Impact of environmental factors on the culturability and viability of Listeria monocytogenes under conditions encountered in food processing plants

Accepted Manuscript Impact of environmental factors on the culturability and viability of Listeria monocytogenes under conditions encountered in food processing plants

Anaïs Overney, Joséphine Jacques-André-Coquin, Patricia Ng, Brigitte Carpentier, Laurent Guillier, Olivier Firmesse PII: DOI: Reference:

S0168-1605(16)30660-2 doi: 10.1016/j.ijfoodmicro.2016.12.012 FOOD 7478

To appear in:

International Journal of Food Microbiology

Received date: Revised date: Accepted date:

12 September 2016 15 December 2016 19 December 2016

Please cite this article as: Anaïs Overney, Joséphine Jacques-André-Coquin, Patricia Ng, Brigitte Carpentier, Laurent Guillier, Olivier Firmesse , Impact of environmental factors on the culturability and viability of Listeria monocytogenes under conditions encountered in food processing plants. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Food(2016), doi: 10.1016/ j.ijfoodmicro.2016.12.012

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ACCEPTED MANUSCRIPT Impact of environmental factors on the culturability and viability of Listeria monocytogenes under conditions encountered in food processing plants

Anaïs Overney - Joséphine Jacques-André-Coquin - Patricia Ng - Brigitte Carpentier -

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Laurent Guillier - Olivier Firmesse*

Université Paris-Est, Anses, Laboratory for Food Safety, 94701 - Maisons-Alfort, France

Corresponding author:

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*

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Tel.: +33 1 49 77 22 67; Fax: +33 1 49 77 46 66; E-mail address: [email protected];

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Postal address: 14, rue Pierre et Marie Curie - 94701 Maisons-Alfort Cedex.

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ACCEPTED MANUSCRIPT Abstract The ability of Listeria monocytogenes to adhere to and persist on surfaces for months or even years may be responsible for its transmission from contaminated surfaces to food products. Hence the necessity to find effective means to prevent the establishment of L. monocytogenes in food processing environments. The aim of this study was to assess, through a fractional

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experimental design, the environmental factors that could affect the survival of

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L. monocytogenes cells on surfaces to thereby prevent the persistence of this pathogen in

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conditions mimicking those encountered in food processing plants: culture with smoked salmon juice or meat exudate, use of two materials with different hygiene status, biofilm of

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L. monocytogenes in pure-culture or dual-culture with a Pseudomonas fluorescens strain, application of a drying step after cleaning and disinfection (C&D) and comparison of two

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strains of L. monocytogenes. Bacterial survival was assessed by culture, qPCR to quantify total cells, and propidium monoazide coupled with qPCR to quantify viable cells and

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highlight viable but non-culturable (VBNC) cells. Our results showed that failure to apply

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C&D causes cell persistence on surfaces. Moreover, the sanitation procedure leads only to a loss of culturability and appearance of VBNC populations. However, an additional daily

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drying step after C&D optimises the effectiveness of these procedures to reduce culturable populations. Our results reinforce the importance to use molecular tools to monitor viable

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pathogens in food processing plants to avoid underestimating the amounts of cells using only methods based on cell culture.

Keywords: Listeria monocytogenes, persistence, survival, drying, cleaning & disinfection, viable but non culturable

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ACCEPTED MANUSCRIPT 1. Introduction Listeria monocytogenes is a zoonotic pathogen causing the rare but serious listeriosis (Donovan, 2015). This foodborne disease primarily affects the developing fetus during pregnancy and immunosuppressed individuals, leading to death in 15-30% of cases (Goulet et al., 2012; Rocourt, 1996). Outbreaks are generally due to consumption of ready-to-eat

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products (Rocourt et al., 2003; Tompkin, 2002), with contamination occurring throughout the

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food chain. L. monocytogenes is in fact distributed in many environments, including food

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processing plants.

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To eliminate this pathogen in factories, cleaning and disinfection (C&D) procedures are performed daily and are some of the most important measures for L. monocytogenes risk

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control (Jessen and Lammert, 2003; Rotariou et al., 2014). However, failure of C&D procedures have been reported to result in cases of survival of L. monocytogenes in several

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food processing environments causing the apparition of persistent strains (Autio et al., 2003;

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Ferreira et al., 2014; Lundén et al., 2003b; Møretrø and Langsrud, 2004; Tompkin, 2002; Wulff et al., 2006). Persistence can be defined as the repeated isolation of organisms of a

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specific molecular subtype from the same processing environment despite the frequent and correct application of C&D procedures (Carpentier and Cerf, 2011; Keto-Timonen et al.,

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2007).

