biomolecules

Article
Advanced Killing Potential of Thymol against a Time and
Temperature Optimized Attached Listeria monocytogenes
Population in Lettuce Broth
Dimitra Kostoglou, Parthena Tsaklidou, Ioannis Iliadis, Nikoletta Garoufallidou, Georgia Skarmoutsou,
Ioannis Koulouris and Efstathios Giaouris *

Laboratory of Food Microbiology and Hygiene, Department of Food Science and Nutrition, School of the
Environment, University of the Aegean, 81400 Myrina, Lemnos, Greece;
dimitra_kostoglou@outlook.com.gr (D.K.); nenatsak13@gmail.com (P.T.); johniliadis1995@gmail.com (I.I.);
nik1595g@gmail.com (N.G.); georgiask95@hotmail.com (G.S.); fns13054@fns.aegean.gr (I.K.)
* Correspondence: stagiaouris@aegean.gr; Tel.: +30-22540-83115

Abstract: Fresh vegetables and salads are increasingly implicated in outbreaks of foodborne infec-
tions, such as those caused by Listeria monocytogenes, a dangerous pathogen that can attach to the
surfaces of the equipment creating robust biofilms withstanding the killing action of disinfectants.



In this study, the antimicrobial efficiency of a natural plant terpenoid (thymol) was evaluated against

 a sessile population of a multi-strain L. monocytogenes cocktail developed on stainless steel surfaces
Citation: Kostoglou, D.; Tsaklidou, incubated in lettuce broth, under optimized time and temperature conditions (54 h at 30.6 ◦ C) as those
P.; Iliadis, I.; Garoufallidou, N.; were determined following response surface modeling, and in comparison, to that of an industrial
Skarmoutsou, G.; Koulouris, I.; disinfectant (benzalkonium chloride). Prior to disinfection, the minimum bactericidal concentrations
Giaouris, E. Advanced Killing (MBCs) of each compound were determined against the planktonic cells of each strain. The results
Potential of Thymol against a Time revealed the advanced killing potential of thymol, with a concentration of 625 ppm (= 4 × MBC) lead-
and Temperature Optimized ing to almost undetectable viable bacteria (more than 4 logs reduction following a 15-min exposure).
Attached Listeria monocytogenes
For the same degree of killing, benzalkonium chloride needed to be used at a concentration of at least
Population in Lettuce Broth.
20 times more than its MBC (70 ppm). Discriminative repetitive sequence-based polymerase chain
Biomolecules 2021, 11, 397.
reaction (rep-PCR) also highlighted the strain variability in both biofilm formation and resistance.
https://doi.org/10.3390/
In sum, thymol was found to present an effective anti-listeria action under environmental conditions
biom11030397
mimicking those encountered in the salad industry and deserves to be further explored to improve
Academic Editors: Pio Maria Furneri the safety of fresh produce.
and Marta Lopez Cabo
Keywords: Listeria monocytogenes; attachment; stainless steel; lettuce; disinfection; thymol; benzalko-
Received: 23 December 2020 nium chloride; strain variability; microbial resistance; response surface methodology
Accepted: 4 March 2021
Published: 8 March 2021

Publisher’s Note: MDPI stays neutral 1. Introduction

with regard to jurisdictional claims in
Listeria monocytogenes is a major foodborne, facultative intracellular, human pathogenic
published maps and institutional affil-
bacterium that causes listeriosis, a relatively rare but particularly serious infection that is
iations.
characterized by both high morbidity and high mortality for the vulnerable population
groups such as the elderly, pregnant, and immunocompromised individuals [1]. Based
on the latest available data for 2018, 14 foodborne outbreaks and 2549 invasive (severe)
human cases of listeriosis were reported in the EU, with a case fatality rate of 15.6% and 229
Copyright: © 2021 by the authors. recorded deaths [2]. That year, vegetables and juices and other products thereof were the
Licensee MDPI, Basel, Switzerland. food vehicles causing the most strong-evidence outbreaks. This is not surprising consider-
This article is an open access article
ing that the microorganism is easily killed by cooking, and thus the most dangerous foods
distributed under the terms and
are those that are contaminated and consumed without heating, such as fresh vegetables
conditions of the Creative Commons
and salads [3]. Alarmingly, 1.5% of the ready-to-eat (RTE) salads tested in Europe in 2018
Attribution (CC BY) license (https://
were reported as positive for this pathogen [2]. The persistence of L. monocytogenes in food
creativecommons.org/licenses/by/
processing areas (sometimes even for years) is believed to be linked, among others, to
4.0/).

Biomolecules 2021, 11, 397. https://doi.org/10.3390/biom11030397 https://www.mdpi.com/journal/biomolecules

Biomolecules 2021, 11, 397 2 of 20

its ability to strongly attach to the surfaces of the equipment (e.g., tanks, cutting tables,
and conveyor belts) and buildings (e.g., walls, ceilings, drains, and floors), creating robust
biofilms on them that can later withstand the typically applied sanitization processes [4,5].
As with other microorganisms, the biofilm-forming ability of L. monocytogenes may depend
on the specific strain(s) employed, including the inherent genotype ability, surface proper-
ties, serotype, and origin, and may also be significantly influenced by the surroundings
(e.g., temperature, nutrients, pH, osmolarity), in addition to the physicochemical properties
of the substratum and the time the cells have available to attach to it and develop the
sessile structure [6–11]. Indeed, several studies have been occupied with the influence of
environmental conditions (especially those of interest to the food industries) on the biofilm-
forming ability of L. monocytogenes and on its subsequent susceptibility to disinfectants,
with the temperature being included among the most studied extrinsic parameters [12–14].
Nowadays, novel, cost-efficient, and sustainable methods need to be developed and
successfully implemented to combat detrimental biofilms, such as those formed by or
containing pathogenic microorganisms, in various areas, including the food industry [15].
This is due to the increased resistance of such microbial communities to many of the
available biocides [5], combined with the potentially toxic effects of some of the latter
and/or their byproducts for the health and the ecosystem [16,17]. In this direction, phy-
tochemicals have been widely explored as anti-biofilm agents in the past years, mainly
due to their great chemical diversity, the relative ease of acquisition, and their multi-target
antimicrobial action [18]. One of the well-studied plant antimicrobial compounds is thymol
(THY), which is the main component of the essential oils (EOs) of thyme, oregano, and
some other widely distributed plants in the Mediterranean region [19]. However, although
several studies have been occupied with the anti-biofilm action of THY against many
microorganisms, including L. monocytogenes [20–24], little is still known on the superior-
ity (if any) of that compound or other phytochemicals over some other classical surface
disinfectants [25–27]. Considering all the above, the main aim of the current study was to
compare the effectiveness of THY to that of benzalkonium chloride (BAC), a well-known
quaternary ammonium compound (QAC) widely used as biocide in many sanitizing for-
mulations applied in industrial, health care, home, and cosmetics settings [28], against
sessile L. monocytogenes bacteria under attachment conditions trying to simulate as much as
possible those encountered in the salad industry. Those latter conditions were initially here
optimized following response surface methodology (RSM) [29] to predict that incubation
time and temperature combination favoring the attachment of a four-strain L. monocytogenes
cocktail to stainless steel (SS) coupons placed fully immersed in diluted sterile lettuce broth
(dLB). The involvement of each strain in the formation of the mixed sessile community
and its antimicrobial recalcitrance was also monitored by a repetitive sequence-based
polymerase chain reaction (rep-PCR) approach [30]. Overall, THY was found to present
an effective anti-listeria action, while the strain variability in both biofilm formation and
resistance is also highlighted.

