Microbial Pathogenesis 96 (2016) 1e9

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Microbial Pathogenesis
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Chemical composition and antifungal activity of Satureja hortensis L.

essentiall oil against planktonic and biofilm growth of Candida
albicans isolates from buccal lesions of HIVþ individuals
Aghil Sharifzadeh a, Ali Reza Khosravi a, *, Shahin Ahmadian b
a
Mycology Research Center, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran
b
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran

a r t i c l e i n f o a b s t r a c t

Article history: Ethnopharmacological relevance: Oral candidiasis is an opportunistic infection of the oral cavity which
Received 13 December 2015 usually occurs in the immunocompromised individuals. Candida albicans (C. albicans) is the most com-
Received in revised form mon species of yeast responsible for oral candidiasis. This study investigated the effects of Satureja
10 April 2016
hortensis L. essentiall oil (EO) on the planktonic, biofilm formation and mature biofilms of C. albicans
Accepted 21 April 2016
Available online 25 April 2016
isolates from buccal lesions of HIVþ individuals.
Materials and methods: MTT reduction assay, broth micro-dilution method and scanning electron mi-
croscopy (SEM) were employed to determine the effect of mentioned EO on the C. albicans planktonic
Keywords:
Candida albicans
and biofilm forms. GC-GC/MS was used to detect the major active compounds of EO.
Biofilm Results: Thymol (45.9%), gamma-terpinen (16.71%), carvacrol (12.81%) and p-cymene (9.61%) were found
Satureja hortensis L. essentiall oil as the most abundant constituents. MIC values ranged from 250 to 400 mg/ml and MFC values ranged
HIV individuals from 350 to 500 mg/ml. All C. albicans isolates formed biofilm on polystyrene plats but the quantity of
biofilm mass (optical density) was different for the isolates ranging from 0.850 to 0.559 nm. The mean of
biofilm formation by C. albicans isolates was reduced by 87.1 ± 3.7%, 73.6 ± 5.1%, 69.4 ± 5.3% and
67 ± 4.2% at 4800, 3200, 2400 and 1600 mg/ml, respectively. In sub-MIC concentration, SEM analysis
revealed loosening of cells, deformity of three dimensional structures of biofilms and shrinkage in cell
membranes of sessile cells.
Conclusions: In conclusion, the substantial anti-fungal activity showed by S. hortensis L. EO suggests
exploitation of this oil as potential natural anti-biofilm product to deal with the problem of buccal cavity
lesion associated with C. albicans.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction majority of superficial and systemic infections [2,3]. Several factors

Candida species are commonly considered harmless commen-
sals, and are isolated from the vagina, mouth and gastrointestinal
tracts. When the host-fungus interaction become unbalanced, the
fungus is able to initiate infection and cause disease [1]. Oral
candidiasis is considered one of the most common fungal infection
of human buccal cavity especially in human immunodeficiency
virus (HIV) infected individuals. Among candida species, Candida
albicans is the most commonly isolated and responsible for the
* Corresponding author. Mycology Research Center, Faculty of Veterinary Medi-
cine, University of Tehran, Azadi St, Tehran, Iran. Tel.: þ98 2161117144; fax: þ98
2166933222.
E-mail address: Khosravi@ut.ac.ir (A.R. Khosravi).
predispose to the development of candidiasis. Among the most
cited factors related to the yeast (virulence factors), the ability of C.
albicans to adapt to a variety of different habitats and the conse-
quent development of surface-attached microbial communities
known as biofilms is considered to be an important virulence at-
tributes for establishing and maintaining candidiasis. The biofilm
lifestyle results in phenotypic characteristics that are markedly
different from that of planktonic existence, such as increased
resistance to antimicrobial agents, blocking or preventing the ac-
cess of antifungals to the more internal biofilm structure and pro-
tection from host defenses [4e7]. This scenario has exacerbated the
need for alternative antifungal therapy. It is expected that com-
pounds with anti-biofilm activities will also reduce the persistence
and increased tolerance to antifungal drugs [3,8]. In recent times,

http://dx.doi.org/10.1016/j.micpath.2016.04.014
0882-4010/© 2016 Elsevier Ltd. All rights reserved.
2 A. Sharifzadeh et al. / Microbial Pathogenesis 96 (2016) 1e9