Thus, strains of L. monocytogenes can remain on equipment or surfaces in niches (Mettler and Carpentier, 1999) for several months or years (Carpentier and Cerf, 2011; Jessen and Lammert, 2003). These strains may be protected by retention areas and thus exposed to sublethal concentrations of sanitisers. What is of concern is that the presence of persistent cells on food-contact surfaces can be a source of recontamination (Lundén et al., 2002; Reij and 3

ACCEPTED MANUSCRIPT Den Aantrekker, 2004). The risk of cross-contamination by transfer of cells from surfaces and equipment to food products is a major threat to the food industry and consumers.

One hypothesis to explain persistence is the ability of bacteria to adapt to and survive environmental stresses such as nutrient deprivation, heat or cold temperatures, sanitisers and preservatives, desiccation, low pH, and high salt concentrations (Carpentier and Cerf, 2011;

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Melo et al., 2015; Thévenot et al., 2006). This ability to persist makes contamination difficult

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to control and eradicate. Elimination is even more difficult as cells adhere to several materials

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found in food processing environments, where they grow and form biofilms (Donlan, 2002; Møretrø and Langsrud, 2004; Pereira da Silva and Pereira De Martinis, 2013; Ronner and

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Wong, 1993). Microbial populations growing in biofilms can be difficult to destroy because they often have greater disinfectant resistance than planktonic organisms (Nakamura et al.,

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2013; Pan et al., 2006). Consequently, it is important to find effective means to prevent the establishment of L. monocytogenes in food processing environments to avoid biofilm

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formation and bacterial persistence.

Moreover, the multitude of adverse environmental conditions in food processing environments can lead to the appearance of viable but non-culturable (VBNC) cells that have

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lost their ability to form colonies on conventional culture media but remain alive (Li et al.,

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2014a; Oliver, 2005). Given that detection of L. monocytogenes in surface samples from food processing environments is based only on conventional microbiological methods, VBNC cells represent a real risk because they are not detected but are potentially a source of contamination of food products.

The aim of this study was to test environmental factors commonly found in food processing plants to determine their impact on the survival and persistence of L. monocytogenes cells. Environmental factors included (i) comparison of two different strains of L. monocytogenes 4

ACCEPTED MANUSCRIPT strongly and weakly resistant to desiccation, (ii) use of two surface materials with a different hygiene status for biofilm formation, (iii) biofilm of L. monocytogenes in pure-culture biofilm or in dual-culture biofilm with a Pseudomonas fluorescens strain that promotes its adhesion, (iv) use of smoked salmon juice or meat exudate as culture medium, and (v) application of a drying step daily for a week or only once a week after C&D. For this purpose, sessile cells

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were subjected daily for a week to sanitation procedure and cultured with a food soil simulating in the laboratory the alternation of C&D and production steps. To study the impact

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of these environmental factors on the survival of L. monocytogenes, bacterial concentration

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evolution (the initial population compared to the remaining population after sanitation procedures applied daily for a week) was assessed by cell culture, quantitative PCR (qPCR) to

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quantify total cells, and propidium monoazide (PMA) coupled with qPCR (PMA-qPCR) to

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quantify viable cells and highlight VBNC cells.

2. Experimental procedures

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2.1. Bacterial strains

Two strains of L. monocytogenes were used: LO28 (human isolate, 1/2c serotype) and EGD-e

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(animal isolate, 1/2a serotype). For dual-culture biofilms, L. monocytogenes was cultured in presence of the Pseudomonas fluorescens 09empf87 strain, which enhances the adhesion of

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L. monocytogenes on the surface (unpublished results).

2.2. Preparation of food soils

Smoked salmon juice and meat exudate were prepared as previously described (Midelet and Carpentier, 2004; Overney et al., 2016). Prior to use, the required volume of food soil was thawed, centrifuged (2100 g, 10 min, 20°C) and the supernatant was then filter-sterilised

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ACCEPTED MANUSCRIPT through a 0.22-µm-pore-size Stericup filter with a 2-µm-pore-size prefilter (Millipore, SaintQuentin-en-Yvelines, France).

The meat exudate contained 9.93 g/100 g protein and less than 0.1 g/100 g lipid. The smoked salmon juice contained 1.93 g/100 g protein and 0.14 g/100 g lipid (ISHA, Institut

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Scientifique d’Hygiène et d’Analyse, Longjumeau, France).

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2.3. Culture conditions

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Long-shelf-life stock cultures of the strains used in this study were stored at −80°C. They were used to inoculate tryptone soya agar (TSA, Biomérieux, Bruz, France) slopes incubated

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for 24 h at 25°C. These were then maintained at 4°C for no more than one month until use. The day before each experiment, the refrigerated culture was transferred to another TSA slope

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and incubated for 24 h at 25°C.