2. Materials and Methods

2.1. L. monocytogenes Strains and Preparation of Their Mixed Working Suspension
The four bacterial strains used in this study were the L. monocytogenes AAL20066
(ser. 1/2a), AAL20074 (ser. 4b), AAL20105 (ser. 1/2c), and AAL20107 (ser. 1/2b), all
isolated from fresh mixed salads and kindly provided by Dr. Andritsos (Athens Analysis
Laboratories S.A.; Metamorfosi, Attica, Greece). All strains were kept frozen (at −80 ◦ C)
in Tryptone Soy Broth (TSB; Lab M, Heywood, Lancashire, UK) containing 15% glycerol
and were resuscitated through streaking on to the surface of Trypticase Soy Agar (TSA;
Condalab, Torrejón de Ardoz, Madrid, Spain) and incubating at 37 ◦ C for 24 h (precultures).
Working cultures were prepared by inoculating a colony from each preculture into 10 mL
of fresh TSB and further incubating at 37 ◦ C for 18 h. Bacteria from those final working
cultures were sedimented by centrifugation (4000× g for 10 min at room temperature),
washed twice with quarter-strength Ringer’s solution (Lab M), and finally suspended
Biomolecules 2021, 11, 397 3 of 20

in the same solution, so as to present an absorbance at 600 nm (A600 nm ) equal to 0.1 (ca.
108 CFU/mL). Those adjusted saline suspensions of each strain were finally mixed together
and used for the subsequent attachment experiments.

2.2. Preparation of the Lettuce Broth

A total of 2 kg of fresh lettuce were bought from a topical greengrocer and immediately
transported to the laboratory where the external leaves were removed, keeping only the
greener parts of the inner leaves, which were washed well in tap water, weighted (ca. 500 g)
and then transferred to a household juicer (Multipress Automatic; Braun AG, Kronberg
im Taunus, Germany). Collected juice (ca. 300 mL) was placed in a glass beaker, covered
with parafilm, and heated at 60 ◦ C (in a water bath) for 30 min to inactivate endogenous
enzymes. Following heating, the juice was kept on ice for 10 min, placed in plastic falcon
tubes (50 mL), and centrifuged at 2000× g for 15 min at 4 ◦ C. The supernatant was carefully
removed, vacuum filtered through paper filter disks (87 g/m2 ; Munktell Filter AB, Falun,
Sweden), and the resulting filtrate was then sterilized by passing through microbiological
syringe filters (0.2 µm diameter; Whatman, Buckinghamshire, UK). Sterile juice broth was
stored at −80 ◦ C till needed for the attachment experiments, on the day of which was
diluted 1:20 with sterile distilled water. That dilution was performed to imitate those
nutritional conditions potentially found in the salad industry, also considering that the
further dilution of the medium (till 1:50) did not much affect the planktonic growth of the
tested strains (data not shown).

2.3. Experimental Design to Study the Combined Influence of Time and Temperature
on Attachment
A central composite rotational design (CCRD) including 10 experiments, each one
twice executed (i.e., 20 experiments in total) (Table 1), was applied to determine the
putative interactive effects of the two independent factors, that is the incubation time (X1 ,
varying between 14.1 and 81.9 h) and temperature (X2 , varying between 3 and 37 ◦ C)
on the concentration of the attached/biofilm cells (Log10 CFU/cm2 ) of the four-strain L.
monocytogenes cocktail on the SS coupons, as previously described [29]. That design also
allowed to determine those values of the two factors that upon concurrently applied would
maximize the final population density (Log10 CFU/cm2 ) of the attached/biofilm bacteria.
Each independent factor was coded at five levels, −1.414, −1, 0, 1, and 1.414, according to
the following equation:
X − X0
xi = i , xi = 1, 2 (1)
∆Xi
where xi and Xi are the dimensionless and the actual value of the independent factor i, X0
the actual value of the independent factor i at its zero level (central point), and ∆Xi the step
change of Xi correlating with a unit alteration of the dimensionless value.
Obtained (measured) data were submitted to least square regression analysis to acquire
the parameters of the derived polynomial mathematical equation. To confirm the ability of
the latter to accurately predict the combined influence of the two independent factors on
the accumulation of the bacteria on the SS surfaces, four additional experiments were also
performed examining different combinations of the two independent factors than those
used to generate the model.
Biomolecules 2021, 11, 397 4 of 20

Table 1. Experimental design with real and coded values of the two independent factors (i.e., time and
temperature) evaluated for their influence on the attached to stainless steel (SS) coupons population
(Log10 CFU/cm2 ) of the four-strain L. monocytogenes cocktail. Measured and predicted values of
Log10 CFU/cm2 , as defined for each individual experiment, are also shown.

Independent Factors a Response b

Incubation Incubation Attached Population

Experiment (Log10 CFU/cm2 )
Time (h) Temperature (◦ C)
(X 1 ) (X 2 ) Measured Predicted
1 14.1 (−1.414) 20 (0) 2.12 ± 0.22 2.61 ± 0.25
2 72 (1) 8 (−1) 2.26 ± 0.32 2.50 ± 0.25
3 48 (0) 37 (1.414) 5.01 ± 0.20 5.41 ± 0.25
4 48 (0) 37 (1.414) 4.74 ± 0.50 5.41 ± 0.25
5 72 (1) 8 (−1) 2.14 ± 0.34 2.50 ± 0.25
6 24 (−1) 8 (−1) −0.05c ± 0.00 −0.20 ± 0.25
7 48 (0) 3 (−1.414) −0.05c ± 0.00 −0.23 ± 0.25
8 24 (−1) 32 (1) 5.69 ± 0.07 4.96 ± 0.25
9 24 (−1) 32 (1) 5.54 ± 0.16 4.96 ± 0.25
10 48 (0) 3 (−1.414) −0.05c ± 0.00 −0.23 ± 0.25
11 48 (0) 20 (0) 4.94 ± 0.09 4.68 ± 0.22
12 81.9 (1.414) 20 (0) 4.90 ± 0.12 4.78 ± 0.25
13 14.1 (−1.414) 20 (0) 2.10 ± 0.30 2.61 ± 0.25
14 72 (1) 32 (1) 5.62 ± 0.02 5.31 ± 0.25
15 48 (0) 20 (0) 4.90 ± 0.38 4.68 ± 0.22
16 24 (−1) 8 (−1) −0.05c ± 0.00 −0.20 ± 0.25
17 48 (0) 20 (0) 4.33 ± 1.04 4.68 ± 0.22
18 81.9 (1.414) 20 (0) 4.94 ± 0.20 4.78 ± 0.25
19 48 (0) 20 (0) 4.53 ± 0.75 4.68 ± 0.22
20 72 (1) 32 (1) 5.41 ± 0.04 5.31 ± 0.25
aCodes values are shown in parentheses. b Measured values are means ± standard deviations, while predicted
ones are means ± standard errors. c Detection limit of the plate counting method.

