Journal of Medical Microbiology (2007), 56, 519–523 DOI 10.1099/jmm.0.

46804-0

Effects of oregano, carvacrol and thymol on

Staphylococcus aureus and Staphylococcus
epidermidis biofilms
Antonia Nostro,1 Andrea Sudano Roccaro,2 Giuseppe Bisignano,1
Andreana Marino,1 Maria A. Cannatelli,1 Francesco C. Pizzimenti,1
Pier Luigi Cioni,3 Francesca Procopio1 and Anna Rita Blanco2
Correspondence 1
Dipartimento Farmaco-Biologico, Facoltà di Farmacia, Università degli Studi di Messina, Villaggio
Antonia Nostro Annunziata, 98168 Messina, Italy
[email protected] 2
Direzione Ricerca, Sviluppo & Innovazione, SIFI SpA, Via Ercole Patti 36, Lavinaio, 95020 Catania,
Italy
3
Dipartimento di Chimica Bioorganica e Biofarmacia, Università di Pisa, Via Bonanno 33, 56126
Pisa, Italy

The aim of this study was to evaluate the effect of oregano essential oil, carvacrol and thymol on
biofilm-grown Staphylococcus aureus and Staphylococcus epidermidis strains, as well as the
effects of the oils on biofilm formation. For most of the S. aureus (n56) and S. epidermidis (n56)
strains tested, the biofilm inhibitory concentration (0.125–0.500 %, v/v, for oregano, and
0.031–0.125 %, v/v, for carvacrol and thymol) and biofilm eradication concentration (0.25–1.0 %,
v/v, for oregano and 0.125–0.500 %, v/v, for carvacrol and thymol) values were twofold or
fourfold greater than the concentration required to inhibit planktonic growth. Subinhibitory
Received 27 June 2006 concentrations of the oils attenuated biofilm formation of S. aureus and S. epidermidis strains
Accepted 18 December 2006 on polystyrene microtitre plates.

INTRODUCTION its major phenolic components, carvacrol [2-methyl-

5-(1-methylethyl)phenol] and thymol (2-isopropyl-5-
Staphylococci are important nosocomial pathogens. Eradi-
methylphenol), are known for their wide spectrum of
cation of these micro-organisms is not always successful
antimicrobial activity, which has been the subject of several
due to their ability to form biofilms. Experimental evidence
investigations in vitro (Dorman & Deans, 2000; Lambert
has shown that micro-organisms in biofilms are less
et al., 2001) and in vivo (Adam et al., 1998; Manohar et al.,
susceptible to conventional treatment (Brown & Gilbert,
2001). They possess multiple biological properties such as
1993) than their planktonic counterparts. Many factors
anti-inflammatory, anti-leishmanial, antioxidant, hepato-
contribute to the lowered susceptibility of bacteria enclosed
protective and anti-tumoral activities (Aeschbach et al.,
in a biofilm, and include the induction of a biofilm pheno- 1994; Alam et al., 1999; Robledo et al., 2005; Skold et al.,
type, the stress response and failure of the antimicrobial 1998; Weber & de Bont, 1996; Zeytinoglu et al., 2003).
agents to penetrate the biofilm (Mah & O’Toole, 2001).
Previously, we have shown the efficacy of oregano oil,
As such, alternative strategies or more effective agents exhib- carvacrol and thymol against planktonic Staphylococcus
iting activity against biofilm-producing micro-organisms aureus and Staphylococcus epidermidis, including meticillin-
are of great interest. Natural drugs could represent an resistant strains (Nostro et al., 2004). The objective of this
interesting approach to limit the emergence and the spread study was to extend the research to evaluate the activity
of these organisms, which currently are difficult to treat. of oregano oil, carvacrol and thymol on biofilm-grown S.
Recently, there has been considerable interest in the study of aureus and S. epidermidis strains, as well as the effects of
plant materials as sources of new compounds for pro- oils on biofilm formation.
cessing into therapeutic agents. One approach may be the
use of essential oils that have been shown to be potential
agents in the treatment of infections, and are safe in terms of METHODS
human and animal health. In this context, oregano oil and Essential oils. The aerial parts of commercial Origanum vulgare L.
(obtained from A. Minardi, Ravenna, Italy) were subjected to
Abbreviations: BEC, biofilm eradication concentration; BIC, biofilm hydrodistillation. The qualitative and quantitative composition of
inhibitory concentration; MBC, minimum bactericidal concentration. the essential oil was analysed by GC and GC/electron impact MS as
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A. Nostro and others