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Two protocols were followed according to the food soil used. For pure-culture biofilm

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experiments with smoked salmon juice, L. monocytogenes cells were suspended in TSB/5m, which is the culture medium developed to mimic this food soil (Overney et al., 2016). The optical density (OD) at 600 nm of the suspension was adjusted to 1.5 (corresponding to a

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concentration of about 109 CFU/ml) in a 1.5-cm-diameter tube. This was the bacterial

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suspension used for biofilm formation. For pure-culture biofilm experiments with meat exudate, bacteria were suspended in 1 g/l peptone water (AES Chemunex, Bruz, France) and the OD adjusted as described above. The volume of the suspension necessary for the experiment was then centrifuged at 2100 g for 10 min at room temperature, the supernatant removed, and the pellet suspended in the same volume of filter-sterilised meat exudate.

For dual-culture biofilm experiments, bacterial suspensions were prepared differently. P. fluorescens cells were suspended in 1 g/l peptone water, the OD adjusted to 1.5, and then 6

ACCEPTED MANUSCRIPT diluted to 1:100 (corresponding to a concentration of about 107 CFU/ml) in smoked salmon juice or in meat exudate. L. monocytogenes cells were suspended in 1 g/l peptone water, the OD adjusted to 0.15, and then diluted to 1:1000 (corresponding to a concentration of about 105 CFU/ml) in smoked salmon juice or in meat exudate.

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2.3. Biofilm development

Biofilms were prepared on coupons (4 cm by 2.5 cm) in stainless steel (SS, 2 RB finish,

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AISI 304, Laser 53, Bazougers, France) or in ceramic clinker tiles dry-pressed unglazed

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(Argelith Bodenkeramik, Bad Essen, Germany). SS coupons were rubbed with a mixture of ethanol at 95% and acetone (3V/1V) to remove any manufacturing process debris and grease.

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As previously described by Peneau et al. (2007), a silicon joint sealant (Fischer, France) was

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applied along the edges of each coupon to obtain an area than could be kept flooded for experiments. Each SS and ceramic coupon was then washed and autoclaved (Aase et al.,

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2000; Leriche and Carpentier, 1995). Lastly, SS surfaces were placed in 90-mm-diameter

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Petri dishes. To prevent dehydration of biofilms during incubation, each 90-mm-diameter Petri dish was then placed in a 120-mm-diameter Petri dish containing 25 ml of sterile water. Ceramic coupons were placed into sterilised boxes measuring 26 cm by 15 cm containing

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50 ml of sterile water.

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For pure-culture biofilms on ceramic and SS surfaces, 1 ml of the L. monocytogenes suspension prepared as described above was left on clean coupons. These were incubated at 25°C for 3 h to allow adhesion of bacteria. Then, the non-adherent bacteria were removed by pouring 25 ml of 1 g/l peptone water over the bacterial deposit. A volume of 1 ml of filtersterilised food soil was deposited so as to cover sessile cells. Lastly, coupons were incubated for 21 h at 25°C.

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ACCEPTED MANUSCRIPT For dual-culture biofilms on ceramic and SS surfaces, 1 ml of the P. fluorescens suspension prepared as described above was deposited on coupons. After incubation at 25°C for 3 h, the non-adherent cells were removed as described above. Then, 1 ml of the L. monocytogenes suspension was left. The coupons were then incubated for 21 h at 25°C.

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2.4. Repeated soiling and chemical treatments of the coupons

Repeating soiling and C&D procedures were applied without mechanical action (Marouani-

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Gadri et al., 2010; Peneau et al., 2007). The total duration of the experiments was 4 days

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taking as the starting point the day on which the first C&D was performed. Topax 66 (Ecolab, Arcueil, France) is a chlorinated alkaline cleaner used at a concentration of 3% (vol/vol).

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Triquart MS (Ecolab) is a quaternary ammonium-based disinfectant with a recommended

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concentration of 2% (vol/vol). Both chemical products are frequently used in the meat and fish industries. To obtain surviving populations on SS surfaces and then to study the impact of

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parameters on the survival of L. monocytogenes, Topax 66 and Triquart MS were diluted to

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model what occurs in retention zones that cannot be reached by mechanical action of cleaning and where some water remains before product application (Marouani-Gadri et al., 2010;

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Peneau et al., 2007).

After biofilm preparation and before treatment, the coupons were first rinsed to eliminate non-

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adhering cells by removing the supernatant with a pipette. Then, 1.5 ml of sterile water at 55°C was left for 2 min. The coupons were then treated with two chemical products. First, 1.2 ml of Topax 66 at 0.3% (vol/vol) was deposited on each coupon and left for 15 min at room temperature. The product was then removed in the same way. Afterwards, a rinsing step was performed with 1.5 ml of sterile water at 55°C for 2 min. Then, 1.2 ml of Triquart MS at 0.2% (vol/vol) was deposited on each coupon and left for 30 min at room temperature. Finally, coupons were rinsed with 1.5 ml of sterile water at room temperature for 2 min. 8

ACCEPTED MANUSCRIPT Immediately after C&D, 1 ml of smoked salmon juice or meat exudate, sterilised by filtration, was deposited onto each coupon to simulate the food processing environment and to allow growth. The coupons were then re-incubated at 25°C.

