foods
Article
Chemical Composition, Antibacterial and Antibiofilm Actions
of Oregano (Origanum vulgare subsp. hirtum) Essential Oil
against Salmonella Typhimurium and Listeria monocytogenes
Sonia Kolypetri 1 , Dimitra Kostoglou 1 , Anastasios Nikolaou 2 , Yiannis Kourkoutas 2
and Efstathios Giaouris 1, *
Citation: Kolypetri, S.; Kostoglou, D.;
Nikolaou, A.; Kourkoutas, Y.;
Giaouris, E. Chemical Composition,
Antibacterial and Antibiofilm
Actions of Oregano (Origanum
vulgare subsp. hirtum) Essential Oil
against Salmonella Typhimurium and
Listeria monocytogenes. Foods 2023, 12,
2893. https://doi.org/10.3390/
foods12152893
Academic Editor: Gary Dykes
Received: 8 June 2023
Revised: 29 June 2023
Accepted: 28 July 2023
Published: 29 July 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Foods 2023, 12, 2893. https://doi.org/10.3390/foods12152893
1 Laboratory of Food Microbiology and Hygiene, Department of Food Science and Nutrition, School of the
Environment, University of the Aegean, 81400 Myrina, Lemnos, Greece
2 Laboratory of Applied Microbiology and Biotechnology, Department of Molecular Biology and Genetics,
School of Health Sciences, Democritus University of Thrace, 68100 Alexandroupolis, Greece
* Correspondence: stagiaouris@aegean.gr; Tel.: +30-22540-83115
Abstract: Essential oils (EOs) are plant mixtures that are known to present strong bioactivities,
including a wide antimicrobial action. Biofilms are microbial sessile structures that represent the
default mode of growth of microorganisms in most environments. This study focused on the
antimicrobial action of the EO extracted from one of the most representative oregano species, that is,
Origanum vulgare (subsp. hirtum), against two important foodborne pathogens, Salmonella enterica
(serovar Typhimurium) and Listeria monocytogenes. For this, the minimum inhibitory concentrations
of the EO against the planktonic and biofilm growth of each bacterium were determined (MICs,
MBICs), together with the minimum bactericidal and biofilm eradication concentrations (MBCs,
MBECs). The EO was also analyzed for its chemical composition by gas chromatography—mass
spectrometry analysis (GC-MS). The influence of EO exposure on the expression of some important
virulence genes (hly, inlA, inlB and prfA) was also studied in L. monocytogenes. Results revealed a
strong antibacterial and antibiofilm action with MICs and MBICs ranging from 0.03% to 0.06% (v/v)
and from 0.06% to 0.13% (v/v), respectively. The application of the EO at 6.25% (v/v) for 15 min
resulted in the total eradication of the biofilm cells of both pathogens. The EO was mainly composed
of thymol, p-cymene, γ-terpinene and carvacrol. The 3 h exposure of L. monocytogenes planktonic cells
to the EO at its MBIC (0.06% v/v) resulted in the significant downregulation of all the studied genes
(p < 0.05). To sum, the results obtained advocate for the further exploitation of the antimicrobial
action of oregano EO in food and health applications.
Keywords: oregano essential oil; foodborne pathogenic bacteria; minimum inhibitory and bacterici-
dal concentrations; biofilms; minimum biofilm eradication concentration; GC-MS; gene expression;
RT-qPCR; virulence; food safety
1. Introduction
The genus Origanum L. comprises several species which differ in morphology and
chemical characteristics, with most of them being originated in the Mediterranean region [1].
One of the most notable species is Origanum vulgare subsp. hirtum, which is a widely used
aromatic plant in foods, feeds, cosmetics, and daily care products, especially due to its
content in essential oil (EO), mainly rich in carvacrol and thymol [2]. An increasing
number of studies have examined various bioactivities of O. vulgare EO, including strong
antimicrobial action against various bacteria and fungi [3]. Such bioactivities may, however,
differ between the EO of the same species due to differences in chemical composition which
in turn are due to parameters such as the soil and climate conditions of plant growth,
cultivation method, harvesting period, anatomical part of the plant used for extraction and
method used to extract the oil [4].
https://www.mdpi.com/journal/foods
Foods 2023, 12, 2893 2 of 14