Table 1 identified in the Pharmacognosy Department, Faculty of Pharmacy,

Chemical compositions (%) of the Satureja hortensis L. essentiall oil analyzed by GC/ University of Tehran, Iran. The essential oil was prepared by the
MS.
Clevenger apparatus, using the hydro-distillation method. The
Number Components RI % Area leaves of Satureja hortensis was chopped and submitted in a
1 a-thujene 829 1.18 distillation apparatus with 1 liter of distilled water and for 5 h. The
2 a-phellandrene 852 0.91 oil was isolated, dried over anhydrous sodium sulfate and stored in
3 a-pinene 858 0.68 a sealed dark glass bottle at 4  C until required.
4 Sabinene 899 2.65
5 b-pinene 905 0.28
6 В-myrcene 916 1.24 2.3. Gas chromatographyemass spectrometry analysis of essential
7 P-cymene 948 9.61
8 L-limonene 951 0.2
oil
9 a-terpinene 968 2.91
10 В-phellandrene 996 0.84 Chromatographic analysis of the essential oil (EO) was per-
11 l-terpinene 1029 16.71 formed using a Gas chromatographyemass spectrometry (GC-MS)
12 Carvacrol methyl ether 1112 0.1
68905973 systems (Agilent Technologies, Palo Alto, CA, USA)
13 Thymol 1195 45.9
14 Carvacrol 1203 12.81 equipped with a HP-5 MS fused silica capillary column
15 Thymol acetate 1268 0.5 (30 m  0.25 mm i. d., 0.25 mm film thickness). The mass spec-
16 Carvacrol acetate 1294 0.1 trometer was operated in the electron ionization mode with an
17 l-Cadinene 1394 0.84 electron energy value 70 eV. Helium gas was used as the carrier gas
18 Cary ophyllene oxide 1431 0.31
19 b-bisabolene 1482 1.21
at a constant flow rate of 1 ml/min. The injector and mass transfer
20 Spathulenol 1512 0.12 line temperatures were set at 250 and 280  C, respectively.
21 Tau-cadinol 1583 0.3 Essential oil solution (1 ml) in hexane was injected and then
analyzed under the following column conditions: initial column
temperature at 40  C for 1 min, which was then increased to
interest in natural products has increased and medicinal plants 250  C at a 3  C/min heating ramp, and then subsequently kept at
have been investigated for various biological activities and thera- 250  C for 20 min.
peutic potentials. The plant derived drugs have been reported to be The Kovats indices were calculated for all volatile components
safe and without side-effects and antimicrobial properties of plant using a homologous series of n-alkanes (C8eC25) on the HP-5 MS
volatile oils have been recognized since antiquity [9e12]. The po- column. The major oil components were identified via co-injection
tential use of essential oils for the prevention and treatment of with standards (whenever possible) and confirmed through the
candida biofilms has been evaluated in several studies [12e17]. Kovats indices using the Wiley (V.7.0) and National Institute of
Furthermore, the exploration of new and effective natural products Standards and Technology (NIST) V.2.0 GCeMS library. The relative
showing antifungal activity against C. albicans biofilm formation concentration of each compound in the essential oil was quantified
and mature biofilms with low cytotoxicity is likely to significantly based on the peak area integrated in the analysis program.
impact the treatment, and the management of biofilm associated
fungal infection [17,18]. Thus, the objective of this study were firstly 2.4. Effect of S. hortensis L. EO on C. albicans planktonic cells
to assess biofilm formation ability and biofilm development ki-
netics of C. albicans isolates, and secondary evaluation of anti- A microdilution broth susceptibility method for yeasts was used,
candida activity of S. hortensis L. EO against planktonic and bio- as recommended by CLSI reference method for the determination
film growth of C. albicans isolates isolated from buccal lesions of of the Minimum inhibitory and fungicidal concentrations (MIC and
HIV þ individuals using MTT reduction assay followed by scanning MFCs) with some modifications (M27-A3). Briefly, different di-
electron microscopy (SEM) observation. lutions (in DMSO 5%) of S. hortensis L. EO (150, 250, 300, 350, 400,
500, 600, 700, 800, 900 and 1000 mg/ml) was prepared in 96-well
2. Materials and methods microtiter plates using RPMI-1640 media (Sigma, St. Louis, MO,
USA) buffered with MOPS (Sigma, St. Louis, MO, USA). Inocula for
2.1. Organisms the assay were prepared using diluting scraped C. albicans cell mass
in to 0.85% NaCL solution, adjusted to McFarland scale 0.5, and
For the development of this study, fifteen oral C. albicans isolates confirmed by spectrophotometric reading at 580 nm (this yields,
with American type culture collection (ATCC 10231) C. albicans stock suspension of 104 CFU ml1 in RPMI-1640 medium for yeasts).
strain, were used. The yeasts were isolated from Iranian HIVþ in- Working inoculums (0.1 ml) was added to the Microtiter plates,
dividuals and identified in our previous study by conventional which were incubated at 30  C for 24e48 h. Non-inoculated me-
methods such as CHROMagar-Candida, morphology and biochem- dium (200 ml) was included as a sterility control (blank). In addition,
ical tests. The pattern of fluconazole resistance of these isolates was growth controls (medium with inoculums but without essential oil)
described earlier [19]. All C. albicans isolates were stored at 80  c were also included. The growth in each well was compared with
in Yeast Extract Peptone Dextrose (YPD) medium (Difco, MD, USA) that of the growth in the control well. MICs were visually deter-
with 15% glycerol and maintained at the Mycology Research Center, mined and defined as the lowest concentration of the essential oil
Faculty of Veterinary Medicine, University of Tehran, Iran. Prior to produced growth inhibition compared with the growth in the
use, frozen stocks were recovered from storage by culture on Sab- control well. Aliquots of the mixture of oil and the Candida sus-
ouraud Dextrose Agar (SDA; Gibco Ltd., Paisley, United Kingdom) pension, which showed negative-visible growth after the first 24 h
medium. of incubation, were inoculated onto the surface of the Sabouraud
dextrose agar. The lowest concentration of the oil giving negative
2.2. Plant materials growth of the Candida was recorded as the minimum fungicidal
concentration (MFC). All experiments were repeated on three
Fresh leaves of Satureja hortensis was purchased from the Na- separate occasions, with triplicate determinations on each
tional Botanical Garden of Iran (NBGI). Plants were taxonomically occasion.
 A. Sharifzadeh et al. / Microbial Pathogenesis 96 (2016) 1e9 3

