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International Journal of Food Microbiology 360 (2021) 109441
Available online 17 October 2021
0168-1605/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Operational culture conditions determinate benzalkonium chloride
resistance in L. monocytogenes-E. coli dual species biolms
Aleksandra Maria Kocot
a
,
b
,
*
, Barbara Wr´
oblewska
a
, Marta Lopez Cabo
c
a
Department of Immunology and Food Microbiology, Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Tuwima 10 Str., 10-748
Olsztyn, Poland
b
Department of Industrial and Food Microbiology, Faculty of Food Science, University of Warmia and Mazury in Olsztyn, Plac Cieszy´
nski 1, 10-726 Olsztyn, Poland
c
Department of Microbiology and Technology of Marine Products (MICROTEC), Instituto de Investigaciones Marinas (IIM-CSIC), Eduardo Cabello 6, 36208 Vigo, Spain
ARTICLE INFO
Keywords:
L. monocytogenes
E. coli
Mixed-biolm
BAC
Foodborne pathogens
Food safety
ABSTRACT
Biolms pose a serious challenge to the food industry. Higher resistance of biolms to any external stimuli is a
major hindrance for their eradication. In this study, we compared the growth dynamics and benzalkonium
chloride (BAC) resistance of dual species Listeria monocytogenes-Escherichia coli 48 h biolms formed on stainless
steel (SS) coupons surfaces under batch and fed-batch cultures. Differences between both operational culture
conditions were evaluated in terms of total viable adhered cells (TVAC) in the coupons during 48 h of the mixed-
culture and of reduction of viable adhered cells (RVAC) obtained after BAC-treatment of a 48 h biolm of L.
monocytogenes-E. coli formed under both culture conditions. Additionally, epiuorescence microscopy (EFM) and
confocal scanning microscopy (CLSM) permitted to visualize the 2D and 3D biolms structure, respectively.
Observed results showed an increase in the TVAC of both strains during biolm development, being the number
of E. coli adhered cells higher than L. monocytogenes in both experimental systems (p <0.05). Additionally, the
number of both strains were higher approximately 2.0 log CFU/coupon in batch conditions compared to fed-batch
system (p <0.05). On the contrary, signicantly higher resistance to BAC was observed in biolms formed under
fed-batch conditions. Furthermore, in batch system both strains had a similar reduction level of approximately 2.0
log CFU/coupon, while signicantly higher resistance of E. coli compared to L. monocytogenes (reduction level of
0.69 and 1.72 log CFU/coupon, respectively) (p <0.05) was observed in fed-batch system. Microscopic image
visualization corroborated these results and showed higher complexity of 2D and 3D structures in dual species
biolms formed in batch cultures. Overall, we can conclude that the complexity of the biolm structure does not
always imply higher resistance to external stimuli, and highlights the need to mimic industrial operational
conditions in the experimental systems in order to better assess the risk associated to the presence of pathogenic
bacterial biolms.
1. Introduction
Biolms are a serious challenge for the food industry. Their
appearance in food-processing environment creates a risk of food
contamination, thus causing a threat to the entire food chain (Srey et al.,
2013). The most undesirable are biolms formed by bacterial pathogens
in food industry surfaces, which can reach humans though consumption
of contaminated food after cross-contamination (Bridier et al., 2015;
Sim˜
oes and Sim˜
oes, 2010). Food contaminated with pathogens such as
Listeria monocytogenes can potentially be fatal to YOPI (young, old,
pregnant, immunocompromised) consumers (Jeyaletchumi et al.,
2010).
The elimination of bacterial pathogens from the food-processing
environment is particularly difcult due to their ability to form bio-
lms. Many previous studies showed that biolms are more resistant to
different antimicrobial substances, including disinfectants used to
cleaning and disinfection in food industry (Amalaradjou et al., 2009;
Chaitiemwong et al., 2010; Cruz and Fletcher, 2011; Rodriguez-Lopez
and L´
opez-Cabo, 2017). Food-processing environments have a favorable
conditions for biolms development, because there are nutrients and
various surface locations such as gouges, scratches, pits, cracks that fa-
cilitates cells adhesion and growth (I˜
niguez-Moreno et al., 2019).
* Corresponding author at: Department of Immunology and Food Microbiology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences
in Olsztyn, Tuwima 10, 10-748 Olsztyn, Poland.
