APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2009, p. 7182–7188 Vol. 75, No.

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0099-2240/09/$12.00 doi:10.1128/AEM.01359-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Listeria monocytogenes Attachment to and Detachment from Stainless

Steel Surfaces in a Simulated Dairy Processing Environment䌤
Sofia Poimenidou,1 Charalambia A. Belessi,1 Efstathios D. Giaouris,2 Antonia S. Gounadaki,1
George-John E. Nychas,2 and Panagiotis N. Skandamis1*
Laboratory of Food Quality Control and Hygiene1 and Laboratory of Microbiology and Biotechnology of Foods,2
Department of Food Science and Technology, Agricultural University of Athens, Athens, Greece
Received 10 June 2009/Accepted 9 September 2009

Downloaded from http://aem.asm.org/ on October 13, 2020 by guest

The presence of pathogens in dairy products is often associated with contamination via bacteria attached to
food-processing equipment, especially from areas where cleaning/sanitation is difficult. In this study, the
attachment of Listeria monocytogenes on stainless steel (SS), followed by detachment and growth in foods, was
evaluated under conditions simulating a dairy processing environment. Initially, SS coupons were immersed
in milk, vanilla custard, and yogurt inoculated with the pathogen (107 CFU/ml or CFU/g) and incubated at two
temperatures (5 and 20°C) for 7 days. By the end of incubation, cells were mechanically detached from coupons
and used to inoculate freshly pasteurized milk which was subsequently stored at 5°C for 20 days. The
suspended cells in all three products in which SS coupons were immersed were also used to inoculate freshly
pasteurized milk (5°C for 20 days). When SS coupons were immersed in milk, shorter lag phases were obtained
for detached than for planktonically grown cells, regardless of the preincubation temperature (5 or 20°C). The
opposite was observed when custard incubated at 20°C was used to prepare the two types of inocula. However,
in this case, a significant increase in growth rate was also evident when the inoculum was derived from
detached cells. In another parallel study, while L. monocytogenes was not detectable on SS coupons after 7 days
of incubation (at 5°C) in inoculated yogurt, marked detachment and growth were observed when these coupons
were subsequently transferred and incubated at 5°C in fresh milk or/and custard. Overall, the results obtained
extend our knowledge on the risk related to contamination of dairy products with detached L. monocytogenes
cells.

Listeria monocytogenes is ubiquitous in nature due to its
inherent ability to survive and grow under a wide range of
adverse environmental conditions, such as refrigeration tem-
peratures, high acidity and salinity, and reduced water activity
(16). This microorganism is a major concern for the food
industry, since it is the causal agent of listeriosis, a severe
disease with high hospitalization and case-fatality rates (ap-
proximately 91% and 30%, respectively) (25). According to the
European Centre for Disease Control and Prevention, listeri-
osis was the fifth most common zoonotic infection in Europe in
2006 (14), while it accounts for approximately 28% of the
deaths resulting from food-borne illnesses in the United States
(34).
In the food industry, inadequately cleaned food-processing
equipment (e.g., stainless steel [SS] surfaces) constitutes a po-
tential source for L. monocytogenes, resulting in contamination
of foods which come in contact with such equipment (36).
Even though adherence to strict sanitation practices should
minimize the risk of survivors on surfaces, existing evidence
suggests that a considerable risk may occur in sites of process-
ing plants which are not easily cleaned or sanitized, such as
those that do not allow direct access of sanitation equipment
* Corresponding author. Mailing address: Laboratory of Food Qual-
ity Control and Hygiene, Department of Food Science and Technol-
ogy, Agricultural University of Athens, Iera Odos 75, Athens 11855,
Greece. Phone: 30 210 529 4684. Fax: 30 210 529 4683. E-mail: pskan
@aua.gr.