Table 2
Susceptibility of planktonic and sessile C. albicans cells isolated from buccal lesions 0.9
of HIV þ individuals to S. hortensis L. EO (SMIC; MIC of sessile or biofilm cells).

Metabolic activity (Absorbance 540 nm)

Test isolates MIC (mg/ml) MFC (mg/ml) SMIC50
(mg/ml) 0.8

C. albicans F81 300 400 1200

C. albicans F94 200 300 1200
C. albicans F87 300 400 1200 0.7
C. albicans F49 400 600 1200
C. albicans F82 400 600 800
C. albicans F95 400 400 800 0.6
C. albicans F92 300 600 1200
C. albicans F60 400 600 800
C. albicans F86 200 300 1200
0.5
C. albicans F91 300 400 1200
C. albicans F69 200 300 800
C. albicans F1 200 300 1200
C. albicans F34 200 300 800 0.4
C. albicans F19 200 300 1200 F81 F94 F87 F49 F82 F95 F92 F60 F86 F91 F69 F1 F34 F19 F78
C. albicans F78 400 600 800
C. albicans strains
MIC: Minimum Inhibitory Concentration.
MFC: Minimum Fungicidal Concentration. Fig. 2. Biofilm forming ability of different isolates of C. albicans. Values represent the
SMIC: Sessile. Minimum Inhibitory Concentration. mean and standard deviation of biofilms formed in wells of microtiter plates (MTT
reduction assay OD550nm).

Fig. 1. Kinetics of biofilm development by C. albicans isolates in wells of microtiter plates as determined by the colorimetric MTT-reduction assay. The X axis represents the time
intervals; the Y axis represents metabolic activity (colorimetric reading at 550 nm) of C. albicans biofilms.
 4
Table 3
Effects of different concentrations of S. hortensis L. EO on biofilm formation (%) in C. albicans isolates.

Concentration (mg/ml) Reduction percent in biofilm formation over untreated control

