Food Control 70 (2016) 371e379

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Inactivation and induction of sublethal injury of Listeria

monocytogenes in biolm treated with various sanitizers
Magdalena A. Olszewska a, b, Tong Zhao a, Michael P. Doyle a, *
a
Center for Food Safety, College of Agriculture and Environmental Sciences, University of Georgia, 1109 Experiment Street, Grifn, GA 30223, USA
b
ski 1, 10-726 Olsztyn, Poland
Chair of Industrial and Food Microbiology, Faculty of Food Science, University of Warmia and Mazury in Olsztyn, Plac Cieszyn

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

Article history: This study determined the effects of different sanitizers (one phenolic-based, one chlorine-based, two
Received 3 May 2016 QACs-based and one levulinic acid and SDS-based) on Listeria monocytogenes biolm. The induction of
Received in revised form the sub-lethal injury state and the biolm formation characteristics as a result of exposure to sanitizers
14 June 2016
were also evaluated. The results revealed that QACs-based and phenolic-based sanitizers most effectively
Accepted 14 June 2016
Available online 16 June 2016
reduced L. monocytogenes, resulting in a reduction of 3.7e6.9 log CFU/ml and 4.9e8.2 log CFU/ml after a
60-min treatment for 37
C- and 15
C-grown biolms, respectively. An enhanced level of sanitizer
resistance was observed in biolms when they were multiply exposed to QACs-based and phenolic-
Keywords:
Listeria monocytogenes
based sanitizers, with a reduction of 0.7e3.5 log CFU/ml and 1.6e>9.3 log CFU/ml for 37
C- and 15
C-
Biolm grown biolms, respectively. As biolm cells became less sensitive, especially to QACs-based sanitizers,
Sublethal injury an increase in the percentage of sublethally injured cells was observed to the levels dependent upon
Sanitizer resistance sanitizer concentration. Confocal laser scanning microscopy (CLSM) analysis revealed that biolm cells
experienced cell membrane damage when exposed to QACs-based and phenolic-based sanitizers,
providing more protection to cells located inside the biolm matrix. This study highlights the ongoing
need for improvement in intervention methods to control L. monocytogenes in food processing plants.
2016 Elsevier Ltd. All rights reserved.

1. Introduction facility, L. monocytogenes can become a long-term resident, being

Bacterial biolms in food processing environments are of
recurrent concern to the food industry, mainly because of their
strong antimicrobial tolerance (Kim, Hahn, Franklin, Stewart, &
Yoon, 2009; Takenaka, Iwaku, & Hoshino, 2001). The limitation of
agent penetration, multiple phenotypes development and exis-
tence of dormant cells, as well as the different reactivities of anti-
microbial agents, are factors that inuence biolm tolerance to
sanitizers (Kim, Pitts, Stewart, Camper, & Yoon, 2008). Furthermore,
as biolm forms, cells can detach and initiate attachment to other
surfaces, providing potential transmission of spoilage- and disease-
causing microorganisms (Yang et al., 2016). Despite improvements
in plant layout, equipment design, and procedures for cleaning and
sanitizing, the phenomenon of biolms in the food industry is still
poorly understood and controlled (Liu et al., 2015).
The presence of Listeria monocytogenes in food processing es-
tablishments is an important consideration. After entering the
* Corresponding author.
E-mail address: mdoyle@uga.edu (M.P. Doyle).
able to persist for months or years in locations such as oor drains
(Berrang, Meinersmann, Frank, Smith, & Genzlinger, 2005;
Tompkin, 2002). The ability of L. monocytogenes to attach and
form a biolm e.g., on different processing plant surfaces, has been
previously documented (Alonso, Perry, Regeimbal, Regan, &
Higgins, 2014; Beresford, Andrew, & Shama, 2001; Gamble &
Muriana, 2007; Zhao et al., 2013), and persistent subtypes are
recognized as strongly adherent and especially capable biolm
producers (Berrang, Frank, & Meinersmann, 2010; Lunde
n,
Miettinen, Autio, & Korkeala, 2000). Moreover, L. monocytogenes
can attach to surfaces previously colonized by other bacteria and
form mixed-species biolms with, e.g., Pseudomonas (Hassan, Birt,
& Frank, 2004). Importantly, L. monocytogenes cells have the po-
tential to cross contaminate processing plant surfaces, even though
a bactericidal treatment was applied (Reij & Den Aantrekker, 2004;
Tompkin, 2002). The mechanisms by which cells survive under
these conditions are not fully understood (Harvey, Keenan, &
Gilmour, 2007). It is therefore believed that biolms of
L. monocytogenes play an important role in the survival of listeriae
in the food processing environment (Tompkin, 2002; Zhao et al.,