䌤
Published ahead of print on 18 September 2009.
for abrasion (e.g., edges, convex surfaces, etc.) (43, 45). At-
tachment to surfaces is believed to be important for the sur-
vival and persistence of this pathogen in such environments,
with some strains able to remain on equipment surfaces for
several years (32, 37). Thus, L. monocytogenes has been shown
to adhere to and form biofilms on various food contact surfaces
under laboratory conditions (3, 42, 44). Furthermore, attached
L. monocytogenes cells are more difficult to mechanically re-
move from surfaces and are more resistant to sanitizers than
their free-living counterparts (15, 40).
Dairy products have been implicated in outbreaks of listeri-
osis (10, 31). However, most of the in vitro studies of the
growth and survival of L. monocytogenes in such products have
used strains previously cultivated planktonically (41). Although
the results obtained in these studies are of great value, such
studies have not taken into consideration that cells contami-
nating a product in a food-processing environment are usually
attached to surfaces enclosed in biofilms. Limited information
is available on the kinetic behavior of L. monocytogenes in
dairy products inoculated with detached cells, although prein-
cubation conditions have been shown to influence subsequent
growth and survival of L. monocytogenes in foods (7, 13, 17,
18). Given the major physiological differences between at-
tached and planktonic cells (15, 27, 48), an effect on subse-
quent growth might be possible.
Considering the above, the main objective of the present
study was to assess the influence of L. monocytogenes preincu-
bation conditions with respect to mode of growth (either at-
tached to SS or grown suspended in dairy products) on the

7182
VOL. 75, 2009 L. MONOCYTOGENES BIOFILM FORMATION AND DETACHMENT
subsequent growth of this pathogen in milk (at 5°C for 20
days). To prepare the two types of inocula, two different
growth media (milk and vanilla custard) and temperatures (5
and 20°C) were studied. The unforced detachment of L. mono-
cytogenes cells from SS coupons and growth in two dairy prod-
ucts (milk and custard) at 5°C for 20 days was also evaluated.
In the latter case, previous attachment of cells to the coupons
was done under especially adverse preincubation conditions
(in yogurt at 5°C for 7 days).
MATERIALS AND METHODS
Bacterial strain and inoculum preparation. L. monocytogenes Scott A (sero-
type 4b, epidemic strain, human isolate), kindly provided by Eddy Smid (Agro-
technological Research Institute ATO-DLO, Wageningen, The Netherlands),
was used throughout the study. Although L. monocytogenes Scott A does not
attach to surfaces as strongly as other strains, its attachment ability has been
ranked in the middle of those of a list of multiple clinical and food isolates (12,
49). This strain was selected due to its clinical origin and the strong epidemio-
logic association of serotype 4b with human listeriosis (25, 29). Moreover, a
single strain was used so that the findings on the comparative growth of plank-
tonic and attached cells following detachment were not affected by the domi-
nance of different strains under different growth conditions. Stock cultures were
maintained in tryptic soy broth supplemented with yeast extract (TSBYE)
(Biolife Italiana Srl, Milan, Italy) supplemented with 20% glycerol at ⫺20°C and
were regenerated by transferring 0.05 ml of the frozen culture into 10 ml of
TSBYE and incubating at 30°C for 24 h. Aliquots (0.1 ml) of activated cultures
were transferred to 10 ml of TSBYE, incubated at 30°C for 18 h, and then
harvested by centrifugation (5,000 ⫻ g for 10 min at 4°C; Heraeus Instruments
Megafuge 1.0 R). The cell pellet was washed and resuspended twice in 10 ml of
Ringer’s solution (Ringer’s tablets; Merck, Darmstadt, Germany) before inocu-
lation. Planktonic growth prior to attachment on abiotic surfaces aimed to
simulate situations where cells from liquid food residues settle on food contact
surfaces.
Commercial dairy products. High-temperature-, short-time-pasteurized whole
cows’ milk (cartons of 1 liter), traditional vanilla custard (packages of 170 g), and
yogurt (packages of 200 g) were purchased from a local market (within 18 to 24 h
of their production) and transferred (at 4°C) to the laboratory for inoculation.