C. F87 F94 F81 F49 F82 F95 F91 F60 F86 F92 F69 F1 F34 F78 F19

50 4 ± 2.8 2 ± 2.2 1 ± 6.8 4 ± 2.8 4 ± 8.4 8 ± 6.1 2 ± 1.4 1 ± 0.2 3 ± 2.6 3 ± 1.4 7 ± 2.1 3 ± 0.9 1 ± 3.1 1 ± 0.7 2 ± 1.3
100 7 ± 0.5 4 ± 5.6 8 ± 3.8 7 ± 5.3 9 ± 5.3 10 ± 5.2 6 ± 1.0 20 ± 6.1 12 ± 1.1 8 ± 5.3 27 ± 1.6 16 ± 1.4 3 ± 0.8 9 ± 1.4 12 ± 0.9
200 8 ± 0.5 16 ± 0.8 16 ± 8.3 14 ± 8.1 15 ± 2.5 15 ± 7.2 9 ± 1.5 21 ± 5.0 20 ± 8.1 16 ± 8.0 36 ± 1.1 20 ± 0.9 7 ± 1.7 17 ± 2.4 23 ± 4
300 12 ± 1.3 19 ± 3.4 33 ± 6.1 17 ± 7.3 19 ± 4.1 22 ± 5.0 16 ± 3.6 22 ± 3.4 24 ± 5.5 19 ± 3.5 44 ± 1.6 23 ± 0.7 25 ± 1.5 23 ± 0.9 31 ± 0.8
400 28 ± 1.7 20 ± 1.6 34 ± 5.6 33 ± 1.6 30 ± 3.3 26 ± 3.2 21 ± 7.2 51 ± 8.2 27 ± 7.3 21 ± 5.1 46 ± 2.8 26 ± 1.6 38 ± 4.6 31 ± 1.1 36 ± 2.5
600 36 ± 0.5 28 ± 7.1 35 ± 3.3 44 ± 6.2 43 ± 4.2 33 ± 4.2 28 ± 7.1 53 ± 2.4 29 ± 7.0 26 ± 7.7 48 ± 1.1 34 ± 7.2 45 ± 3.1 40 ± 0.9 46 ± 1.8
800 38 ± 2.7 44 ± 1.4 49 ± 8.4 46 ± 8.3 50 ± 2.5 52 ± 5.4 31 ± 8.4 58 ± 5.1 37 ± 1.4 32 ± 1.3 57 ± 1.5 49 ± 2.8 51 ± 0.9 58 ± 2.4 49 ± 1.9
1200 46 ± 2.9 60 ± 6.7 60 ± 2.9 58 ± 2.2 61 ± 6.2 60 ± 5.2 58 ± 6.0 59 ± 5.3 52 ± 1.0 58 ± 1.5 66 ± 1.3 63 ± 0.6 57 ± 2.3 64 ± 0.8 68 ± 3.1
1600 64 ± 2.4 68 ± 2.2 65 ± 5.3 63 ± 5.3 70 ± 8.1 64 ± 1.3 70 ± 7.2 62 ± 3.8 66 ± 8.2 66 ± 2.3 71 ± 3.1 70 ± 2.0 61 ± 0.8 69 ± 3.7 77 ± 0.2
2400 66 ± 5.6 69 ± 6.5 67 ± 3.3 65 ± 3.3 73 ± 9 64 ± 8.5 72 ± 4.4 63 ± 3.3 67 ± 4.3 67 ± 8.8 73 ± 2.6 75 ± 0.6 63 ± 3.6 76 ± 1.7 81 ± 4.3

A. Sharifzadeh et al. / Microbial Pathogenesis 96 (2016) 1e9

3200 69 ± 4.9 70 ± 5.3 71 ± 8.1 69 ± 8.4 75 ± 5.3 66 ± 3.2 76 ± 6.1 75 ± 6.2 69 ± 4.4 69 ± 6.3 76 ± 8.5 82 ± 4.2 76 ± 2.4 79 ± 5.3 83 ± 2.6
4800 86 ± 2.1 90 ± 8.2 91 ± 1.2 92 ± 7.1 89 ± 1.6 81 ± 1.8 83 ± 8.4 80 ± 0.8 87 ± 2.2 90 ± 2.2 91 ± 1.8 87 ± 1.4 86 ± 1.5 84 ± 1.4 90 ± 0.8

Table 4
Effect of different concentrations of S. hortensis L. EO on mature biofilm in C. albicans isolates.

Concentration (mg/ml) Reduction percent in mature biofilms over untreated control