Biofilms are microbial sessile (surface-attached) structures that represent the dominant
mode of growth of almost all microorganisms in most environments, both natural and
man-made [5]. Biofilm formation by bacterial pathogens is a worrying ability due to
the increased tolerance and resistance of the enclosed cells to antimicrobials and other
physicochemical stresses [6]. Such recalcitrance of biofilm cells is the result of many factors
that act in concert and are related to biofilm growth, such as the limited penetration and/or
the inactivation of some antimicrobials through the extracellular biofilm matrix, the reduced
growth rate, the altered gene expression and physiology of biofilm cells, together with the
increased presence of dormant and super-durable cells called persisters [7]. Alarmingly,
biofilm formation is considered a key virulence factor for a wide range of microorganisms
that cause human infections [8].
Salmonella enterica and Listeria monocytogenes are two well-known pathogenic bacteria
that are responsible for foodborne infections [9]. Based on the latest epidemiological
data for Europe, in 2021, salmonellosis remained the second most common foodborne
zoonosis in humans after campylobacteriosis. That year, Salmonella provoked 773 foodborne
outbreaks (FBOs), which is the largest number of FBOs recorded for 2021 [10]. As in
previous years, the three most commonly reported Salmonella serovars were S. Enteritidis
(54.6%), S. Typhimurium (11.4%) and monophasic S. Typhimurium (1,4,(5),12:i:-) (8.8%),
representing 74.8% of the 50,817 confirmed human cases. On the other hand, listeriosis
concerned 2183 confirmed human cases, provoked 196 deaths, and resulted in a case fatality
ratio of 13.7% which is the highest compared to all the other zoonoses monitored. Both
S. enterica and L. monocytogenes can form robust biofilms on various surfaces encountered
within the food production chain and in this way persist and contaminate foods [11,12].
Thus, the ability of these bacteria to form biofilms and/or be included in those formed by
other species (e.g., those of spoilage microflora) is considered a strong contributing factor
to their prevalence in foods and their virulence potential [13–15].
The increased tolerance and resistance of several pathogenic bacteria to antibiotics
and/or disinfectants has led to the continuous search for novel antimicrobial agents [16–18].
Plants may be a promising source for such compounds mainly due to their great biological
diversity, abundance, and safe and ecofriendly nature [19,20]. In the past few years,
there have been numerous studies published on the antimicrobial and antibiofilm actions
of various EOs including those of oregano species [21–24]. Given that the EOs have
multiple targets against microbial cells (e.g., rupture of the cell membrane, coagulation
of the cytoplasm, interference with DNA transcription, protein synthesis and enzymatic
activity, disturbance of the proton motive force etc.), it is believed that this lowers the
possibility for microorganisms to develop resistance [1]. In this study, we evaluated the
antimicrobial action of the EO extracted from plants of O. vulgare subsp. hirtum, organically
cultured on Lemnos Island (north-eastern Greece), against both planktonic and biofilm
cells of S. Typhimurium and L. monocytogenes. The EO was also analyzed for its chemical
composition by gas chromatography—mass spectrometry analysis (GC-MS). The influence
of EO sublethal exposure on the expression of some important virulence genes (hly, inlA,
inlB, and prfA) was also studied in planktonic L. monocytogenes cells through reverse
transcription quantitative polymerase chain reaction (RT-qPCR).

2. Materials and Methods

2.1. Bacterial Strains and Preparation of Their Working Cultures
The bacterial strains that were used in this study were S. enterica str. FMCC_B137,
which was kindly provided by Prof. George-John Nychas (Agricultural University of
Athens, Athens, Greece), and L. monocytogenes str. AAL 20107, which was kindly supplied
by Dr. Nikolaos Andritsos (Athens Analysis Laboratories S.A., Microbiology Laboratory,
Metamorfosi, Greece). The strain of S. enterica belongs to serovar Typhimurium; it is an epi-
demic strain of phage type DT193 and was originally isolated from a human salmonellosis
outbreak [25]. The strain of L. monocytogenes belongs to serovar 1/2b and was originally
isolated from a mixed green salad [26]. Before use, both strains were maintained frozen
Foods 2023, 12, 2893 3 of 14

at −80 ◦ C in BHI broth (Lab M, Heywood, Lancashire, UK) containing 15% v/v glycerol.
When needed, each strain was streaked on Tryptone Soya Agar (TSA; Oxoid, Thermo Fisher
Specialty Diagnostics Ltd., Hampshire, UK) and incubated at 37 ◦ C for 24 h (preculture).
Working cultures were prepared by inoculating a single colony from each preculture into
10 mL of fresh TSB (Condalab, Torrejón de Ardoz, Madrid, Spain) and then incubating at
37 ◦ C for 18 h. The final concentration of each working culture was ca. 109 CFU/mL and
its purity was always confirmed by streaking on TSA and incubating it at 37 ◦ C for 24 h.

2.2. O. vulgare EO
The EO that was used in this work was extracted from dried plant material by hydro-
distillation procedure as previously described [27] and was kindly donated by Dr. Nikolaos
Paterakis, owner of the essential oil production and trading company Aegean Organics
(Agios Dimitrios, Lemnos, Greece). The year of harvest was 2022. Besides oregano, this
company produces EOs and decoctions of various other aromatic plants that are all grown
under a certified organic culture. Upon receipt, the EO was kept in a dark glass vial in the
refrigerator (4 ◦ C).

2.3. Determination of Minimum Inhibitory and Bactericidal Concentrations of EO against

Planktonic Bacteria (MICs, MBCs)
The MIC of the EO against the planktonic growth of each bacterial strain was deter-
mined following the broth microdilution method as previously described [27]. In brief,
ten different concentrations were tested for the EO which were prepared by successive
two-fold dilutions in TSB and ranged from 0.5 to 0.001% v/v (a stock solution of the EO
at 1% v/v was initially prepared in TSB also containing 9% v/v ethanol). Then, 360 µL of
each of those dilutions were placed in duplicate in the wells of a sterile 100-well Bioscreen
honeycomb plate (Oy Growth Curves Ab Ltd., Turku, Finland), and inoculated with the
tested bacteria so as to have an initial concentration of ca. 104−5 CFU/mL. The plate was
incubated at 37 ◦ C for 24 h in the Bioscreen Co Pro instrument, which was adjusted to
record the absorbance of the contents of each well every 30 min at 600 nm (A600nm ). Pure
sterile growth medium (TSB) was used as a negative control (NC). For each bacterial strain,
the MIC of the EO was calculated as its lowest concentration, resulting in no increase in
absorbance with respect to the NC. To determine the MBC, 10 µL of broth cultures were
aspirated from all the non-growth wells of the MIC assay and spotted (in duplicate) on
TSA plates, which were then incubated at 37 ◦ C for 24 h. For each bacterial strain, the MBC
of the EO was determined as its lowest concentration that reduced the initial inoculum by
more than 99.9% (no appearance of colonies). Each experiment was repeated three times
starting from independent bacterial cultures.

