LWT - Food Science and Technology 92 (2018) 477–483

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LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Listeria innocua and Listeria monocytogenes strains from dairy plants behave T
similarly in biofilm sanitizer testing
Annalisa Costaa,b, Antonio Lourencob,1, Tiziana Civeraa, Luísa Britob,∗
a
Dipartimento di Scienze Veterinarie, University of Turin, Grugliasco, TO, Italy
b
LEAF-Linking Landscape, Environment, Agriculture and Food/DRAT-Departamento dos Recursos Naturais, Ambiente e Território, Instituto Superior de Agronomia,
University of Lisbon, 1349-017, Lisbon, Portugal

A R T I C L E I N F O A B S T R A C T

Keywords: Five L. innocua and five L. monocytogenes, including persistent and non-persistent isolates collected from
Listeria innocua Gorgonzola processing plants, were compared regarding their biofilm-forming ability and biofilm susceptibility
Listeria monocytogenes to two hydrogen peroxide (HP) based disinfectants in use at the plants. No significant difference in biofilm-
Biofilms forming ability by both species was observed (P > 0.05) in crystal violet and viable count assays. The sus-
Disinfectants
ceptibility to disinfectants was determined in simulated clean and soiled biofilm forming conditions, by growing
Surrogate
biofilms, respectively, in 1/10 diluted TSB-YE and in TSB-YE. The results showed no significant differences
between species or conditions (P > 0.05) regardless of whether the isolates were classified as persistent or non-
persistent. A hierarchical clustering based on Principal Component Analysis performed on the tested variables,
indicated the presence of two major clusters. Isolates from both species were allocated in both clusters, sug-
gesting that they behaved in a similar way in response to the tested conditions. This study showed that biofilms
of L. innocua could monitor the effectiveness of HP-based disinfectants. Moreover, biofilms of L. innocua from the
same environment could be used as surrogates of L. monocytogenes in sanitizer-based biofilm eradication trials
simulating dairy processing environments, whenever the use of the pathogen is not an option.

1. Introduction sanitizer susceptibility rendering valuable data to predict L. mono-

cytogenes behavior. In fact, L. innocua has already been proposed as a
Listeria innocua and Listeria monocytogenes have been isolated from surrogate, although not in biofilm state, in order to predict the response
the same foods (Lappi et al., 2004; Moshtaghi & Mohamadpour, 2007; of L. monocytogenes to chemical and physical stresses (Delaquis, Stanich,
Simmons et al., 2014; Vongkamjan, Fuangpaiboon, Turner, & & Toivonen, 2005; Fairchild & Foegeding, 1993; Friedly et al., 2008;
Vuddhakul, 2016) and food processing environments (Chambel et al., Silva-Angulo et al., 2015).
2007; Lappi et al., 2004; Nucera, Morra, & Grassi, 2011; Rørvik, L. monocytogenes is a concern in the production of Gorgonzola
Caugant, & Yndestad, 1995). Sauders et al. (2012) found that both cheese, an Italian blue-veined cheese made of pasteurized cow's milk.
species were associated with urban environments, in contrast with Some L. monocytogenes strains can persist in Gorgonzola processing
Listeria seeligeri and Listeria welshimeri that were associated with natural plants, suggesting niche adaptation to the dairy environment
environments. Also phylogenetic analysis showed that L. innocua and L. (Lomonaco et al., 2009). The reasons for persistence are not known, but
monocytogenes are closely related species (den Bakker et al., 2010; strong biofilm forming ability and disinfectant susceptibility do not
Glaser et al., 2001; Schmid et al., 2005). seem to be prerequisites (Costa, Bertolotti, Brito, & Civera, 2016). The
Reviews on L. monocytogenes sanitizer susceptibility and persistence present study aimed to investigate whether L. innocua biofilms may be
have pointed out the difficulty to extrapolate from laboratory-based used as surrogates of the pathogenic L. monocytogenes biofilms. Persis-
results to a food processing environment and the need to better un- tent and non-persistent L. innocua and L. monocytogenes isolates from
derstand the involved mechanisms (Carpentier & Cerf, 2011; Ferreira, Gorgonzola processing environments were selected and their biofilm-
Wiedmann, Teixeira, & Stasiewicz, 2014). Since L. innocua is non- forming ability as well as biofilm susceptibility to two hydrogen per-
pathogenic, it might be possible to employ this species directly in trials oxide (HP) based disinfectants in use at the dairies were compared.
in processing plant environments to investigate its persistence and