2.4. Disinfectants and Other Chemicals

Thymol (THY) was bought from Penta Chemicals (Radiová, Prague, Czech Republic)
(powder min. 99.0%, molar mass: 150.22 g/mol; product code: 27450-30100), while benza-
lkonium chloride (BAC) was purchased from Acros Organics (Thermo Fisher Scientific,
Geel, Belgium) (liquid, alkyl distribution from C8H17 to C16H33; product code: 215411000).
The stock solution of THY (10% w/v) was prepared in absolute ethanol, while that of BAC
(1% v/v) in sterile distilled water. Both stock solutions were maintained at 4 ◦ C for up to
two weeks. All other chemicals and reagents used for the experiments were purchased
from Merck KGaA (Darmstadt, Germany) unless otherwise stated.

2.5. Determination of the Minimum Inhibitory and Bactericidal Concentrations (MICs, MBCs)
The minimum inhibitory concentration (MIC) and minimum bactericidal concentra-
tion (MBC) of each disinfection chemical (i.e., THY, BAC) against the planktonic cells of
each L. monocytogenes strain were specified using the broth microdilution and agar spot
methods, respectively, as previously described [26]. Briefly, to calculate the MICs, broth
cultures (in TSB), inoculated with bacteria (ca. 105 CFU/mL) and also containing 10 differ-
ent increasing concentrations of each chemical, were statically incubated at 37 ◦ C for 24 h.
The tested concentrations for THY ranged from 10,000 to 19.5 ppm (two-fold dilutions),
while those for BAC from 1 to 10 ppm (in 1 ppm increments). The MIC of each chemical
was determined as its lowest concentration inhibiting the visible bacterial growth (i.e., no
increase in broth’s turbidity), while the MBC was calculated as its lowest concentration
reducing the initial inoculum by at least 3 logs (≥99.9%). Each experiment was thrice
repeated starting from independent bacterial cultures.
Biomolecules 2021, 11, 397 5 of 20

2.6. Attachment of L. monocytogenes to SS under Various Time and Temperature Combinations

and Quantification of the Sessile and Planktonic Populations
The four-strain L. monocytogenes cocktail was left to attach to or form biofilms on
rectangular SS coupons (3 × 1 × 0.1 cm, type AISI 304), as previously described [31]. Briefly,
individual, cleaned, and sterilized coupons were fully and vertically immersed in 1:20
dLB (5 mL in glass tubes), which was also inoculated with the mixed bacterial suspension,
so as to have an initial concentration of ca. 103 CFU/mL. This setup was then incubated
under the 10 different combinations of time (varying from 14.1 h to 81.9 h) and temperature
(varying from 3 ◦ C to 37 ◦ C) as these had been predetermined by the applied CCRD (Table
1). Following attachment/biofilm formation, the loosely attached cells were removed from
the surfaces by placing each coupon in 5 mL of quarter-strength Ringer’s solution, under
agitation for 5 min. This rinsing procedure was repeated once more and then each coupon
was placed into a new sterile glass test tube containing 6 mL of quarter-strength Ringer’s
solution and 10 sterile glass beads (diameter 3.0 mm), where it was thoroughly vortexed
for 2 min (at 3000 rpm using ZX3 Advanced Vortex Mixer (VELP Scientifica Srl, Usmate,
Italy) in order to detach from surfaces the strongly attached/biofilm bacteria. These were
finally enumerated by counting colonies on (duplicate) spread inoculated (100 µL) TSA
plates, following 10-fold serial dilutions in quarter-strength Ringer’s solution, plating, and
incubation at 37 ◦ C for 24 h. Upon no appearance of colonies on TSA plates (in some of the
experiments combining short times with low temperatures), and to lower the detection
limit, 1 mL was spread plated on four Petri dishes (i.e., 250 µL/plate) resulting thus in a
detection limit of 1 CFU/mL corresponding to an attached population of 0.9 CFU/cm2
(equal to −0.05 Log10 CFU/cm2 ), given that the total surface area of each SS coupon
was 6.8 cm2 . For each experiment and at the end of incubation, the mixed planktonic
population (Log10 CFU/mL) found in the dLB where the coupons had been placed was
also enumerated through plate counting.

2.7. Disinfection of the Mixed Sessile Community and Calculation of the Log Reductions
The four-strain L. monocytogenes cocktail was initially left to attach to/form biofilm
on the SS coupons incubated in dLB under time and temperature conditions previously
determined/verified to maximize the concentration (Log10 CFU/cm2 ) of the sessile bac-
teria (i.e., for 54 h at 30.6 ◦ C). At the end of this incubation, the loosely attached cells
were removed from the surfaces, as previously described (Section 2.6), and the coupons
were then placed in the appropriate aquatic disinfectant solution (5 mL in glass tubes).
Each disinfectant was left to act for 15 min at 20 ◦ C and tested at three different concen-
trations, based on the previous determination of the MBCs (Section 2.5). Thus, THY was
applied at two, three, and four times more than its MBC (i.e., 312.5 ppm, 468.8 ppm, and
625 ppm, where MBC = 156.3 ppm), while BAC at 4.7, 11.7, and 23.3 times more its MBC
(i.e., 14 ppm, 35 ppm, and 70 ppm, where MBC = 3 ppm). Sterile distilled water was
used as the negative disinfection control. This also contained 0.6% v/v ethanol when
THY was used as the disinfectant, given that this low ethanol concentration was the one
existing in the highest tested concentration for that terpenoid (i.e., 625 ppm). Following
disinfection, each coupon was removed from the disinfectant solution and placed in 5 mL
of quarter-strength Ringer’s solution, under agitation for 5 min, to remove disinfectant
residues and was then immersed for 10 min in a 10 mL-plastic falcon tube containing 6
mL of Dey-Engley (D-E) Neutralizing broth (Lab M) and 10 sterile glass beads (3 mm in
diameter). Strongly attached/biofilm cells were removed from surfaces and enumerated,
as previously described (Section 2.6). Plate counts were converted to Log10 CFU/cm2 and
for each disinfectant and tested concentration, the logarithmic reductions (Log10 CFU/cm2 )
of bacteria following disinfection were calculated by subtracting the log10 of the survivors
from that counted following disinfection with water (negative control).
Biomolecules 2021, 11, 397 6 of 20