2.4. Determination of Minimum Biofilm Inhibitory Concentrations (MBICs)

The MBIC of the EO against the biofilm growth of each bacterial strain was determined
following the crystal violet (CV) staining method as previously described [27]. In brief,
eight different concentrations were tested for the EO which were prepared by successive
two-fold dilutions and ranged from 0.25 to 0.002% v/v. These dilutions were done in the
respective growth medium for each strain (see below). Each bacterium was left to form
biofilm on 96-well polystyrene (PS) microtiter plates (transparent, flat, Cat. No. 30096,
SPL Life Sciences, Gyeonggi-do, Korea) for 96 h in the presence of each EO concentration.
For that assay, biofilm formation took place under the optimum (biofilm-maximizing)
conditions for each strain (with respect to medium, time and incubation temperature), as
these had been previously determined [27]. Thus, S. Typhimurium was grown in 1/10
diluted TSB (dTSB) at 20 ◦ C, whereas L. monocytogenes was grown in BHI broth at 37 ◦ C.
For both bacteria, the growth medium was renewed at 48 h of incubation. At the end of
incubation, the accumulated biomasses were quantified through their staining with CV
(0.1% w/v) and subsequently measuring absorbance at 590 nm (A590nm ). For each bacterial
strain, the MBIC of the EO was determined as its lowest concentration that completely
Foods 2023, 12, 2893 4 of 14

inhibited biofilm formation (biomass accumulated did not significantly differ from the
negative control). Each experiment was repeated three times starting from independent
bacterial cultures.

2.5. Determination of Minimum Biofilm Eradication Concentrations (MBECs)

The MBEC of the EO against the already 96 h preformed biofilms of each bacterial strain
was determined as previously described [27]. In brief, five different concentrations were
tested for the EO which were all prepared in sterile distilled water (dH2 O) by successive
two-fold dilutions and ranged from 6.25 to 0.39% v/v. Each EO disinfection solution was
left to act against the biofilm cells for 15 min at room temperature. For each disinfection
treatment, the survivors were enumerated by plate counting on TSA following their removal
from surfaces through scratching (with a plastic pipette tip). For each bacterial strain, the
MBEC of the EO was determined as its lowest concentration leading to at least six log
reductions of the biofilm population compared to that of the negative control (dH2 O also
containing 10% v/v ethanol). Each experiment was repeated three times starting from
independent bacterial cultures.

2.6. Chemical Analysis of EO (GC-MS)

Oregano EO was diluted in pure hexane (1:9) and the mixture was then chemically
characterized by GC-MS analysis on a 6890N system (Agilent Technologies, Santa Clara,
CA, USA) equipped with an HP-5MS column (30 m, 0.25 mm inner diameter, 0.25 µm film
thickness) (Agilent Technologies) and coupled with a 5973Network mass detector (Agilent
Technologies), as previously described [27].

2.7. Sublethal Exposure of Planktonic L. monocytogenes Cells to EO and RNA Extraction

L. monocytogenes bacteria from the final 18 h working culture were used to inoculate
10 mL of sterile BHI broth at 1:100 dilution (so as to have an initial concentration of ca.
107 CFU/mL). The new culture was incubated at 37 ◦ C for 6 h and its cells were then
collected by centrifugation (5000× g for 10 min at 4 ◦ C) and the obtained pellet was
subsequently suspended in 4 mL of quarter-strength Ringer’s solution (Lab M). That
freshly saline cellular suspension was aliquoted in two Eppendorf® tubes (2 mL in each
one) and centrifuged (5000× g for 10 min at 4 ◦ C). One of the two bacterial pellets was then
suspended in 1 mL of the EO solution (0.063% v/v; equal to the MBIC), while the second
pellet was suspended in 1 mL of dH2 O to be used as the untreated control sample. The
dH2 O of the control sample also contained absolute ethanol at 0.56% v/v. This latter was the
concentration that existed in the EO solution (and was used to disolve the oil). Both samples
were incubated in a heating dry block for 3 h at 37 ◦ C (sublethal EO exposure) and were
then immediately centrifuged (5000× g for 10 min at 4 ◦ C). Supernatants were discarded
and each pellet was washed with dH2 O through an additional centrifugation step (5000× g
for 10 min at 4 ◦ C) to remove any EO residues. Washed pellets were then suspended in 1
mL of RNAlater™ Stabilization Solution (AM7021, Invitrogen™, Thermo Fisher Scientific,
Carlsbad, CA, USA) and incubated overnight at 4 ◦ C. Next day, RNAlater™ Solution was
removed by centrifugation (5000× g for 10 min at 4 ◦ C) and each bacterial pellet was then
resuspended in 100 µL of TE buffer (10 mM Tris-HCl, 1 mM EDTA; pH 8) also containing
2.5 mg/mL lysozyme (native, chicken egg white, 4403, Calbiochem® , Merck KGaA) and
incubated at 37 ◦ C for 10 min. Following this enzymatic digest of the bacterial cell walls,
the resulting homogenates were directly used for RNA extraction using the NucleoSpin®
RNA Mini Kit (740955, MACHEREY-NAGEL GmbH & Co., KG, Düren, Germany).