After a C&D procedure and before enumeration, the disinfectant residues were inactivated by depositing 1.5 ml neutraliser, as recommended by ISO Standard 18593 (Anonymous, 2004)

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for 5 min.

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2.5. Drying step

The drying step was performed as described by Zoz et al. (2016). To simulate desiccation in a

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food processing plant, hermetic plastic boxes (20 cm by 13 cm by 6 cm, Lock&Lock, France) were used. Drying chambers were disinfected with 70% ethanol. Then, two small ventilators

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of 4.1 W with a diameter of 80 mm and a height of 25 mm comprising seven blades (Sunon, Radiospares, France) were placed in each box with a voltage of 5 V. A saturated salt solution

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of sodium chloride (VWR, Fontenay-sous-Bois, France) was added at the bottom of the

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drying chambers to adjust the relative air humidity (RH) to 75%. Before each experiment, the container was treated for 15 min under UV light.

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After the rinsing step with water at 55°C as described above, the coupons were deposited in a

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90-mm-petri dish and then placed in a drying chamber on a rack to keep them above the salt solution. Therefore, the drying chamber was opened for just a short time to avoid high variation of the RH. Afterwards, the container was incubated for 3 h at 25°C and the ventilators were turned on for that time. A probe (EL-USB-2-LCD data logger, Dataq Instruments, Akron, USA) was placed in the container to measure the %RH and the temperature during the experiment.

2.6. Sonication 9

ACCEPTED MANUSCRIPT Sessile cells were removed from the surface by sonication which is able to remove more sessile cells than with swabbing with no harm to the cells (Midelet and Carpentier, 2002). Each coupon was immersed in a plastic bottle containing 1 g/l peptone water (20 ml for ceramic coupons and 30 ml for SS coupons) and placed in a Delta220 sonication bath (Deltasonic, Meaux, France). Ultrasounds were generated for 4 min at 30°C (28 kHz, 150 W)

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as described by Asséré et al. (2008).

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2.7. Enumeration of culturable cells

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Suspensions from ultrasound treatments were diluted with 1 g/l peptone water, plated by spiral plating (WASP 2 spiral plater, AES Chemunex) on TSA in the case of pure-culture

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biofilms or on ALOA (Agar for Listeria according to Ottaviani and Agosti, Biomérieux) in

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the case of coculture. Culturable cells (CFU/coupon) were enumerated after incubation of 24 h on TSA or 48 h on ALOA at 37°C.

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2.8. Quantification of total L. monocytogenes cells by qPCR

Samples of 2 ml of suspension obtained after sonication were centrifuged at 13000 g for 3 min at room temperature. The supernatant was removed and the pellet suspended in 200 µl

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of phosphate buffered saline 1X (PBS, Dulbecco A, Oxoid Limited, Hampshire, England,

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UK). Cellular DNA was then extracted using the MagNa Pure LC DNA isolation III kit (Roche Diagnostics, Meylan, France).

Quantitative real-time PCR amplification and detection were performed with a LightCycler® 2.0 System (Roche Diagnostics). Each reaction mixture (25 µl) was composed of 12.5 µl of Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix (Agilent Technologies), 1.5 µl of molecular grade water, 0.5 µl of each hlyA gene DNA primers (Forward:

5’-TGCAAGTCCTAAGACGCCA-3’;

Reverse:

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ACCEPTED MANUSCRIPT CACTGCATCTCCGTGGTATACTAA-3’) (Nogva et al., 2000) used at a final concentration of 200 mM, and 10 µl of DNA solution extracted from the bacterial suspensions. The hlyA gene, encoding listeriolysin O, is a specific gene of L. monocytogenes. The amplification programme consisted of one cycle at 95°C for 10 min and 40 amplification cycles (95°C for 10 s, 60°C for 1 min and 70°C for 1 min). Water was included as a negative control in each

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PCR run.

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For quantification, standard curves were obtained using DNA extracted from serial decimal

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dilutions of an L. monocytogenes suspension prepared as described above in 1 g/l peptone water with known CFU counts (Marouani-Gadri et al., 2010). Standard curves were generated

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by plotting the threshold cycles (Ct) obtained after amplification against the CFU counts of the samples. The efficiency of amplification was at least 85%. The quantification limit was

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4.0 log10(cells/coupon).

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2.9. Quantification of viable L. monocytogenes cells by PMA-qPCR

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Before DNA extraction, propidium monoazide (PMA, Biotium, Hayward, United States) was added to the samples obtained after sonication at a final concentration of 100 µM, following

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the method used by Nocker et al. (2007). PMA is a DNA intercalating agent that penetrates only bacteria with altered membranes (dead cells). Only the PMA-free DNA of viable cells is

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amplified by PCR. Samples were then incubated in the dark at room temperature for 5 min and exposed to light for 5 min, using a PMA-liteTM LED Photolysis Device (Biotium). Samples were then centrifuged at 13000 g for 3 min at room temperature, the supernatant was removed, and the pellet suspended in 200 µl of PBS 1X. Cellular DNA was then extracted using the MagNa Pure LC DNA isolation III kit and amplification by qPCR was performed as described above.