http://dx.doi.org/10.1016/j.foodcont.2016.06.015
0956-7135/ 2016 Elsevier Ltd. All rights reserved.
372 M.A. Olszewska et al. / Food Control 70 (2016) 371e379
2013).
Cleaning and sanitizing procedures are widely used to inactivate
and remove biolms in the food industry (Yang et al., 2016).
However, standardized methods for antimicrobial selection and for
the design of effective strategy for biolm control do not exist
(Simo~ es, Simo~ es, & Vieira, 2009). Thus, in order to design an
effective strategy, a better understanding of biolm behaviour in
response to various single and combined antimicrobials of different
categories is essential. It is necessary not only to apply appropriate
antimicrobials but also adjust the dosing, as bacterial cells may
exhibit different sensitivity to a certain bactericidal concentration,
depending on the mode of persistence (Kim et al., 2008; Simo ~es
et al., 2009). Accordingly, the development of adaptive response
by biolm cells as a result of bactericidal misuse should also be
addressed with regard to the performance and maintenance of a
strategy (Kim et al., 2008).
Because for the food industry, the control of L. monocytogenes in
the food chain and the plant environment is of major concern, we
examined L. monocytogenes LM101, serotype 4 isolated from food
(salami isolate). Moreover, most listeriosis outbreaks have been
caused by serotype 4b (Borucki & Call, 2003; Laksanalamai et al.,
2014), hence L. monocytogenes LM101 is of the serotype most
often associated with greater virulence; however further charac-
terization is needed. In this study, the primary objective was to
further elucidate the effects of different sanitizers on a
L. monocytogenes biolm. For this purpose, ve sanitizers were
selected and several parameters were tested, including tempera-
ture of biolm growth, concentration of the sanitizers, time of
exposure, and intervals of treatment application. Induction of the
sublethal injury state and the biolm formation characteristics as a
result of exposure to sanitizers were also evaluated.
2. Materials and methods
2.1. Bacterial strain and preparation of single-species culture
Listeria monocytogenes strain LM101 (serotype 4, salami isolate)
was used in this study. A culture was rst grown at 37
C in Tryptic
soy broth (TSB, Becton Dickinson, Sparks, MD) for 24 h, and then
subcultured into sterile TSB for another 5 h at 37
C in order to
reach mid-log phase. The single-species culture was prepared by
diluting a mid-log phase culture in sterile TSB to a nal concen-
tration of ca. 2 log units of CFU/ml. The cell number was conrmed
by plating on Tryptic soy agar (TSA, Becton Dickinson).
2.2. Biolm growth and sanitizer treatment
The diluted mid-log phase culture of L. monocytogenes LM101
was added into 24-well at-bottom polystyrene plates (Costar,
Corning, NY) at 1 ml per well. The plates were incubated statically
at 37
C for 72 h and at 15
C for additional 48 h to obtain ca. 8 log
units of CFU. To maintain bacterial viability, the TSB was changed
every 24 h by aspirating old medium from the walls of each well
and dispensing fresh medium along the walls. After incubation, the
wells were washed with 0.1 M phosphate buffer saline (PBS, Sigma,
St. Louis, MO), and air-dried for 10 min. Two concentrations, in-use
concentration (IUC) recommended by the manufacturer for use on
hard non-porous surfaces and 1:1 in-use concentration (1:1 IUC), of
commercially available surface cleaners and sanitizers: Vesphene
IIse (Steris, St. Louis, MO), FS Formula 12167 (Zep, Atlanta, GA),
Micronex (Zep, Atlanta, GA), Fit-L Antibacterial Produce Cleaner e
at the concentration typically used for washing produce, not sani-
tizing equipment (HealthPro Brands, Cincinnati, OH), and Zep-
amine A (Zep, Atlanta, GA) were used in this study. The compo-
sition and concentration of each sanitizer are listed in Table 1. All
sanitizers were prepared with Milli-Q water (Millipore, USA) ac-
cording to the manufacturers instruction. Sterile PBS was used as a
control. Sanitizers or PBS were removed by aspiration after 5, 15, 30
and 60-min treatment and then wells were lled with D/E
neutralizing broth (Becton Dickinson), held for 10 min, and washed
with sterile PBS. The content of each well was harvested by scraping
the surface carefully with a sterile polyester-tipped swab (15.2 cm;
Fisher Scientic). The swabs were placed into 9-mL PBS tubes, and
vigorously (250 rpm) agitated by a Vortex mixer (G-560, Scientic
Industries, Bohenia, NY) for 30 s. Cell suspension (100 mL) or
appropriate dilutions in 0.01 M PBS were spread plated in duplicate
onto TSA plates. The colonies on the plates were counted after 24 h
of incubation at 37
C. The level of inactivation was expressed as the
log10 reduction in the cell survival ratio for the sanitizer treatments
(log N/N0). N refers to the bacterial counts after sanitizer treat-
ments, whereas N0 refers to the bacterial counts following PBS in-
cubation (control biolms). Sublethal injury was determined by
simultaneously spread plating the treated cells on TSA supple-
mented with sodium chloride (NaCl; Fisher Scientic, Pittsburgh,
PA) plates. In a preliminary study, several concentrations of NaCl
(1e6% w/v) were tested to determine the maximum concentration
that did not affect the growth of healthy, untreated cells (Ghate
et al., 2013) (data not shown). Based on the results obtained, 3%
NaCl was selected for use in the sublethal injury test. The following
formula was used to calculate the percentage of sublethal injured
cells:
 