E-mail address: a.kocot@pan.olsztyn.pl (A.M. Kocot).
Contents lists available at ScienceDirect
International Journal of Food Microbiology
journal homepage: www.elsevier.com/locate/ijfoodmicro
https://doi.org/10.1016/j.ijfoodmicro.2021.109441
Received 16 August 2021; Received in revised form 8 October 2021; Accepted 13 October 2021
International Journal of Food Microbiology 360 (2021) 109441
2
In the context of food industry, L. monocytogenes is one of the mi-
croorganisms of food safety concern, causing listeriosis with high mor-
tality rate (EFSA, 2015; Rodriguez-Lopez et al., 2015). Indeed, L.
monocytgenes is considered a weak biolm former, but in co-existence
with other species, the ability to biolm formation increased (Mos-
quera-Fern´
andez et al., 2014). Interspecies interactions occurring be-
tween cohabiting strains in the same biolm are not the only factor
affecting the number of cells and their resistance, the conditions in
which the biolm is formed are also important, such as access to nu-
trients (Van der Veen and Abee, 2011). Another problem with the
elimination of L. monocytogenes from food-processing plants is that it can
persist for months or even years on equipment items, in drains or on the
oor (Ferreira et al., 2014). In addition, L. monocytogenes is character-
ized by the ability to survive in an environment of food processing, i.e.
refrigerated temperatures, heat, desiccation or the presence of a high
amount of salt, whereas for other microorganisms these conditions are
limiting factors (Gardan et al., 2003; Zoz et al., 2017).
The study of biolms is even more complex if we take into account
that biolms can be found in very different locations along the food-
processing plants (Rodriguez-Lopez and L´
opez-Cabo, 2017). Biolms
are formed in spaces of different geometry, different external conditions
and exposed or not to different hydrodynamic conditions caused by
continuous and discontinuous ow of liquids such as water, milk or
juices, depending on the food sector. However, the number of articles
comparing biolms formed under different modalities of culture that
could mimic different industrial conditions is limited (Cunault et al.,
2015; Le Gentil et al., 2010). Most of the available research focuses on
comparing mono- and dual- or multi-species biolms formed in different
surfaces present in food processing plants under batch conditions (Kocot
and Olszewska, 2019; Rodriguez-Lopez and L´
opez-Cabo, 2017; Van der
Veen and Abee, 2011). The importance of different operational condi-
tions and the inclusion of these parameters in the research results e.g.
from the fact that L. monocytognes-carrying contamination sites were
found in very different locations of a food-processing plant (Rodríguez-
L´
opez et al., 2020).
Therefore, in this study, we compared growth dynamics, BAC resis-
tance and microscopic 2D and 3D structures of L. monocytogenes-E. coli
biolms formed on SS coupons under batch and fed-batch culture
conditions.
2. Materials and methods
2.1. Bacterial strains
L. monocytogenes A1 and E. coli A14 used in this study were previ-
ously isolated from a sh industry processing plant and genetically
characterized (Rodriguez-Lopez and L´
opez-Cabo, 2017). Stock cultures
were maintained at −80 ◦C in brain heart infusion broth (BHI; Biolife,
Milan, Italy) with 50% glycerol (1:1, v/v). Working cultures were pre-
pared similarly but maintained at −20 ◦C until use.
2.2. Inocula preparation
L. monocytogenes A1 and E. coli A14 stock working cultures were
activated by twice cultured (100
μ
L each time) in 5 mL of TSB (Tryptic
Soy Broth, Cultimed, S.L., Spain) at 37 ◦C. In both species, inocula was
prepared by adjusted activated cultures (Abs
700
) to 0.1 ±0.001 with
using sterile PBS and spectrophotometer (Cecil Instruments, Cambridge,
England) to obtain a bacterial concentration of 8 log CFU/mL accord-
ingly with previous calibrations. Adjusted cultures were further diluted
in TSB to obtain a nal concentration of each species of 4log CFU/mL
and used as inoculum for dual-species biolm formation on SS coupons
surface under batch and fed-batch conditions.
2.3. Dual-species biolms formation
SS coupons (Ø 12.7 mm) (Comevisa, Vigo, Spain) were washed with
a detergent solution for 10 min and next with 70% ethanol (v/v) for 1
min in ultrasonic cleaner (BRANSOR 250, Thermo Fisher, USA), rinsed
with deionized water, air-dried and autoclaved at 121 ◦C for 20 min.