2.8. cDNA Synthesis and qPCR

The cDNA synthesis was executed starting from 500 ng of each RNA sample (i.e.,
exposed and not exposed to the EO) using PrimeScript™ RT reagent Kit (RR037A, Takara
Bio Inc., Shiga, Japan), according to the manufacturer’s instructions. Each cDNA template
was then used as substrate in qPCR prepared using the FastGene® IC Green 2 × qPCR
Foods 2023, 12, 2893 5 of 14

Universal Mix (LS4005, NIPPON Genetics EUROPE GmbH). Four virulence genes of
L. monocytogenes were selected (hly, inlA, inlB and prfA) for this analysis, which was done as
previously described [28]. Two reference (internal control) genes (tuf, gap) were included
in each assay for the normalization of the qPCR data. Each experiment was repeated
three times starting from independent bacterial cultures and each reaction was performed
in triplicate.

2.9. Statistics
Biofilm plate counts (CFU/cm2 ) were transformed to logarithms before means and
standard deviations were computed. The derived data on biofilm biomasses (A590nm ),
planktonic absorbances (A600nm ), and biofilm populations (log10 CFU/cm2 ) were all sub-
mitted to analyses of variance (ANOVA), followed by Tukey’s multiple range post hoc hon-
estly significant difference (HSD) tests for mean comparison, using the statistical software
STATISTICA® (StatSoft Inc., Tulsa, OK, USA). In ANOVA, the dependent variables were
the biofilm biomasses (A590nm ), planktonic absorbances (A600nm ) and biofilm populations
(log10 CFU/cm2 ), while the categorical factor was always the treatment (EO concentration).
Regarding gene expression, the data derived from a total of 108 reactions were analyzed.
These were the result of six different cDNA samples (i.e., one bacterial strain × two treat-
ments (exposed and not exposed to the EO) × three biological repetitions) × six genes (four
targets plus two references)/sample × three technical replicates/gene. For each bacterial
strain and target gene, unpaired two-tailed Student’s t-tests were applied to check for
any significant difference in gene expression (expressed as log2 (fold difference)) between
the two treatments. These tests were performed using the relevant function of Excel®
module of the Microsoft® Office 365 suite (Redmond, WA, USA). For all statistical analyses,
significant differences were reported at a p level of <0.05.

3. Results and Discussion

3.1. Antimicrobial Actions of O. vulgare EO against Planktonic and Biofilm Cells
The minimum inhibitory and minimum bactericidal concentrations (MICs, MBCs)
of O. vulgare EO against the planktonic cells of S. Typhimurium and L. monocytogenes
were initially determined. The MICs were found equal to 0.06 and 0.03% v/v against S.
Typhimurium and L. monocytogenes, respectively (Table 1; equal to 0.55 and 0.27 mg/mL,
respectively). No differences between the MICs and MBCs were observed, indicating the
strong bactericidal action of the EO.

Table 1. Antibacterial (MIC and MBC values) and antibiofilm (MBIC and MBEC values) actions of O.
vulgare EO against S. Typhimurium and L. monocytogenes strains expressed as either EO percentages
by volume (% v/v) or µL/mL.

Antibacterial Action Antibiofilm Action

Bacterial Species MIC 1 MBC 2 MBIC 3 MBEC 4
% (v/v) µL/mL % (v/v) µL/mL % (v/v) µL/mL % (v/v) µL/mL
S. Typhimurium 0.06 0.6 0.06 0.6 0.13 1.3 6.25 (=104.2 × MBC) 62.5
L. monocytogenes 0.03 0.3 0.03 0.3 0.06 0.6 6.25 (=208.4 × MBC) 62.5
1Minimum Inhibitory Concentration; 2 Minimum Bactericidal Concentration; 3 Minimum Biofilm Inhibitory
Concentration; 4 Minimum Biofilm Eradication Concentration.

Oregano EO, such as many other EOs, may present multiple modes of action against
microbial cells. The reader is referred to specialized reviews on the mode of actions of
EOs for more info [1,2]. Many other studies have in the past analyzed the antimicrobial
action of O. vulgare EOs against planktonic bacteria. For instance, Mazzarrino et al. (2015)
determined the MICs of Italian O. vulgare EO against Salmonella spp. and L. monocytogenes
and found values ranging from 0.6 to 1.2 µL/mL [29]. Assiri et al. (2016) determined the
minimal lethal concentration (MLC) of O. vulgare EO from Saudi Arabia against S. Enteri-
Foods 2023, 12, 2893 6 of 14