Colonies on TSA NaCl
Sublethal injury % 1 x 100
Colonies on TSA
The second treatment using the same exposure times was
conducted after a 24-h biolm recovery in TSB at 37
C or 15
C for
all sanitizers. Biolm cell inactivation and cell injury after the 2nd
treatment were determined by enumeration and the sublethal
injury test as described above. The most effective sanitizer treat-
ments were repeated at one-week intervals during a three-week
biolm incubation at 37
C or 15
C by exposing biolms for
60 min to sanitizers, and then the same enumeration procedure
and sublethal injury test were performed.
2.3. Confocal laser scanning microscopy (CLSM)
For CLSM, the inoculum of L. monocytogenes LM101 was pre-
pared as described in Section 2.1 and added to 8-well chamber
slides (Nunc Lab-Tek, Fisher Scientic) at 400 mL per well. The
plates were incubated at 37
C for 72 h or at 15
C for an additional
48 h before sanitizer treatments. The medium was changed every
24 h to maintain bacterial viability. Prior the treatment step, the
wells were washed with 0.1 M PBS and air-dried for 10 min, and
after treatments were lled with N/E neutralizing broth to
neutralize the residual sanitizer solutions. To visualize cells, uo-
rescent dyes were used by applying the LIVE/DEAD BacLight
viability kit (Molecular Probes, LifeTechnologies, Eugene, OR). The
staining mixture was prepared by adding 3 mL of component A and
3 mL of component B per 1 mL of sterile saline. Freshly prepared
staining mixture (400 mL) was added to each well and incubated at
21
C for 15 min in the dark. After incubation, the wells were
washed with PBS as described above. The wells were further lled
with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (Sigma) and
incubated for 30 min at room temperature to x the specimen. The
slides were removed from the chambers and then covered with
coverslips with the use of BacLight Mounting Oil (Molecular
Probes, LifeTechnologies, Eugene, OR). The samples were analyzed
with a Zeiss LSM 700 CLSM (Carl Zeiss Microscopy, Thornwood, NY)
using Plan-Apochromat 63  oil-immersion, numerical aperture 1.4
 M.A. Olszewska et al. / Food Control 70 (2016) 371e379 373

Table 1
Composition of tested sanitizers.

Cleaner/Sanitizer Composition In-use concentration/dilution

Vesphene IIse 7.66% amylphenol 1:128

9.09% phenylphenol
FS Formula 12167 5.25% sodium hypochlorite 200 ppm
Micronex 10.14% didecyl dimethyl ammonium chloride 660 ppm
6.76% n-alkyl (C1450%, C1240%, C1610%) dimethyl benzyl ammonium chloride
Fit-L Antibacterial Produce Cleaner 0.5% levulinic acid 1:88
0.05% sodium dodecyl sulfate
Zep-amine A 5.0% n-alkyl (60%C14, 30%C16, 5%C12, 5%C18) dimethyl benzyl ammonium chlorides 195 ppm
5.0% n-alkyl (68% C12, 32% C14) dimethyl ethylbenzyl ammonium chlorides