2.8. Recovery of L. monocytogenes Colonies and DNA Extraction

In total, 100 colonies were randomly selected and recovered from the highest dilutions
of the TSA plates used to enumerate the strongly attached/biofilm viable bacteria found
on the SS coupons both before and after their disinfection and from those plates used to
enumerate the planktonic bacteria existing in the surrounding dLB at the end of incubation
(i.e., for 54 h at 30.6 ◦ C). More specifically, 20 colonies were recovered following the quantifi-
cation of the planktonic population (treatment A), while other 20 colonies represented the
sessile population being found on the SS coupons at the end of incubation and just before
disinfection (treatment B). The other 60 colonies were recovered after disinfection and
following the quantification of the remaining viable sessile bacteria. For this, one interme-
diate sub-lethal concentration per disinfectant was selected (out of the three concentrations
tested). In particular, 20 colonies were isolated after each disinfection treatment, that is
disinfection with water (negative control), 468.8 ppm THY, or 35 ppm BAC (treatments C,
D, and E, respectively). All isolated colonies were suspended in TSB (1 mL) containing 15%
(v/v) glycerol and stored at –80 ◦ C till the extractions of their DNAs.
The genomic DNA of each isolate (×100) was extracted following the enzymatic
method described by Doulgeraki et al. [32], with some minor adaptations. Analytically, a
loopful of the frozen suspension of each isolate was streaked on to the surface of TSA and
incubated at 37 ◦ C for 24 h (precultures). Working cultures were prepared by inoculating
a colony from each preculture into 5 mL of fresh TSB and further incubating at 37 ◦ C for
18 h. Bacteria from those final working cultures were harvested by centrifugation (4000× g
for 10 min at room temperature) and resuspended in 0.5 mL of buffer lysis solution (1 M
sorbitol, 0.1 M ethylenediaminetetraacetic acid, pH 7.5) containing 25 mg/mL lysozyme
(Applichem GmbH, Darmstadt, Germany) and 5 U mutanolysin (from Streptomyces glo-
bisporus ATCC 21553; Merck), and incubated at 37 ◦ C for 2 h. Following this incubation
period, the mixture was centrifuged (5000× g for 10 min at 4 ◦ C) and the new pellet was
suspended in 0.5 mL of buffer solution (50 mM Tris-HCl, 20 mM EDTA, pH 7.4), 50 µL of
10% sodium dodecyl sulfate (SDS) were also added and incubated at 65 ◦ C for 30 min. Each
sample was then mixed with 0.2 mL of 5 M potassium acetate, placed on ice for 30 min,
and centrifuged at 20,000× g for 10 min at 4 ◦ C. The supernatant was precipitated with
1 mL of ice-cold isopropanol and centrifuged at 10,000× g for 15 min at 4 ◦ C. Each new
pellet was washed by suspending it in 1 mL of ice-cold absolute ethanol and centrifuging
at 20,000× g for 15 min at 4 ◦ C. Each pellet was then air-dried (at 40 ◦ C for ca. 1.5 h) and
resuspended in 120 µL of Tris-EDTA (TE) buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8).
The absorbances of each DNA solution were finally measured at 260 and 280 to determine
the concentration of the extracted nucleic acids and their purity [33]. A 5 µL aliquot of each
extracted DNA sample was also submitted to electrophoresis (1.5% w/v TBE agarose gel;
50 V for 1 h) to verify its integrity, while the rest of each sample was maintained at –20 ◦ C
to be used as a substrate in the subsequent rep-PCRs.

2.9. Discrimination of L. monocytogenes Strains through Rep-PCR

Each recovered isolate (×100) was discriminated/typed to the strain level following a
previously described rep-PCR approach [30], with some minor adaptations. Briefly, the
Kapa Taq PCR Kit with dNTPs (KK1016, 500 U; Kapa Biosystems, Wilmington, MA, USA)
was used for the PCRs. Each reaction mixture contained 2.5 µL of 10× kapa Taq Buffer
A (1.5 mM final MgCl2 concentration at 1×); 0.5 µL of 10 mM dNTP Mix (0.2 mM final
concentration); 2.5 µL of 10 µM GTG5 primer (GTG GTG GTG GTG GTG; 1 µM final
concentration); 3 µL of DNA template (≈ 300 ng); 0.2 µL of Kapa Taq DNA polymerase
(5 U/µL) and 16.3 µL of high-performance liquid chromatography (HPLC) grade water
(Applichem) to a total volume of 25 µL. Following their preparation, the mixtures were
placed in a PeqStar 96 HPL Gradient Thermocycler (Peqlab, VWR International GmbH,
Darmstadt, Germany). The PCR program consisted of an initial denaturation step at 95 ◦ C
for 5 min, followed by 30 cycles of denaturation at 95 ◦ C for 30 s, primer annealing at
40 ◦ C for 1 min, and primer extension at 72 ◦ C for 8 min, and this was concluded by a final
Biomolecules 2021, 11, 397 7 of 20

extension step at 72 ◦ C for 16 min. For each recovered isolate, the rep-PCR protocol was
twice repeated on different days.
Resulting amplicons were separated in a 1.5% (w/v) Tris-Borate-EDTA (TBE) agarose
gel, also containing 0.05 µg/mL of ethidium bromide (EtBr), in 0.5× TBE buffer, at 50
V for 2 h, using the Mupid-One electrophoresis system (NIPPON Genetics EUROPE
GmbH, Dueren, Germany). FastGene® 100 bp DNA Ladder (110 µg/1 mL; MWD100,
NIPPON Genetics) was used as the molecular weight marker (5 µL/well), while DNA
bands were detected by visualizing gels after electrophoresis under UV trans-illumination
using the Quantum ST4 gel documentation imaging system (Vilber Lourmat, Marne-la-
Vallée, France). To be sure for the correct discrimination/typing of the isolates (×100),
the rep-PCR amplicons of DNAs extracted from pure cultures of each one of the four L.
monocytogenes strains were always loaded on each gel (as controls, ×4), together with and
next to those amplicons resulting from the rep-PCRs using as substrates the DNAs of the
recovered isolates (samples).

2.10. Statistics and Graphics

Each attachment experiment included two replicate SS coupons and was twice re-
peated starting from independent bacterial cultures. Disinfection experiments also included
two coupons, but these were thrice repeated. Bacterial counts (CFU/mL and CFU/cm2
for planktonic and sessile populations, respectively) were converted to logarithms before
calculating the means and standard deviations. Experimental design and statistical analysis
were performed using JMP™ v10 (SAS Institute Inc., Cary, NC, USA). Analysis of variance
(ANOVA) was applied to estimate the significance of the derived model at a p-value of 0.05,
while the good fit of the derived equation was also assessed by its adjusted regression coef-
ficient (R2 adj ). In addition, the predictive ability of the model was evaluated by determining
the bias and accuracy factors (Bf and Af , respectively) of the four additional confirmation
experiments, together with that one predicted maximizing sessile population, as previously
reported [34]. The contour plot of the predicted data was constructed using SigmaPlot
for Windows v11 (Systat Software Inc., Chicago, IL, USA). Pearson correlation analysis
was finally applied to determine any possible correlation existing between planktonic and
sessile populations for the various tested time and temperature combinations.