Milk had a typical composition of 88% water, 3.5% fat, 3.2% protein, and 4.6%
carbohydrate, with a pH ranging from 6.6 to 6.7. Vanilla custard, otherwise called
“vanilla cream,” is a traditional milk-based dessert which is very popular to
Mediterranean countries (41). It is made from cows’ milk, sugar, modified starch
of tapioca, wheat and corn starch, egg yolk, and vanillin (41). To ensure its
microbiological safety, the custard is subjected to thermal treatment (82°C for 30
min) before packing. According to the manufacturer’s specifications, the final
product contains 4.6% fat, 2.9% protein, and 17.8% carbohydrate and has a pH
ranging from 6.3 to 6.7. The yogurt was made of cows’ milk and contained 4% fat,
4.5% protein, and 6.5% carbohydrate according to the manufacturer’s specifi-
cations, with a pH ranging from 4 to 4.4.
Preparation of test surface. SS coupons (2 by 5 cm, type AISI-304, no. 2b
finish, 0.1 cm thick; Halyvourgiki Inc., Athens, Greece) were the abiotic sub-
strates used for L. monocytogenes adhesion studies, since SS is a material com-
monly used for the manufacture of food-processing equipment (4). The coupons
were initially soaked in acetone (overnight) to remove any debris and grease
from the manufacturing process. Coupons were then washed in commercial
detergent solution, rinsed thoroughly with distilled water, air dried, and finally
sterilized by autoclaving at 121°C for 15 min before use.
Experimental design. SS surfaces exposed to different types of dairy products
(milk, vanilla custard, and yogurt) were used to simulate harborage sites within
processing plants that cannot be easily cleaned and sanitized. Bacterial attach-
ment was evaluated at 5 and 20°C to simulate the environmental conditions
typically encountered in dairy processing environments. To investigate the hy-
potheses described above, three main studies were carried out. Initially, the
adhesion of L. monocytogenes on SS coupons immersed in milk, custard, and
yogurt for 7 days at 5 and 20°C was evaluated. The selection of 7 days of
habituation was made in order to allow sufficient time for several cycles of
dissociation events and subsequent regrowth of the biofilm to occur (47). This
experiment aimed to determine the effect of the environment (i.e., growth me-
dium and temperature) on the ability of L. monocytogenes to attach and to form
biofilm on food-soiled SS coupons. At the next stage, two types of L. monocy-
togenes cells (i.e., detached from food-soiled surfaces or suspended in surround-
7183
ing product) were used to inoculate milk in order to evaluate the effect of the
previous environment on the subsequent growth kinetics of L. monocytogenes at
5°C. The experiment focused directly on the comparative growth of the two types
of cells in case of different cross-contamination scenarios. Specifically, cells
growing in suspension aimed to simulate cross-contamination of milk from prod-
uct waste, purge, or residues, whereas attached cells represented contamination
from soiled surfaces. Finally, the ability of L. monocytogenes cells to detach
(unforced) from SS coupons soiled with yogurt and to disperse in noninoculated
dairy products, such as milk and vanilla custard, at 5°C was evaluated. All three
studies were performed at least twice, and in each replicate study the samples
were analyzed in duplicate (n ⫽ 4).
Inoculation of dairy products and adhesion experiments. Prior to inoculation,
portions (40 ml) of freshly pasteurized milk were aseptically transferred into
50-ml sterile plastic centrifuge tubes. Inoculation of custard and yogurt was
performed in their commercial packages (170 g for vanilla custard and 200 g for
yogurt). Specifically, a 0.5-ml aliquot of appropriately diluted L. monocytogenes
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culture was added into the center of each package and/or tube in order to obtain
an initial population density of approximately 107 CFU/g or CFU/ml as deter-
mined in preliminary trials. To ensure uniform distribution of the inoculum, milk
samples were mixed by vortexing (30 s), whereas samples of custard and yogurt
were thoroughly stirred with a sterile spatula. Following inoculation, individual
sterile SS coupons were vertically immersed in the center of the inoculated
packages and/or tubes, which were stored in high-precision (⫾0.5°C) incubation
chambers (MIR-153; Sanyo Electric Co., Osaka, Japan) for 7 days at 5°C or 20°C.