C. F87 F94 F81 F49 F82 F95 F91 F60 F86 F92 F69 F1 F34 F78 F19

50 31 ± 1.3 2 ± 1.3 1 ± 6.8 4 ± 2.8 4 ± 8.4 8 ± 6.1 2 ± 1.4 1 ± 0.2 3 ± 2.6 3 ± 1.4 2 ± 0.8 7 ± 1.3 3 ± 0.8 8 ± 1.2 3 ± 0.5
100 49 ± 2.1 9 ± 2.2 8 ± 3.8 7 ± 5.3 9 ± 5.3 10 ± 5.2 6 ± 1.0 20 ± 6.1 12 ± 1.1 8 ± 5.3 27 ± 4.1 14 ± 2.0 9 ± 1.2 10 ± 0.8 20 ± 2.3
200 57 ± 0.9 14 ± 2.1 16 ± 8.3 14 ± 8.1 15 ± 2.5 15 ± 7.2 9 ± 1.5 21 ± 5.0 20 ± 8.1 16 ± 8.0 35 ± 2.8 25 ± 1.9 30 ± 1.2 31 ± 3.4 23 ± 3.7
300 62 ± 2.1 28 ± 4.2 33 ± 6.1 17 ± 7.3 19 ± 4.1 22 ± 5.0 16 ± 3.6 22 ± 3.4 24 ± 5.5 19 ± 3.5 45 ± 2.6 25 ± 6.2 46 ± 1.8 43 ± 1.5 38 ± 0.1.6
400 70 ± 0.7 55 ± 2.3 34 ± 5.6 33 ± 1.6 30 ± 3.3 26 ± 3.2 21 ± 7.2 51 ± 8.2 27 ± 7.3 21 ± 5.1 52 ± 3.7 41 ± 3.4 53 ± 0.6 68 ± 1.5 54 ± 3.3
600 72 ± 1.6 66 ± 4.1 35 ± 3.3 44 ± 6.2 43 ± 4.2 33 ± 4.2 28 ± 7.1 53 ± 2.4 29 ± 7.0 26 ± 7.7 58 ± 0.9 49 ± 0.9 64 ± 1.1 40 ± 3.1 74 ± 5.2
800 73 ± 0.7 70 ± 2.3 49 ± 8.4 46 ± 8.3 50 ± 2.5 52 ± 5.4 31 ± 8.4 58 ± 5.1 37 ± 1.4 32 ± 1.3 74 ± 6.7 57 ± 1.1 76 ± 2.4 58 ± 1.1 76 ± 7.1
1200 73 ± 1.4 74 ± 4.4 60 ± 2.9 58 ± 2.2 61 ± 6.2 60 ± 5.2 58 ± 6.0 59 ± 5.3 52 ± 1.0 58 ± 1.5 77 ± 1.1 72 ± 2.6 78 ± 4.4 64 ± 0.7 81 ± 5.4
1600 74 ± 1.1 75 ± 5.3 65 ± 5.3 63 ± 5.3 70 ± 8.1 64 ± 1.3 70 ± 7.2 62 ± 3.8 66 ± 8.2 66 ± 2.3 78 ± 4.1 76 ± 0.8 81 ± 0.9 69 ± 2.1 81 ± 6.2
2400 74 ± 3.8 75 ± 6.2 67 ± 3.3 65 ± 3.3 73 ± 9 64 ± 8.5 72 ± 4.4 63 ± 3.3 67 ± 4.3 67 ± 8.8 78 ± 4.4 76 ± 1.4 82 ± 1.6 76 ± 3.2 81 ± 6.9
3200 74 ± 5.3 76 ± 4.8 71 ± 8.1 69 ± 8.4 75 ± 5.3 66 ± 3.2 76 ± 6.1 75 ± 6.2 69 ± 4.4 69 ± 6.3 78 ± 7.5 77 ± 3.9 86 ± 3.8 79 ± 2.1 82 ± 5.6
4800 76 ± 5.6 80 ± 6.7 91 ± 1.2 92 ± 7.1 89 ± 1.6 81 ± 1.8 83 ± 8.4 80 ± 0.8 87 ± 2.2 90 ± 2.2 80 ± 6.8 79 ± 5.4 91 ± 2.5 84 ± 5.4 85 ± 4.8
 A. Sharifzadeh et al. / Microbial Pathogenesis 96 (2016) 1e9
Biofilm development
Mature biofilm
100
90
Reduction in metabolic activity %
80
70
60
50
40
30
20
10
0 described above. Briefly, after biofilm formation, the media was
Rela ve EO concentra on (μg/mL)
Fig. 3. In vitro inhibitory effects of S. hortensis L. EO on biofilm development and
mature biofilms produced by oral isolates of C. albicans. (Values are expressed as
mean ± SD).
2.5. Evaluation of biofilm formation ability by C. albicans isolates
Biofilm formation capacity was appraised according to the
method described by Pierce [20] with slight modifications. Briefly,
C. albicans isolates were grown on SDA plates at 37  C for 18 h and
then inoculated into Yeast Nitrogen Base (YNB) medium (Fulka)
containing 50 Mm glucose and pH 7. After overnight incubation at
30  C and in a rotary shaker at 75 rpm, the cells were harvested at
3000 g for 10 min at 4  C and washed twice with sterile Phosphate
Buffered Saline (PBS) pH 7.2. Pellets were then diluted in RPMI 1640
supplemented with L-glutamine and buffered with 0.165 M
morpholine-propanesulfonic acid (MOPS) (Sigma-Aldrich, Milan,
Italy) and the cellular density adjusted to 1  106 cells ml1 using a
Neubauer counting chamber (working solution). C. albicans bio-
films were formed on commercially available, pre-sterilized, poly-
styrene, flat-bottomed, 96-well microtitre plates (JetBiofil). Outset,
wells of these plates was coated for at least 30 min at 37  C with
100 ml 50% Fetal Bovine Serum (FBS). The wells were washed once
with 200 ml PBS and 100 ml of working solution were seeded in
selected wells for 90 min at 37  C. Non-adherent yeasts were
removed by gentle washing twice with 200 ml PBS. Subsequently,
biofilm formation was propagated by incubating selected wells in
200 ml RPMI over a series of time intervals (0, 3, 6, 12, 18, 24, 48, 72
and 96 h) at 37  C to serve as controls for biofilm development. At
each time interval, biofilm development was quantified using a 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) (Sigma-Aldrich-USA) reduction assay. An aliquot of MTT
