Biofouling

The Journal of Bioadhesion and Biofilm Research

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Anti-biofilm activities of essential oils rich in

carvacrol and thymol against Salmonella Enteritidis

Ivana Čabarkapa, Radmilo Čolović, Olivera Đuragić, Sanja Popović, Bojana

Kokić, Dubravka Milanov & Lato Pezo

To cite this article: Ivana Čabarkapa, Radmilo Čolović, Olivera Đuragić, Sanja Popović,
Bojana Kokić, Dubravka Milanov & Lato Pezo (2019) Anti-biofilm activities of essential oils
rich in carvacrol and thymol against Salmonella Enteritidis, Biofouling, 35:3, 361-375, DOI:
10.1080/08927014.2019.1610169

To link to this article: https://doi.org/10.1080/08927014.2019.1610169

Published online: 14 May 2019.

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BIOFOULING
2019, VOL. 35, NO. 3, 361–375
https://doi.org/10.1080/08927014.2019.1610169

Anti-biofilm activities of essential oils rich in carvacrol and thymol against

Salmonella Enteritidis


Ivana Cabarkapaa

, Radmilo Colovica - uragica
, Olivera D , Sanja Popovica , Bojana Kokica ,
b
Dubravka Milanov and Lato Pezoc
a
University of Novi Sad, Institute of Food Technology, Novi Sad, Serbia; bScientific Veterinary Institute “Novi Sad”, Rumena
cki put bb,
Novi Sad, Serbia; cUniversity of Belgrade, Institute of General and Physical Chemistry, Belgrade, Serbia

ABSTRACT ARTICLE HISTORY

The aim of the present study was to determine the bioactive compounds in four essential oils Received 4 December 2018
(EO’s) from Origanum heracleoticum, Origanum vulgare, Thymus vulgaris and Thymus serpyllum Accepted 16 April 2019
and to assess their antimicrobial and anti-biofilm activity against Salmonella Enteritidis. Strains
KEYWORDS
were previously characterized depending on the expression of the extracellular matrix compo-
Biofilm; essential oil;
nents cellulose and curli fimbriae as rdar (red, dry and rough) and bdar morphotype (brown, dry antimicrobial and
and rough). This study revealed that the EO’s and EOC’s (carvacrol and thymol) investigated antibiofilm activity
showed inhibition of biofilm formation at sub-minimum inhibitory concentration. Comparing the
efficacy of EO’s and EOC’s in the inhibition of biofilm formation between the strains with differ-
ent morphotype (rdar and bdar) did not show a statistically significant difference. Results related
to the effectiveness of EO’s and EOC’s (the essential oil components, carvacrol and thymol) on
eradication of preformed 48 h old biofilms indicated that biofilm reduction occurred in a dose-
dependent manner over time.

Introduction Stoodley et al. 2002). The biofilm can be defined as a

Salmonella spp. are the second most commonly
reported bacterial pathogens in the European Union
and it has also been associated with numerous cases
of food-borne infections worldwide. S. Enteritidis is
the most frequently (44.0%) reported serotype, fol-
lowed by S. Typhimurium (17.4%), in confirmed cases
of human infections. Moreover, in 2014 the propor-
tion of salmonellosis caused by S. Enteritidis was sig-
nificantly higher when compared to the previous year
(EFSA 2015). The frequent involvement of serotype
Enteritidis in human salmonellosis may be a conse-
quence of the strain’s ability to form biofilms, but this
still remains to be proved.
It is widely recognized that, in response to different
environmental conditions, bacteria have developed a
variety of strategies to adapt and survive. The forma-
tion of multicellular communities, known as biofilms,
is one such strategy, which is generally associated
with the survival and persistence in different environ-
mental conditions (de la Fuente-N ~ez et al. 2013;
un
Hall-Stoodley et al. 2004). It is also well-accepted that
the biofilm represents the dominant lifestyle of bac-
teria in all environments (Flemming et al. 2016;
highly structured community of microorganisms that
are attached to a surface or an interface, embedded in
a self-produced extracellular matrix (ECM) (Donlan
and Costerton 2002; Flemming et al. 2016).
Biofilms formed in food processing environments
may represent a long-term source of food contamin-
ation, not only with food spoilage bacteria, but also
with food-borne pathogens such as Salmonella spp.
(Chia et al. 2009), Campylobacter spp. (Hanning et al.
2008; Joshua et al. 2006), Escherichia coli (Dourou
et al. 2011; Sheen and Hwang 2010) and Listeria
monocytogenes (Møretrø and Langsrud 2004;
Stepanovic et al. 2004). Furthermore, the existence of
bacteria in biofilms in the food industry may cause
cross- and post- process contamination and economic
losses by reducing the shelf life of food products,
increasing food spoilage, impairing heat transfer, and
increasing corrosion rate (Garrett et al. 2008; Sim~ oes
et al. 2010). Also, it should be noted that the existence
of bacteria in biofilms contributes to the acquisition
of tolerance to cleaning and disinfection agents
(Brooks and Flint 2008; Mariscal et al. 2009; Srey
et al. 2013). Previous studies indicated that bacterial

CONTACT Ivana Cabarkapa ivana.cabarkapa@fins.uns.ac.rs

ß 2019 Informa UK Limited, trading as Taylor & Francis Group
362 I. CABARKAPA ET AL.
exposure to sub-lethal concentrations of sanitizers
may increase their tolerance to antibiotics, that can
lead to concern and implications for public health
(Condell et al. 2012). Due to the high tolerance of
biofilms to current treatment techniques, higher con-
centrations of sanitizers and more effective treatment
measures are required in order to remove biofilm
forming strains (Kroupitski et al. 2009; Soni et al.
2013). However, the occurrence of disinfectant resi-
dues may have damaging effects on human health
along with undesirable sensory characteristics, which
has shifted consumer preference towards natural
€
ingredients (Olmez and Kretzschmar 2009).
In this respect, it is very important to acquire
knowledge about the nature and behavior of food-
borne pathogens as biofilm-associated cells, in order
to optimize cleaning and disinfection procedures in
the food industry. Over the last decades, a large
number of studies have focused on the evaluation of
plant-based compounds as potential sources of anti-
microbial agents, since they are effective, safe and
eco-friendly. The anti-biofilm activity of essential
oils (EO’s) against different food-borne pathogens
such as Listeria spp. (Jadhav et al. 2013; Upadhyay
et al. 2013), Vibrio spp. (Manju et al. 2016),
Staphylococcus aureus and E. coli (Bazargani and
Rohloff 2016), S. Typhimurium (Miladi et al. 2016;
Soni et al. 2013) and mixed biofilm of S. aureus and
S. Typhimurium (Knowles et al. 2005) has been
reported, however little attentions has been given to
S. Enteritidis.
The aim of the present study was to investigate the
antimicrobial and anti-biofilm activity of EO’s from
Origanum heracleoticum, Origanum vulgare, Thymus
vulgaris and Thymus serpyllum, as well as their active
components carvacrol and thymol, against S.
Enteritidis using in vitro assays.
Materials and methods
Plant material and extraction
The plant materials (O. heracleoticum, O. vulgare,
T. vulgaris and T. serpyllum) used in this study were
obtained from the Institute of Medicinal Plant
Research “Dr. Josif Pan
cic”, Belgrade, Serbia and vou-
cher specimens (IC1312, IC1212, IC1112 and IC1012)
were deposited in the herbarium of the mentioned
Institute. EO’s were extracted from dried plant mate-
rials (100 g) by hydro-distillation using Clevenger type
apparatus for 4 h. The extracted EO’s were dried
using anhydrous sodium sulfate and stored in sealed
dark vials at 4
C. The yield of each EO was expressed
as a volume percent (% v/w), calculated relative to
100 g of dried plant material. Identification of essential
oil components (EOC’s) was done by gas chromatog-
raphy mass spectrometry (GC-MS) analysis following a