objective, a 488-nm argon laser and a 555-nm diode-pumped solid- 3. Results

state laser, with band-pass 490e550 nm for detection of green cells
and 560e700 nm for red cells. Z-stack scanning was performed to
visualize the spatial distribution of cells in the biolm structure.
Serial images were captured and processed by Zeiss Zen 2012
software (Carl Zeiss) using the transparent and surface projection
modes.
2.4. Statistical analysis
Mean values were obtained from three independent trials with
duplicate plating (n 6). Data were analyzed using one-way
analysis of variance (ANOVA). Differences were considered signif-
icant at p < 0.05 level of probability. Principal component analysis
(PCA) was performed to discriminate the inuence of different
factors: biolm growth temperatures (37
C/15
C), sanitizer
treatment episodes (1st/2nd) and exposure times (5/15/30/60 min)
on biolm inactivation and injury induction. This was performed
with Statistica software ver. 12 (StatSoft Inc., Tulsa, OK).
3.1. Biolm inactivation and injury induction after sanitizer
treatments
The effectiveness of the tested sanitizers on the inactivation of
L. monocytogenes biolm grown at 37
C and 15
C is shown in
Figs. 1 and 2, respectively. The chlorine-based (Formula), and
QAC-based (Zep-amine) sanitizers, and levulinic acid/SDS-based
(Fit-L) produce wash reduced biolm populations by 1.1-, 1.4- and
1.5-log CFU/ml, respectively, after a 60 min treatment of the 37
C-
grown biolm, whereas the phenolic-based (Vesphene) and QAC-
based (Micronex) sanitizers yielded the signicantly (P < 0.05)
greatest reduction of 6.9 and 3.7 log CFU/ml, respectively (Fig. 1).
Vesphene differed from the other sanitizers, as the only one that
exhibited signicant inactivation after a 5-min exposure. Similar
inactivation levels were observed when sanitizer treatments were
repeated after a 24 h biolm recovery (Fig. 3). However, compared
to the 1st treatment, after the 2nd treatment, sublethal injury was
observed for most of the sanitizers.

Fig. 1. The effect of ve sanitizers on inactivation of L. monocytogenes LM101 in the 37
C-grown biolm after treatment for 5, 15, 30, and 60 min. The bars represent the mean
values standard deviations (n 6). * indicates signicant reduction (P < 0.05). 1:1 IUC; IUC (in-use concentration).
374 M.A. Olszewska et al. / Food Control 70 (2016) 371e379

Fig. 2. The effect of ve sanitizers on inactivation of L. monocytogenes LM101 in the 15
C-grown biolm after treatment for 5, 15, 30, and 60 min. The bars represent the mean
values standard deviations (n 6). * indicates signicant reduction (P < 0.05). ** indicates values below the detection limit. 1:1 IUC; IUC (in-use concentration).

Fig. 3. The effect of ve sanitizers on inactivation of L. monocytogenes LM101 in the 37
C-grown biolm after the 2nd treatment episode for 5, 15, 30, and 60 min. The bars represent
the mean values standard deviations (n 6). * indicates signicant reduction (P < 0.05). 1:1 IUC; IUC (in-use concentration).
 M.A. Olszewska et al. / Food Control 70 (2016) 371e379 375

Fig. 4. Principal component analysis (PCA) diagrams showing the discrimination of mean score factors for biolm inactivation (A) and injury induction (B) of L. monocytogenes
LM101. The PCA evaluates the inuence of growth temperatures (37
C/15
C), treatment episodes (1st/2nd) and exposure times (5/15/30/60 min) on L. monocytogenes LM101
biolm.