Uninoculated control samples were also held under the same conditions and
analyzed frequently to confirm the absence of L. monocytogenes using the ISO
11290 enrichment protocol (23).
Detachment of the attached population from SS coupons and quantification of
the attached and/or suspended population. Detachment of attached cells from
the SS coupons was performed by using the bead vortexing method (19), which
has been established as the most suitable method for removal of attached bac-
teria (30). Briefly, after 7 days of incubation, each SS coupon was carefully
removed from the dairy product, using sterile forceps, and was thoroughly rinsed
with 25 ml of Ringer’s solution in order to remove both food residue and loosely
attached cells. The coupon was then transferred to a new 50-ml centrifuge tube
containing 40 ml of Ringer’s solution and 12 sterile glass beads (diameter, 5 mm)
and was subsequently vortexed for 2 min at maximum intensity in order to detach
the cells from the coupon. Quantification of attached L. monocytogenes cells and
total microflora (TVC) was performed by surface plating 0.1-ml aliquots of
appropriate 10-fold serial dilutions on duplicate plates of Palcam Listeria selec-
tive agar (Palcam; Biolife) and tryptic soy agar (Biolife) supplemented with 0.6%
yeast extract (TSAYE), respectively. Formed colonies were counted after incu-
bation of plates at 37°C for 48 h (Palcam) or at 30°C for 72 h (TSAYE).
The populations of suspended L. monocytogenes and TVC in the dairy prod-
ucts from which the SS coupons had been removed were also determined.
Specifically, portions (1 ml for milk and 25 g for custard and yogurt) from the
dairy samples were homogenized and serially diluted (1:10 dilution) in Ringer’s
solution. Aliquots (0.1 ml) of the appropriate dilution were surface plated in
duplicate as described above. Moreover, typical L. monocytogenes colonies from
Palcam and TSAYE plates were biochemically confirmed according to ISO
11290 (23).
Evaluation of L. monocytogenes growth kinetics in milk inoculated with cells
previously grown under different conditions. Milk and custard incubated at 5°C
and 20°C for 7 days were the environmental conditions used to harvest attached
and suspended L. monocytogenes cells as described above. Aliquots (1 ml) of the
appropriate dilution of each bacterial suspension (attached or suspended) were
used to inoculate freshly pasteurized milk (100 ml), as described above, in order
to obtain an initial population density of approximately 102 CFU/ml. All inocu-
lated milk samples were statically incubated at 5°C for 20 days, and the sus-
pended L. monocytogenes population was quantified every 24 h following the
procedures described above.
Incubation of coupons bearing attached bacteria in vanilla custard and milk.
SS coupons bearing attached L. monocytogenes cells habituated in yogurt for 7
days at 5°C as described above were transferred to uninoculated custard and
milk. Specifically, by the end of habituation, SS coupons were aseptically re-
moved, rinsed thoroughly with 25 ml of Ringer’s solution, and finally transferred
into either custard (170 g in its original package) or freshly pasteurized milk (50
ml in a plastic centrifuge tube). The population of coupon-attached cells before
their transfer was also determined. Samples were statically incubated at 5°C for
20 days, while the L. monocytogenes population in the two dairy products was
quantified every 48 h following the procedures described above.
Microscopic observation of attached cells on SS coupons. Attached cells were
also examined using epifluorescence microscopy (N-400F; Optika Microscopes,
7184 POIMENIDOU ET AL. APPL. ENVIRON. MICROBIOL.

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FIG. 1. Populations of Listeria monocytogenes (f) and TVC (䡺) attached to SS coupons (log CFU/cm2) or of Listeria monocytogenes (o) and
TVC (t) suspended into different dairy products (milk, custard, and yogurt) (log CFU/ml or log CFU/g) and incubated for 7 days at 5°C and 20°C.
Each bar represents the mean value ⫾ standard deviation (n ⫽ 4). Bars of the same color, regardless of product and storage temperature, sharing
at least one common capital letter are not significantly different (P ⱖ 0.05). Bars of different colors, at each product and storage temperature,
sharing at least one common lowercase letter are not significantly different (P ⱖ 0.05). *, count below the detection limit (1.3 log CFU/cm2); in
this case the value of the detection limit was used for statistical analysis.