Cleaned coupons were disposed in both experimental systems as follows:
i) Batch system: sterilized coupons were individually placed in 24-well
polystyrene at-bottomed plates (Falcon, Corning, NY, USA) and
inoculated with 1 mL of adjusted co-culture of L. monocytogenes and
E. coli and incubated at 25 ◦C for 48 h.
ii) Fed-batch system; CDC bioreactor (Fig. 1): sterilized coupons were
placed in coupons holder available in the CDC biolm reactor.
Initially, 3 mL of inocula were added to 300 mL of TSB and culture
was maintained under static conditions. After 3 h the CDC culture
was performed under stirring (60 rpm) until the end of the experi-
ment. Fed batch cultivation was performed by continuously uxing of
3 mL of TSB to maintain glucose concentration accordingly with
preliminary experiments (data not shown). The CDC culture was
incubated at 25 ◦C for 48 h.
Coupons were removed from both experimental systems for TVAC
and RTVC determinations. For the experiments carried out to determine
the kinetic of growth of both species (Section 2.4.), coupons were
removed after 8, 12,17, 24, 36 and 48 h of culturing. BAC-resistance
experiments (Section 2.5) were carried out with coupons removed
after 48 h culturing.
2.4. Determination of total viable adhered cells (TVAC) in the SS coupons
First, each coupon was gently rinsed with PBS to eliminate non-
adhered cells and then placed in Falcon tube with 1 mL of buffered
peptone water (BPW; Cultimed, Barcelona, Spain). Tubes were vortexed
for 1 min and then sonicated for another 1 min (38 kHz, 30 ◦C)
(BRANSOR 250, Thermo Fisher, USA). Finally, samples were serially
diluted in BPW and in duplicate spread-plated on ALOA (Cultimed,
Barcelona, Spain) and HiCrome™ Coliform agar (Sigma-Aldrich, St
Louis, MO, USA) to quantify L. monocytogenes and E. coli, respectively.
Plates were incubated at 37 ◦C for 48 h. Automatic counting was per-
formed by using Flash & Go Automatic Colony Counter (SCAN 500,
Interscience). TVAC were expressed in log CFU/coupon (cm
2
). Experi-
ments were done in triplicate.
2.5. Resistance to BAC of L. monocytogenes-E. coli biolms formed under
batch and fed-batch conditions
2.5.1. BAC and neutralizing solution preparation
Benzalkonium chloride (BAC, Guinama, Alboraya, Spain) was pre-
pared at 200 ppm by dissolving the stock solution in sterile distilled
water and kept at 4 ◦C until use. Neutralizing solution was prepared with
following composition, per liter: 10 ml of a 34 g l
−1
KH
2
PO
4
solution
adjusted to pH =7.2 with NaOH (
aq
), 3 g soy lecithin, 5 g Na
2
S
2
O
3
, 1 g L-
histidine, 30 mL Tween 80 and deionized water (Rodriguez-Lopez et al.,
2017). This solution was sterilized by autoclaving at 121 ◦C for 20 min
and kept at 4 ◦C until use.
2.5.2. BAC treatment
The effect of 200 ppm of BAC against 48 h old L. monocytogenes-E. coli
biolms formed under both experimental conditions was assayed. SS
coupons removed from both culture conditions were placed separately
in another 24-bottom well microtiter plates. Next, 1 mL of 200 ppm BAC
solution was added to each well and allowed to remain for 10 min at
room temperature. Treated coupons were transferred to new wells
containing 1 mL of neutralizing solution and immersed for 30 s, which
was considered the minimum time necessary for proper neutralization
A.M. Kocot et al.
International Journal of Food Microbiology 360 (2021) 109441
3
according to a previous assay. Untreated biolm samples were used as
controls. Finally, TVAC in controls and treated samples was calculated as
described above and RVAC was calculated as the difference between
TVAC present in mixed biolms formed in SS coupons in controls and
after BAC treatment. RVAC is expressed in log CFU/coupon.
2.6. Microscopic assays
The 48 h L. monocytogenes-E. coli biolms from both experimental
systems before and after BAC-inactivation were rinsed with 1 mL of
0,9% NaCl and stained for examination under epiuorescence (EFM)
and confocal scanning microscopy (CLSM).