previously described procedure (Cabarkapa et al. 2011).
EOC’s (carvacol and thymol) were obtained from
Sigma-Aldrich (Switzerland).
Bacterial strains
All tests in this study were performed using two
strains of S. Enteritidis isolated from poultry. Tested
strains were obtained from the Veterinary Institute in
Kraljevo. Serological typing and verification of
Salmonella strains were carried out in the National
Reference Laboratory for Salmonella, Shigella, Vibrio
cholera and Yersinia enterocolitica, Institute of Public
Health of Serbia “Dr. Milan Jovanovic Batut”,
Belgrade, Serbia. Strains of S. Enteritidis were previ-
ously characterized depending on the expression of
ECM components as rdar (cellulose þ curly fimbriae)


and bdar morphotypes (curly fimbriae) (Cabarkapa
et al. 2015). Strains were cultured on Tryptone Soya
agar (TSA) (Lab M International Diagnostics Group
Plc, Bury, Lancashire, UK) and incubated at 37
C
for 24 h. Isolated colonies were picked and transferred
to 5 ml of Tryptone Soy Broth (TSB) (Lab M,
International Diagnostics Group Plc, Bury, Lancashire,
UK) and incubated at 37
C for 18 h. After incubation,
suspensions were diluted 1:40 with fresh TSB. The
density of the suspensions used for biofilm formation
was adjusted to 0.5 Mc Farland units (1-2 x 108 CFU
ml1) using a densitometer DEN-1 (Biosan, Riga,
Latvia) and standard plate counts.
Determination of the minimum inhibitory
concentration (MIC) against planktonic cells and
the Checkerboard assay
Prior to the examination of the efficacy of EO’s and
EOC’s on biofilms, their effectiveness on planktonically
grown microorganisms was determined according to
the Clinical Laboratory Standards (CLSI 2018) with
slight modifications. The bacterial suspensions were
prepared using overnight cultures and adjusted to 0.5
Mc Farland standard turbidity. All tests were per-
formed in Muller-Hinton Broth (MHB) (Lab M,
International Diagnostics Group Plc, Bury, Lancashire,
UK). Propylene glycol (2-(2- hydroxypropoxy)-1-pro-
panol) (Applichem) was used for dissolving the EO’s/
EOC’s, as well for their dilution to the concentration
ranging from 50 to 0.024 ll ml1. Twenty-microliter