3. Results
3.1. Combined Influence of Time and Temperature on Sessile and Planktonic Cell Numbers
The measured and predicted values for the concentrations of the attached populations
(Log10 CFU/cm2 ), of the four-strain L. monocytogenes cocktail to SS coupons, at the vari-
ous selected time and temperature combinations, are presented in Table 1. Thus, under
the current test conditions, the measured sessile populations were found to range from
–0.05 ± 0.00 Log10 CFU/cm2 to 5.69 ± 0.07 Log10 CFU/cm2 . Regarding the lowest sessile
population, this was always observed at those experiments where the incubation tempera-
ture was ≤8 ◦ C, and in parallel the attachment time ≤48 h (i.e., experiments 6, 7, 10, and
16). Indeed, under such short time and low-temperature conditions, the attached cells
recovered from surfaces were always lower than the detection limit of the plate counting
method (i.e., 0.9 CFU/cm2 ). On the other hand, the maximum observed sessile counts were
recorded upon incubation at 32 ◦ C for 24 h (experiment 8), with seemingly no significant
differences upon increasing the incubation time up to 72 h at that specific temperature
(experiments 14 and 20).
The above presented measured data were submitted to multiple regression analysis to
derive the polynomial mathematical equation, which is shown in Table 2. Equation fitting
was estimated by determining its square regression coefficient (R2 ), its adjusted value
(R2 adj ), and p-value, which were 0.97, 0.96, and < 0.0001, respectively. Those values clearly
demonstrate an acceptable agreement between the measured and predicted data. The
derived model could thus well predict the combined effects of the two factors (i.e., time and
Biomolecules 2021, 11, 397 8 of 20

temperature) on the attached SS coupons population (Log10 CFU/cm2 ) of the four-strain L.

monocytogenes cocktail.

Table 2. Polynomial mathematical equation and statistical parameters (R2 , R2 adj, and p-value)
characterizing the influence of the two independent factors evaluated (i.e., time and temperature) on
the attached to SS coupons population (Log10 CFU/cm2 ) of the four-strain L. monocytogenes cocktail.

Response Polynomial Equation R2 R2 adj p

4.68 + 0.77 × t + 1.99 × T − 0.59(t
Log10 CFU/cm2 0.97 0.96 <0.0001
× T) − 0.49 × t2 − 1.04 × T2
Refers to the coded values of the independent factors.

Indeed, a satisfactory linear relationship was recorded between the measured and
predicted data for the concentrations of the attached-to-SS coupons populations (Log10
CFU/cm2 ) of the four-strain L. monocytogenes cocktail (Figure 1).

Figure 1. Linear relationship between the measured and predicted data for the concentrations of the
attached to SS coupons populations (Log10 CFU/cm2 ) of the four-strain L. monocytogenes cocktail.
The mathematical equation of the regression plot, together with its regression coefficient (R2 ), are also
shown. Dots represent the mean values of all experiments included in the CCRD (n = 20; i.e., those
shown in Table 1). For more clarity, the bars of standard deviations have been omitted.

The determined values and fitting statistics (95% confidence limits, p-values, and t
ratios) for the parameters of the derived model are presented in Table 3.

Table 3. Calculated values and fitting statistics for the parameters of the polynomial mathematical
equation predicting the attached to SS coupons population (Log10 CFU/cm2 ) of the four-strain L.
monocytogenes cocktail as a function of time (t) and temperature (T).

95% Confidence Limits

Parameter Estimated Value a p-Value t Ratio
Lower Upper
Intercept 4.68 ± 0.22 4.20 5.15 <0.0001 21.32
t 0.77 ± 0.11 0.53 1.00 <0.0001 6.98
T 1.99 ± 0.11 1.76 2.23 <0.0001 18.18
t*T −0.59 ± 0.16 −0.92 −0.25 0.002 −3.79
t2 −0.49 ± 0.15 −0.80 −0.18 0.004 −3.38
T2 −1.04 ± 0.15 −1.35 −0.73 <0.0001 −7.18
a Values are means ± standard errors.
Biomolecules 2021, 11, 397 9 of 20

Following extraction of the mathematical equation, the application of the desirability

function allowed the determination of that specific time and temperature combination
which could maximize the sessile population of the four-strain L. monocytogenes cocktail
to SS coupons. Thus, under those specified conditions (i.e., 54 h at 30.6 ◦ C) the equa-
tion predicted a maximal value for the concentration of the attached/biofilm population
equal to 5.65 ± 0.40 Log10 CFU/cm2 . Indeed, the confirmation experiment performed
under those specific conditions resulted in a measured attached/biofilm population of
5.46 ± 0.31 Log10 CFU/cm2 (Table 4).

Table 4. Confirmation experiments of the mathematical equation describing the concentration of the
attached to SS coupons population (Log10 CFU/cm2 ) of the four-strain L. monocytogenes cocktail as
a function of time and temperature. Measured and predicted values of sessile populations (Log10
CFU/cm2 ) for each individual experiment, together with the bias and accuracy factors of the model,
are also shown. The 5th experiment was the one executed under conditions (i.e., 54 h at 30.6 ◦ C)
predicted to result in the maximum sessile population (5.65 ± 0.40 Log10 CFU/cm2 ).

Independent Factors a Response b

Incubation Incubation Attached Population (Log10

Experiment CFU/cm2 )
Time (h) Temperature (◦ C)
(X1 ) (X2 ) Measured Predicted
1 36 (−0.5) 26 (0.5) 5.39 ± 0.14 5.05 ± 0.40
2 60 (0.5) 26 (0.5) 5.40 ± 0.09 5.52 ± 0.40
3 66 (0.75) 11 (−0.75) 2.96 ± 0.37 3.22 ± 0.40
4 66 (0.75) 29 (0.75) 5.58 ± 0.05 5.55 ± 0.40
5 (max) 54 (0.25) 30.6 (0.89) 5.46 ± 0.31 5.65 ± 0.40
Factors
Bias 1.01
Accuracy 1.04
a Codes values are shown in parentheses. b Values are means ± standard deviations.