For EFM, biolms were stained with ViaGram ™ Red
+
Bacterial
Gram Stain and Viability Kit (Life Technologies, Eugene, OR) according
to the manufacturer's instructions. This stain allows in a mixed popu-
lation the distinction of Gram-positive cells (red emitting) with a
counterstain with DAPI. After incubation, the staining mixture was
removed and coupons were mounted on microscopic slides with using
one drop of mounting medium (FluoroShield; Life Technologies,
Eugene, OR). Microscopic visualization was conducted with a Leica DM
6000 epiuorescence microscope (Leica, Wetzlar, Germany) using a
40×objective and 10×ocular lens Metamorph MMAF software (Mo-
lecular Devices, Sunnyvale, CA, USA).
For CLSM, biolms were stained with LIVE/DEAD® viability kit (Life
Technologies, Eugene, OR, USA) following manufacturer instructions in
order to observe the nal biolm morphology and the distribution of
live (green-emitting) cells and damaged/dead (red-emitting) cells. Next,
stained biolms were xed with 1 mL of 4.0% formaldehyde (Sigma-
Aldrich, Lisbon, Portugal) and incubated for 30 min at room tempera-
ture. Then, the samples were washed with PBS and then treated with 1
mL of 50 mM ammonium chloride and incubated for 3 min at room
temperature. Fixed biolms were placed on microscopic slides covered
with coverslips using Baclight immersion oil (ThermoFisher Scientic,
Massachusetts, USA). Confocal images were acquired using an upright
laser scanning microscope SPE with a Plan-apochromatic 63×/NA 1.4
objective (Leica Microsystems, Wetzlar, Germany). Green uorescence
from Syto 9 was acquired exciting samples with 488 nm laser and
emission signal was detected between 490 and 550 nm. Red uores-
cence from PI was acquired exciting samples with 561 nm laser and
emission signal was detected between 590 and 650 nm. All images
shown correspond to a maximal intensity projection of a confocal z-
stack. Images were processed with Adobe Photoshop CS.
2.7. Statistical analysis
Statistical analysis was conducted with the use Statistica software
ver. 13.1 (StatSoft Inc., Tulsa, OK). Inactivated samples were tested by
one-way ANOVA test. Differences were considered signicant at p <
0.05 level of probability.
3. Results
3.1. Kinetics of growth of L. monocytogenes-E. coli under batch and fed-
batch culture
Dynamics of biolm formation on SS coupons in batch and fed-batch
conditions are shown in Fig. 2. The number of cells of both strains
increased in the following hours of biolm growth, regardless of the
experimental conditions and after 48 h of biolm development reached
the values of 6.98 and 8.03 log CFU/coupon and 4.98 and 6.35 log CFU/
Fig. 1. The scheme showing the CDC bioreactor presenting the setup of the fed-batch experiment.
A.M. Kocot et al.
International Journal of Food Microbiology 360 (2021) 109441
4
coupon for L. monocytogenes and E. coli under batch and fed-batch con-
ditions, respectively. It was shown that the number of cells of both
strains had higher numbers under batch conditions as compared to the
fed-batch system – these differences were about 2.0 log CFU/coupon and
they were statistically signicant (p <0.05). Moreover, it was observed
that in both experimental systems the number of E. coli cells was higher
than of L. monocytogenes. Initially, these differences were small, but from
17 h of biolm formation, the number of cells of both strains differed
signicantly (p <0.05).
3.2. Resistance to BAC of dual-species biolm formed under batch and
fed-batch conditions
The efcacy of BAC was evaluated in terms of reduction of viable
adhered cells (RVAC). Reduction of viable adhered cells of L. mono-
cytogenes and E. coli in dual-species biolms formed under batch and fed-
batch conditions with treatment of BAC-based disinfectant are showed in
Fig. 3. The reduction levels were 2.18 and 2.20 log units per coupon and
1.72 and 0.69 log units per coupon for L. monocytogenes and E. coli under
batch and fed-batch conditions, respectively. Both L. monocytogenes and
E. coli achieved similar reduction levels when biolm was formed in a
batch system, while E. coli was denitely more resistant than L. mono-
cytogenes in a fed-batch system (p <0.05).
3.3. Microscopic images evaluation
The efcacy of BAC against dual-species biolm of L. monocytogenes-
E. coli was also evaluated using microscopic assays.
3.3.1. Epiuorescence microscopy – EFM
The ViaGram™ protocol allows for cell differentiation based on the
integrity of the membranes in bacterial cells and based on Gram-signs.