The level of inactivation of L. monocytogenes biolm grown at
15
C by Vesphene and Micronex was signicant (P < 0.05),
irrespective of exposure time, resulting in a 8.2- and 4.9-log CFU/ml
reduction after a 60 min treatment, respectively (Fig. 2). After
receiving the 2nd treatment, a smaller and greater reduction was
observed for Vesphene and Micronex, respectively (6.2 and 6.3
log units). A signicant reduction (P < 0.05) at all exposure times
was also observed for Zep-amine, resulting in an inactivation
level of 2.9 log CFU/ml after a 60-min treatment (Fig. 2). Formula
and Fit-L yielded a signicant reduction (P < 0.05) of ca. 1 and ca. 2
log CFU/ml, respectively after 30- and 60-min treatments; however,
only when in-use concentrations were applied. After the 2nd

Fig. 5. Reduction and sublethal injury of L. monocytogenes LM101 cells in a 37
C-grown biolm when multiple-exposed to Vesphene and Micronex for 60 min at 1-week
intervals. * indicates signicant reduction (P < 0.05). The threshold line is set at 35% sublethally injured cells.
376 M.A. Olszewska et al. / Food Control 70 (2016) 371e379
Fig. 6. Reduction and sublethal injury of L. monocytogenes LM101 cells in a 15
C-grown biolm when multiple-exposed to Vesphene , Micronex
60 min at 1-week intervals. * indicates signicant reduction (P < 0.05). ** indicates values below the detection limit. The threshold line is set at 18% sublethally injured cells.
treatment the inactivation level for these two sanitizers did not
differ from the 1st treatment (data not shown).
A principal component analysis (PCA) was conducted to deter-
mine if temperature, treatment episode, and exposure time inu-
ence biolm inactivation and injury induction of L. monocytogenes.
The PCA revealed that environmental temperature and exposure
time were the dominant factors affecting biolm inactivation and
injury induction, respectively. As shown on Fig. 4, two clusters were
obtained for each PCA diagram. Fig. 4A presents cluster 1 and 2,
which are a collection of all treated samples at 37
C and at 15
C,
respectively, regardless of exposure time and treatment episode.
This PCA score plot comprised 75.49% of the total variance and
revealed a temperature-dependent behaviour to sanitizers. In
contrast, the injury test results were discriminated based on
exposure time (Fig. 4B). Cluster 1 was comprised primarily of the 5-
and 15-min treated samples and cluster 2 comprised the 30- and
60-min treated samples; however, the total variance was 38.27%.
Still, the separation between exposure times suggests that the
increased exposure time of the biolms to sanitizers induces
L. monocytogenes injury.
3.2. Biolm resistance after continuing exposure to sanitizers
The most effective sanitizers, Vesphene and Micronex for the
37
C-grownbiolm and Zep-amine for the 15
C-grown biolm,
were further tested to determine if Listeria cells in biolms have
sanitizer resistance. This was determined by exposing the biolm to
and Zep-amine for
the sanitizer at one-week intervals for three weeks (Fig. 5; Fig. 6).
When the biolm was incubated at 37
C, the continuing exposure
to 1:1 IUC/IUC Vesphene and Micronex signicantly enhanced
sanitizer resistance, with the average reduction of 3.5 and 0.7 log
CFU/ml for IUCs after 3rd week, respectively (Fig. 5). Results also
revealed that the percentage of sublethally injured cells increased
as the sanitizing exposure to IUC of Micronex continued, subse-
quently resulting in 32% cell injury. Hence, the longer the incuba-
tion time at 37
C, the greater the percentage of injured cells, but
the percentage of injured cells receiving the multiple Vesphene
treatments did not reach 35%.
Results of the sanitizer resistance of 15
C-L. monocytogenes
biolm when multiply exposed to Vesphene, Micronex, and Zep-
amine is presented in Fig. 6. The continuing exposure to
Vesphene resulted in almost complete inactivation of Listeria cells
in biolms, with the reduction level below the detection limit for
IUC. Moreover, Vesphene 1:1 IUC results revealed that the Listeria
cells were either dead or injured. For Micronex and Zep-amine
at IUC, the biolm cells inactivation was an average of 4.4 and 1.6
log CFU/ml, respectively, after the 3rd week. Even though the
effectiveness of Micronex was greater after continuing exposure
within 2 weeks compared to the 1st or 2nd treatments, the greatest
resistance (ca. 5 log units) of the biolm was observed after the 3rd
week of incubation. For both of the QAC-based sanitizers, the
smaller the cell reduction, the greater the percentage of injured
cells, irrespective of concentration used.
 M.A. Olszewska et al. / Food Control 70 (2016) 371e379 377

Fig. 7. Representative CLSM images of L. monocytogenes LM101 biolm. The biolm was grown in TSB at 37
C (A), and treated for 60 min with Vesphene (B), Formula (C),
Micronex (D), Fit-L (E), and Zep-amine (F). The biolm was grown in an 8-well chamber slide system and stained with the Live/Dead BacLight. A1, B1, C1, D1, E1, and F1 are 3D-
images obtained by the transparent projection mode, and A2, B2, C2, D2, E2, and F2 are images obtained by the surface projection mode with the Zeiss Zen software. The scale bar is
10 mm.