The alterations in the concentration of sessile cells (Log10 CFU/cm2 ) as a function

of the concurrent change of each possible pair of the two factors evaluated (i.e., time and
temperature) are presented in the form of a contour plot in Figure 2. It is clear that the
concentration of the attached/biofilm cells tends to be maximized (>5.4 Log10 CFU/cm2 )
at temperatures above 26 ◦ C combined with incubation time ranging from approximately
45 h to 65 h. At the same time, the further increase of incubation temperature (>26 ◦ C)
seems to decrease the time needed for the cells to maximize their accumulation onto the
surfaces. For instance, at 37 ◦ C, attached cells surpass the density of 5.4 Log10 CFU/cm2
as early as 30 h of incubation. However, at this high temperature, the sessile population
seems to slightly decrease earlier as incubation time passes (i.e., <5.4 Log10 CFU/cm2 from
60 h and more). This is probably due to detachment phenomena.
Biomolecules 2021, 11, 397 10 of 20

Figure 2. Contour plot describing the interactive influence of incubation time and temperature
on the concentration of the attached SS coupons population (Log10 CFU/cm2 ) of the four-strain L.
monocytogenes cocktail. This plot was constructed by considering the predictions of all experiments
included in the central composite rotational design (CCRD) (n = 20; i.e., those shown in Table 1).
Dotted lines illustrate time and temperature conditions predicted maximizing sessile population
(i.e., 54 h at 30.6 ◦ C).

Figure 3 shows the positive linear correlation between the concentrations of the
measured planktonic (Log10 CFU/mL) and attached populations to the SS coupons (Log10
CFU/cm2 ) of the four-strain L. monocytogenes cocktail. It is obvious that these two factors
are related to each other with the increase of one factor leading to (or perhaps caused by)
the increase of the other factor.
Biomolecules 2021, 11, 397 11 of 20

Figure 3. Correlation between the concentrations of the measured planktonic (Log10 CFU/mL) and
attached populations to the SS coupons (Log10 CFU/cm2 ) of the four-strain L. monocytogenes cocktail.
The solid line represents the linear regression equation, while the dashed parallel lines represent the
prediction intervals (α = 0.95). The mathematical equation of the linear regression, together with its
regression coefficient (R2 ), Pearson correlation coefficient (rp ), and p-value are also presented. Dots
represent the mean values of all experiments included in the CCRD (n = 20; i.e., those shown in
Table 1). For more clarity, the bars of standard deviations have been omitted. The horizontal dotted
line illustrates the detection limit of the plate counting method of the sessile cells (i.e., −0.05 Log10
CFU/cm2 ).

3.2. Calculation of MICs and MBCs of THY and BAC against Planktonic Cells and Disinfection of
the Mixed Sessile Community
The MIC of THY was found equal to 78.1 ppm against all four L. monocytogenes
strains, while a double concentration was always needed to kill their planktonic cells
(i.e., MBC = 156.3 ppm). The MIC of BAC was found equal to 2 ppm against all L. mono-
cytogenes strains, quite close to the MBC for that compound, which was determined at
3 ppm.
The log reductions of attached/biofilm population on SS coupons of the four-strain
L. monocytogenes cocktail (5.46 ± 0.31 Log10 CFU/cm2 ), following the 15 min disinfection
exposure to each chemical (i.e., THY, BAC) being applied at three different concentrations
(ppm), are presented in Figure 4. As expected, log reductions increased as the chemicals’
concentrations increased, meaning that more bacteria died upon increasing the chemical’s
concentration. More importantly, the results revealed the significant disinfection efficiency
of THY, with a concentration of 625 ppm (= 4 × MBC), leading to almost undetectable
(<0.95 Log10 CFU/cm2 ) viable bacteria (i.e., more than 4 logs reduction; 99.99% killing rate).
On the other hand, the reduction did not exceed 3.5 logs even when BAC was applied at
23.3 times more than its MBC (i.e., 70 ppm).
Biomolecules 2021, 11, 397 12 of 20

Figure 4. Log reductions of attached/biofilm cells on SS coupons (Log10 CFU/cm2 ), of the four-strain L. monocytogenes
cocktail, following the 15 min disinfection exposure to either thymol (THY) or benzalkonium chloride (BAC), each applied
at three different concentrations (ppm). The bars represent the mean values ± standard deviations (n = 6).

3.3. Strain Variability on Planktonic Growth, Attachment, and Disinfection Resistance

As expected, the four L. monocytogenes strains used in this study to prepare the cellular
attachment cocktail (i.e., AAL20066, AAL20074, AAL20105, and AAL20107), were found to
present distinct rep-PCR profiles (Figure 5), thus enabling their easy discrimination through
the applied rep-PCR approach. After all, those strains had been deliberately chosen so that
each belongs to a different serovar (i.e., 1/2a, 4b, 1/2c, and 1/2b, respectively).

Figure 5. Characteristic repetitive sequence-based polymerase chain reaction (rep-PCR) profiles of 10

L. monocytogenes isolates, where it can be observed that each strain (AAL20066, AAL20074, AAL20105,
and AAL20107) presents a distinct amplicon pattern enabling this way its easy discrimination from
the other ones.

The contribution of each strain in the composition of the mixed planktonic population,
which existed at the end of incubation (i.e., for 54 h at 30.6 ◦ C) in the dLB in which
the SS coupons had been placed as substrata for the bacterial attachment, in addition to
that encountered in the mixed sessile communities found on the surfaces, both before
and after their disinfection (i.e., 15-min exposure to water as control, 468.8 ppm THY, or
35 ppm BAC), are depicted in Figure 6. In general, the different strains were found to
behave differently regarding their either planktonic or sessile growth and their disinfection
resistance; the latter also found to be affected by the applied disinfectant. Thus, for
instance, strain AAL20066 was not at all detected in the planktonic population (treatment
Biomolecules 2021, 11, 397 13 of 20

A), whereas this strain still represented the 15% of the isolates (colonies) recovered from
those plates used to quantify the attached/biofilm population (treatment B). As another
example of this variability, this time on resistance, strain AAL20105 was not at all detected
following disinfection with either water (control) or THY (468.8 ppm) (treatments C and D,
respectively), whereas the 20% of colonies appearing on the plates following disinfection
with BAC (70 ppm) belonged to that strain (treatment E). That heterogeneity in the behavior
of each strain is also evident when someone considers the average overall distribution of
each one in which, for instance, strain AAL20074 presented an appearance rate of 41%,
whereas strain AAL20105 appeared approximately four times less exhibiting an average
overall distribution equal to 11%.

Figure 6. Distribution percentages (%) of each L. monocytogenes strain (i.e., AAL20066, AAL20074,
AAL20105, and AAL20107) for each of the five examined treatments (i.e., A, B, C, D, and E; previously
described in Section 2.8). The average overall distribution of each strain is also shown.

