcus biofilm formations was observed lower than carvacrol. In the presence of thymol (MIC), the mean biofilm formation values were equal to 35.7 % for S. epidermidis MU 30 and 49.2 % for S. aureus MU 47. There are no studies in the literature on the anti-biofilm effect of T. sipyleus subsp. sipyleus var. davisianus essential oil, however, there is one study showing the anti-biofilm effect of carvacrol and thymol, the major constituents of T. sipyleus subsp. sipyleus var. davisianus, on S. aureus and S. epidermidis biofilms, at concentrations of 0.031–0.125 %, v/v, for carvacrol and thymol (Nostro et al. 2007). In contrast to Staphylococcus spp., antibiofilm activity of essential oil was more pronounced against Pseudomonas
spp. (Table 4 and Fig. 2). For essential oil, significant antibiofilm inhibition for P. aeruginosa was observed at MIC, inhibiting *65 % biofilm formation (Fig. 2). 5 ll/ml essential oil showed 67 % inhibition against P. aeruginosa MU 188. In the presence of 25 ll/ml essential oil (MIC), the mean biofilm formation values were inhibited to 63.7 % for P. aeruginosa MU 189 and 60.3 % for P. aeruginosa MU 187, respectively. According to our findings, essential oil was more effective on biofilm formation of P. aeruginosa than thymol and carvacrol. In the presence of thymol and carvacrol (MIC), the mean biofilm formations values were equal to 42.7 and 54.1 % for P. aeruginosa MU 187, 61.7 and 41.2 % for P. aeruginosa MU 188, and 54.7 and 47.7 % for P. aeruginosa MU 189, respectively. Treatment with essential oil with MIC gave rise to a 65.1 % inhibition of the P. fluorescens MU 181 biofilm formation. On the other hand, essential oil at concentration of MIC caused at 27.5 % decrease in P. fluorescens MU 180 biofilm formation. Thymol showed the strongest antibiofilm activity against the P. fluorescens MU 181 and it was induced 68.6 % inhibition of the biofilm when used at MIC. In the presence of carvacrol and thymol (MIC), the mean inhibition values were equal to 61.4 and 47.2 % for P. fluorescens MU 180 biofilm formation. Our results pointed out the in vitro potential of T. sipyleus subsp. sipyleus var. davisianus essential oil and major compounds such as thymol and carvacrol as antibiofilm agents and clearly demonstrate the feasibility of using essential oil to prevent biofilm formation. In addition, plant essential oil showed significant antimicrobial activity against Staphylococcus spp. planktonic cells and antibiofilm activity against Pseudomonas spp. biofilms. These results showed that the essential oil of T. sipyleus subsp. sipyleus var. davisianus could be considered as a natural alternative to treat biofilms of medical relevance and be used as an innovative plant-derived antimicrobial agent. Acknowledgments Authors thank Ass. Prof. Dr. M.D. Sahin, Botanist, Usak University, Usak, Turkey, for providing plant taxonomic identification. Conflict of interest
The author declare no conflict of interest.
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