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ACCEPTED MANUSCRIPT 2.10. Statistical analysis

For each experiment, at least two independent replicates were tested. Within each replicate, cell quantification was performed from three coupons.

First, to verify C&D effectiveness and define the conditions needed to obtain surviving cells

using

Statgraphics

Centurion

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software,

(Statpoint

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Technologies Inc., SigmaPlus, Levallois-Perret, France).

version 15.2.00

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(ANOVA)

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on SS surfaces, data were processed statistically through a multi-way analysis of variance

Then, a fractional factorial design was used to assess the impact of environmental factors on

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the ability of L. monocytogenes to survive. A full factorial design corresponds to 64 experiments for the five environmental factors: L. monocytogenes strain (2 levels), surface

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material (2 levels), presence of a strain enhancing L. monocytogenes adhesion (2 levels), food soil (2 levels), and scenario of sanitation procedure (4 levels). Using a fractional factorial

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design of resolution III, 16 experiments were carried out. The asymmetric fractional design

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(as the number of levels differs between factors) was generated with the Planor R package (Kobilinsky et al., 2015; Monod et al., 2012) using R 3.2.1 software. The sixteen

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combinations of factors generated are shown in Table 1; two additional experiments were carried out (Table 1). The lm function of the R 3.2.1 software was used to fit a linear model

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that quantifies the effect of the five environmental factors on δ d1-d4 values. The Anova.lm function was used to test for the significance of effect of factors at the 0.05 level. The package effect for R (Fox et al., 2016) was used to test for differences within levels of each factor. The fitdistcens function of the R fitdistrplus package (Delignette-Muller and Dutang, 2015) was used for levels of cell amounts where at least one of the enumerated coupons present had cell amounts under the detection limits.

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ACCEPTED MANUSCRIPT 3. Results & discussion 3.1. surviving cells on SS surfaces after repeated soiling and C&D treatments daily for a week

3.1.1. Conditions to obtain surviving cells

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To verify the effectiveness of C&D products used for this study in laboratory conditions,

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C&D procedures were carried out daily for a week at the concentrations recommended by the

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manufacturer. Results are presented in Fig. 1. When undiluted C&D products were applied daily for a week on SS surfaces, the decrease of the CFU amount was around

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5 log10(CFU/coupon), and all enumeration values were below the detection threshold

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(2.8 log10(CFU/coupon)).

To obtain surviving populations on SS surfaces and then to study the impact of parameters on

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the survival of L. monocytogenes, Topax 66 and Triquart MS were diluted. With the C&D

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products diluted to 1:10, 44% of the values were higher than the detection threshold (Fig. 1). Moreover, this dilution was the smallest for which we had a population on SS surfaces after

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the application of C&D procedures daily for a week (data not published). The use of C&D products diluted to 1:10 enabled us to obtain surviving cells on SS surfaces in laboratory

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conditions. As reviewed by Martínez-Suárez et al. (2016), in food processing environments, dosage failure, such as disinfection of wet surfaces, can lead to sub-inhibitory concentrations of disinfectants, thus reducing their effectiveness.

3.1.2. Physiological state of sessile cells after repeated soiling and C&D treatments daily for a week

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ACCEPTED MANUSCRIPT Each day of the week, the physiological state of sessile cells before and after C&D was assessed by culturable cells enumeration, qPCR to quantify total cells, and PMA-qPCR to quantify viable cells.

Changes in sessile L. monocytogenes EGD-e populations on SS coupons after daily C&D treatments with C&D products diluted to 1:10 and soiling with smoked salmon juice are

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presented in Fig. 2. Initially, all viable sessile cells were culturable and the total and

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culturable sessile cell amounts reached more than 8 log10(cells/coupon). The total and viable

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cell amounts decreased around 1 log10(cells/coupon) with the first C&D treatment of the week, and remained stable on the following days at around 7.2 and 6.5 log10(cells/coupon),

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respectively. The number of viable cells was always below that of total cells. We observed that there was a range between 0.3 and 1.4 log10(cells/coupon) between the total and viable

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cells amounts, meaning that some sessile cells were dead. The culturable cell count decreased throughout the week. After the first C&D treatment, a significant difference between the

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viable and the culturable cell amounts appeared and remained on the following days. As

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already shown by Firmesse et al. (2012), our results indicate that C&D treatments lead only to a loss of culturability of cells and represent a stress inducing the appearance of VBNC cells.