tidis and L. monocytogenes and found values equal to 0.16 and 0.32 mg/mL [30]. Elansary
et al. (2108) found MIC and MBC of Egyptian O. vulgare EO against L. monocytogenes
equal to 0.40 and 0.83 mg/mL, respectively [31]. Barbosa et al. (2020) found maximum
sublethal concentration (MSC) of Brazilian O. vulgare EO against S. Enteritidis equal to
0.13 mg/mL [32]. Torabian Kakhki et al. (2020) found MIC and MBC of Iranian O. vulgare
EO against L. monocytogenes equal to 1.28 and 2.56 mg/mL, respectively [33]. Solarte et al.
(2020) determined the MIC of Spanish O. vulgare EO against one reference S. Typhimurium
strain and two porcine isolates equal to 0.31 mg/mL [34]. It is clear that the EO from the
Greek organically cultured oregano plants tested in the current study presents strong an-
timicrobial action that is similar or even superior to that previously described for O. vulgare
EOs from some other countries.
Following the calculation of the MICs and MBCs of the O. vulgare EO against the
planktonic S. Typhimurium and L. monocytogenes bacteria, we determined its minimum
biofilm inhibitory concentrations (MBICs) against the biofilm growth of those bacteria. This
is because both pathogens are known to form robust biofilms on various surfaces (both food-
contact and non-food-contact ones) that display increased tolerance to antimicrobial agents
and other stresses [35,36]. The protocol we followed to form biofilms had been previously
optimized to maximize the sessile growth by each of these two bacterial strains [27].
However, and despite that optimization, the determined MBICs were still quite small and
equal to 0.13 and 0.06% v/v against S. Typhimurium and L. monocytogenes, respectively
(Table 1). This indicates the ability of the O. vulgare EO to efficiently inhibit biofilm
formation by the tested pathogens at very low concentrations and thus its high antibiofilm
potential. Figure 1 presents the biofilm biomasses (A590nm ) accumulated on the PS surface
for each one of the eight tested EO concentrations (0.25–0.002% v/v). The densities of
the surrounding planktonic suspensions (A600nm ) found in the same wells at the time of
sampling (96 h) are also shown for each treatment (as dotted curved lines). It is worth
noting that in the case of L. monocytogenes, the application of the EO at its MBIC (0.06% v/v)
resulted in the total inhibition of biofilm formation, whereas the surrounding planktonic
cells were still able to multiply, although to a lesser extent compared to the positive control
(Figure 1B). This is quite interesting since it probably indicates that the observed inhibition
of biofilm formation should not be a sole result of the reduced growth rate of the bacteria
(before and after their attachment to the surfaces), but it could also be associated with the
inhibition of some other biofilm-specific mechanisms, such as intercellular interactions and
aggregation, motility, production of extracellular polymeric substances (EPS), altered gene
expression etc. It is indeed known that various phytochemicals and plant extracts may
display antivirulence abilities and anti-quorum sensing behavior upon their application
at sub-MICs [37,38]. Such targeted antibiofilm action at sub-MICs is believed to limit the
possibilities of the bacteria to develop resistance [39].
Besides the determination of the MBICs, we also determined the Minimum Biofilm
Eradication Concentrations (MBECs) of O. vulgare EO against the 96 h preformed biofilms
of each target pathogen. Results revealed that the oil needed to be applied at 6.25% v/v
to destroy the biofilm communities of both pathogens (Figure 2). Undoubtedly, this is
a quite high concentration that clearly denotes the great recalcitrance of biofilm cells.
This is however not surprising since it is well known that the inclusion of bacteria in
biofilm structures can greatly increase their tolerance and resistance to biocides, including
known antibiotics [40,41]. Thus, in the current experimental setup, O. vulgare EO needed
to be applied more than one hundred times more than its MBC to kill S. Typhimurium
biofilm cells, while in the case of L. monocytogenes biofilm cells, the required increase in
concentration was even more spectacular (more than two hundred times more than its
MBC) (Table 1). Another probably interesting observation is that a dose-response behavior
does not seem to exist in the case of the killing of biofilm cells (Figure 2). This is more
evident in the case of L. monocytogenes where the application of O. vulgare EO at 0.39%
v/v provoked a ca. five-log reduction in the number of biofilm cells, whereas the further
ten-fold increase of the EO concentration to 3.13% v/v did not further improve its killing
 Foods 2023, 12, 2893 7 of 14

action. The reasons lying behind this last observation are not known but we speculate that
these may be somehow related to the presence of EPS and/or the complex biofilm tertiary
structure [42,43]. A similar behavior has been previously observed in another similar older
Foods 2023, 12, 2893 study of our group where an increase in the concentrations of sage and spearmint 7 of EOs
14
from 5% to 15% v/v did not improve their efficiency against Staphylococcus aureus biofilm
bacteria [44].

c c c c c 0.25
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3.5
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Planktonic culture (A600 nm)

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Figure
Figure 1. 1. Biofilm
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590nm
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formation) is indicated by the vertical arrow. The absorbances of planktonic suspensions (A600nm )
suspensions (A600nm) found in the same wells at the time of sampling (96 h) are also shown for each
found in the same wells at the time of sampling (96 h) are also shown for each treatment (dotted
treatment (dotted curved lines). The bars of standard deviations of the planktonic means were
curvedfor
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each graph (bacterial strain), mean biofilm values
superscript letters (abc) differ significantly (p < 0.05). followed by different superscript letters (abc) differ
significantly (p < 0.05).
Besides the determination of the MBICs, we also determined the Minimum Biofilm
Eradication Concentrations (MBECs) of O. vulgare EO against the 96 h preformed biofilms
of each target pathogen. Results revealed that the oil needed to be applied at 6.25% v/v to
destroy the biofilm communities of both pathogens (Figure 2). Undoubtedly, this is a quite
high concentration that clearly denotes the great recalcitrance of biofilm cells. This is
however not surprising since it is well known that the inclusion of bacteria in biofilm
structures can greatly increase their tolerance and resistance to biocides, including known
antibiotics [40,41]. Thus, in the current experimental setup, O. vulgare EO needed to be
applied more than one hundred times more than its MBC to kill S. Typhimurium biofilm
cells, while in the case of L. monocytogenes biofilm cells, the required increase in
 further ten-fold increase of the EO concentration to 3.13% v/v did not further improve its
killing action. The reasons lying behind this last observation are not known but we
speculate that these may be somehow related to the presence of EPS and/or the complex
biofilm tertiary structure [42,43]. A similar behavior has been previously observed in
another similar older study of our group where an increase in the concentrations of sage
Foods 2023, 12, 2893 and spearmint EOs from 5% to 15% v/v did not improve their efficiency against 8 of 14
Staphylococcus aureus biofilm bacteria [44].