Italy) according to the protocol described by Pan et al. (40). Briefly, SS coupons
habituated for 7 days in the different dairy products were aseptically removed
from the products, rinsed thoroughly with 25 ml of Ringer’s solution, and trans-
ferred to a small petri dish (diameter, 5 cm). Samples were stained with 0.01%
acridine orange (Sigma-Aldrich Ltd., Greece) for 5 min at room temperature.
Subsequently, coupons were rinsed three times with Ringer’s solution to remove
excess stain. Images of attached cells taken with a digital (charge-coupled device)
camera (E-330; Olympus, Greece) were processed using Image-Pro Plus image
analysis software (version 4.5; Media Cybernetics, Silver Spring, MD).
Fitting and statistical analysis. The data from plate counts (CFU per gram or
per ml) of duplicate samples from two independent experiments (n ⫽ 4) were
transformed to log10 values. The log10-transformed data for attached and sus-
pended cells (from SS coupons) of L. monocytogenes were fitted to the Baranyi
model with the in-house program DMFit version 2.1 (available at http://www.ifr
.ac.uk/Safety/DMfit/default.html) in order to estimate the growth kinetics of the
two populations. For each growth curve, the maximum specific growth rate
(␮max; days⫺1), the lag time (tlag; days), the lower asymptote (yo; log CFU/ml),
and the upper asymptote (yend; log CFU/ml) were determined. The growth data
as well as the parameter estimates of the fitting then were analyzed by analysis
of variance by the general linear model procedure of the SPSS statistical package
(SPSS 10.0.1 for Windows; SPSS, Inc., Chicago, IL). Tukey’s multiple-range test
was used to compare means. All differences are reported at a significance level
of alpha 0.05.
RESULTS
Attachment of L. monocytogenes and TVC on food-soiled SS
coupons. Bacterial attachment on SS coupons and prolifera-
tion in different food matrices were more favorable (P ⬍ 0.05)
at 20°C than at 5°C (Fig. 1). At 20°C, significantly (P ⬍ 0.05)
higher numbers of attached populations were recovered from
coupons placed in custard (5.30 and 5.42 log10 CFU/cm2 for L.
monocytogenes and TVC, respectively) than from those placed
in milk (4.49 and 4.55 log10 CFU/cm2 for L. monocytogenes and
TVC, respectively). It should be noted that at 20°C for both
dairy products, similar levels of attached L. monocytogenes and
TVC were observed, suggesting that the pathogen dominated
TVC. At 5°C, the lowest level of attached cells was observed in
yogurt, whereas milk and custard resulted in similar levels of
attachment (approximately 3.7 log10 CFU/cm2 and 3.9 log10
CFU/cm2 for L. monocytogenes and TVC, respectively). Fur-
thermore, incubation of milk and yogurt at 5°C resulted in
significantly (P ⬍ 0.05) higher levels of attached TVC than of
attached L. monocytogenes, indicating the existence of a mixed
attached population on the SS coupons. Moreover, micro-
scopic observation of the coupons placed in yogurt revealed
the presence of attached cells (Fig. 2). Regardless of storage
temperature, the bacterial populations growing in suspension
were significantly greater (P ⬍ 0.05) than the attached popu-
lations (Fig. 1). Except for in yogurt, no major differences were
seen between the suspended populations of L. monocytogenes
and TVC (Fig. 1).