∗
Corresponding author. Laboratório de Microbiologia, DRAT, Instituto Superior de Agronomia, University of Lisbon, Tapada da Ajuda, 1349-017, Lisbon, Portugal.
E-mail addresses: annalisa.costa@unito.it (A. Costa), antonio.lourenco@teagasc.ie (A. Lourenco), tiziana.civera@unito.it (T. Civera), lbrito@isa.ulisboa.pt (L. Brito).
1
Current address: Food Safety Department, Teagasc Food Research Centre, Moorepark, Fermoy, Cork, Ireland.

https://doi.org/10.1016/j.lwt.2018.02.073
Received 28 November 2017; Received in revised form 22 January 2018; Accepted 28 February 2018
Available online 01 March 2018
0023-6438/ © 2018 Elsevier Ltd. All rights reserved.
A. Costa et al. LWT - Food Science and Technology 92 (2018) 477–483

Table 1 two distinct biofilms.

Listeria innocua and Listeria monocytogenes isolates from Gorgonzola cheese processing
plants used in this study.
2.3. Data analysis
Species Isolate ID Collection year Reference
Agreement to a normal distribution of the data generated by the CV
Listeria innocua 1 2008 Nucera et al. (2011) (A600), enumeration on SSC (Log CFU/cm2), and biofilm susceptibility
2 2009
(reduction of log CFU/cm2) assays was checked using the Shapiro-Wilk
3 2009
4 2011 This work test, and the homogeneity of the variance was confirmed by Levene's
5 2011 test. Comparisons between means were then performed via one-way
Listeria monocytogenes GI 2007 Nucera et al. (2013); Costa ANOVA, using Scheffé test. These analyses were performed with the
G39 2005 et al. (2016) OriginPro 8 SR0 (Northampton, MA, USA) software.
99 2005
Using JMP Pro 13.2.1 (SAS, Cary, NC USA) software, a principal
GN 2007
GR 2007 component analysis (PCA) was performed in order to compare the
isolates, based on their response to the ten following conditions of log
Isolates in bold represent persistent isolates showing the same or highly correlated ge- CFU/cm2 reduction on SSC after disinfectant (P3 or MS) treatment of
netic profile (similarity level ≥95%) in different visits within the collection period. biofilms grown in clean (C) and soiled (S) conditions, and of biofilm
formation: 1) P3 2.5 min 0.2% C; 2) P3 2.5 min 0.2% S; 3) P3 5 min
2. Materials and methods 0.2% C; 4) P3 5 min 0.2% S; 5) MS 2.5 min 0.5% C; 6) MS 2.5 min 0.5%
S; 7) MS 5 min 0.5% C; 8) MS 5 min 0.5% S; 9) biofilm formation by CV;
2.1. Bacterial isolates and 10) biofilm formation by enumeration on SSC. A hierarchical
clustering using the Ward method was performed based on the com-
The bacterial isolates used in this work, five L. innocua and five L. ponent 1, which explained the majority of the variance.
monocytogenes, were isolated from Gorgonzola cheese processing plants For all tests, the confidence level for significance was 95%
and in their milk supplying farms, located in Piedmont and Lombardy, (P < 0.05).
Italy (Table 1). Isolates were classified as persistent if they were found
repeatedly within the collection periods indicated in Table 1, and were 3. Results
at least 95% similar according to repetitive element sequence-based
PCR assays (rep-PCRs) (Nucera, Lomonaco, Costa, Morra, & Grassi, 3.1. Comparison of the biofilm-forming ability of the isolates
2013; Nucera et al., 2011) using ERIC and REP primers (Versalovic,
Koeuth, & Lupski, 1991). The biofilm formation of L. innocua isolates ranged from 0.117 to
0.170 (A600) using the CV method while the enumeration method re-
2.2. Evaluation of biofilm-forming ability and biofilm susceptibility gistered values between 6.20 and 6.69 log CFU/cm2 (Fig. 1). When L.
innocua isolates biofilm-formation values are presented alongside L.
The evaluation of biofilm-forming ability was performed by quan- monocytogenes values (Costa et al., 2016), it is possible to observe that L.
tification of biofilm biomass on microtiter plates (polystyrene), using innocua presented a similar range for both the CV method