10
A
9

8 d A d S. Typhimurium

Biofilm (Log10CFU/cm2)
7
L. monocytogenes
6

5 bc
c
4 B B
ab
B a
3 B

1
detection limit e C
(0.21 log10 CFU/cm2) 0
before NC 0.39% 0.78% 1.56% 3.13% 6.25%
disinfection EO concentration (% v/v)
after disinfection
Treatment
Biofilmpopulations
Figure2.2.Biofilm populations(log (log CFU/cm 2 ) found in the wells of the 96-well microtiter plates
Figure CFU/cm
10 10 2) found in the wells of the 96-well microtiter plates for
the
fortwothe bacterial strains
two bacterial (S. Typhimurium
strains (S. Typhimurium and L.and monocytogenes) beforebefore
L. monocytogenes) and after
anddisinfection with
after disinfection
the
withO.thevulgare EO. For
O. vulgare each
EO. Forbacterial strain,strain,
each bacterial the EOthe was
EO tested in fivein
was tested different concentrations,
five different two-
concentrations,
fold dilutions
two-fold ranging
dilutions fromfrom
ranging 0.390.39
to 6.25% v/v.v/v.
to 6.25% TheThebiofilm population
biofilm population of of
the negative
the negativedisinfection
disinfection
control
control (NC; sterile distilled water also containing 10% v/v ethanol) is also shown.The
(NC; sterile distilled water also containing 10% v/v ethanol) is also shown. Thebars
barsrepresent
represent
the mean values ± standard deviations. For each bacterial strain separately, mean values followed
the mean values ± standard deviations. For each bacterial strain separately, mean values followed
by different superscript letters differ significantly (p < 0.05). For each bacterial strain, the MBEC of
by different superscript letters differ significantly (p < 0.05). For each bacterial strain, the MBEC
the EO (resulting in more than six log reductions of its biofilm population with respect to that of the
of the
NC) is EO (resulting
indicated withinthe
more than arrow.
vertical six log The
reductions of its
detection biofilm
limit of thepopulation with respect
plate counting methodto(0.21
that
of 10the
log NC) is
CFU/cm 2) indicated
is indicated with
withthe vertical
the arrow.
horizontal The detection
dashed line. limit of the plate counting method
(0.21 log10 CFU/cm2 ) is indicated with the horizontal dashed line.
A few other studies have been published in the previous years testing the antibiofilm
A few other studies have been published in the previous years testing the antibiofilm ef-
efficiency of O. vulgare EO against biofilm formation and/or preformed biofilms of
ficiency of O. vulgare EO against biofilm formation and/or preformed biofilms of Salmonella
Salmonella spp. or L. monocytogenes. In such a study, Čabarkapa et al. (2019) tested the
spp. or L. monocytogenes. In such a study, Čabarkapa et al. (2019) tested the effectiveness
effectiveness of O. vulgare EO against planktonic and biofilm cells of two strains of S.
of O. vulgare EO against planktonic and biofilm cells of two strains of S. Enteritidis [21].
Enteritidis [21]. In that study, O. vulgare EO demonstrated (as expected) a significant
In that study, O. vulgare EO demonstrated (as expected) a significant antimicrobial action
antimicrobial action against the planktonic cells (MIC/MBC = 0.16/0.31 μL/mL).
against the planktonic cells (MIC/MBC = 0.16/0.31 µL/mL). Supplementation of EO at
Supplementation of EO at concentration 0.25xMIC caused 50% inhibition of biofilm
concentration 0.25xMIC caused 50% inhibition of biofilm formation for both the tested
formation for both the tested strains, while its supplementation at 2xMIC resulted in 90%
strains, while its supplementation at 2xMIC resulted in 90% inhibition of their biofilm
inhibition
formation. ofContrary
their biofilm formation.
to our Contrary
results, the study to
onour
theresults, the study
effectiveness on the
of EO effectiveness
on eradication of
of EO on eradication of preformed 48 h-old biofilms indicated that biofilm
preformed 48 h-old biofilms indicated that biofilm reduction occurred in a dose-dependent reduction
occurred
manner overin a dose-dependent manner over
time (the time of exposure time
ranged (the15time
from to 60ofmin).
exposure ranged
In another fromDi
study, 15Vito
to
60 min).
et al. In compared
(2020) another study, Di Vito
the in vitro et al. (2020)
antibiofilm compared
effectiveness the in
of Italian vitro antibiofilm
O. vulgare EO against
effectiveness
29 strains of Salmonella spp. isolated from poultry and pig farms belonging toisolated
of Italian O. vulgare EO against 29 strains of Salmonella spp. from
two serotypes
poultry and pig farms belonging to two serotypes (S. Typhimurium and
(S. Typhimurium and S. Infantis) [45]. O. vulgare EO was active on the disaggregation S. Infantis) [45].
O. vulgare EO was active on the disaggregation of mature biofilms of both
of mature biofilms of both serotypes, but no inhibiting activity was exerted on biofilm serotypes, but
no inhibiting activity was exerted on biofilm formation. Soni et al. (2013)
formation. Soni et al. (2013) evaluated EOs of thyme and oregano and their antimicrobial evaluated EOs
phenolic constituent carvacrol for their ability to inhibit biofilm formation and inactivate
preformed Salmonella biofilms [46]. The presence of nonbiocidal concentrations of thyme
oil, oregano oil and phenolic carvacrol at 0.006 to 0.012% v/v suppressed Salmonella spp.
biofilm formation two- to four-fold, but could not completely eliminate biofilm formation.
Treatment of 1-day-old biofilms with thyme oil, oregano oil or carvacrol at 0.05 or 0.1%
v/v significantly reduced the amount of preformed biofilms. In a recent study, Vidaković
Knežević et al. (2023) determined the antibiofilm activity of O. vulgare EO from Serbia
against five L. monocytogenes strains [47]. The MIC values of oregano EO ranged from 0.09
to 0.72 µL/mL. The exposure of 48 h-old L. monocytogenes biofilms on PS to the oregano
EO at its MBC (2xMIC) for 48 h reduced L. monocytogenes biofilm biomasses in the range of
42.9% to 78.6%. Desai et al. (2012) tested oregano EO for its ability to eliminate L. monocyto-
Foods 2023, 12, 2893 9 of 14