4. Discussion
Various phytochemicals, either in pure form or as components of plant extracts, have
been last years tested as anti-biofilm agents to overcome antimicrobial resistance (AMR)
against various harmful microorganisms, including significant foodborne pathogenic
bacteria, such as Salmonella enterica, Campylobacter spp., L. monocytogenes and Escherichia coli
O157:H7 [35,36]. In this study, a natural terpenoid found in rich quantities in the EOs of
thyme and other aromatic plants, already authorized as a food additive in many countries,
THY, was tested against a sessile cocktail of four foodborne L. monocytogenes strains, all
previously isolated from mixed salads and each belonging to a different serovar. Three of
the four serovars here tested (i.e., 1/2a, 1/2b, and 4b) are known to cause the vast majority
of human listeriosis cases [37]. Similarly, most of the L. monocytogenes strains isolated from
foods and food processing environments belong to one of those three serovars, although the
relative abundance of each serovar differs from that observed in clinical cases [37]. Strains
belonging to serovar 1/2c are also pathogenic and isolated from retail foods, including
RTE ones [38,39].
A mixed bacterial suspension, containing equal cell numbers for each strain, was
initially left to attach to SS coupons incubated in sterile dLB under various time and
temperature combinations, to extract a mathematic model which could be able to predict
the density of the attached population (Log10 CFU/cm2 ) as a combined function of time
and temperature, and as thus be able to define that specific combination of those two
environmental factors (i.e., 54 h at 30.6 ◦ C) that would maximize that density [40]. This last
was desired so that the subsequent disinfection experiments could be executed following a
Biomolecules 2021, 11, 397 14 of 20

worst-case scenario in which the environmental conditions would be quite favorable for the
bacterial attachment. Surely, in addition to temperature, several other environmental factors
could influence that attachment (e.g., pH, nutrients, osmolarity). Although some of those
could be incorporated into the model, the reason for not doing so was because our primary
aim was to comparatively evaluate the effectiveness of the two studied disinfectants
(i.e., THY and BAC) against a well-established sessile L. monocytogenes population left to
be formed in a specific plant-based growth medium, that is the dLB, and not to study
the influence of environmental factors in general on attachment/biofilm formation by
that pathogen. In addition, this incorporation could probably result in an inability for
that specific CCRD design to accurately predict the combined (complex quite probably)
influence of all those many interacting parameters. To deliver the model, RSM was applied,
which is, in general, a collection of mathematical and statistical techniques based on the
fit of a polynomial equation to a set of experimental data, with the ultimate objective of
making statistical previsions [41]. The generation of large amounts of information from
even a small number of experiments, decreasing thus time, labor, and expenses, is the main
advantage of this multivariate technique, together with the possibility of evaluating the
interaction effect between the tested variables on the studied response. This latter effect is
not fully depicted when someone follows a one-variable-at-a-time approach, as is usually
the case in most of the studies published so far regarding the influence of environmental
factors on attachment and/or biofilm formation [42]. So far, RSM has mainly been used as
a tool for optimization in analytical chemistry; however, it can be well applied whenever a
response (e.g., biofilm formation), or even a set of responses of interest, may be influenced
by several variables (e.g., environmental factors). Thus, this technique has also been used
in the field of microbiology, specifically in inactivation applications [43,44], and, in recent
years, also in biofilm research [31,45–47].
It is well-known that the nutrients that are available in a medium can affect both the
attachment of the bacteria to surfaces and their subsequent sessile proliferation [7,8,14]. A
diluted lettuce broth (dLB) was here used to simulate nutritional conditions potentially
encountered within the fresh salad industry. However, it should still be noted that this
broth was initially heated (at 60 ◦ C for 30 min). Although this was just carried out to
inactivate the endogenous enzymes of the plant tissue, this mild heating may slightly
change the nutritional and physicochemical characteristics of that broth. However, if not
inactivated, the action of those enzymes could still alter those parameters as well over
time, considering that the lettuce broth was de facto impossible to be directly used on the
day of its preparation. After all, previous studies that had also used vegetables’ broths
as substrates for bacterial growth, have conducted similar (or even quite more intense)
heating steps during their preparation [48–50]. Until now, however, few other studies have
been published using such model food systems and dealing with the sessile behavior of L.
monocytogenes [51,52], although these studies could better imitate the “real” scenario. Before
diluting the lettuce broth (i.e., 1:20), this was also here sterilized through filtration. This
sterilization was performed to simplify the experimental approach, choosing to initially
work with pure (mono-species) cultures of the tested pathogen and be easier able to make
observations and deliver conclusions. However, it should be noted that in the “real” world,
bacteria belonging to different species and genera may be found together in the same
niche, even together with other microorganisms (e.g., fungi, protozoa, bacteriophages),
interacting with each other, with these intra/inter-species and inter-kingdom interactions
be particularly evident in most of the natural biofilm communities, affecting their overall
physiology and resistance [53,54].
It was here found that the increase of the incubation time above 45 h and in parallel of
temperature above 30 ◦ C had a favorable effect on the accumulation of the bacteria on the SS
surfaces, with the sessile population being maximized following growth for 54 h at 30.6 ◦ C.
Surely, the temperature is one of the most significant factors affecting microbial growth,
either planktonic or sessile. Not surprisingly, this is an environmental parameter widely
investigated in biofilm research, including L. monocytogenes; the results obtained, however,
Biomolecules 2021, 11, 397 15 of 20