Effect of habituation conditions on the subsequent growth of
L. monocytogenes in milk. Multivariate statistical analysis re-
vealed that the preincubation conditions (storage temperature,
growth medium, and mode of bacterial growth) significantly
(P ⬍ 0.05) affected the subsequent growth kinetics of L. mono-
cytogenes in milk at 5°C. Habituation at 20°C delayed (P ⬍
0.05) subsequent growth of both attached and suspended cells
of L. monocytogenes in milk, as seen by an increased lag phase,
compared to habituation at 5°C (Table 1; Fig. 3). At 5°C, no
significant difference (P ⬍ 0.05) was observed between the two
types of inocula in the subsequent growth kinetics (lag phase
and growth rate) of L. monocytogenes in milk (Table 1). Re-
gardless of temperature, habituation of L. monocytogenes in
milk decreased the lag phase of cells previously attached to SS
coupons compared to cells previously grown in suspension
(Table 1; Fig. 3). However, for populations habituated in milk,
a slightly higher growth rate was observed for suspended cells
than for attached cells (Table 1). In contrast, habituation in
custard increased the lag phase of cells previously attached to
SS coupons compared to cells previously grown in suspension
(Table 1; Fig. 3) In general, the highest maximum specific
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FIG. 2. Photomicrographs of cells attached to SS coupons after 7 days of habituation in yogurt at 5°C.

growth rates were observed when the habituation conditions
were identical to the subsequent storage conditions (Table 1).
Moreover, total bacterial counts reached approximately 6 to 7
log10 CFU/ml, regardless of preincubation conditions (data not
shown).
Unforced detachment of L. monocytogenes cells from cou-
pons and subsequent growth in dairy products. Although L.
monocytogenes was not detectable on SS coupons after 7 days
of incubation in yogurt (Fig. 1) the transfer of whole coupons
into uninoculated milk and custard resulted in the detachment
and proliferation of the pathogen in both products (Fig. 4).
More specifically, detectable growth of detached L. monocyto-
genes cells occurred after 9 and 10 days in custard and milk,
respectively (Fig. 4). However, by the end of incubation (21
days), higher growth was obtained in custard (4.4 log10 CFU/g)
than in milk (3.7 log10 CFU/ml) (Fig. 4).
DISCUSSION
The ability of bacteria, including L. monocytogenes, to ad-
here to and colonize surfaces is influenced by many environ-
mental factors (26) that affect the physiological characteristics
of bacteria (6, 11, 33) and/or alter the chemistry of the surface
(26). The type and the composition of food residue on food-
processing equipment have been suggested to influence both
the population levels of attached cells and their resistance to

TABLE 1. Kinetic parameters of Listeria monocytogenes growth in milk at 5°C following preincubation under different conditionsa
Mean ⫾ SD (n ⫽ 4)b
Preincubation Preincubation Preincubation mode
Growth rate yend r2
growth medium temp (°C) of growth Lag time (days) yo (log CFU/ml)
(day⫺1) (log CFU/ml)

Milk 5 Attached 0.00 a ⫾ 0.00 0.51 c ⫾ 0.02 1.66 ab ⫾ 0.02 5.98 b ⫾ 0.10 0.982–0.987
Suspended 1.85 ab ⫾ 0.19 0.57 c ⫾ 0.02 1.54 ab ⫾ 0.09 5.26 a ⫾ 0.06 0.977–0.991
20 Attached 3.04 b ⫾ 0.95 0.23 a ⫾ 0.03 1.61 ab ⫾ 0.43 — 0.938–0.981
Suspended 6.23 c ⫾ 1.31 0.26 a ⫾ 0.00 2.07 b ⫾ 0.16 — 0.963–0.975

Custard 5 Attached 1.41 ab ⫾ 0.30 0.25 a ⫾ 0.01 2.12 b ⫾ 0.16 6.42 c ⫾ 0.18 0.983–0.991
Suspended 0.00 a ⫾ 0.00 0.24 a ⫾ 0.01 2.09 b ⫾ 0.32 6.62 d ⫾ 0.00 0.986–0.965
20 Attached 9.07 d ⫾ 1.87 0.41 b ⫾ 0.08 1.15 a ⫾ 0.21 — 0.975–0.976
Suspended 5.71 c ⫾ 0.65 0.27 a ⫾ 0.03 1.89 b ⫾ 0.46 — 0.975–0.979
a
Food-soiled surfaces from which cells were detached or food in which the suspended population was grown prior to transfer to the new environment.
b
Means within a column sharing at least a common letter are not significantly different at a P value of ⬍0.05. yo, upper asymptote; yend, lower asymptote; —, no upper
asymptote occurred.