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These results highlight the importance of taking into account the VBNC state, and could lead to new ways of improving sanitation procedures, to better characterisation of the

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L. monocytogenes risk in food processing plants by perfected detection methods, and could help to explain unsolved cases of outbreaks. Importantly, there is no reason to assume that VBNC bacteria cannot regain their ability to develop in food products, grow to reach a critical concentration, and cause disease (Cappelier et al., 2007; Oliver, 2005; Rowan, 2004). This can be discussed but few data are available to evaluate the real risk that VBNC cells produced under stresses can regain their culturability. Indeed, some authors did not highlights that

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ACCEPTED MANUSCRIPT stressed L. monocytogenes cells were able to recover to the culturable state in non-stressful environmental conditions (Dreux et al., 2007).

3.2. Impact of environmental factors commonly encountered in food processing plants on the survival of L. monocytogenes

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After defining the conditions that enabled the survival of L. monocytogenes populations on SS surfaces, we studied the influence of five environmental factors found in food processing

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environments (L. monocytogenes strain, surface material, presence of a strain enhancing

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L. monocytogenes adhesion, food soil, and scenario of sanitation procedure) on these populations, through a fractional design (Table 1). To assess the effect of each environmental

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factor, we compared the number of cells recovered by sonication between the first day (day 1)

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before stress application and the last day of the week (day 4) after stress application, corresponding to the Delta-values (δ d1-d4 values). The narrower the gap between initial and

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final cell amounts, the more the environmental factor promoted the survival of

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L. monocytogenes. Bacterial survival was assessed by culturable cells enumeration, qPCR and PMA-qPCR.

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3.2.1. Impact of the L. monocytogenes strain

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Two L. monocytogenes strains were used: L. monocytogenes EGD-e which is resistant to desiccation and L. monocytogenes LO28 which is relatively more sensitive to desiccation than L. monocytogenes EGD-e (Zoz et al., 2016). According to our results, δ d1-d4 values were not significantly different between the two L. monocytogenes strains concerning the culturable, viable and total populations (p-value = 0.30, 0.23 and 0.51, respectively) (Table 2).

Some studies suggested that surviving and persistent bacteria are genetically distinct from transient strains (Autio et al., 2003; Holch et al., 2013; Wulff et al., 2006). Based on this 15

ACCEPTED MANUSCRIPT hypothesis, many studies focused on looking for the presence or absence of specific genes to explain the ability to persist (Mazza et al., 2015). Studies have demonstrated by molecular typing that persistent strains all belonged to the same genetic group (Wulff et al., 2006), corresponding to the clonal complex CC121 (Holch et al., 2013). This suggests that some genotypes of L. monocytogenes are more likely than others to persist.

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For other authors, bacterial persistence is a stochastic event that cannot be explained by

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specific genetic characteristics (Carpentier and Cerf, 2011; Ferreira et al., 2014; Stasiewicz et

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al., 2015). On the basis of this hypothesis, bacteria persist because they can adhere to surfaces, be protected in retention niches, grow and adapt to stress conditions. It is sometimes

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explained that bacteria are persistent because they are more sessile than non-persistent bacteria (Borucki et al., 2003; Norwood and Gilmour, 1999; Wang et al., 2015), but this

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difference is disputed (Djordjevic et al., 2002). Other results suggest that strains can persist because they are more resistant to chemical products used for C&D (Aase et al., 2000;

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Lundén et al., 2003a). However, the difference in sensitivity to disinfectants between

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persistent and transient bacteria is not always demonstrated (Holah et al., 2002; Kastbjerg and Gram, 2009; Lourenço et al., 2009; Magalhães et al., 2016; Wang et al., 2015).

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3.2.2. Impact of the surface material

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No significant difference was found between SS and ceramic surfaces. After sanitation procedures applied daily for a week, the quantity of culturable, viable and total populations was not influenced by the surface material (p-value = 0.06, 0.14 and 0.10, respectively) (Table 2), although SS is considered one of the most hygienic materials (Verran et al., 2008). This result is probably the consequence of the dilution of C&D products.

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ACCEPTED MANUSCRIPT It is well established that the effectiveness of C&D depends on the surface material, because surfaces have different degrees of roughness (Garry 1994; Bang et al, 2014). Each irregularity represents a potential retention zone for water, organic matter and microorganisms, and can enhance bacterial persistence (Verghese et al., 2011). For example, holes formed during the manufacturing process of materials are important retention areas for microorganisms (Mettler

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and Carpentier, 1999). Moreover, Beltarme et al. (2015) showed that worn materials are less hygienic than new materials. The harbourage sites cannot be reached by mechanical action

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and some water may collect before product application. This is why, in our study, chemical

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products were diluted to obtain a surviving population on surfaces and were applied without

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mechanical action.

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3.2.3. Impact of the presence of a strain enhancing L. monocytogenes adhesion

The presence of Pseudomonas fluorescens 09empf87 which enhances L. monocytogenes

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adhesion did not significantly impact the survival of L. monocytogenes, whatever the

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physiological state of cells (p-value = 0.08 for culturable cells, 0.71 for viable cells and 0.26 for total cells) (Table 2).