described previously (Nostro et al., 2004). The oregano oil was in the presence or absence of subMIC concentrations of carvacrol
characterized principally by carvacrol and thymol (14 and 24.7 %, v/v, (0.5 and 0.25 MIC) were fixed in 2 % glutaraldehyde in 0.1 M PBS for
respectively) and by their two precursors, c-terpinene and p-cymene 2 h at 4 uC and then post-fixed for 1 h at 4 uC in 1 % osmium tetroxide
(11.7 and 14.6 %, v/v, respectively). Carvacrol (¢97.0 % pure) and in the same buffer. After thorough washing with PBS, samples were
thymol (¢99.0 % pure) were purchased from Aldrich. Stock dehydrated in a series of ethanol solutions (30–100 %). Specimens were
solutions of 50 % (v/v) essential oils were prepared in ethanol mounted on aluminium stubs with conductive carbon cement, allowed
(EtOH) and used following dilution. to dry and then coated with a gold film. Samples were observed with an
S-400 scanning electron microscope (Hitachi).
Bacterial strains. The bacteria used were S. aureus (n56) and S.
epidermidis (n55) isolated from ocular infections belonging to our Statistical analysis. The biofilm formation values were analysed by a
private collection, and the reference strain S. epidermidis ATCC 35984 hierarchical analysis of variance test following angular transforma-
(slime producing). Each isolate was characterized for biofilm-related tion. The differences between groups (different oil concentrations)
properties as reported previously (Blanco et al., 2005). The isolates were considered significant when P,0.05. Where significant differ-
were capable of forming biofilms with an OD492 ranging from 0.52 ences existed, the Duncan test was performed to verify the significant
to 1.55 (Blanco et al., 2005). difference levels.

Efficacy of oregano oil on planktonic cells. The MIC and minimum

bactericidal concentration (MBC) of oregano, carvacrol and thymol on
planktonic cells were determined in tryptic soy broth (TSB) using a RESULTS AND DISCUSSION
broth dilution micromethod in polystyrene flat-bottomed microtitre In this study, the in vitro effects of O. vulgare essential oil,
plates (Costar Corning) according to CLSI guidelines (Clinical
carvacrol and thymol on staphylococcal biofilms were
Laboratory Standards Institute, 2000). The data from at least five
replicates were evaluated and modal results were calculated. Two evaluated. Remarkably, the in vitro activity of the oils on
growth controls consisting of TSB medium and TSBEtOH were included biofilm was only slightly lower than that on planktonic
culture (Table 1). For most of the strains tested, the BIC
Effect on established biofilms. The effect on established biofilms was (0.125–0.500 %, v/v, for oregano, and 0.031–0.125 %, v/v,
verified as described by Johnson et al. (2002) with some modifica- for carvacrol and thymol) and BEC (0.25–1.0 %, v/v, for
tions. All isolates were grown as biofilms using polystyrene flat- oregano, and 0.125–0.500 %, v/v, for carvacrol and
bottomed microtitre plates. After 24 h of incubation at 37 uC, the
planktonic-phase cells were gently removed and the wells were washed
thymol) values were twofold or fourfold greater than the
three times with PBS and filled with 200 ml twofold dilutions of the concentration required to inhibit growth in suspension.
oils, ranging from the MIC to a 16-fold dilution of the MIC. The Despite a different inhibitory effect among the strains, a
plates were incubated for 24 h at 37 uC. The OD492 was measured
at time 0 and after incubation for 24 h. The biofilm inhibitory
general attenuated level of biofilm formation in the pres-