Live cells (interact membranes) stain blue (DAPI), while dead cells
(damaged membranes) stain green (SYTOX) and Gram-positive cells
stain red due to selective binding. The EFM images showed a denser
biolm structure and architecture under batch conditions when
comparing with the fed-batch system (Fig. 4A and C). Under batch sys-
tem, cells were organized into clusters, forming interconnected colonies,
a signicant part of the surface was covered with biolm (Fig. 4A), while
under fed-batch conditions, the cells were more dispersed, it was possible
to observe free space as well as single cells whereas colonies and clusters
are not dominant in the microscopic image (Fig. 4C). After BAC treat-
ment, a clear damage in the structure of the biolm formed under batch
conditions can be appreciated in the images (Fig. 4B). Indeed, cell ag-
gregates and single cells are more separated and so a less denser struc-
ture can be appreciated. Contrary, the structure of biolm after BAC in
fed-batch system changed relative to the control, but these changes were
not as noticeable as in batch system. The main difference between two
experimental set ups is the number of cells, not their location, which
indicates the stages of biolm development. In the case of the control
and after BAC treatment in fed-batch system, we observe single cells,
without aggregates and microcolonies. Looking at the proportion of cells
(separately in control and after BAC treatment), after BAC, the blue-
stained cells are superior to red cells (Fig. 4D).
3.3.2. Confocal laser scanning microscopy – CLSM
EFM study was complemented by the visualization of L. mono-
cytogenes-E. coli stained with LIVE/DEAD under CLSM (Fig. 5). A general
look at microscopic images of biolms formed in a batch and fed-batch
experimental system obtained with the use of LIVE/DEAD staining
allowed to observe a dense, complex structure covering almost the entire
eld of view during the development of biolm in a batch system, while
in a fed-batch system a greater dispersion of cells was observed (Fig. 5A
and C). Cells in the control biolms showed differences depending on
the conditions of biolm development – in the batch system the cells had
intact cytoplasmic membranes as evidenced by the green color of the
cells (Fig. 5A), while in the fed-batch system the cells were characterized
by damaged cytoplasmic membranes and absorption of both dyes, which
made them greenish-yellowish color (Fig. 5C). This observation testies
the viability of bacterial cells. BAC inactivation affected the structure of
the biolm formed under batch conditions (Fig. 5B). Additionally, a
subpopulation of dead cells (red), with damaged cytoplasmic mem-
branes was distinguished. Moreover, the remaining cells were stained
yellow, thus, despite the physiological activity of the cells, their cyto-
plasmic membranes were disturbed. Inactivation of the biolm formed
Fig. 2. The cell counts of L. monocytogenes A1 ( ) and E. coli A14 ( ) in dual-
species biolms formed in batch (A) and fed-batch (B) system for 48 h. The bars
represent the mean values ±standard deviations. Asterisks indicate signicant
differences between counts of L. monocytogenes A1 and E. coli A14 (p <0.05).
Fig. 3. The effect of BAC-based disinfectant on inactivation of 48 h old dual-
species biolm of L. monocytogenes A1 ( ) and E. coli A14 ( ) formed under
batch and fed-batch conditions after 10 min of BAC treatment. The bars repre-
sent the mean values ±standard deviations. Asterisks indicate signicant dif-
ferences between log reduction levels of L. monocytogenes A1 and E. coli A14 (p
<0.05).
A.M. Kocot et al.
International Journal of Food Microbiology 360 (2021) 109441
5
Fig. 4. Representative EFM images stained
with ViaGram™ of L. monocytogenes A1 and
E. coli A14 in 48 h old dual-species biolms
formed in batch (A, B) and fed-batch (C, D)
system. Images represent cells in control
biolm (A, C) and after 10 min of BAC-
based disinfectant treatment (B, D). Red
cells represent live cells of L. monocytogenes
A1 and blue cells represent live cells of E.
coli A14 as described in Materials and
methods according the procedure Via-
Gram™. (For interpretation of the refer-
ences to color in this gure legend, the
reader is referred to the web version of this
article.)