both the crystal violet (CV) method (Borucki, Peppin, White, Loge, & (0.087–0.270) and enumeration method (5.65–6.74 log CFU/cm2)
Call, 2003) and a method of enumeration of viable cells on stainless (Fig. 1).
steel coupons (SSC), as described by Costa et al. (2016). Biofilms were The CV method showed no significant differences between isolates
grown in tryptic soy broth with 0.6% yeast extract (TSB-YE, Biokar nor between species (P > 0.05, Fig. 1B). The same outcome was ob-
Diagnostics, Beauvais, France) at 25 °C for 24 (CV) and 48 h (SSC). The tained for the enumeration on SSC, except for L. monocytogenes isolate
CV approach was replicated at least three times on biologically in- GR, that was significantly different from L. monocytogenes isolates GI
dependent cultures on distinct days (biological replicates), with six and GN and from L. innocua isolate 2, with P-values of 0.011, 0.009 and
repetitions under identical conditions each (technical replicates). For 0.016, respectively (Fig. 1A).
the cell enumeration on SSC, two biological replicates were performed,
with two technical replicates each. 3.2. Comparison of the antibiofilm activity of the disinfectants
The evaluation of the biofilm susceptibility to the disinfectants was
performed on biofilms grown on SCC in nutrient-limiting (1/10 diluted The log reductions obtained after the treatment of L. innocua bio-
TSB-YE) and in nutrient-rich (TSB-YE) medium at 25 °C for 48 h, as films (light dots) with the disinfectants P3 and MS are shown in Fig. 2.
described by Costa et al. (2016). Two commercial HP-based disin- In order to allow comparison between species, data from L. mono-
fectants, commonly employed at the dairies where the isolates were cytogenes isolates (dark dots) (Costa et al., 2016) are also shown. Among
collected, were used: P3-oxonia active (ECOLAB S.r.l) containing acetic the isolates that did not reach the 4-log reduction threshold, and for
acid and peracetic acid (designated herein as P3), and Mida San 315 that reason were consequently exposed to longer treatments and/or
(Christeyns Food Hygiene S.r.l) containing citric acid (designated higher concentrations of P3 and MS, L. monocytogenes and L. innocua
herein as MS). Disinfectants were diluted using sterile hard water, persistent and non-persistent isolates were found. This was observed
prepared according to EN 13697 (European Standard EN 13697, 2001), when biofilms were grown both in nutrient-limiting (1/10 diluted TSB-
to achieve the concentrations recommended by the manufacturers: YE) and in nutrient-rich (TSB-YE) conditions (Fig. 2A-I, quadrants II, III
0.2% (v/v) and 0.5% (v/v) for P3, and 0.5% (v/v) and 1% (v/v) for MS. and IV). Moreover, when the log reduction values obtained with both
Disinfectant efficiency was defined according to EN 13697 (European disinfectants at all the tested concentrations and contact times were
Standard EN 13697, 2001), that states that a minimum 4-log reduction compared, there were no significant differences between isolates nor
in viable cells is required. If that threshold was obtained in 2.5 min with between species (P > 0.05). The exception was the comparison of
the lowest concentration, no additional treatments were performed. susceptibility to P3 at the mildest exposure conditions (0.2% [v/v] for
When a 4-log reduction was not achieved, 5- and 7.5-min treatments 2.5 min) in which L. monocytogenes presented a higher log reduction (P-
with higher disinfectant concentrations, when needed, were carried out value of 0.0497).
until a 4-log reduction was observed. For each isolate, a control was The disinfectant P3 showed a greater efficacy than MS at 7.5 min of
exposed to sterile hard water and used for the calculation of the log exposure and at the lowest concentration indicated by the manufacturer
reduction. Each treatment was repeated under identical conditions on (0.2% [v/v]), in fact P3 was effective for all the isolates Fig. 2A-C) and