genes biofilms on PS and stainless steel (SS) surfaces [48]. For 1-day old biofilms of mixed
L. monocytogenes strains produced at 22 ◦ C on PS microtiter plates, only 0.1% v/v of EO
was needed to eliminate 7 log CFU per well. On the SS coupons, 0.5% v/v was adequate
to completely eliminate 4-day-old biofilms at 7 log CFU per coupon. However, in that
older study, the EO was left to act for 24 h against the preformed biofilms, something that
probably explains its high efficiency. In our study, we chose a short exposure time (15 min)
to try to imitate a short disinfection/biocidal protocol that could be applied in the food
industry and/or clinical settings.

3.2. Chemical Composition of O. vulgare EO

The GC–MS analysis of the O. vulgare EO revealed the presence of 28 compounds
representing 98.1% of the total oil (Table 2). The EO was mainly composed of the monoter-
penes thymol (31.5%), p-cymene (23.7%), γ-terpinene (13.9%), and carvacrol (8%). All these
compounds are commonly found as main constituents of O. vulgare EOs and contribute to
their bioactivities [2,3,49]. However, in most other studies, the EOs are mainly composed of
carvacrol, whereas the EO analyzed in the current study was mainly composed of thymol.
This is not strange since some other studies have also described O. vulgare EOs of thymol
chemotypes [50–54].

Table 2. Chemical composition of O. vulgare EO, as characterized by GC-MS analysis.

Compounds Detected LRI % Area

Methyl-cyclopentane <700 0.8
Methyl α-methylbutanoate 781 0.1
α-Thujene 930 2.6
α-Pinene 936 2.3
Camphene 949 0.6
β-Pinene 977 0.4
1-Octen-3-ol 997 0.2
β-Myrcene 999 3.4
α-Phellandrene 1008 0.7
3-Carene 1012 0.2
α-Terpinene
1020 4.4
(1-methyl-4-(1-methylethyl)-1,3-cyclohexadiene)
p-Cymene
1034 23.7
(1-methyl-4-(1-methylethyl)-benzene)
Sylvestrene
1035 2.4
((R)-1-methyl-5-(1-methylethenyl)-cyclohexene)
p-Cymenene
1044 0.1
(1-methyl-4-(1-methylethenyl)-benzene)
β-cis-Ocimene 1062 0.1
γ-Terpinene
1070 13.9
(1-methyl-4-(1-methylethyl)-1,4-cyclohexadiene)
α-Terpinolene
1095 0.3
(1-methyl-4-(1-methylethylidene)-cyclohexene)
Linalool
1123 0.1
(3,7-dimethyl-1,6-octadien-3-ol)
Borneol 1183 0.4
Terpinen-4-ol
1191 0.7
(4-methyl-1-(1-methylethyl)-3-cyclohexen-1-ol)
α-Terpineol
1209 0.1
(α,α-4-trimethyl-3-cyclohexene-1-methanol)
1-methoxy-4-methyl-2-(1-methylethyl)-benzene 1262 0.8
Thymol 1341 31.5
 Foods 2023, 12, 2893 10 of 14

Table 2. Cont.

Compounds Detected LRI % Area

Carvacrol
1346 8.0
(2-methyl-5-(1-methylethyl)-phenol)
Caryophyllene 1439 0.8
α-Caryophyllene 1475 0.1
β-Bisabolene
((S)-1-methyl-4-(5-methyl-1-methylene-4-hexenyl)- 1532 0.1
cyclohexene)
Caryophyllene oxide 1610 0.1
Total 98.1
LRI: Linear retention index.