concentration (BIC) was determined as the lowest concentration ence of subinhibitory concentrations of oregano, carvacrol
where no growth occurred in the supernatant fluid, confirmed by no and thymol was observed (Table 2). Significant differences
increase in optical density compared with the initial reading. Samples (P,0.05) in biofilm formation values were observed
of biofilms from the bottom of these wells were scarified by a metal between groups (different oil concentrations). Doses of
loop, spread over the surface of tryptic soy agar (TSA) plates and 0.5 MIC produced a greater influence than doses of 0.25
incubated for 72 h at 37 uC. The biofilm eradication concentration
and 0.125 MIC (Duncan test). This effect was more evident
(BEC) was determined as the lowest concentration at which no
bacterial growth occurred on the TSA plates. Data from at least five for S. aureus than for S. epidermidis strains. In the presence
replicates were evaluated and modal results were calculated. Two of oregano, carvacrol and thymol (0.5 MIC), the mean
biofilm controls consisting of TSB medium and TSBEtOH were biofilm formation values were equal to 46.7, 28.3 and
included. 30.1 % for S. aureus, and 58.9, 57.1 and 54.4 % for S.
epidermidis, respectively. Oregano showed a slight inhibi-
Effect on biofilm formation. The effect of different concentrations of tory effect because carvacrol and thymol represent only a
oil (ranging from 0.5 to 0.125 MIC) on biofilm-forming ability was
tested on polystyrene flat-bottomed microtitre plates as described by
fraction (38.7 %) of the entire essence and they interact in
Cramton et al. (1999) with some modifications. Cultures were grown an additive rather than a synergistic way (Lambert et al.,
overnight in 10 ml TSB with 1 % glucose, diluted in growth medium 2001).
to 56105 c.f.u. ml21 and 100 ml was dispensed into each well of 96-
well polystyrene flat-bottomed microtitre plates in the presence of Direct observation by scanning electron microscopy of S.
100 ml subinhibitory concentrations (subMIC) of oregano, carvacrol aureus 815 showed that, after 24 h, in the absence of
and thymol (0.5, 0.25 and 0.125 MIC) or 100 ml medium (control). carvacrol (Fig. 1a, b), bacterial cells formed evident
After incubation for 24 h at 37 uC, each well was washed twice with biofilms with matrix material. In the presence of carvacrol
sterile PBS (pH 7.4), dried, stained for 1 min with 0.1 % safranin and at concentrations of 0.5 and 0.25 MIC (Fig. 1c, d, e),
washed with water. The stained biofilms were resuspended in 200 ml bacterial cells grew as looser colonies, and the amount of
PBS and OD492 was measured by spectrophotometry using an ELISA
biofilm was reduced, being almost absent at 0.5 MIC.
reader. Two biofilm controls consisting of TSB medium and TSBEtOH
were included. Each assay was performed in quadruplicate and Having more-effective antimicrobial agents that are also
repeated at least three times. As a measure of efficacy, relative biofilm active against biofilm, and are able to prevent or at least
formation was defined as follows: (mean OD492 of treated well/mean
OD492 of control well)6100.
interfere with biofilm formation, would be a considerable
achievement. As an extension of our earlier work on the
Scanning electron microscopy. Biofilms of a strong biofilm producer efficacy of oregano, carvacrol and thymol against plank-
(S. aureus 815) formed on polystyrene flat-bottomed microtitre plates tonic meticillin-resistant staphylococci (Nostro et al.,
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 Biofilm inhibition by oregano, carvacrol and thymol