aliquots of each tested EO/EOC were added to 96-well
microtiter plates. Afterward, aliquots of 160 ml of
MHB, were added to each well. As the final step, 20 ml
of the standardized bacterial suspension were inocu-
lated into each well. The test was performed in a total
volume of 200 ml with final EO’s/EOC’s concentrations
ranging from 5 to 0.0024 ml ml1. Plates were incu-
bated at 37
C for 24 h. The same tests were performed
simultaneously for the growth control (MHB þ test
organism), the negative control (MHB þ propylene gly-
col þ test organism), sterility control (MHB þ test oil),
and the positive control (MHB þ streptomycin þ test
organism). Streptomycin was prepared in sterile water
and diluted in MHB.
Following incubation, 20 ml of the resazurin solu-
tion (0.01%) (Sigma-Aldrich) were added to each
well. Subsequently, the plates were further incubated
at 37
C for 6 h (in darkness). After visual examin-
ation, the plates were additionally incubated for 18 h.
A change of color from blue (oxidized) to pink
(reduced) indicated the growth of bacteria. On com-
pletion of the incubation, wells without color change
(blue color of resazurin remained unchanged) were
scored as above the MIC value. The MIC was defined
as the lowest concentration at which a color change
occurred (Elshikh et al. 2016).
Referring to the results of the MIC assay, the
wells showing the complete absence of growth were
identified and 100 ml of the solutions from each well
were transferred to Plate count agar plates (PCA)
(Lab M, International Diagnostics Group Plc, Bury,
Lancashire, UK) and incubated at 37
C for 24 h.
The minimal bactericidal concentration (MBC) was
defined as the lowest concentration of the EO’s/
EOC’s at which 99.9% of the inoculated microorgan-
isms were killed.
To detect any synergism or antagonism between
the carvacrol and thymol, a checkerboard assay was
carried out. Different concentration of carvacrol and
thymol were mixed in 1:1 rations and added to the
cell suspension.
The test was performed in a total volume of 200 ml
with final carvacrol and thymol concentrations rang-
ing from 5 to 0.0024 ml ml1. Plates were incubated
at 37
C for 24 h.
Color indicators were added as for the colorimetric
assay. MICs were determined for each component in
the presence of the second component, and fractional
inhibitory concentrations (FICs) were calculated from
these as follows:
MIC a þ b
FIC ðaÞ ¼ (1)
MIC a
BIOFOULING 363
MIC b þ a
FIC ðbÞ ¼ (2)
MIC b
FIC index ¼ FICðaÞ þ FICðbÞ (3)
where MIC aþb, - MIC (a in the presence of b); MIC
bþa - MIC (b in the presence of a); MICa - MIC a
alone; and MICb - MIC b alone.
The results were interpreted as synergistic when
the FIC index was 0.5, as additive when the index
was between 0.5 and 1.0, as indifferent when the
index was between 1.0 and 2.0, and antagonistic when
the index was 2.0
Biofilm biomass formation (crystal violet) assay
The biofilm formation assay applied in the present
study was based on the method previously described
by Lianou and Koutsoumanis (2012), with slight
modifications. More specifically, three wells of sterile
flat-bottomed 96-well polystyrene microtiter plates
(Greiner Bio-One) were filled with 180 ml of TSB.
Afterward, aliquots of 20 ml of each bacterial suspen-
sion were added to each well. Negative control wells
contained only 200 ml of TSB per well. The plates
were sealed and incubated statically for 48 h at 25
C.
Following incubation, the contents of each well were
removed and the wells were washed three times with
250 ll of phosphate-buffered saline (PBS) (Sigma-
Aldrich). Each plate was air dried in an inverted pos-
ition at room temperature for 30 min. Subsequently,
each well was stained with 250 ll of 0.5% crystal violet
(CV) (Sigma-Aldrich) for 20 min. Excess stains were
rinsed off by filling the wells with PBS and emptying
them by inversion of the plates for a total of six times.
The microtiter plates were air dried for 1 h. The bound
CV was solubilized in 250 ll of solution (ethanol/
acetone, 80:20%) for 15 min. The optical density of
wells was measured at 595 nm (OD595 nm) using an
automated microtiter plate reader (ChemWel,
Awareness Technology).
Effect of EO’s/EOC’s on biofilm formation
The influence of EO’s/EOC’s on biofilm formation
were assessed as previously described by Jadhav et al.
(2013) with slight modifications. In brief, three wells
of a sterile flat-bottomed 96-well polystyrene micro-
titer plates (Greiner Bio-One) were filled with 20 ml
of EO’s/EOC’s at the appropriate concentration
(0.25MIC, 0.5MIC, 1MIC, and 2MIC). Afterward, a 160
ml aliquot of TSB was added into each well. As the
final step, 20 ml of bacterial suspension adjusted to
0.5 Mc Farland standard turbidity was inoculated into
364 I. CABARKAPA ET AL.
each microplate. The final volume of each well was
200 ml. Simultaneously, the following controls were
positioned; positive control: TSB (160 ml), bacterial
suspension (20 ml) and propylene glycol (20 ml); nega-
tive control I: TSB (200 ll); negative control II: TSB
(160 ll) and the appropriate concentration of EO in
propylene glycol (20 ml). The plates were incubated at
25
C for 48 h. Following incubation, the content of
the plate wells was discarded. Afterward, the wells
were washed three times with 250 ll of PBS. Further
determination of EO’s/EOC’s effectiveness on biofilm
formation was assessed using the CV assay.
Efficacy of EO’s/EOC’s on preformed biofilms
Biofilms were formed according to the previously
described protocol until the drying step. Thereafter, dif-
ferent solutions of EO’s/EOC’s were added (equivalent
to 0.5MIC, 1MIC, 2MIC and 4MIC). Simultaneously, the
following controls were positioned; positive control:
TSB (180 ll) and bacterial suspension (20 ml); negative
control I: TSB (200 ml); negative control II: TSB
(180 ml) and an appropriate concentration of EO in
propylene glycol (20 ml).
After the desired contact times (15, 30 and 60 min)
at room temperature, the test product was discarded
and treated biofilms were washed with PBS.
Quantification of the total biomass of biofilm was car-
ried out using the CV assay.
Biofilm metabolic activity assay
The experimental setup for the determination of
metabolic activity levels using resazurin was per-
formed according to the protocol described by Tote
et al. (2010). In brief, after incubation, rinsing and
drying (previously described in protocols 2.5 and 2.6),
each well was inoculated with 190 ml of PBS and 10
ml of resazurin (0.01%). Afterwards, plates were incu-
bated at 37
C for 6 h. Subsequently, fluorescence
measurement was performed using a fluorescence
microplate reader (Fluroscan AscentTM FL Thermo
Labsystems) using filters at wavelengths for excitation
(544 nm) and emission (590 nm).
All assays were performed in triplicate, over two
sessions, but under the same conditions on different
testing days. The effect of EO’s on the initial adhesion
and preformed biofilm was determined using
Equation (4) (Kwasny and Opperman 2010).


A1A2
% reduction ¼ 1 x100 (4)
Apc  Anc
where: A1 – absorbance (OD595)/fluorescence
(OD544ex/590em) test wells; A2 - absorbance (OD595)/
fluorescence (OD544ex/590em) wells with negative con-
trol II; Apc – absorbance (OD595)/fluorescence
(OD544ex/590em) positive control and Anc- absorbance
(OD595)/fluorescence (OD544ex/590em) wells with nega-
tive control I (broth only).
In order to more easily evaluate the effectiveness of
EO’s, the following definition was introduced:
MBIC50/MBIC90 (Minimum Biofilm Inhibition
Concentration) - the lowest concentration of EO
required to prevent biofilm formation by 50% and
90%, respectively; MBEC50/MBEC90 (Minimum
Biofilm Eradication Concentration) - the lowest con-
centration of EO required to eradicate preformed bio-
film by 50% and 90%, respectively.
Statistical analysis
The data were processed statistically using the soft-
ware package Statistica 12 (StatSoft, Inc., Tulsa, OK,
USA; 2012). Analysis of variance (ANOVA) was con-
ducted to show the significant effects of independent
variables to the responses, and to show which of the
responses were significantly affected by the varying
treatment combinations.
Analysis of variance and post hoc Tukey’s HSD test
for comparison of means were used to analyse the
biofilm forming reduction and the reduction in meta-
bolic activity during biofilm formation for both the
tested strains (rdar and bdar), according to the type
of EO’s/EOC’s, concentration (Conc) and the time of
exposition (t).
The second order polynomial (SOP) models in the
following form were developed to relate responses (Y)
and process variables (X):
X
l X
l
Yk ¼ bk0 þ bki  Xi þ bkii  Xi2
i¼1 i¼1
(5)
X
l X
l1
þ bkij  Xi  Xj ; k ¼ 18
i¼1 j¼iþ1
where bk0 ; bki ; bkii ;bkij were constant regression coef-
ficients; Yk - the biofilm forming reduction for both
the tested strains (Y1 and Y2,), for rdar and bdar or
the reduction in metabolic activity, during biofilm
formation for both the tested strains (Y3,andY4), and
the biofilm forming reduction for both the tested
strains (Y5 and Y6,), for rdar and bdar and the
reduction in metabolic activity during biofilm forma-
tion for both the tested strains (Y7,andY8), in the
incubation period. The influential factors were labeled
 BIOFOULING 365

Table 1. Chemical composition (%) of O. heracleoticum (O1), Table 1. Continued.