solution (0.5 mg/ml in PBS containing 0.1% glucose; 100 ml) was
added to each of the prewashed wells and the plates were then
incubated for 6 h in the dark at 37  C. Following the incubation,
plates were washed once with PBS. Acid-isopropanol (200 ml) was
then added to solubilize the MTT formazan product. The color in-
tensity of acid-isopropanol dissolved formazan was determined by
a microplate reader (Bio-Tek Instruments Inc., USA) at 550 nm. For
each time interval, four biofilms replicate were evaluated.
5
2.6. Effect of S. hortensis L. EO on biofilm formation
The biofilm formation in 96-well microtiter plates was per-
formed as described previously [20]. Briefly, 100 ml of the various
concentration including 50, 100, 200, 300, 400, 600, 800, 1200,
1600, 2400, 3200 and 4800 mg/ml of S. hortensis L. EO diluted with
RPMI-1640 in the microtiter plates. An equal volume of the candida
suspension (106 cell/ml) was added and mixed with the EO, the
plates were incubated for 48 h at 37  C. After biofilm formation the
inhibitory effect of the EO on biofilm formation was determined by
MTT reduction assay. The percentage of inhibitory effect was
calculated by using the formula [1- (OD550 Sample/OD
Control]  100%. The test was performed in three independent
experiments, each in triplicate.
2.7. Effect of S. hortensis L. EO on preformed C. albicans biofilms
C. albicans biofilms were formed for 24 h at 37  C on the surface
of the wells of microtiter plates using the previously protocol
aspirated and non-adherent cells were removed by thoroughly
washing the biofilms three times in sterile PBS. Residual PBS was
removed by blotting with paper towels before the added of EO.
Different S. hortensis L. EO concentrations (25, 50, 100, 200, 300,
400, 500, 800, 1000, 1500, 2000 and 3000 mg/ml in RPMI-1640)
were added to selected wells and the plates were incubated for
24 h at 37  C. A series of EO-free wells and biofilm-free wells were
also incubated as positive and negative control, respectively. Effect
of EO on preformed biofilms was estimated using the MTT reduc-
tion assay. The percentage of inhibitory effect was calculated by
using the formula [1-(OD550 Sample/OD Control]  100%. Testing
was performed in triplicate.
2.8. Scanning electron microscopy (SEM)
For SEM, C. albicans biofilms were formed on sterile poly (vinyl
chloride) coverslips (with thickness of 0.18 mm and radius of
7.5 mm) within 24-well microtiter plates (Biofil) in the presence of
½ and ¼  MIC (Sub MIC) of S. hortensis L. EO for 48 h at 37  C as
described in the preceding section. Biofilm grown in the absence of
EO served as control. For SEM observation, the biofilms were
assayed as previously described by Priester [21]. Briefly, the cov-
erslips were removed, washed twice with sterile PBS (0.1 M and
PH ¼ 7.2) and placed in primary fixative solution (glutaraldehyde
0.15 M 2.5% (VOL/VOL) in PBS) for 60 min at 4  C. After this pro-
cedure, the coverslips were rinsed 2 times for 5 min each in PBS.
Then, the coverslips were treated with secondary fixator (Osmium
tetroxide (OsO4 1% W/V) for 1 h. Samples were subsequently
washed with distilled water, dehydrate in a series of ethanol so-
lutions (70% for 10 min, 95% for 10 min and 100% for 20 min) and air
dried overnight in a desiccator containing silica gel prior to ion
sputter coating with gold (KYKY Technology Development Ltd.
SBC-12). After gold coating the samples were viewed under SEM
(KYKY-EM3200, China).
2.9. Statistical analysis
Results were compared by One-Way analysis of variance
(ANOVA) using SPSS software. All tests were performed with a
confidence level of 95%.
3. Results
S. hortensis L. EO was analyzed using GC-MS and the compo-
nents were identified on the basis of their RI values as well as by
6 A. Sharifzadeh et al. / Microbial Pathogenesis 96 (2016) 1e9