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These were the treatments applied to the 10 isolates, since isolates that
after these treatments reached the 4-log reduction threshold were not
subsequently exposed.
By PCA, the initial 10-dimensional space (10 variables) was reduced
to a plane F1F2, defined by the two first principal components. This
plane accounts for about 68.5% of the variance of the original data
(Fig. 3). The projection of the 10 original variables on the first two
principal components is presented in Fig. 3A. Except for the treatment
with P3 for 5 min at 0.2% in both clean and soiled conditions, in gen-
eral, disinfectant susceptibility (log reduction) shows a positive corre-
lation with the first component increasing along it. The second prin-
cipal component is positively correlated with both CV and SSC values,
i.e. biofilm production increase along this axis.
The projection of the different isolates in the plane F1F2 is pre-
sented in Fig. 3B. The isolates in quadrant I (2, GN, GI and 4) and in
quadrant II (1) are more susceptible to P3 as all of them reached the 4-
log reduction threshold after exposure for 5 min to 0.2% (v/v) (Figs. 3,
and Fig. 2B). Isolates in quadrants III (99, 5, 3 and G39) and IV (GR)
were less susceptible to P3. These isolates only achieved the 4-log re-
duction after a 7.5 min exposure to the same concentration (Figs. 3, and
Fig. 2C). Isolate GR can be seen isolated on quadrant IV as it presented
the higher log reductions in both clean and soiled condition for the
eight treatments used to perform the PCA (Figs. 3, and Fig. 2A, B, D and
E). Moreover, isolate GR is worst biofilm producer than three (2, GN
and GI) of the four isolates positioned in quadrant II (Figs. 3 and 1).
A hierarchical clustering, based on the first principal component
which explains the majority (43.9%) of the variance (Fig. 3) was per-
formed and allowed to confirm the presence of the three clusters of
isolates (C1, C2 and C3) (Fig. 4), as suggested by PCA. A two-way
clustering was performed. According to the intensity of the response of
each isolate in every experiment, the treatments with P3 for 5 min at
0.2%, in both clean and soiled conditions, were the ones that allowed
the most differentiation of the isolates. It is also possible to observe
that, persistent and non-persistent isolates from both species L. innocua
and L. monocytogenes were allocated in both clusters, confirming that
both species respond in a similar way to the conditions tested in this
work.