Fig. 5. Representative CLSM images stained
with LIVE/DEAD viability kit of L. mono-
cytogenes A1 and E. coli A14 in 48 h old dual-
species biolms formed in batch (A, B) and
fed-batch (C, D) system. Images represent
cells in control biolm (A, C) and after 10
min of BAC-based disinfectant treatment (B,
D). Images represent live (green) and dead
(red) cells as described in Materials and
methods. (For interpretation of the refer-
ences to color in this gure legend, the
reader is referred to the web version of this
article.)
A.M. Kocot et al.
International Journal of Food Microbiology 360 (2021) 109441
6
in the fed-batch system did not show the effect of BAC on the structure of
the biolm compared to the control (Fig. 5D). In turn, a slight decrease
in the proportion of living cells (green), with intact cytoplasmic mem-
branes, was observed. Remaining adhered cells were greenish-yellowish,
without a distinct subpopulation of cells with damaged membranes
(red). This may indicate a higher resistance to BAC-based disinfectant of
cells formed in a fed-batch system compared to a biolm formed in a
batch system.
4. Discussion
In the present study, the operational culture conditions in the context
of BAC-resistance of L. monocytogenes-E. coli dual-species biolm, was
determined. The comparison of growth dynamic in two operational
cultures demonstrated a higher number of cells in the biolm formed in
the batch system when comparing with the fed-batch system. In addition,
it was shown that the number of E. coli was higher than L. monocytogenes
regardless of the experimental set-up. These results are in agreement
with previous studies carried out in our laboratory, in which higher E.
coli adherence viable cell counts in mixed biolms formed in stainless
steel compared to L. monocytogenes were observed (Rodriguez-Lopez
et al., 2017). Indeed, microscopic analyzes (EFM as well as CLSM)
showed a higher cell density and a more developed biolm structure
under batch conditions.
Similar results regarding the adhesion of bacterial cells under static
and dynamic conditions were obtained by Song et al. (2017), who
cultured biolm of periodontal pathogens on hydroxyapatite discs in
test plates and in a CDC bioreactor. The amount of adhered bacterial
cells, determined by scanning electron microscopy (SEM) and confocal
laser scanning microscopy (CLSM) was higher in biolms formed in a
static system. The authors stated that this effect is because under static
conditions, cell adhesion was due to the force of gravity, while in a
dynamic system, cell adhesion is somehow hindered by shaking and for
the vertical placing of the coupons. Interestingly, the authors showed no
signicant differences in thickness or resistance against chlorhexidine
when comparing biolms formed in a static and dynamic system. The
limiting effect of hydrodynamic conditions on biolm formation was
shown by Fonseca and Sousa (2007). In their study, it was shown that
the shear force (shaking) reduced the adhesion and biolm formation
capacity of P. aeruginosa compared to static conditions. In turn, Cotter
et al. (2010) demonstrated a limiting effect of rotational speed on bio-
lm formation of S. epidermidis.
The above-mentioned studies indicate that the dynamic system
achieved by mixing or shaking is a stressful condition for cells. This has
consequences in hampering cell adhesion and slow biolm formation,
thus giving rise to a simpler 2D and 3D biolm structure. But the most
important results is that mixed biolm formed under fed-batch condi-
tions was more resistant to BAC, even although the number of cells in
this biolm was smaller and it had a less dense structure than biolm
formed in a batch system. Additionally, it was also observed that L.
monocytogenes was more sensitive to BAC than E. coli under dynamic
conditions, which is in agreement with previous studies carried out to
characterize the effects of combined enzyme-BAC treatments against the
removal of this dual-species biolm (Rodriguez-Lopez and L´
opez-Cabo,
2017).
In general, in the literature it is accepted that complex-structured
biolms are more resistant (de Oliveira et al., 2010; Liu et al., 2017;
Sa´
a Ibusquiza et al., 2012; Sengupta et al., 2013). Several factors
contribute to this general assumption: i) Biolms acquire a complex 3D
structure, in which cells of the outer layers protect the cells located in
the inner layers to external stimuli and to the penetration of antimi-
crobial agents (Gebreyohannes et al., 2019), ii) Development of a
intercellular communication system between bacteria inside the biolm,
quorum sensing. This cell-to-cell communication regulates the behavior
of bacteria, including the regulation of gene expression responsible of
cells resistance (Singh et al., 2017; Tang and Zhang, 2014), iii)
Production of EPS (extracellular polymeric substances), which sur-
rounds the biolm and can also play a protective role (Uhlich et al.,
2006) and iv) the effect of the presence of bacterial dead cells in the
outer layers of the biolm, which are most exposed to stress factors and
protect the cells located in the inside of the biolm structure (de Oliveira
et al., 2010).