3.3. Effect of O. vulgare EO on Virulence Gene Expression of Planktonic L. monocytogenes Cells

The expression of the four target genes (hly, inlA, inlB, and prfA) by the planktonic
L. monocytogenes bacteria following their sub-lethal exposure to the O. vulgare EO is shown
in Figure 3. All the studied genes were significantly downregulated following the exposure
of the cells to the EO at its MBIC (0.06% v/v). This is an intriguing result and seems to agree
with and is probably related to the inhibition of biofilm formation that was noticed for
that EO concentration (Figure 1B). Interestingly, in a previous study of our group, all these
four genes were found to be significantly overexpressed when the same L. monocytogenes
strain (AAL 20107) was left to form biofilm on a PS surface [28]. The hly gene encodes in
L. monocytogenes the hemolysin Listeriolysin O (LLO) which is crucial in the manifestation
of listeriosis by boosting the cell-to-cell spread and thus successful dissemination of the
pathogen during infection [55]. Both inlA and inlB genes encode in L. monocytogenes for
important surface proteins that are known to interact with distinct host receptors to cause
infection of human cells and crossing of the intestinal, blood–brain or placental barriers [56].
PrfA encodes in L. monocytogenes for the master regulator of virulence that controls the
expression of multiple virulence factors that altogether facilitate the transition from extra- to
intracellular environments [57]. Rather in agreement with our results, some other previous
studies have shown the involvement of all these four genes in attachment, sessile growth,
Foods 2023, 12, 2893
and biofilm formation by L. monocytogenes [58–61]. In addition, the reduced expression 11 of 14
of hly and prfA genes in L. monocytogenes following exposure to some other EOs has been
shown in some other previous studies as well [62–64].

2
log2 (fold difference)

-1
*
* *
-2
*
-3
hly inlA inlB prfA
gene

Figure 3. Relative quantification (log (fold differences)) of the expressions of the four target genes (hly,
Figure 3. Relative quantification (log22(fold differences)) of the expressions of the four target genes
inlA,
(hly, inlB,inlB,
inlA, and prfA) by theby
and prfA) planktonic L. monocytogenes
the planktonic bacteria bacteria
L. monocytogenes followingfollowing
their sub-lethal exposure to
their sub-lethal
the O. vulgare EO at its MBIC (0.06% v/v). Each bar represents the mean ± standard errors
exposure to the O. vulgare EO at its MBIC (0.06% v/v). Each bar represents the mean ± standard errors (n = 9).
The statistically significant differences in genes’ expressions between the two treatments
(n = 9). The statistically significant differences in genes’ expressions between the two treatments (exposed
(exposed
and not and not exposed
exposed to the
to the EO) are EO) are indicated
indicated with asterisks
with asterisks (*) (p < 0.05).
(*) (p < 0.05).

4. Conclusions
Origanum vulgare subsp. hirtum EO, extracted from organically cultivated plants on
the Greek island of Lemnos was found to present strong antimicrobial and antibiofilm
actions against two important foodborne pathogenic bacteria (S. Typhimurium and L.
monocytogenes). Its application at 0.13% v/v and 0.06% v/v was sufficient to inhibit biofilm
Foods 2023, 12, 2893 11 of 14

4. Conclusions
Origanum vulgare subsp. hirtum EO, extracted from organically cultivated plants on the
Greek island of Lemnos was found to present strong antimicrobial and antibiofilm actions
against two important foodborne pathogenic bacteria (S. Typhimurium and L. monocyto-
genes). Its application at 0.13% v/v and 0.06% v/v was sufficient to inhibit biofilm formation
by S. Typhimurium and L. monocytogenes, respectively, whereas this needed to be applied
at 6.25% v/v (for 15 min) to eradicate the preformed biofilms of the same species. The EO
was mainly composed of thymol, p-cymene, γ-terpinene and carvacrol. The sub-lethal
exposure of L. monocytogenes planktonic cells to the EO at its MBIC (0.06% v/v) resulted in
the significant downregulation of four important virulence genes (hly, inlA, inlB, and prfA).
To sum, the results obtained advocate for the further exploitation of the antimicrobial action
of oregano EO in food and health applications. Future studies could be conducted on some
such applications. In addition, the specific mode of action of this EO against microbial cells
could be tested by applying either the crude extract or some of its individual components.

Author Contributions: Conceptualization, E.G.; methodology, D.K., A.N. and E.G.; validation, S.K.,
D.K. and A.N.; formal analysis, S.K, D.K., A.N. and E.G.; investigation, S.K. and A.N.; resources,
Y.K. and E.G.; data curation, D.K., A.N. and E.G.; writing—original draft preparation, E.G.; writing—
review and editing, A.N., Y.K. and E.G.; visualization, D.K. and E.G.; supervision, D.K. and E.G.;
project administration, E.G.; funding acquisition, Y.K. and E.G. All authors have read and agreed to
the published version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement: The data presented in this study are available upon reasonable request
form the corresponding author.
Acknowledgments: We thank George-John Nychas (Agricultural University of Athens, Greece) for
providing us with S. enterica str. FMCC_B137, and Nikolaos Andritsos (Athens Analysis Laboratories
S.A., Microbiology Laboratory, Metamorfosi, Greece) for providing us with L. monocytogenes str.
AAL 20107. We also thank Nikolaos Paterakis from Aegean Organics for providing us the oregano
essential oil.
Conflicts of Interest: The authors declare no conflict of interest.

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