4. Discussion

Fig. 1. Biofilm forming ability of Listeria monocytogenes (grey bars) and Listeria
The use of Listeria spp. as an indicator of a possible contamination
innocua (white bars). Oblique line bars represent persistent isolates; A - assessed by
by L. monocytogenes has been suggested by some authors and guidelines
cell enumeration on SSC (grown in TSB-YE for 48 h at 25 °C). The isolate GR, only on
region 1, is statistically different from isolates 2, GI and GN, on region 3 (regions marked (Food and Drug Administration [FDA], 2008; Pennsylvania State
on the right). The isolates on region 2 are neither statistically different from isolates on University [Penn State], 2003; Tompkin, Scott, Bernard, Sveum, &
region 1 nor from region 3. Two biological replicates with two technical replicates each Gombas, 1999). This may be a conservative approach since in Gor-
were performed; B - assessed by crystal violet (CV) method in polystyrene 96-well mi- gonzola processing plant L. innocua was far more frequent than L.
crotiter plates (grown in TSB-YE for 24 h at 25 °C). Isolates were not statically different monocytogenes (Nucera et al., 2001). In fact, repeated positive testing
(P > 0.05). At least three biological replicates were performed, with six technical re-
for Listeria spp. requires more stringent cleaning and disinfecting pro-
plicates, each. Results from L. monocytogenes were previously published by Costa et al.
(2016) and are shown here only for comparison. Error bars represent standard deviations.
cedures and indicates the need to elucidate the reasons for these posi-
tive results.
Meylheuc, Giovannacci, Briandet, and Bellon-Fontaine (2002) have
the treatment with the higher concentration (0.5% [v/v]) was not re- compared the bioadhesive behavior of both species and concluded that
quired. For the same exposure time, the lowest concentration of MS the non-pathogenic strain exhibited a more marked electronegative
(0.5% [v/v]) was not enough to achieve a 4-log reduction by all the character and a slightly more hydrophilic nature than L. monocytogenes.
isolates (Fig. 2D-F) and a higher concentration (1% [v/v]) was needed Nevertheless, to our knowledge, only two studies have previously
(Fig. 2G-I). compared the biofilm production of L. monocytogenes and L. innocua:
Zhou et al. (2011) used the CV method and concluded that L. innocua is
3.3. Comparison of L. monocytogenes and L. innocua by PCA a weaker biofilm former compared to L. monocytogenes; Koo,
Ndahetuye, O'Bryan, Ricke, and Crandall (2014) used the cell enu-
Principal Component Analysis (PCA) of data from the 10 isolates meration method on aluminum and stainless steel and concluded that
regarding biofilm forming ability (CV and SSC) and disinfectant (P3 and after 24 h the attachment of L. monocytogenes was significantly higher
MS) susceptibility was performed. Data were from biofilms produced in than that of L. innocua, though no significant differences were observed
nutrient-limiting (clean) (1/10 diluted TSB-YE) and in nutrient-rich between both species biofilms after 72 h. In the present work, two
(soiled) (TSB-YE) conditions. The log reduction values of the 10 isolates methods were used (CV in polystyrene P96 microtiter plates and enu-
exposed to the mildest disinfectant concentrations (0.2% for P3 and meration on SSC) to evaluate the biofilm forming ability of a set of L.
0.5% for MS) for 2.5 and 5 min were considered (Fig. 2A, B, D and E). innocua and L. monocytogenes isolates fairly representative of the

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Fig. 2. Log reductions (log CFU/cm2) obtained after disinfectant treatment of L. innocua (light dots) and L. monocytogenes (dark dots) biofilms grown for 48 h at 25 °C on SSC,
in clean conditions (1/10 TSB-YE; x-axis) and soiled conditions (TSB-YE; y-axis), using P3 at 0.2% or MS at 0.5% and 1%. The isolates placed in quadrant III did not reach the 4-
log reduction neither with biofilms formed in soiled conditions nor with biofilms formed in clean conditions and further treatments with extended contact time/increased disinfectant
concentration were performed, as needed. The isolates placed in quadrant I reached the reduction threshold of 4 logs, with biofilms formed in both conditions and, for this reason, no
other treatments were performed. The isolates placed in quadrant II and IV reached the 4 log reduction threshold, respectively, only in soiled or clean biofilm forming conditions. Further
treatments respectively with biofilms grown in clean and soiled conditions, were carried out in order to achieve the 4-log reduction in both conditions. Isolates that underwent treatment
only with biofilms produced under clean/soiled condition are marked with an asterisk (*) and placed on x- or y-axis, respectively. Isolates' ID in bold represent persistent isolates. Results
from L. monocytogenes were previously published by Costa et al. (2016) and are shown here only for comparison. Error bars represent standard deviations. For each treatment, two
technical replicates were performed.