O. vulgare (O2), T. vulgaris (T1) and T. serpyllum (T2) EO’s. Origanum
Origanum Plants spp. Thymus spp.
No
Plants spp. Thymus spp. Compound K.I.a O1 O2 T1 T2
No
Compound K.I.a O1 O2 T1 T2 cadalene 1674.2 0.07 – 0.04 0.02
tryciclene 917.3 -b 0.06 0.13 0.14 Total 98.32 98.08 98.26 97.26
a-thujene 923.0 0.63 0.25 0.19 0.36 Monoterpene hydrocarbons 20.38 28.49 28.67 45.58
a-pinene 928.5 0.73 1.35 0.76 1.22 Oxygenated monoterpenes 73.65 66.18 66.35 48.37
camphene 942.5 0.24 0.39 0.27 0.66 Sesquiterpene hydrocarbons 2.67 2.22 1.88 1.84
sabinene 971.0 0.21 – – – Oxygenated sesquiterpenes 0.45 0.3 0.66 0.53
b-pinene 971.2 0.16 0.55 0.03 0.04 Hydrocarbones 0.44 0.75 0.54 0.7
1-octen-3-ol 982.0 0.73 0.14 0.16 0.24 Others 0.73 0.14 0.16 0.24
a
myrcene 989.1 1.38 0.87 0.79 1.02 Kovats retention index
a- phellandrene 1001.2 0.16 0.13 0.92 0.95 b
Below threshold level of <0.01, or not detected
D-3-carene 1006.5 0.08 0.1 0.13 0.11 Levels of major compounds are marked in bold.
a-terpinene 1013.0 1.33 1.05 1.25 1.1
p-cymene 1023.1 7.7 19.9 18.71 34.84
b-phellandrene 1025.5 0.39 0.51 0.48 0.67 as follows: X1 - EO’s/EOC’s type, X2 - Conc and
1.8-cineole 1027.2 0.08 0.16 0.61 0.05 X3 - time of exposition (t).
(Z)-b-ocimene 1037.6 0.26 0.04 – –
(E)-b-ocimene 1047.4 0.02 0.02 – –
trans-decahydro-naphthalene 1047.4 – – 0.04 0.02
v-terpinene 1055.5 6.85 3.11 4.34 4.38
cis-sabinene hydrate 1067.3 – – 0.14 0.25
terpinolene 1084.9 0.16 – 0.06 0.04 Results
p-menth-2.4(8)-diene 1085.5 – 0.02 – –
cis-decahydro-naphthalene 1091.5 – 0.03 0.21 0.02 Chemical composition of the essential oils
undecane 1096.9 0.07 0.18 0.05 0.21
linalool 1100.7 0.23 0.26 2.52 2.95 The EO’s isolated by hydro-distillation from the aerial
camphor 1141.2 – 0.1 0.08 0.35
menthone 1152.8 – – 0.17 0.21 parts of O. heracleoticum, O. vulgare, T. vulgaris and
borneol 1165.0 0.58 0.58 0.74 1.21 T. serpyllum were found to be yellow liquids charac-
terpinen-4-ol 1176.3 0.71 0.78 1.27 1.12
a-terpineol 1192.7 – – 0.24 0.22 terized by a typical odor for each plant and were
trans-dihydrocarvone 1199.4 0.07 0.12 0.17 0.45 obtained with a yield of 2.0, 1.8, 0.8 and 0.8% (based
dodecane 1200.1 0.26 0.39 0.12 0.12
dihydrocitronellol 1215.2 – – 0.11 0.08 on v/w), respectively. The relative percentage compos-
thymol methyl ether 1233.7 0.04 – 0.2 0.41 ition of EO’s and retention indices calculated for indi-
carvacrol methyl ether 1242.5 0.32 0.54 0.49 0.51
carvone 1252.7 – – 0.05 0.05 vidual components of the oils are presented in
tetrahydrocitronellene 1269.2 0.05 0.04 0.03 0.07 Table 1. All EO’s samples were dominated by oxygen-
carvenone 1272.0 – – 0.18 0.23
1-tridecene 1283.8 – – – 0.04 ated monoterpenes, with the highest content observed
bornyl acetate 1289.3 – – 0.1 0.12 in O. heracleoticum (73.65%), followed by EO’s of O.
isobornyl acetate 1289.3 0.05 0.14 – –
tridecane 1296.3 0.06 0.1 0.07 0.28 vulgare, T. vulgaris and T. sperpyllum (66.18%,
thymol 1309.1 1.62 4.76 43.23 34.48 66.35% and 48.37%, respectively).
carvacrol 1317.1 69.98 58.84 16.57 5.56
thymol acetate 1355.4 – – 0.03 0.03 The GC–MS analysis of EO originating from O.
carvacrol acetate 1355.6 – – 0.06 0.07 heracleoticum revealed the presence of 46 compounds
alpha-ylangene 1367.0 – – 0.04 0.03
a-copaene 1371.6 – – 0.05 0.08 representing 98.32% of the total oil and 43 com-
a-cubebene 1371.6 0.08 0.08 – – pounds representing 98.08% of the O. vulgare oil. The
b-bourbonene 1380.7 0.04 0.05 0.04 0.07
tetradecane 1396.5 0.05 0.05 0.05 0.01 major constituent in both EO’s was carvacrol (69.98%
trans-b-caryophyllene 1414.6 1.29 1.04 1.01 0.88 and 58.84%, respectively), followed by p-cymene
aromadendrene 1439.0 0.06 0.05 – –
a-humulene 1449.1 0.14 0.14 0.08 0.12 (7.7%, 19.9%), c-terpinene (6.85%, 3.11%) and thymol
v-muurolene 1472.8 0.02 – 0.06 0.09 (1.62%, 4.76%).
germacrene-D 1484.0 0.04 0.04 – –
v-amorphene 1490.9 – – 0.06 0.04 A total of 56 compounds were identified in the EO
viridiflorene 1496.0 0.08 0.05 – – of T. vulgaris representing 98.26% of the total oil, and
a- muurolene 1496.7 0.08 – 0.05 0.07
b-bisabolene 1506.0 0.66 0.63 0.21 0.08 58 compounds were identified in the EO of T. serpyl-
v-cadinene 1510.0 0.04 0.04 0.12 0.17 lum oil, representing 97.26% of the total oil (Table 1).
d-cadinene 1519.9 0.14 0.1 0.16 0.21
spathulenol 1577.0 0.08 – – – The major constituent in EO’s of T. vulgaris and T.
caryophyllene oxide 1579.2 0.3 0.3 0.53 0.26 serpyllum was thymol (43.23% and 34.48%, respect-
1.10-diepicubenol 1612.4 – – 0.02 0.03
10-epi-v-eudesmol 1616.9 – – 0.02 0.05 ively) followed by p-cymene (18.71% and 34.84%).
s-cadinol (epi-alpha-cadinol) 1639.2 – – 0.05 0.11 Among other constituents found in significant
epi-alpha-muurolene 1653.0 – – – 0.06
amounts were carvacrol (16.57% and 5.56%) and
(continued)
c-terpinene (4.34% and 4.38%).
366 I. CABARKAPA ET AL.