3.3. Biolm formation characteristics determined by CLSM analysis
Confocal laser scanning microscopy (CLSM) was used to study
L. monocytogenes biolm characteristics after a 60-min exposure to
the ve sanitizers. For this purpose, the biolm was grown in an 8-
well chamber slide system and stained with Live/Dead BacLight,
wherein live and dead cells were visualized based on cell mem-
brane integrity. CLSM analysis revealed the three-dimensional
development of L. monocytogenes biolm, with an average height
of ca. 6 mm after 72 h at 37
C and an additional 48 h at 15
C. Biolm
cells growing at 37
C experienced cell membrane perturbations
when exposed to Vesphene and Micronex (Fig. 7), as did cells
growing at 15
C and exposed to Zep-amine. Even though, there
was considerable cell membrane damage within the population,
some cells located inside biolm clusters were still viable after the
60-min treatment, regardless of the environmental temperature.
4. Discussion
Despite the application of best cleaning and sanitation practices,
Listeria is continuously reintroduced in food processing environ-
ments (Tompkin, 2002). The presence of L. monocytogenes in single-
or mixed-biolms in food processing establishments is of great
concern, due to the risk of cross-contamination of other surfaces,
including those that contact edible products (Berrang et al., 2010).
In general, biolms are known to have enhanced resistance to
antimicrobial agents (Kim et al., 2009). The mechanisms of biolm
resistance to sanitizers have not been fully elucidated (Yang et al.,
2016); however, it was determined that L. monocytogenes is more
resistant when grown in mixed-species communities than when
grown as single-species biolms (Saa
Ibusquiza, Herrera, Va
zquez-