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Fig. 3. Principal Component Analysis. A - Projection of the 10 variables on the plane F1F2 (first and second principal components). B- Projection of the 10 isolates on the plane F1F2.
Variables of biofilm disinfectant (P3 or MS) susceptibility in clean (C) and soiled (S) conditions: P3 2.5 min 0.2% C; P3 2.5 min 0.2% S; P3 5 min 0.2% C; P3 5 min 0.2% S; MS 2.5 min
0.5% C; MS 2.5 min 0.5% S; MS 5 min 0.5%C; MS 5 min 0.5% S. Variables of biofilm formation: by CV and by enumeration on SSC.
Isolates: five L. innocua (1, 2, 3, 4 and 5) (light dots) and five L. monocytogenes (GI, G39, 99, GN and GR) (dark dots). Isolates in bold represent persistent isolates.

contaminant microorganisms, collected from Gorgonzola processing suggest the effectiveness of the sanitation procedures in place in the
plants. The CV assay relies on the property of the dye to bind to ne- processing plant. Conversely, the detection of positive samples for Lis-
gatively charged surface molecules and polysaccharides in the matrix. teria spp. would indicate a need of improving the procedures to keep the
This assay may be influenced by the amount of exocellular polymer and environmental contamination under control, as suggested by Tompkin
by cell sedimentation, which increases with planktonic growth et al. (1999). Moreover, Zitz, Zunabovic, Domig, Wilrich, and Kneifel
(Lourenco, Rego, Brito, & Frank, 2012). This may explain data varia- (2011) also verified reduced detectability of L. monocytogenes in the
bility obtained with this method. However, the comparison performed presence of L. innocua mainly due to the overgrowth of L. monocytogenes
here using the data obtained with both methods indicated that the two by L. innocua during the selective enrichment, leading to false-negative
species produced similar values, suggesting an equivalent biofilm pro- results. Furthermore, the use of Listeria spp. as an indicator of a po-
duction. tential L. monocytogenes contamination represents lower costs for rou-
Regarding disinfectant susceptibility, Best, Kennedy, and Coates tine laboratory analysis, due to the higher cost of chromogenic media
(1990) tested the efficacy of 14 disinfectants against both species after used for L. monocytogenes (Tompkin, 2002).
been spotted onto the surface of stainless steel disks and dried for The presented work tried to mimic food industry conditions.
30 min. The obtained results showed that the pathogenic species was Consequently, susceptibility testing was conducted with biofilms pro-
slightly less susceptible to disinfection than L. innocua. A few other duced on stainless steel, in soiled and in clean conditions, and testing
comparative studies have assessed differences in the susceptibility of two commonly used HP-based disinfectants at the dairy plants where
planktonic cells of both species to disinfectants finding no differences the isolates were collected from. Nevertheless, in the real industrial
between species (Margolles, Mayo, & de los Reyes-Gavilán, 2000) or environment the strains will form multi-species biofilms and that may
highlighting an higher resistance of planktonic L. innocua (Yeater, become relevant to the response of both L. monocytogenes and L. innocua
Kirsch, Taylor, Mitchell, & Osburn, 2015). to disinfectants.
In this work, using L. innocua and L. monocytogenes collected from
the same environment, a similar susceptibility to the tested disin-
fectants was found for biofilms of both species. Moreover, the sus- 5. Conclusion
ceptibility to P3 and to MS of biofilms grown in conditions mimicking
clean and soiled environment, showed no significant differences in This study has shown that biofilms of L. innocua isolated from the
terms of log reduction between persistent and non-persistent isolates. same environment could be employed for the validation and monitoring
According to these results, L. innocua could be used as a surrogate for L. of HP-based disinfectant efficacy and proper sanitation procedures in
monocytogenes, not only regarding the biofilm production, but also the Gorgonzola processing plants. In fact, not only L. innocua susceptibility
biofilm susceptibility to HP-based disinfectants. to HP-based disinfectants is similar to L. monocytogenes biofilms, but
L. innocua was the only Listeria species besides L. monocytogenes also both species, collected from the same food industry environment,
detected in the Gorgonzola processing plants from where the isolates showed no differences in biofilm forming ability. The common origin of
analyzed in this work were collected (Nucera et al., 2011). Our results the isolates is probably fundamental when looking for the adequacy of
demonstrate that the presence of L. innocua could indicate a con- L. innocua as a surrogate of L. monocytogenes. The convenience and
tamination by L. monocytogenes, since the disinfectant susceptibility of safety in using a non-pathogenic surrogate will certainly contribute to
the two species was similar. In fact, as concluded in the review by clarify the factors that contribute to L. monocytogenes persistent colo-
Milillo et al. (2012), if the two species have adapted to fit different nization not only in some Gorgonzola processing plants but also in
environmental niches, they may not always respond to stress the same other food industry environments.
way. Therefore, the absence of Listeria spp. (i.e. L. innocua and L.
monocytogenes) on food contact surfaces, equipment and floors would