Table 2. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of the EO’s/EOC’s against
tested strains of S. Enteritidis.
EO/EOC S. Enteritidis rdar MIC/MBC ml ml-1 S. Enteritidis bdar MIC/MBC ml ml-1
Origanum heracleoticum (O1) 0.078/0.156 0.078/0.156
Origanum vulgare (O2). 0.156/0.3125 0.156/0.3125
Thymus vulgaris (T1) 0.156/0.3125 0.156/0.3125
Thymus serpyllum (T2) 0.3125/0.625 0.3125/0.625
Carvacrol (C) 0.156/0.3125 0.156/0.3125
Thymol (T) 0.156/0.3125 0.156/0.3125
CþT 0.078/0.156 0.078/0.156
Streptomycin (S) 0.0195/0.039 0.0195/0.039

Figure 1. Effectiveness of different concentration of EO’s/EOC’s on the biofilm forming ability of S. Enteritidis strains with
expressed bdar and rdar morphotypes determined using the CV assay. [O. heracleoticum (O1), O. vulgare (O2), T. vulgaris (T1), T. ser-
pyllum (T2), carvacrol (C) and thymol (T)]. Each bar represents the mean ± standard deviation (SD). MBIC50/MBIC90 (Minimum
Biofilm Inhibition Concentration) - the lowest concentration of EO required to prevent biofilm formation by 50% and 90%,
respectively.

Activity of the EO’s/EOC’s against
planktonic bacteria
MIC/MBC values of the EO’s/EOC’s are presented in
Table 2. In response to the treatment with EO’s,
EOC’s and streptomycin there was no established dif-
ference between the tested strains. The values of MIC
and MBC for streptomycin (MIC/MBC ¼ 0.0195/
0.039 ml ml1) were lower in comparison to the tested
EO’s. The highest antimicrobial activity against strains
was demonstrated by the EO of O. heracleoticum
(MIC/MBC ¼ 0.078/0.156 ml ml1). O. vulgare and
T. vulgaris EO’s also demonstrated a significant anti-
microbial property (MIC/MBC ¼ 0.156/0.3125 ml ml1),
while the EO of T. serpyllum showed the least effect
on the tested strains (MIC/MBC ¼ 0.3125/0.625ml
ml1) in comparison to other EO’s. In response to
the thymol and carvacrol treatment there was no
established difference between the tested strains.
Thymol and carvacrol demonstrated a significant anti-
microbial property (MIC/MBC ¼ 0.156/0.3125 ml ml1).
The checkerboard assay revealed that the effects of
thymol and carvacrol were additive (FIC 1.0). A com-
bination of thymol and carvacrol showed higher anti-
microbial activity (MIC/MBC ¼ 0.078/0.156 ml ml1).
Inhibition of biofilm formation
Biomass reduction
The results obtained for the inhibition of biofilm for-
mation are shown in Figures 1 and 2. The data show
that the investigated EO’s/EOC’s possess inhibitory
effects on biofilm formation and impairment of meta-
bolic activity. Also, the investigated EO’s/EOC’s
exhibited an inhibitory effect on biofilm formation in
a dose-dependent manner.
Supplementation of O1 and O2 at concentration
0.25MIC caused 50% inhibition of biofilm formation
for both the tested strains (rdar and bdar) marked by
MBIC50 values compared to the non-treated control.
For the same effect with T1 and T2, a 0.5MIC concen-
tration was required. MBIC50 when using carvacrol
and thymol was established at a concentration of
 BIOFOULING 367

Figure 2. Effectiveness of different concentration of EO’s/EOC’s on the metabolic activity of S. Enteritidis strains with expressed
bdar and rdar morphotypes. [O. heracleoticum (O1), O. vulgare (O2), T. vulgaris (T1), T. serpyllum (T2), carvacrol (C) and thymol (T)].
Each bar represents the mean ± SD. MBIC50/MBIC90 (Minimum Biofilm Inhibition Concentration) - the lowest concentration of EO
required to prevent biofilm formation by 50% and 90%, respectively.

0.25MIC on biofilm formation by the bdar strain com- Table 3. ANOVA calculation of the reduction in biofilm
pared to the non-treated control. In the case of their formation and metabolic activity.
application on biofilm of the rdar strain, the same Biofilm Reduction in
effect was recorded after using a two times higher forming reduction metabolic activity
Term df
concentration 0.5MIC (Figure 1). bdar rdar bdar rdar
Supplementation of O1, O2, and C at a concentra- EO/EOC 1 543þ 993þ 101þ 41
EO/EOC 2 1 608þ 718þ 293þ 227þ
tion 2MIC prevented 90% of biofilm formation Conc 1 14169þ 16172þ 57353þ 61677þ
marked by MBIC90 values for both the tested strains Conc2 1 2352þ 2880þ 11578þ 13295þ
EO/EOC Conc 1 21 0 18 10
(rdar and bdar). On the other hand, when using T1, Error 66 1073 1534 509 509
T2 and T at same concentration (2MIC), MBIC90 was r2 0.938 0.926 0.992 0.993
adj r2 0.934 0.921 0.991 0.992
not reached. þ
Significant at p < 0.01 level, Significant at p < 0.05 level, error terms
have been found statistically insignificant, df - degrees of freedom
Metabolic activity reduction EO/EOC - type of Essential oils (EO’s), or carvacrol and thymol (EOC’s),
Conc - concentration, t - the time of exposition.
The effectiveness of EO’s/EOC’s on metabolic
(respiratory) activity was confirmed using a resazurin The data presented in Table 3. show the significant
reduction assay. The results obtained provide prelim- effects of the independent variables to the responses,
inary evidence that the EO’s/EOC’s affected metabolic while Figure 1. and Figure 2. show which of the
activity in both the tested groups. responses were significantly affected by the varying
Inhibition of bdar morphotype metabolic activity variable combinations. The evaluation of the reduc-
by 50% (MBIC50) was obtained in the treatment tion in preformed biofilms and metabolic activity dur-
with O1, O2 and carvacrol at a concentration 0.5MIC. ing biofilm formation for both tested strains (rdar
In the case of T1 and T2 application, in order to and bdar) were mostly affected by the linear and the
obtain the same inhibition, a higher concentration of quadratic terms of EO’s/EOC’s type and concentra-
EO’s (1MIC) was required for both the tested strains. tion, statistically significant at the p < 0.01 level. SOP
For rdar strains, MBIC50 has been shown only in models representing the reduction in metabolic activ-
the treatment with O1 at a concentration of 0.5MIC. ity during the biofilm growth for both tested strains
On the other hand, this borderline limit in the treat- (rdar and bdar) had an insignificant lack of fit tests.
ment with other tested EO’s/EOC’s has been obtained The influences of EO/EOC type and concentration
at a concentration of 1MIC. In the case of all the were found statistically significant (at p < 0.01 level),
EO’s/EOC’s used for both tested strains (rdar and while predicted and observed responses corresponded
bdar), MBIC90 has been shown using a contentarion well, with coefficients of determination between 0.921
twice as high, 2MIC (Figure 2). and 0.992 for the rdar and bdar strains, respectively.
368 I. CABARKAPA ET AL.