Fig. 4. Scanning electron microscopy of C. albicans isolates biofilms formed at 48 h. This figure indicate the structural difference among C. albicans isolates biofilms.
 A. Sharifzadeh et al. / Microbial Pathogenesis 96 (2016) 1e9
comparison of their mass spectra with those reported in literature.
Based on GC-MS results, 21 components were identified, repre-
senting 99.4% of the S. hortensis L. EO. As Table 1, thymol (45.9%),
gamma-terpinen (16.71%), carvacrol (12.81%) and p-cymene
(9.61%) were found as the most abundant constituents.
The MICs and MFCs values of S. hortensis L. EO towards
C. albicans isolates using broth microdilution method are illustrated
in Table 2. MIC values ranged from 250 to 400 mg/ml
(mean ¼ 306.7 ± 53 mg/ml) and MFC values ranged from 350 to
500 mg/ml (mean ¼ 407.1 ± 72.8).
In this study, the kinetics of biofilm formation over a period of
96 h was determined by quantitative assessment of biofilm mass
metabolic activity after the attachment of C. albicans cells to the
surface of polystyrene plates. As it shown in Fig. 1, overall, biofilm
formation curves demonstrated that the kinetics of biofilm for-
mation among all isolates of C. albicans was similar. In all C. albicans
isolates the maximum cell density or uttermost growth rate of
biofilm was performed in 48 h after the cell attachment with the
exception of F60 isolate. Afterwards (72e96 h), the cell density of
biomass began to decrease. Kinetics of biofilm formation showed a
similar trend in four separate replication. Fig. 2 presents the results
of biofilm quantification using MTT reduction assay. It was evidence
that all C. albicans isolates formed biofilm on polystyrene plats after
48 h but the quantity of biofilm mass (OD at 550 nm) was different
for the isolates ranging from 0.850 to 0.559 nm (average
Abs ¼ 0.668 ± 0.06).
Biofilm formation was highest in C. albicans F87 and F94
Fig. 5. Scanning electron micrographs of mature C. albicans biofilms in the presence S. hortensis L. EO.
7
followed by F19 and F49 isolates (Fig. 2). However, no significant
differences were observed between biofilm mass of C. albicans
isolates (p ¼ 0.095). The inhibitory effect of EO on biofilm formation
is depicted in Table 3. Fig. 3 revealed that the inhibitory effect of EO
on biofilms appeared to be dose dependent. The mean of biofilm
formation by C. albicans isolates was reduced by 87.1± 3.7%,
73.6± 5.1%, 69.4± 5.3% and 67± 4.2% at 4800, 3200, 2400 and
1600 mg/ml, respectively. In less than 1200 mg/ml dilutions, the
metabolic activity reduction rate was less than 50%. The average
inhibition percentage of S. hortensis L. EO (50e4800 mg/ml) on
metabolic activity of mature C. albicans biofilms ranged from 7.1 ± 7
to 80± 2.8% Table 4. Fig. 3 the analyses of MIC and SMIC indicated
that the SMIC of S. hortensis L. EO for 33.3% (n ¼ 5) isolates of
C. albicans was twofold to the MIC and in 66.7% (n ¼ 10) isolates
SMIC was three times higher than the MIC (Table 2). The action of
S. hortensis L. EO upon C. albicans biofilm morphology, evaluated by
SEM analysis. Results showed that, although some of the differ-
ences were observed in the three-dimensional structure of biofilms
produced by different C. albicans isolates, but there was no evident
different between ultra-structural visages of biofilms. Treated bio-
film cells with EO showed obvious morpho-structural changes
compared to non-treated cells (Fig. 4). Untreated sessile cells
showed smooth cell membrane whereas treatment with EO
exhibited shrinkage in cell membranes of sessile cells. in treated
groups, filamentation is inhibited and destruction of three-
dimensional structure of biofilm was observed (Fig. 5).
Untreated sessile cells resulted in intact biofilm formation with
8 A. Sharifzadeh et al. / Microbial Pathogenesis 96 (2016) 1e9
a dense one or multilayered network (depending on isolate) of
yeast cells and filamentous forms scaffold “true and pseudo-hy-
phae” (Fig. 3) (see Fig. 5). Some of which was covered with dense
extracellular matrix leading to a compact three-dimensional
structure that covered the entire surface of the polyvinyl chloride
coverslips (Fig. 3). Meanwhile, in treated cells with EO the biofilm
mass density was reduced and in any of isolates extracellular ma-
trix was not observed (Fig. 3). Untreated sessile cells showed
smooth cell membrane whereas treatment with EO exhibited