7186 POIMENIDOU ET AL.
FIG. 3. Comparative growth of Listeria monocytogenes detached
from SS coupons (a) or grown in suspension (b) in custard (triangles)
or milk (squares) at 5°C (open symbols) or 20°C (closed symbols) and
transferred to milk at 5°C. Error bars represent standard deviations for
duplicate samples from two independent experiments (n ⫽ 4).
disinfectants (6, 20, 24, 33, 44). Moreover, in accordance with
previous studies, attachment of L. monocytogenes was found to
increase with increasing temperature (6, 19, 33, 38). It has been
suggested that these differences in attachment are independent
of increases in cell density (33). Generally, enhanced attach-
ment has been attributed to the increased production of fla-
gella, which are highly related to cell attachment on surfaces
under static conditions (9, 50). In contrast, Lemon et al. (28)
showed that flagellum-mediated motility, and not flagella, is
critical for both adhesion and biofilm formation of L. mono-
cytogenes on abiotic surfaces. It has been speculated that the
differences between studies are mainly due to different exper-
imental approaches which in turn result in bacterial changes,
including changes in pH, oxygen tension, and nutrient avail-
ability (47).
In contrast to temperature, low pH has been shown to stim-
ulate initial adhesion of L. monocytogenes to SS, as negative
groups on the cell surface become protonated at low pH (6).
APPL. ENVIRON. MICROBIOL.
Thus, the limited attachment of L. monocytogenes in yogurt-
soiled surfaces as found in the current study (Fig. 1) could be
indicative of the inhibiting effect of low pH on the subsequent
steps in the development of biofilm (i.e., microcolony forma-
tion, maturation, and regrowth of biofilm). Therefore, further
studies are needed to elucidate the effect that such acidic
environments may have on the different steps of biofilm devel-
opment. Moreover, survival of the pathogen in the surrounding
medium (Fig. 1) may be attributable to acid adaptation mech-
anisms induced by habituation in yogurt for 7 days (2).
The natural flora of milk and custard did not seem to inhibit
attachment of L. monocytogenes, since the enumerated popu-
lations on surfaces immersed in these products ranged from 3.5
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to 5.5 log CFU/cm2 (Fig. 1). Listeria commonly exists as a part
of multispecies biofilms with other bacteria in food-processing
facilities, and competitive microflora was shown to either en-
hance or inhibit L. monocytogenes attachment to surfaces (5, 8,
52). The presence of other bacterial species may also render
the pathogen more resistant to stress conditions (5). On the
other hand, Zhao et al. (52) demonstrated that some bacterial
species may inhibit L. monocytogenes by producing antilisterial
metabolites. Thus, the impact that the resident microorgan-
isms may have on the biofilm-forming capacity of L. monocy-
togenes needs further investigation.
Detached L. monocytogenes cells from soiled surfaces may
contaminate foods and proliferate under refrigeration. Our
findings also suggested that habituation of L. monocytogenes on
surfaces at 20°C delayed subsequent growth of the detached
cells in milk at 5°C compared to previous habituation at 5°C.
Such findings are supported by studies demonstrating that ex-
posure to temperature downshifts increased the lag time of
planktonic L. monocytogenes cells (7, 13). In addition to tem-
perature, the variability of food matrices for bacterial habitu-
ation and attachment markedly affected the growth behavior of
suspended and detached cells. Geornaras et al. (17, 18) inves-
tigated the effect of inoculum origin on the survival and growth
kinetics of L. monocytogenes during postprocess control of
commercial frankfurters and smoked sausages. They found
that the ecological background of the cells (i.e., attached to SS
or grown in suspension) affected the growth behavior of the
pathogen, including the lag time and the general response to
various antimicrobial solutions (17, 18).