Figure 3a. The effect of different concentrations of O. heracleoticum (O1), O. vulgare (O2) and carvacrol (C) on preformed 48 h old
biofilms of S. Enteritidis strains with expressed bdar and rdar morphotypes determined using the CV assay. b. The effect of differ-
ent concentrations of T. vulgaris (T1), T. serpyllum (T2) and thymol (T) on preformed 48 h old biofilms of S. Enteritidis strains with
expressed bdar and rdar morphotypes determined using the CV assay. Each bar represents the mean ± SD. MBEC50/MBEC90
(Minimum Biofilm Eradication Concentration) - the lowest concentration of EO required to eradicate preformed biofilm by 50%
and 90%, respectively.

Eradication of preformed biofilms
Biomass reduction of preformed biofilms
The EO’s/EOC’s showed time and concentration
dependent effects on the growth and development of
preformed 48 h biofilm, as presented in Figures 3a,
3b, 4a and 4b. For preformed biofilms of strain with
expressed rdar morphotype, MBEC50 was achieved
after 60 min exposure to O1 and O2 at the
concentration 2MIC, and 4MIC after exposure to car-
vacrol and thymol. In the case of T1 and T2 applica-
tion on preformed biofilms of this morphotype,
MBEC50 was not achieved despite the exposure to the
highest concentrations of EO’s for 60 min. A consid-
erably higher efficiency of the applied treatments was
shown in the case of their applications to the pre-
formed biofilms by the bdar morphotype (Figure 3a).
 BIOFOULING 369

Figure 4a. The effect of different concentrations O. heracleoticum (O1), O. vulgare (O2) and carvacrol (C) on the metabolic activity
of preformed 48 h old biofilms of S. Enteritidis strains with expressed bdar and rdar morphotypes. b. The effect of different con-
centrations of T. vulgaris (T1), T. serpyllum (T2) and thymol (T) on the metabolic activity of preformed 48 h old biofilms of S.
Enteritidis strains with expressed bdar and rdar morphotypes. Each bar represents the mean ± SD. MBEC50/MBEC90 (Minimum
Biofilm Eradication Concentration) - the lowest concentration of EO required to eradicate preformed biofilm by 50% and 90%,
respectively.

From Figure 3b it can be seen that the MBEC50
for strains with an expressed bdar morphotype was
obtained after exposure to O1 for 15 min at the con-
centration 2MIC, while the same was achieved for
O2, carvacrol and thymol at the concentration 4MIC.
For the same effect, application of T1 and T2 at
concentrations 2MIC and 4MIC, respectively, for
30 min was required. MBEC90 was not achieved even
after exposure of preformed biofilms for 60 min
on any of the tested concentrations of EO’s for
both groups.
Reduction of metabolic activity in preformed
biofilms
Inhibition of the metabolic activity in preformed bio-
films by strains with an expressed rdar morphotype
(by  50%) was observed after exposure of preformed
biofilm to O1, O2 and carvacrol at the concentration
4MIC for 30 min. For T1, T2 and thymol, MBEC50
was observed after exposure of preformed biofilm for
60 min at the same concentration (Figure 4b.).
MBEC90 was not achieved with applied treatments
on preformed biofilm of the rdar morphotype. For
370 I. CABARKAPA ET AL.

Table 4. ANOVA calculation of the reduction in biofilm formation and metabolic activity in
48 h old biofilms.
Reduction in metabolic
Biofilm forming reduction activity in 48 h old biofilms
Term df
bdar rdar bdar rdar
EO/EOC 1 1511þ 1032þ 1598þ 1217þ
EO/EOC2 1 3411þ 2400þ 3133þ 1336þ
t 1 13815þ 11133þ 16722þ 13131þ
t2 1 816þ 232þ 70 70
Conc 1 4034þ3 26125þ 37284þ 27100þ
Conc2 1 4678þ 2413þ 3392þ 1789þ
EO/EOC t 1 33 545 79 160þ
EO/EOC Conc 1 4 9 24 0
t Conc 1 7 0 1 14
Error 206 6690 5477 4792 3132
r2 0.904 0.887 0.928 0.934
adj r2 0.900 0.882 0.925 0.931
þ
Significant at p < 0.01, Significant at p < 0.05, Significant at p < 0.10, error terms have been found statistic-
ally insignificant, df - degrees of freedom
EO/EOC - type of Essential oils (EO’s), or carvacrol and thymol (EOC’s), Conc - concentration, t - the time
of exposition.

preformed biofilms of the bdar morphotype MBEC90
was obtained after exposure to O1, O2 and carvacrol
for 60 min at concentration 4MIC (Figure 4a.). The
results indicate that in addition to reducing biomass,
most of the EOs had an effect on metabolic activity.
The data presented in Table 4. show the significant
effects of the independent variables on the responses
at the end time of exposition. The evaluation of the
biofilm forming reduction and the reduction in meta-
bolic activity of preformed 48 h old biofilms at the
end of the exposure time for both the tested strains
(rdar and bdar) was mostly affected by the linear
term and quadratic terms of concentration in SOP
models, the linear term of time of exposure and the
linear and the quadratic terms of EO/EOC type, and
statistically significant at the p < 0.01 level. The quad-
ratic term of time of exposition was influential for the
prediction of the biofilm forming reduction (for both
strains). The SOP models representing the biofilm
forming reduction and the reduction in metabolic
activity of preformed 48 h old biofilms for both the
tested strains (rdar and bdar) at the time of expos-
ition had an insignificant lack of fit tests. The influen-
ces of EO’s/EOC’s, t and Conc were found to be
statistically significant, while the predicted and
observed responses corresponded well, with coeffi-
cients of determination between 0.882 and 0.931 for
the preformed 48 h old biofilms and a reduction in
metabolic activity for both the tested strains (bdar
and rdar), respectively.
Discussion
The composition of the tested EO’s can explain their
efficiency. A high percentage of phenolic compounds
(carvacrol and thymol constitute 71.60% of the tested
EO) contributes to the antimicrobial effect of O. hera-
cleoticum EO (MIC/MBC ¼ 0.078/0.156 ml mml1).
On the other hand, the EO’s of O. vulgare and T. vul-
garis also showed antimicrobial activity at very low
concentrations, MIC/MBC ¼ 0.156/0.3125 ml ml1.
The equal antimicrobial effect of these EO’s can be
explained by the almost similar amounts of phenolic
compounds (carvacrol and thymol) in the total oil,
63.60% for O. vulgare and 59.77% for T. vulgaris. The
lowest percentage of phenolic compounds was
detected in T. serpyllum EO (40.04%), which resulted
in a lower antimicrobial effect compared with the
other tested EO’s. Several studies have demonstrated
that the phenolic compounds carvacrol and thymol
have both bacteriostatic and bactericidal activity
against food-borne microorganisms including
Salmonella enterica (Burt 2004; Burt et al. 2005;
Veldhuizen et al. 2006).
The results obtained in this research point to a
negative correlation between the amount of phenolic
compounds in the total oil and the MIC of the EO’s
(Spearman’s correlation coefficient rs ¼ -0.95,
p < 0.001), indicating that as the content of phenolic
compounds in the EO’s is higher, the MIC is lower.
Therefore, it was noted that the antimicrobial activ-
ities of the examined EO’s were directly proportional
to the total content of phenolic components in
the EO’s.
Generally, the main constituents of the tested EO’s
are represented by monoterpenes (carvacrol, thymol,
c -terpinene and p-cymene). In particular, from the
scientific literature, carvacrol and thymol show a sub-
stantial inhibitory effect against food borne pathogens
(Burt et al. 2005; Du et al. 2015; Gavaric et al. 2015)