shrinkage in cell membranes of sessile cells the treated cells
exhibited loosening of cells and disappearance of matrix. Fila-
mentation is inhibited and destruction of three-dimensional
structure of biofilms were observed. S. hortensis L. EO led to
sorely destruction of 3D biofilm network leaving only a few wrinkle
yeasts and sometimes disintegrated filamentous forms.
4. Discussion
C. albicans is one of the most frequently isolated human fungal
pathogen from oral cavity lesions especially in HIVþ patients [1,2].
Ability to formation of structured surface-attached microbial
communities known as biofilm is one of the major contributions
virulence factors related to the C. albicans. Biofilms can increase the
resistance to antifungal drugs, blocking or preventing the access of
antifungals to the more internal biofilm tree dimensional structure
[5,6,22]. In this context, new agents such as plant essential oils can
inhibit the growth of microorganisms with ability of biofilm
development are greatly needed and would enhance the number of
effective therapeutic alternative strategies [17,23,24]. Taking into
account this, the present study was carried out to assess the ki-
netics, ability of biofilm formation and antifungal properties of
S. hortensis L. EO upon planktonic and sessile cells of C. albicans oral
isolates. In this study, the biofilm formation ability of different
isolates of C. albicans was evaluated and the results demonstrated
that all isolates studied formed biofilms, although the biofilm for-
mation ability varied among C. albicans isolates. These results were
in agreement with those of other authors, who reported that ability
of candida species to develop biofilm structures [24e26]. Results of
current study demonstrated that biofilm forming potential of
C. albicans species was highly strain dependent. SEM analysis also
demonstrated structural and morphological differences among
biofilms of the studied isolates. Biofilms of C. albicans oral isolates
consisted of a compact network cells with different morphological
characteristics. All C. albicans isolates expect F60 were constructed
yeasts, pseudo and true hyphae but, biofilm of F60 was subtended
only of yeast and pseudo hyphae cells. It is known that biofilm
structures is dependent on several environmental including growth
conditions, nature of surface colonization and microbial species
[25,27e29]. Such finding probably reflects inherent physiological
differences among isolates and could have significance with respect
to pathogenic potential.
As revealed by GC-MS analysis, thymol was the major com-
pounds of S. hortensis L. EO followed by g-Terpinene, carvacrol and
P-cymene. These results found to be in agreement with the litera-
ture [30,31].
In this study, we used the MTT reduction assay to measure the
metabolic activity of C. albicans biofilms treated with S. hortensis L.
EO. According to the results this EO inhibited all C. albicans isolates
in planktonic and sessile forms with different concentrations in a
dose dependent manner (mean of MIC, MFC and SMIC50 were
306 ± 53, 407 ± 72 and 1040 ± 202.8 mg/ml, respectively).
These results can be explained on the basis of chemical
composition of this EO. High standard deviations in relation to
SMIC might be due to the difference in biofilm mass created by
different C. albicans isolates. Thymol was the major active
ingredient of S. hortensis L. EO. This indicates that inhibitory activity
of this oil is largely due to the thymol. However, minor constituents
may also play a key role in the biological activities of this EO. Braga
et al. demonstrated that thymol interfered with the starting phases
of biofilm formation by reducing the amount of metabolically active
yeast as well as the dimorphic switching from yeast to filamentous
forms [32,33]. In summary, the current study demonstrated that
S. hortensis L. EO has potential antifungal activity against biofilm or
planktonic cultures of C. albicans obtained from patients with HIV
and may find use in future therapeutic strategies. Finally, further
work involving In vitro and In vivo experiments is needed in order
to determine the validity of our observations.
Acknowledgments
This work was supported by the Research Council of the Uni-
versity of Tehran. The authors would like to thanks Mr. Ali Ghayour
who helped us in this study.
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