Contrary to this assumption, our results demonstrated no correlation
between the complexity of the biolm structure and its level of resis-
tance to BAC. We hypothesize that fed-batch bioreactor operation im-
plies limiting adhesion factors derived of the vertical orientation of the
SS-coupons in presence of continuous gentle mixing that could promote
an increase in individual cell resistance. In other words, at the time cells
overcome unfavorable adhesion conditions they acquire individual
resistance that could imply cross-resistance to BAC. This is in agreement
with previous studies in which higher resistant biolms were formed
under dynamic conditions (Cotter et al., 2010; Fonseca and Sousa, 2007;
Song et al., 2017). Moreover, fresh nutrient supply could have an impact
on the physiological state of a single cell, shaping its well-being
(“health”), which may contribute to the higher cell resistance to BAC in
biolms formed under fed-batch conditions. Furthermore, the supply of
fresh medium could result not only in better “health” of individual
bacterial cells in the biolm, but also on the metabolic activity of the
entire biolm community, including parameters determining better
resistance. The high metabolic activity of the biolm formed under fed-
batch conditions was stimulated by the inow of fresh medium, which in
turn increases the rate of extracellular polymeric substances (EPS)
synthesis, regulation of the individual genes expression, as well as the
synthesis of molecules participating in quorum-sensing (QS) (Flemming
et al., 2007; Nadell et al., 2015; Wingender et al., 2001). By the contrary,
the depletion of nutrients in biolms formed in batch system might have
a limiting effect on the metabolic activity of bacterial cells, and so they
stay sensitive to BAC despite the complex 3D structure. The next factor
which could determine the higher resistance of bacterial cells under fed-
batch conditions is vertical alignment of coupons in CDC bioreactor. In
order to produce biolm on such a surface, bacterial cells have to
overcome more factors than cells in batch system, where the force of
gravity facilitates the adhesion of bacterial cells to the surface of the
coupons. So, our hypothesis is that this “adhesion effort” of bacteria
results in their higher resistance.
Our results unplug somehow the assumed relation between biolm
structure and biolm resistance to external stimuli. Moreover, they lead
to reect on the meaning and implications of a classied “poor biolm
former bacteria” and highlight the importance of considering resistance
related parameters for a correct evaluation of the risk associated to
cross-contamination from biolms formed in food industry surface.
5. Conclusions
In summary, our results indicated that dynamic conditions, repre-
sented in this study by the fed-batch system, limited the adhesion of
bacterial cells, which affects the architecture and BAC-resistance of the
L. monocytogenes-E. coli biolm. Microscopic analysis of both EFM and
CLSM showed a denser structure, composed of interconnected clusters in
biolms formed in batch system, while scattered and even single cells
were observed in fed-batch system. However, biolms formed in the fed-
batch system were more resistant to BAC than biolms formed in batch
system. Our study indicates that a complex 3D structure does not always
mean the higher resistance of biolms. This observation sheds new light
on the problem of biolm in the food industry and indicates the need of
considering operational conditions of the culture like hydrodynamic
factors, shaking or the inow of fresh medium and even concentration of
the culture medium by e.g. mixing, which will better reect the situation
in food industry plants.
A.M. Kocot et al.
International Journal of Food Microbiology 360 (2021) 109441
7
Funding
This research did not receive any specic grant from funding
agencies in the public, commercial, or not-for-prot sectors.
CRediT authorship contribution statement
Aleksandra Maria Kocot (AMK) and Marta Lopez Cabo (MLC):
conceptualization and methodology; AMK: carried out the experiments,
performed data collection and analysis, interpreted and visualization the
data, writing the original draft; MLC: scientic direction and coordina-
tion of the study, data analysis and discussion; MLC, Barbara
Wr´
oblewska (BW): reviewing and editing. All authors approved the nal
version of the manuscript.
Declaration of competing interest
The authors declare no conict of interest.
Acknowledgements
The research was carried out during student internships under the
Erasmus+program during the implementation of doctoral studies at
University of Warmia and Mazury in Olsztyn. The authors would like to
thank L. Sanchez for her help throughout confocal analysis and S.
Rodríguez and T. Blanco for their help throughout all the study.
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