Table 1. Susceptibility of planktonic and biofilm organisms to Table 2. Effects of oregano (O), carvacrol (C) and thymol (T)
oregano (O), carvacrol (C) and thymol (T) on biofilm formation

Strain Agent MIC BIC MBC BEC Strain Agent Biofilm formation*
(%, v/v) (%, v/v) (%, v/v) (%, v/v)
0.5 MIC 0.25 MIC 0.125 MIC
S. aureus
6ME O 0.062 0.250 0.125 0.500 S. aureus
C 0.015 0.062 0.062 0.250 6ME O 52.45±6.3 73.3±8.2 103±10
T 0.031 0.062 0.062 0.250 C 34.8±5.1 90.8±9.5 96.8±9.8
810 CT O 0.125 0.500 0.250 0.500 T 49.9±3 98.4±9.6 96.9±7.2
C 0.031 0.125 0.062 0.250 810 CT O 50.3±6.1 63.4±7.3 93.9±9
T 0.031 0.062 0.062 0.125 C 41.2±5.6 75.6±8.4 76.5±8.4
815 CT O 0.062 0.250 0.125 0.500 T 20.9±1.2 72.6±8 76.8±8.4
C 0.031 0.125 0.125 0.500 815 CT O 10.6±1.4 17.1±4.1 79.33±8.6
T 0.031 0.062 0.062 0.125 C 3.1±4.2 19.9±5 62±3.2
808 CT O 0.125 0.250 0.125 1 T 3.48±3.2 20±2 71.3±8.4
C 0.031 0.125 0.062 0.250 808 CT O 6.23±5.2 30.7±3.6 95.9±9.7
T 0.031 0.062 0.062 0.125 C 8.3±2.5 25.9±2.7 35±7.4
5 ME O 0.062 0.250 0.125 0.500 T 14.9±1.5 20±2 48±5.9
C 0.031 0.062 0.125 0.250 5 ME O 50±1.2 78.1±2.3 95±6
T 0.031 0.125 0.062 0.250 C 40.1±3 75.2±4 98.4±10.1
74CCH O 0.062 0.250 0.125 0.500 T 49.1±2.3 72±1.3 96.9±2.3
C 0.015 0.031 0.062 0.125 74CCH O 24.4±2.3 53.4±6.4 83±8.8
T 0.062 0.125 0.125 0.250 C 32.3±4.6 66.4±7.8 101±8.2
S. epidermidis T 23.2±2 73.27±8.1 118±11.2
ATCC 35984 O 0.125 0.500 0.250 1 S. epidermidis
C 0.031 0.125 0.125 0.500 ATCC 35984 O 65±4.9 72.4±8 98.8±9.6
T 0.062 0.125 0.125 0.500 C 70.9±8.2 99±9.9 98.16±9.9
14 ME O 0.062 0.125 0.125 0.500 T 71.67±8 102±10 102±10.1
C 0.031 0.125 0.125 0.500 14 ME O 33.46±2 81.9±8.5 95.88±7.6
T 0.031 0.062 0.062 0.125 C 40±3.2 64.6±6.8 104±10.3
807 CT O 0.125 0.500 0.250 1 T 31.24±4 36±2 71.5±7.5
C 0.031 0.062 0.062 0.125 807 CT O 58±6.9 85.6±9 103±10
T 0.031 0.125 0.062 0.125 C 22.9±1.9 41.6±5.2 81.7±8.78
813 O 0.125 0.250 0.250 0.500 T 22.56±1.8 28.5±3 64.1±7.4
C 0.031 0.125 0.062 0.125 813 CT O 25.5±2.6 72.4±8.1 111±10.6
T 0.031 0.062 0.062 0.125 C 48±3 79.2±8.6 87.5±9.2
23 S O 0.125 0.250 0.250 0.250 T 40.1±1.3 75.8±8.3 98.1±9.8
C 0.015 0.031 0.031 0.125 23 S O 81.8±8 107.3±10 101±10
T 0.031 0.031 0.062 0.125 C 47.2±5.8 93.9±9.6 106±10.4
809 O 0.125 0.250 0.250 0.500 T 25.2±2.5 60.5±7 76.8±8.4
C 0.015 0.031 0.062 0.250 809 O 70±7.9 94.4±9.6 97.3±9.8
T 0.031 0.062 0.062 0.125 C 43.4±5.4 65.3±7.5 86±4.1
T 24±2.2 73.9±8.2 82.6±8.8

*Biofilm formation values were calculated as: (mean OD492 treated

2004), we show here that these oils inhibited growth of
well)/(mean OD492 control well)6100. Values are expressed as
preformed biofilm and interfered with biofilm formation means±SD.
during planktonic growth. The reasons for this could be
due to many factors acting either synergistically or alone.
The antimicrobial activity of oregano oil is mostly attri-
buted to the action of its principal phenolic components, 1999). The extent of membrane damage induced by a
carvacrol and thymol, which exhibit significant bactericidal compound can be related to its intrinsic hydrophobicity,
activity when tested separately (Friedman et al., 2002; which can be determined experimentally by its partition
Juven et al., 1994; Lambert et al., 2001; Ultee et al., 1998). coefficient in octanol/water (Po/w). Carvacrol and thymol
Due to their hydrophobic nature, carvacrol and thymol have a log Po/w of 3.64 and 3.30, respectively (Griffin et al.,
interact with the lipid bilayer of cytoplasmic membranes 1999; Ultee et al., 2002). Weber & de Bont (1996) reported
causing loss of integrity and leakage of cellular material that compounds with a log Po/w value higher than 3 will
such as ions, ATP and nucleic acid (Helander et al., 1998; partition deeply in the cell membrane. However, carvacrol
Lambert et al. 2001; Trombetta et al., 2005; Ultee et al., and thymol have been reported to possess a relative
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A. Nostro and others

polystyrene microtitre plates. In this context, Juven et al.

(1994) suggested a reaction between phenolic compounds
(such as thymol) and bacterial membrane proteins.
The findings of the present study highlight the promising
role of oregano, carvacrol and thymol as new lead struc-
tures in the search for novel antibacterial agents. Data in
the literature on the availability and pharmacokinetics of
carvacrol and thymol (Bhattaram et al., 2002; De Vincenzi
et al., 2004), and on acute and short-term in vivo effects,
(a) 700 mm (b) 2 mm suggest that they may not pose a risk for human and
animal health (Chami et al., 2005; Stammati et al., 1999).
Therefore, further experiment may be worthy of evaluation.

ACKNOWLEDGEMENTS
We thank Pino Mondio of the Biomedical Science Department,
Section of General and Cellular Biology and Molecular Genetics of the
University of Catania, Italy, for expert collaboration in the realization
of the scanning electron microscopy artwork.
(c) 20 mm

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