In the present study, the observed variations in growth of
detached L. monocytogenes cells from surfaces soiled with dif-
ferent products likely depended on the temperature, the state
(e.g., liquid or solid), and the composition of the food as well
as the available nutrients. Specifically, detached cells from
milk-soiled surfaces had better subsequent growth in milk than
those from custard-soiled surfaces at both high and low tem-
peratures. Presumably, detachment and dispersal of cells in a
food ecosystem different from that where attachment took
place increased the adaptive response. Adaptation to a new
environment is analogous to the lag time (35), and studies with
liquid laboratory media have shown that adaptation of plank-
tonically grown L. monocytogenes cells increases with abrupt
environmental shifts (i.e., pH and water activity) (35, 46). Cell
adaptation is an important issue in challenge studies, since
preparation of inocula under conditions different from those
encountered in the targeted food could result in underestima-
tion of bacterial growth and survival. In addition, the observa-
VOL. 75, 2009 L. MONOCYTOGENES BIOFILM FORMATION AND DETACHMENT
FIG. 4. Detachment of Listeria monocytogenes cells from SS coupons habituated in yogurt for 7 days at 5°C and subsequent growth in milk (f)
or custard (Œ) at 5°C. Error bars represent standard deviations for duplicate samples from two independent experiments (n ⫽ 4). Dashed lines
indicate sampling points at which counts were below the detection limit (1 log CFU/ml and 2 log CFU/g for milk and custard, respectively) by direct
plating.
tion that detached cells from milk exhibited faster growth than
their suspended counterparts underlines the importance of the
physiological or metabolic history of cells for their subsequent
growth (35, 46). It can be speculated that in the case of con-
tamination, the risk of L. monocytogenes growth may be higher
when bacteria are derived from biofilms rather than from re-
sidual liquids or product waste (e.g., whey or purge). There-
fore, studies evaluating the effect of preservation treatments or
modeling of microbial responses should also consider cells
detached from biofilms.
Attached L. monocytogenes cells habituated in yogurt
seemed to detach from the SS coupons and migrate to the
favorable medium of milk or custard at 5°C (Fig. 4). This
observation suggests that the bead vortexing procedure and/or
the conventional plating technique used in this study could
underestimate the attached microbial population on SS cou-
pons and thus the actual risk of contaminated surfaces (1). It
has also been suggested that in order to survive under adverse
environmental conditions, many food-borne pathogens enter a
viable but nonculturable state (i.e., a dormant state) (51) in
which they maintain metabolic activity and pathogenicity but
cannot be cultured by common laboratory methods (39). This
conclusion is supported by a previous study demonstrating that
cells that were metabolically active but not culturable by con-
ventional plating techniques (detection limit, 1.3 log10 CFU/
cm2) remained attached to the SS coupons after the bead
vortexing (19). Furthermore, custard was more favorable than
milk for detachment of L. monocytogenes from soiled surfaces
and proliferation (Fig. 4). This may be associated with the
structural heterogeneity and difference in nutrient composition
that contributed to the faster detachment and proliferation of
custard-derived cells. Moreover, the density, structure, and
strength of the attached microbial population are among the
factors influencing bacterial detachment (21, 22, 36).
In conclusion, the results of this work indicate that L. mono-
cytogenes cells attached to equipment surfaces may constitute a
7187
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significant risk due to possible detachment and multiplication
in food products. Moreover, detached cells may exhibit a
higher tolerance to stressful conditions than cells in suspen-
sion, which depends on the growth environment before and
after their transfer. Detached cells, although old or injured,
still pose a major public health threat to the food industry.
Food soil, as ecological background, does affect both the
growth and behavior of L. monocytogenes, and this effect differs
between planktonic and biofilm states of growth. However,
future research should evaluate similar contamination scenar-
ios with multiple L. monocytogenes strains, also assessing ge-
notypic changes due to attachment and detachment of the
bacterium. This would improve understanding of the mecha-
nisms that allow persistence of L. monocytogenes in dairy
plants.
ACKNOWLEDGMENTS
This work was funded by the European Union Integrated project
“BIOTRACER: Improved bio-traceability of unintended microorgan-
isms and their substances in food and feed chains” (proposal/contract
no. 036272). E. D. Giaouris acknowledges the Greek State Scholar-
ships Foundation (I.K.Y.) for providing a fellowship for postdoctoral
studies.
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