Carvacrol and thymol are structural isomers and
have a phenolic hydroxyl at a different location on
the phenolic ring. The hydroxyl group increases their
hydrophilic ability, which could help them dissolve in
the microbial membrane and impair them (Nazzaro
et al. 2013; Sikkema et al. 1995; Xu et al. 2008).
Compared with carvacrol, thymol has similar anti-
microbial activity, even though its hydroxyl group is
located in a different position (Ultee et al. 2000).
Similar to carvacrol, the antimicrobial activity of thy-
mol results in structural and functional alterations in
the cytoplasmic membrane that can damage the outer
and inner membranes; it can also interact with
membrane proteins and intracellular targets. The
interaction of thymol with the membrane affects
membrane permeability and results in the release of
K þ ions and ATP (Lambert et al. 2001; Xu et al.
2008). Thymol integrates within the polar head-
groups of the lipid bilayer, inducing alterations in the
cell membrane. In contrast to the efficiency of mono-
terpenes with added oxygen molecules (carvacrol and
thymol), the monoterpene hydrocarbons p-cymene
and c-terpinene used separately do not show substan-
tial inhibitory effect against bacteria (Dorman and
Deans, 2000, Burt et al., 2005).
However, a number of studies have demonstrated
that p-cymene can enhance the inhibitory effects of
carvacrol when these two compounds are used
together (Cristani et al. 2007; Ultee et al. 2000). It has
been shown that p-cymene is hydrophobic in nature
and causes swelling of the cytoplasmic membrane to a
greater extent. These studies suggested that this effect
occurred when p-cymene enabled carvacrol to be
more easily transported into the cell.
Other terpenes, such as limonene, a-pinene,
b-pinene, d-3-carene, (þ)-sabinene and a-terpinene
showed a very low or no antimicrobial activity against
25 genera of bacteria (Dorman and Deans 2000).
Some studies have demonstrated stronger anti-
microbial activities of EO mixtures, compared to
when they are used alone (Garcia-Garcia et al. 2011;
Zhou et al. 2007). In most synergy testing studies
combinations of carvacrol and thymol were found to
have additive effects expressed through fraction inhib-
ition concentration (FIC ¼ 0.5-1.1) (Burt et al. 2005;
Du et al. 2015; Gavaric et al. 2015). In contrast,
Gallucci et al. (2009) reported antagonistic activities
of binary mixture of carvacrol and thymol (FIC ¼
4.0). Due to the inconsistent results reported in the
literature regarding EO combinations (synergistic,
additive and antagonistic), before drawing any conclu-
sions more studies should be conducted in this area.
BIOFOULING 371
Based on the results obtained, regarding the impact
of EO’s and individual components on the biofilm
growth, which are shown in Figures 1 and 2, it can
be noticed that the applied treatments based on EO’s/
EOC’s at concentrations below the MIC caused
attenuation of the adhesion ability and consequently a
reduction in biofilm formation for both the tested
strains. The same concentration of EO’s/EOC’s also
led to a reduction in metabolic activity of adherent
cells, but at a lower percentage. This finding indicates
a potential suppressor effect of sub-lethal concentra-
tions of EO’s on the mechanisms of bacterial adhe-
sion and biofilm matrix synthesis.
The results presented related to the effects of higher
concentrations of EO’s/EOC’s (1MIC and 2MIC) indi-
cate that the use of these concentrations led to a sig-
nificantly greater reduction in biofilm growth and
metabolic activity. The application of these concentra-
tions also caused a high-level reduction in metabolic
activity. Due to the high efficiency of concentrations
1MIC and 2MIC, the reduction in adhesion and meta-
bolic activity could be a consequence of growth inhib-
ition and subsequent cell death.
Comparing the ability of EO’s to inhibit biofilm
growth and metabolic activity between the strains of
the rdar and bdar morphotype, statistically significant
differences were not established (p > 0.05).
A survey of other studies also pointed to the exist-
ence of inhibitory effects of EO’s on the initial adhe-
sion and consequently on biofilm formation (Burt
et al. 2014; Jadhav et al. 2013; Soni et al. 2013).
Soni et al. (2013) found that the application of
sub-lethal concentrations of oregano and thyme EO’s,
as well as carvacrol, which is constituent of these
EO’s, in the range from 0.006 to 0.012% (0.06-
0.12 ll ml1) reduced the ability of three strains of S.
Typhimurium to form biofilms. In addition, investiga-
tions by Burt et al. (2014) also showed a significant
reduction in biofilm growth by S. Typhimurium using
carvacrol in concentrations ranging from 0.75 mM
to 1.25 mM (0.11 – 0.19 ll ml1). Considering that
treatments with sub-lethal concentrations of carvacrol
did not record significant reductions in the number
of bacteria, these authors assumed that in this case,
the inhibitory effect of carvacrol on the biofilm devel-
opment included mechanisms different from growth
inhibition and cell death. A similar effect was shown
in the case of application of yarrow EO on the initial
adhesion of L. monocytogenes and L. innocua (Jadhav
et al. 2013).
Recent studies indicate that sub-lethal concentra-
tions of carvacrol can reduce the mobility of bacteria
372 I. CABARKAPA ET AL.
due to interference using a "quorum sensing" (QS)
mechanism between the bacterial cells, and reducing
their ability to form biofilm (Burt et al. 2014; Inamuco
et al. 2012). The precise mechanism by which carvacrol
inhibits biofilm formation is still not fully understood.
Results from current preliminary research indicate that
carvacrol, thymol and a mixture of carvacrol/thymol
may inhibit QS signaling on the AHL-based system,
on biosensor strain of Chromobacterium violaceum
CV026. Carvacrol, thymol and a mixture of carvacrol/
thymol displayed different active principles, i.e., anti-
microbial activity indicated by inner clear ring and
anti-QS activity indicated by an outer non-pigmented
ring with a visually detectable inhibition of violacein.
Contrary to the current results, natural compound
antibiotics have shown only antimicrobial effects.
The results presented in Figures 3a, 3b, 4a and 4b.
indicate that within the first 15 min the EO’s/EOC’s
used caused the eradication of preformed biofilms in
the range of 11.3% to 67.0%, and impaired metabolic
activity in the range of 9.6% to 70.5% depending on
the concentration of EO’s/EOC’s used compared to
the non-treated preformed biofilm. The best efficacies
were obtained after treatmen for 60 min. Based on the
results presented related to the impact of the EO’s
carvacrol and thymol on the preformed biofilm
and metabolic activity, the applied EO’s caused a
reduction in the total biomass/metabolic activity in
the preformed biofilm in a dose-dependent manner
over time.
As previously mentioned, the antimicrobial effect
of EO’s depends on their chemical composition and
the applied concentration. Application of EO’s at
lower concentrations mainly initiates an increase in
cell membrane permeability, resulting in disorders of
its structure without consequences for viability, while
the use of higher concentrations leads to severe
membrane damage and complete disruption of
homeostasis inducing cell death (Nazzaro et al. 2013).
Consequently, a prolonged contact time between the
EO’s and microorganisms could cause more severe
damage that inevitably leads to death of the
bacterial cells.
This mode of action of EO’s can be explained by
the greater efficiency of EO’s after a long time expos-
ure (60 min) compared to treatment with a short time
of exposure (15 min) (p < 0.05). Therefore, it is highly
probable that the greater efficiency of EO’s with pro-
longed time of action contributes to the fact that by
extending the time of exposure greater diffusion of
applied substances through the biofilm matrix occurs.
This mode of action of EO’s has been shown in
studies conducted by other authors (Oliveira et al.
2010; Soni et al. 2013; Valeriano et al. 2012).
A study by Burt et al. (2014) based on the treatment
of 24 h old biofilms with carvacrol at concentrations of
2, 4, 6 and 8 mM (0.3, 0.6, 0.9 and 1.2 ll ml1)
showed a reduced effect on the reduction in the total
biomass of biofilm in comparison with the effects of
the same concentrations on the initial adhesion.
Surveys conducted by other authors also point to the
weaker effect of EO’s on preformed biofilms (Jadhav
et al. 2013; Szczepanski and Lipski 2014). The weaker
effect of the EO’s/EOC’s used on preformed biofilms
could be explained by the presence of ECM, which in
varying levels could act as a diffusion barrier.
The results obtained indicate the existence of a
lower efficacy of EO treatment in the case of pre-
formed biofilms by strains expressing the rdar mor-
photype compared to strains expressing the bdar
morphotype (p < 0.05). It is assumed that this is a
consequence of the presence of more complex biofilm
matrix (cellulose þ curly fimbriae) in relation to the
biofilm matrix of strains with expressed bdar mor-
photype (curly fimbriae). It is well known that bacter-
ial cellulose within the biofilm has a structural role
providing mechanical, chemical and biological protec-
tion within the natural habitat. Investigations carried
out by other authors also suggest that the presence of
cellulose within the biofilm matrix, contributes to
enhanced survival capacity and is directly responsible
for the cells tolerance to treatments with different
antimicrobial agents (Solano et al. 2002; White et al.
2006; Yang et al. 2016). Also, it is noteworthy that
biofilms are microbial communities known to present
great structural and physiological heterogeneity, even
for those formed by the same microorganism under
different environmental conditions. This observation
is confirmed in the research conducted by Solano
et al. (2002), which is based on the influence of disin-
fectants on bacteria in biofilms. Specifically, these
authors examined the effect of chlorine at concentra-
tions 100-200 times higher than the concentrations
applied for water sanitation. When testing preformed
biofilms produced by strains that produced cellulose
and thin aggregative fimbriae (rdar) and cellulose-
deficient strain (bdar) they found that the morpho-
type bdar which produced cellulose had a significantly
greater tolerance to the treatments. This supports the
claims that bacterial cellulose is directly responsible
for the tolerance to chlorine for strains that produces
cellulose and fimbriae. On the other hand, the investi-
gation by Vestby et al. (2009) which dealt with the
effect of hypochlorite and benzalkonium chloride on