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In food processing environments, biofilms are generally composed of several bacterial species promoting interactions between microorganisms (Burmølle et al., 2014). The growth of

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L. monocytogenes and its adherence to surfaces may be favoured or, conversely, reduced by the presence of other bacteria. Guðbjörnsdóttir et al. (2005) showed that colonisation of SS surfaces by L. monocytogenes was enhanced by the presence of several species of Pseudomonas. In contrast, Staphylococcus sciuri reduced adhesion of L. monocytogenes probably because of competition for nutrients and the anti-adhesive properties of S. sciuri (Leriche and Carpentier, 2000). However, adhesion is only one of the events that occur in processing plants and an increase in the number of sessile cells does not give any indication of 17

ACCEPTED MANUSCRIPT the fate of L. monocytogenes after C&D when associated with another bacterial species. Nevertheless, according to our results, L. monocytogenes was identically surviving with or without the presence of a strain which favoured its adhesion on surfaces.

3.2.4. Impact of the food soil

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The difference between initial and final cell amounts was not significantly different when cells were cultured in presence of smoked salmon juice or meat exudate. This result was true

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for culturable, viable and total populations (p-value = 0.89, 0.60 and 0.13, respectively)

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survival of L. monocytogenes in the same way.

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(Table 2). From these results, we concluded that the two tested food soils influence the

Overney et al. (2016) demonstrated that the food soil had an impact on L. monocytogenes

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growth and its behaviour (spatial distribution, adhesion forces and physiological state) on SS surfaces. Furthermore, the presence of organic matter on surfaces greatly reduced the

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effectiveness of C&D (Jaudon et al., 2000; Lourenço et al., 2009; Nyati et al., 2012; Ruiz-

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Cruz et al., 2007). For instance, the presence of milk protected bacteria from disinfectants (Kim et al., 2007; Li et al., 2014b). Moreover, the presence of organic matter had a protective

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al., 2010).

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effect on L. monocytogenes cells against other types of stress, such as desiccation (Vogel et

3.2.5. Impact of sanitation procedure

The scenario of the sanitation procedure was the only environmental factor that had a significant impact on the δ d1-d4 values of the culturable, viable and total populations (pvalue = 0.004, 0.02 and 0.05, respectively) (Table 2). To assess the influence of each sanitation procedure on L. monocytogenes survival, we can calculate the difference between δ d1-d4 values obtained after daily C&D and δ d1-d4 values obtained with other scenarios of 18

ACCEPTED MANUSCRIPT sanitation procedures. Daily C&D is used as a reference because it is currently the most common procedure used in food processing plants.

According to our results, a daily drying step was less effective than the daily C&D to reduce the surviving populations. The δ d1-d4 values for daily drying step were lower by -0.95, -1.74 and -0.88 log10(cells/coupon) respectively for culturable, viable and total populations (Fig. 3)

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compared to daily C&D. For viable and total populations, δ d1-d4 values were even negative

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when only daily drying was applied, indicating that cells accumulated on surfaces.

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C&D with a drying step was more effective in decreasing sessile populations than C&D without a drying step. All δ d1-d4 values were higher when C&D were followed by a drying

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step (Fig. 3). Moreover, the efficacy on culturable populations was enhanced when the drying

drying step was

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step was applied daily for a week rather than just once at the end of the week. When the applied daily after C&D,

the δ d1-d4

value was

higher by

5.87 log10(cells/coupon) for the culturable populations compared to the value obtained for the

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procedure with no drying step after C&D (Fig. 3). The difference of δ d1-d4 values for viable populations was only about 0.07 log10(cells/coupon) (Fig. 3).

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To our knowledge, very few studies on bacterial survival are based on reproducing the conditions encountered in food processing plants in the laboratory by alternating C&D

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procedures and culture steps with food soils over several days, to mimic the succession of C&D and production steps (Marouani-Gadri et al., 2010; Peneau et al., 2007). As described by Campdepadrós et al. (2012), sanitation procedures were able to reduce the L. monocytogenes load, but not to eradicate the microorganisms completely. It is particularly important to improve C&D in order to reduce and prevent bacterial persistence. Drying surfaces during production and/or after C&D enables greater control of bacterial growth on surfaces. The presence of water on surfaces favours the development of biofilms in 24 h, 19

ACCEPTED MANUSCRIPT whereas drying can significantly reduce the culturable population (Fuster-Valls et al., 2008; Rückerl et al., 2014). By drying surfaces, bacteria surviving C&D undergo an additional stress that reduces their growth. It can be assumed that the accumulation of stresses can potentially lead to reduce the risk of return to cell growth. Fluctuations in relative humidity are an effective way to prevent the persistence of L. monocytogenes cells on surfaces (Zoz et

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al., 2016). Furthermore, Bang et al. (2014) also showed that the decrease of the culturable population was higher when C&D were followed by a drying step, rather than when C&D

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Conclusion

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4.