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Fig. 4. Hierarchical clustering based on the component 1 of the principal component analysis of the 10 variables in study: Biofilm formation by CV; Biofilm formation by
enumeration on SSC; Biofilm susceptibility to disinfectant (P3 or MS) in clean (C) and soiled (S) conditions for different exposure times (2.5 and 5 min) and concentrations (0.2% and
0.5%). The five isolates of L. innocua are 1, 2, 3, 4 and 5, and the five L. monocytogenes isolates are GI, G39, 99, GN and GR. Isolates 1, 2, G39 and GI are persistent isolates. The intensity of
the colors is proportional to the value registered for each isolate (increasing values from light to dark).

Funding formation among strains of Listeria monocytogenes. Applied and Environmental

Microbiology, 69, 7336–7342.
Carpentier, B., & Cerf, O. (2011). Review-Persistence of Listeria monocytogenes in food
This work was supported by the Italian Ministry of University and industry equipment and premises. International Journal of Food Microbiology,
Research (Progetto Giovani) and Piedmont Region (Progetto BIOPACK- 145, 1–8.
175-1). It was also partially supported by funds allocated to the LEAF/ Chambel, L., Sol, M., Fernandes, I., Barbosa, M., Zilhão, I., Barata, B., et al. (2007).
Occurrence and persistence of Listeria spp. in the environment of Ewe and cow's milk
ISA-University of Lisboa and to the University of Turin. cheese dairies in Portugal unveiled by an integrated analysis of identification, typing
and spatial-temporal mapping along production cycle. International Journal of Food
Declarations of interest Microbiology, 116, 52–63.
Costa, A., Bertolotti, L., Brito, L., & Civera, T. (2016). Biofilm formation and disinfectant
susceptibility of persistent and nonpersistent Listeria monocytogenes isolates from
None. Gorgonzola cheese processing plants. Foodborne Pathogens and Disease, 13, 602–609.
Delaquis, P., Stanich, K., & Toivonen, P. (2005). Effect of pH on the inhibition of Listeria
spp. by 3 vanillin and vanillic acid. Journal of Food Protection, 68, 1472–1476.
Acknowledgments
European Standard EN 13697 (2001). Chemical disinfectants and antiseptics – qualitative
non-porous surface test for the evaluation of bacteriocidal and/or fungicidal activity of
The authors thank to Ana Carla Silva for the support with the work chemical disinfectants used in food, industrial, domestic and institutional areas – test
performed in Laboratory of Microbiology (ISA/ULisboa). method and requirements without mechanical action (phase 2, step 2).
Fairchild, T. M., & Foegeding, P. M. (1993). A proposed nonpathogenic biological in-
dicator for thermal inactivation of Listeria monocytogenes. Applied and Environmental
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