nchez, & Cabo, 2012). Moreover, it was observed that
Sa
L. monocytogenes can integrate itself to the extracellular poly-
saccharides and biolms of other bacteria, and attached in greater
numbers to surfaces with Pseudomonas putida biolms than to
Pseudomonas-free surfaces (Hassan et al., 2004). Additionally, pre-
vious studies revealed that persistent strains of L. monocytogenes
can adhere better to surfaces and have increased antimicrobial
resistance than transient strains, thereby facilitating their survival
in processing plants (Gilbert, Allison, & Mcbain, 2002; Lunde
n,
Autio, Markkula, Hellstrom, & Korkeala, 2003; Lunde
n et al.,
2000). Hence, cleaning and sanitizing strategies must be effective
against Listeria.
A wide range of chemical antimicrobials are available for the
food industry, among which chlorine-based sanitizers are most
widely used, exhibiting broad-spectrum bactericidal activities
(Yang et al., 2016). To be an effective sanitizer against biolms, a 3-
log reduction of L. monocytogenes must be demonstrated (Somers &
Wong, 2004). Norwood and Gilmour (2000) determined that for a
substantial reduction of L. monocytogenes in a mixed-species
378 M.A. Olszewska et al. / Food Control 70 (2016) 371e379
biolm, a 100-times greater chlorine concentration is required than
for a planktonic culture (1000 ppm and 10 ppm, respectively).
L. monocytogenes is easily isolated from poultry and poultry pro-
cessing plants, and poultry have also been implicated in foodborne
outbreaks of listeriosis, hence L. monocytogenes has become a
concern for poultry processing facilities (Berrang et al., 2010). In
poultry processing plants, quaternary ammonium compounds
(QACs) are commonly used for cleaning and disinfection (Zhao
et al., 2013). They exhibit greater antimicrobial activity against
Gram-positive bacteria than Gram-negative bacteria, likely due to
breakdown of the cell wall and cell membrane of Gram-positive
bacteria (Parish et al., 2003). Therefore, L. monocytogenes is
considered to be more sensitive to QACs than coliforms or Salmo-
nella spp. However, the potential resistance to QACs due to the
common spread of Class 1 integrons among bacteria has been re-
ported (Parish et al., 2003). Accordingly, Mereghetti, Quentin,
Marquet-Van Der Mee, and Audurier (2000) determined that
some L. monocytogenes strains isolated from the food processing
environment and food products were resistant to QACs, suggesting
an intrinsic resistance is easily transferred among L. monocytogenes
strains. In addition, the use of QACs in a facility has limitations, as
their effectiveness is generally reduced in the presence of organic
material, metallic ions, anionic surfactants and soaps (Zhao et al.,
2013). Considering these issues, there is an increasing need for
developing novel, safe and effective disinfectants, e.g., based on
levulinic acid and SDS (Chen, Zhao, & Doyle, 2015). For example,
Chen et al. (2015) reported that the combination of 3% levulinic acid
plus 2% SDS is effective in substantially (>6.9 log CFU) reducing
L. monocytogenes levels in a single-species biolm.
Our results revealed that IUCs of Vesphene, and Micronex and
Zep-amine sanitizers most effectively reduced L. monocytogenes
LM101 in a biolm, and that Listeria cells were more sensitive to
sanitizers when the cells were grown at 15
C. However, two con-
centrations of most effective sanitizers, in-use and 1:1 in-use,
signicantly reduced biolm cells, irrespective of growth temper-
ature. Chorine-based (Formula) sanitizer and levulinic acid/SDS-
based (Fit-L) produce wash (not evaluated at sanitizer concentra-
tions) did not yield more than a 2-log reduction for in-use con-
centrations, regardless of environmental temperature, treatment
episode or exposure time tested. We also observed enhanced levels
of sanitizer resistance when biolm cells were multiply exposed to
Vesphene, Micronex, and Zep-amine, with only one exception,
i.e., increased sanitizer resistance did not occur for IUC of
Vesphene when the biolm was formed at 15
C. However, results
of treatments with 1:1 IUC Vesphene exposure on 15
C-grown
L. monocytogenes biolm as well as the Micronex, and Zep-
amine treatments results suggests there is a potential for
increased sanitizer resistance and the presence of injured cells. It is
recognized that bacteria, including L. monocytogenes can enter the
injured state as a result of exposure to a variety of treatments, such
as heating, freezing, sanitizers (Busch & Donnelly, 1992). Suble-
thally injured cells may repair and regain pathogenic potential.
Moreover, injured cells are not recoverable on selective media,
which are typically employed in standard detection procedures.
Therefore, in order to ensure sanitizer effectiveness, detection of
injured cells of L. monocytogenes within food processing plants
should be considered.
Images captured by CLSM enabled us to reveal spatial organi-
zation and effectiveness of sanitizers on L. monocytogenes biolm.
To our knowledge, reports on the investigation of the three-
dimensional structure of L. monocytogenes biolms by CLSM are
limited, including observing the direct effect of antimicrobials on
biolm behaviour. Few studies have been conducted focused on the
structure of L. monocytogenes biolm (e.g. Guilbaud, Piveteau,
Desvaux, Brisse, & Briandet, 2015). L. monocytogenes on
Pseudomonas uorescens biolms were studied by Puga, Orgaz, and
SanJose (2016) using CLSM, whereby they determined that species
interactions or biolm aging could be used as targets for cleaning
and disinfection procedures. Our study revealed that Listeria cells
were distributed regularly in a biolm consortium, which consisted
of multiple layers of cells, and the average height of the biolm was
ca. 6 mm. Biolm cells experienced cell membrane perturbations
when exposed to the most effective sanitizers evaluated at IUCs,
with cells located inside biolm clusters being more protected.
Hence, it is a challenge in selecting suitable sanitizers to disrupt
Listeria cell aggregates in biolms. These ndings should be
considered in designing Listeria control strategies in food process-
ing facilities. Considering that increased sanitizer resistance can
occur for Listeria with some sanitizers, the constant use of the same
sanitizers would not be advisable to control L. monocytogenes in
biolms at different locations of a food processing facility. Of
greatest concern are locations where moisture accumulates and
cleaning and sanitizing procedures are inaccessible or the accessi-
bility is hindered, e.g., inner surfaces of oor drains, which pose a
risk of listeriae escaping and contaminating other surfaces by bio-
lm cells.
In conclusion, this study contributes to a better understanding
of the bactericidal interactions between Listeria biolm cells and
sanitizers, and may help to inform better strategies for the use of
sanitizers against Listeria biolms through suitable antimicrobial
selection and the prevention of listeriae adaptation.
Acknowledgments
The authors thank Ping Zhao for excellent technical assistance.
This study was supported by Kosciuszko Foundation Fellowship for
Magdalena Olszewska for the academic year 2015/16 at University
of Georgia.
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