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were applied without drying.

Our study increases the understanding of the behaviour of microorganisms such as

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L. monocytogenes on surfaces in conditions encountered in food processing plants, and highlights the difficulty in reducing the occurrence of viable persistent sessile cells. Sanitation

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procedures alone lead to a loss of culturability and appearance of VBNC populations.

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However, an additional daily drying step after C&D optimises the effectiveness of C&D to reduce culturable populations. Our study underline the importance of using methods

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independent of the culturability of the cells to monitor viable pathogens in food processing plants since culture may underestimate the amounts of cells.

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Acknowledgments

This study is part of the EcoSec project to reduce the environmental impact of C&D procedures in refrigerated food processing plants through optimal use of air drying. This project is funded by the French National Research Agency within the framework of the ALID programme (ANR-12-ALID-0005) and supported by the Vitagora competitiveness cluster.

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ACCEPTED MANUSCRIPT The authors are grateful to Denis Boudot from Ecolab (Arcueil, France) for providing C&D

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products.

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ACCEPTED MANUSCRIPT TABLES

Table 1: Experimental design followed to study the impact of L. monocytogenes strain, surface material, the presence of a strain enhancing L. monocytogenes adhesion, food soil, and

1

Presence of a strain enhancing L. monocytogene s adhesion Pure-culture

2

Pure-culture

SS

C&D

3

Dual-culture

Ceramic

C&D

4

Dual-culture

Ceramic

C&D

5

Dual-culture

SS

6

Dual-culture

SS

7

Pure-culture

Ceramic

8

Pure-culture

Ceramic

9

Pure-culture

Ceramic

10

Pure-culture

11

Dual-culture

SS

12

Dual-culture

SS

13

Dual-culture

Ceramic

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scenario of sanitation procedure on the survival of L. monocytogenes.

Dual-culture

Ceramic

Pure-culture

SS

16

Pure-culture

SS

17

Pure-culture

SS

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Pure-culture

SS

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Surface material

Scenario of sanitation procedure

SS

C&D

Lm EGD-e Lm EGD-e

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Strain

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C&D + daily drying step C&D + daily drying step C&D + daily drying step C&D + daily drying step Daily drying step Daily drying step Daily drying step Daily drying step C&D + final drying step C&D + final drying step C&D + final drying step C&D + final drying step C&D + daily drying step C&D + final drying step

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Lm LO28 Lm LO28

Food soil Meat exudate Smoked salmon juice Meat exudate Smoked salmon juice

Lm EGD-e

Meat exudate

Lm EGD-e

Smoked salmon juice

Lm LO28

Meat exudate

Lm LO28

Smoked salmon juice

Lm EGD-e

Meat exudate

Lm EGD-e

Smoked salmon juice

Lm LO28

Meat exudate

Lm LO28

Smoked salmon juice

Lm EGD-e

Meat exudate

Lm EGD-e

Smoked salmon juice

Lm LO28

Meat exudate

Lm LO28 Lm EGD-e Lm EGD-e

Smoked salmon juice Smoked salmon juice Smoked salmon juice

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Environmental factor

Viable cells

Total cells

L. monocytogenes strain

0.30

0.23

0.51

Surface material

0.06

0.14

0.10

of

a

strain

enhancing

L. monocytogenes adhesion

0.08 0.89

Scenario of sanitation procedure

0.004

0.26

0.60

0.13

0.02

0.05

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Food soil

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Presence

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Culturable cells

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ACCEPTED MANUSCRIPT FIGURE LEGENDS

Figure 1: Culturable population of L. monocytogenes EGD-e cultured on SS coupons with smoked salmon juice, obtained after five days without daily C&D treatment, and after daily C&D treatments with undiluted C&D products and products diluted to 1:10. Culturable cells detection threshold 2.8 log10(CFU/coupon) (n = 6, 9 or 12, i.e. 2, 3 or 4 experiments). Bars

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represent standard deviation.

Figure 2: Influence of daily C&D treatments with C&D products diluted to 1:10 on

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culturable, viable and total L. monocytogenes EGD-e cells cultured on SS with smoked salmon juice. Culturable cells detection threshold 1.3 log10(CFU/coupon) and viable/total

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cells detection threshold 4 log10(cells/coupon). For the measures obtained after C&D treatment, the symbols are slightly to the right of those obtained before C&D treatment (n = 6,

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i.e. 2 experiments). Bars represent standard deviation.

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Figure 3: δ d1-d4 values for culturable, viable and total cells for each tested scenario of

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sanitation procedure. Bars represent confidence intervals.

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ACCEPTED MANUSCRIPT Highlights:

Total and viable cells were weakly affected by C&D

-

Use of PMA-qPCR highlighted the appearance of VBNC populations after C&D

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Additional drying step after C&D optimised the effectiveness of C&D to reduce CFU

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