cell suspensions derived from similar morphotypes,
showed no significant differences in tolerance to the
applied disinfectants. Based on the results obtained
and the literature data, it could be argued that the
greater tolerance of preformed biofilms formed by
strains expressing the rdar morphotype is due to a
large extent to the presence of a biofilm matrix, pri-
marily cellulose, but the possible influence of other
components of the biofilm matrix as well as other
possible tolerance mechanisms of bacteria in the bio-
film should not be excluded.
Conclusions
In conclusion, the results obtained in the current study
reveal that bioactive phytochemicals possess antibacter-
ial and anti-biofilm activity. The findings indicate that
EO’s, as well as carvacrol and thymol are capable of
preventing, or at least interfering with biofilm forma-
tion and they may also eradicate preformed biofilms.
Particularly, in relation to Salmonella Enteritidis bio-
films, the EO of O. heracleoticum showed the strongest
effect, followed by O. vulgare, T. vulgaris, carvacrol and
thymol, while the EO of T. serpyllum showed the
weakest effect regarding the prevention of biofilm
growth and eradication of preformed biofilms.
Therefore, the EO’s of selected plants, as well as bio-
active phytochemicals obtained from EO’s, could be
used for the development of effective eco-friendly strat-
egies to combat Salmonella biofilms.
Disclosure statement
The authors declare that they have no conflicts of interest.
Acknowledgements
The authors wish to express their sincere gratitude for financial
support from the Provincial Secretariat for Higher Education
and Scientific Research of the Autonomous Province of
Vojvodina, Republic of Serbia, in the framework of the project
“Application of novel and conventional processes for removal
of most common contaminants, mycotoxins and salmonella, in
order to produce safe animal feed in the territory of AP
Vojvodina” No. 114-451- 2505/2016-02.
ORCID


Ivana Cabarkapa http://orcid.org/0000-0003-2215-4281


Radmilo Colovi c http://orcid.org/0000-0002-0689-9147
Olivera D- uragic http://orcid.org/0000-0003-3189-5057
Sanja Popovic http://orcid.org/0000-0001-9383-4984
Bojana Kokic http://orcid.org/0000-0002-9960-0393
Dubravka Milanov http://orcid.org/0000-0003-1987-8211
Lato Pezo http://orcid.org/0000-0002-0704-3084
BIOFOULING 373
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