are not always consistent. Thus, there are studies showing the increase of biofilm formation
by that pathogen upon increasing the temperature toward that optimum for planktonic
growth (i.e., 30–37 ◦ C) [9,55,56], something that was also observed in this study, whereas
other studies demonstrated an increase in sessile growth upon decreasing temperatures to
sub-optimal ranges for planktonic growth, such as 15–20 ◦ C [13] or even lower [57]. These
differences are probably explained by the different strains employed in the various studies,
combined with the rather complex nature of biofilm formation, even for mono-species
consortia. Thus, the biofilm-forming ability of a given strain may be influenced by several
other parameters, such as the morphological and physicochemical characteristics of the
attachment substrate, available nutrients, shear stress, pH, and osmolarity, with interactive
effects more often being observed between some of those parameters [58]. It is hence not
strange for a given microbial strain to alter its biofilm-forming capacity in response to
changing environmental conditions [59]. With all this in mind, a cocktail of four different
strains was here employed in the attachment and the subsequent disinfection experiments.
The MICs that were here determined for the two tested chemicals (i.e., THY, BAC)
were close to the ones having been previously reported against L. monocytogenes. Thus,
the MIC of THY for that bacterial species is usually ranging between 78.1 ppm and 1024
ppm [22,23,60], while that of BAC is generally lower than 10 ppm, except for some BAC-
tolerant isolates for which this value may be even higher than 20 ppm [61–63]. Although no
critical breakpoints for disinfectant resistance have been defined, unlike for antibiotics, the
four isolates here tested do not seem to present resistance to either of those two chemicals,
based on the data available screening collections of more strains. The MBCs previously
recorded for both chemicals are usually slightly higher compared to the respective MICs,
considering that both present a strong bactericidal activity, something that was also here
verified. Thus, the MBCs here recorded were equal to 156.3 ppm and 3 ppm, for THY
and BAC, respectively. Following the calculation of the MBCs against the planktonic cells
of each strain, each chemical was tested at three different concentrations, all higher than
the MBCs, against the mixed sessile community on the SS surfaces. The results revealed
the significant anti-biofilm potential of THY, considering that its application at just four
times its MBC (i.e., 625 ppm) was sufficient to kill almost all sessile bacteria (> 4 logs
reduction). On the other hand, the application of BAC at even 23.3 times more than its
MBC (i.e., 70 ppm) reduced the sessile population by 3.28 logs. However, it should be
noted that although sessile bacteria were here found to be quite more resistant than the
planktonic ones (against both chemicals and especially against BAC), it is still alleviative
that the application of BAC at the concentration this is usually applied in food industries
(i.e., 200 ppm) resulted in the complete killing of the sessile population (data not shown).
This is still not always the case for many other pathogenic bacteria being enclosed in
biofilm structures. For instance, in a previous related study, the application of BAC at 200
ppm caused only a 1.5 log reduction of an Staphylococcus aureus biofilm population (> 107
CFU/cm2 ) on polystyrene surfaces [26]. Another aspect, however, that should be always
also considered is the potential for bacterial cells surviving disinfection to enter the viable
but not-culturable (VBNC) state, being thus unable to be enumerated by traditional plating
methods, such as those here applied. As it seems, this is not a so rare phenomenon and has
also been described for L. monocytogenes following the action of QAC disinfectants [64,65]
and some EOs [66]. If this is indeed the case, the log reductions here recorded might be
even lower.
THY and other components of EOs are known to kill the microbial cells mainly due
to their interaction with cellular membranes causing their collapse [19]. Considering the
significantly better anti-biofilm potential of THY over the classical surface disinfectant that
was here observed, this may be due to the better ability of the terpenoid to diffuse through
the biofilm matrix, and thus, kill the cells. Noteworthily, in a previous related study, the
hydrosol of the Mediterranean spice Thymbra capitata, consisting a plant mixture that also
contains THY, was visualized in real-time, through confocal microscopy, to easily penetrate
the biofilm structure of S. Typhimurium, killing much more quickly the enclosed bacteria,
Biomolecules 2021, 11, 397 16 of 20

compared to BAC [25]. In another recent study, evaluating and comparing the disinfection
efficiency of THY and BAC against preformed biofilms of either S. aureus or Staphylococcus
epidermidis, it was again shown that the phytochemical presented a significant lower
resistance coefficient (Rc) than the synthetic biocide, meaning that the required increase
in its concentration to be equally effective against biofilm cells as this was against the
planktonic ones was much lower compared to the synthetic biocide [26].
The involvement of each strain in the formation of the mixed sessile community and
its antimicrobial recalcitrance was also here monitored by recovering many colonies (both
before and after disinfection) and discriminating/typing them to the strain level through
rep-PCR. This is a rather classical and broad range genomic fingerprinting technique that
has been widely applied to genotype bacteria through PCR amplification of various lengths
fragments of their genomic DNA, using primers that are complementary to repetitive
sequences occurring in prokaryotic genomes and resulting in strain-specific amplicon pat-
terns [67]. Results revealed heterogeneity in the behavior of each strain, with the recorded
distribution percentages found to vary depending on the mode of growth (planktonic
vs biofilm) and the tested disinfectant. This strain variability in biofilm formation and
disinfection resistance is not something peculiar, and it has to do with the inherent differ-
ences in molecular and physiological aspects of microbial behavior between the different
strains [68]. In addition, this has already been previously observed in multi-strain biofilms
of L. monocytogenes and other species submitted or not to disinfection [69–71]. Considering
that the planktonic cells of all here tested strains presented equal sensitivity to the two
antimicrobials (based on the MIC and MBC results) and in parallel displayed similar plank-
tonic growth rates in the dLB (data not shown), the differences observed in the strains’
prevalence at the different examined treatments should be associated with other param-
eters affecting sessile growth and resistance (e.g., production of extracellular polymeric
substances [EPS], motility, coaggregation), such as those determining the final placement
of each strain within the developed sessile structure, and thus its antimicrobial exposure,
if this is not a coincidence. It should be finally stated that although someone could have
tested the biofilm-forming ability and resistance of each strain one by one, the rep-PCR
approach that was here followed allowed the evaluation of any strain variability with
respect to those attributes somehow in situ. This is because any observation and conclusion
made on single-strain cultures could not be safely extended to a mixed-strain cocktail, since
in that latter case, each strain can interact with each other possibly affecting its sessile and
resistance behavior. Indeed, a previous relevant study has shown quite complex patterns
of bacterial interactions within mixed-culture L. monocytogenes biofilms [70].

5. Conclusions
A well-known natural terpenoid of plant origin (i.e., THY) was found to present
strong killing efficiency against a sessile population of a multi-strain L. monocytogenes
cocktail found on SS surfaces. Thus, its application at 625 ppm (= 4 × MBC) resulted
in the almost elimination of the attached bacteria (> 4 logs reduction). On the other
hand, the application of a widely used synthetic biocide (i.e., BAC) at a concentration that
was more than 20 times higher than its MBC (i.e., 70 ppm) caused a significantly lower
reduction (3.28 logs). The attached population was quite more resistant to the action of both
disinfectants compared to the planktonic cells. That population has been initially optimized
for the maximum cellular density (5.46 ± 0.31 log10 CFU/cm2 ), leaving bacteria to attach
to surfaces under the most favorable time and temperature incubation conditions (i.e., for
54 h at 30.6 ◦ C). Those conditions were determined by applying response surface modeling,
delivering a mathematical model capable of predicting the interactive influence of both
factors (i.e., time and temperature) on the accumulation of the pathogenic bacteria on the SS
surfaces, under other environmental conditions mimicking those encountered in the salad
industry. Strain variability in biofilm formation and resistance was also recorded. This
study, hopefully together with some future studies that will also consider and incorporate
the potentially rich natural microbiota found in these food environments, offer knowledge
Biomolecules 2021, 11, 397 17 of 20

on the sessile behavior of this important foodborne pathogenic bacterium, highlighting

an alternative sustainable way for its elimination. Phytochemicals such as THY deserve
to be further studied to improve the safety of the fresh produce limiting possibilities of
infections following their consumption.

Author Contributions: Conceptualization, E.G.; methodology, D.K., P.T., I.K., and E.G.; validation,
D.K., P.T., I.I., N.G., and G.S.; formal analysis, E.G.; investigation, D.K., P.T., I.I., N.G., G.S., and I.K.;
resources, E.G.; data curation, E.G.; writing—original draft preparation, E.G.; writing—review and
editing, E.G.; supervision and project administration, E.G. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: We are grateful to Andritsos (Athens Analysis Laboratories S.A.; Metamorfosi,
Attica, Greece) for isolating and providing the four L. monocytogenes strains used in this study and his
will for some useful preliminary discussions.
Conflicts of Interest: The authors declare no conflict of interest.

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