lable at ScienceDirect

Food Control 51 (2015) 94e107

Contents lists avai

Food Control

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

Environmental sampling for Listeria monocytogenes control in foodprocessing facilities reveals three contamination scenarios

Meryem Muhterem-Uyar a, b, Marion Dalmasso c, Andrei Sorin Bolocan d,Marta Hernandez e, Anastasia E. Kapetanakou f, Tom�a�s Kuchta g, Stavros G. Manios f,Beatriz Melero e, Jana Minarovi�cov�a g, Anca Ioana Nicolau d, Jordi Rovira e,Panagiotis N. Skandamis f, Kieran Jordan c, David Rodríguez-L�azaro e, Beatrix Stessl a,Martin Wagner a, *

a Institute of Milk Hygiene, Milk Technology, and Food Science, Department for Farm Animals and Veterinary Public Health,University of Veterinary Medicine, Veterin€arplatz 1, A-1210 Vienna, Austriab Department of Nutritional Sciences, Faculty of Life Sciences, University of Vienna, Althanstraße 14, 1090 Vienna, Austriac Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Irelandd Faculty of Food Science and Engineering, Dunarea de Jos University of Galati, str. Domnesca 47, 800008, Galati, Romaniae University of Burgos, Department of Biotechnology and Food Science, Hospital del Rey, s/n, 09001 Burgos, Spainf Agricultural University of Athens, Iera odos 75, 118 55, Athens Greeceg VUP Food Research Institute, National Agricultural and Food Centre, Priemyseln�a 4, SK 824 75 Bratislava, Slovak Republic

a r t i c l e i n f o

Article history:Received 27 June 2014Received in revised form21 October 2014Accepted 27 October 2014Available online 13 November 2014

Keywords:Listeria monocytogenesFood processing environmentCross contaminationHygieneCritical control area

* Corresponding author. Tel.: þ43 (1)25077 3500; fE-mail address: martin.wagner@vetmeduni.ac.at (

http://dx.doi.org/10.1016/j.foodcont.2014.10.0420956-7135/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Listeria monocytogenes enters the food processing facility via environment, or contaminated raw mate-rials. To increase the understanding of L. monocytogenes environmental contamination in the meat anddairy food sector, six European scientific institutions sampled twelve food processing environments(FPEs) in a harmonized methodological approach. The selection of six previously assumed uncontami-nated (UC) FPEs and six contaminated (C) FPEs was based on the L. monocytogenes occurrence infor-mation originating from the time prior to the current study. An aim of the study was to highlight, thatFPEs regarded for years as uncontaminated, may also become L. monocytogenes contaminated andrepeated environmental sampling could help to identify the potential sources of contamination.

From a total of 2242 FPE samples, L. monocytogenes was present in 32% and 8.8% of meat and dairyprocessing environments, respectively. In the actual study, each FPE was contaminated withL. monocytogenes on at least one sampling occasion. Three contamination scenarios could be observed: (i)sporadic contamination in the interface of raw material reception and hygienic areas, (ii) hotspotcontamination in the hygienic processing areas (iii), and widely disseminated contamination in theentirely FPE. These data demonstrate that L. monocytogenes are common colonizers of FPEs in the Eu-ropean processing facilities sampled and that a consistent cross-contamination risk exists. To avoid foodcontamination, a risk assessment approach should assign risk levels to critical control areas (CCAs) andidentify those where cross-contamination should be essentially excluded.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The transmission of Listeria monocytogenes via food vehiclesaffects immunocompromised patients, pregnant women, and theelderly leading to a high fatality rate (20e30%). The consumption ofsmoked fish, fermented and fully cooked meat, poultry and dairy

ax: þ43 (1)25077 3590.M. Wagner).

products have been most frequently implicated in listeriosis cases(Lambertz, Ivarsson, Lopez-Valladares, Sidstedt, & Lindqvist, 2013;Lomonaco et al., 2013; Todd & Notermans, 2011).

L. monocytogenes is present in food processing environments(FPEs) due to a saprophytic lifestyle (Ferreira, Wiedmann, Teixeira,& Stasiewicz, 2014). Temporal breakdowns in hygiene barrier effi-ciency, poor hygiene practices and unhygienic design of equipmentmay trigger L. monocytogenes food plant contamination (Almeidaet al., 2013; Carpentier & Cerf, 2011; Fox, Hunt, O'Brien, & Jordan,

M. Muhterem-Uyar et al. / Food Control 51 (2015) 94e107 95

2011; Ibba et al., 2013; Keto-Timonen, Tolvanen, Lunden, & Kor-keala, 2007; Lomonaco et al., 2009;Wagner andMcLaughlin, 2008).Having colonized a FPE, L. monocytogenes can spread throughoutthe facility due to contaminated contact materials, inappropriatepersonnel movements and foodworkflows (Alali& Schaffner, 2013;Di Ciccio et al., 2012; Hoelzer et al., 2012; Viswanath et al., 2013).Such contamination can be an intermediate step in transmissionfrom their original habitat in the environment (in biofilms, waterand organic soil residues) to food contact surfaces (FCS) and foodunder processing (Reij, Den Aantrekker,& ILSI Europe Risk Analysisin Microbiology Task Force, 2004; da Silva & De Martinis, 2013;Stessl et al., 2014).

Food business operators (FBOs) manufacturing ready-to-eatfoods are required to test processing areas and equipment for theabsence/presence of L. monocytogenes as part of their samplingscheme (Anonymous, 2005). Supporting guidelines for sampling offood processing areas and equipment for the detection ofL. monocytogenes have already been published (Carpentier & Barre,2012). Large food companies have the resources to establish aneffective self-monitoring sampling plan and employ a well-educated quality management board. However, this is more diffi-cult for smaller enterprises (Luber et al., 2011; Vitas, Díez-Leturia,Tabar, & Gonz�alez, 2013; Winkler & Freund, 2011.)

Monitoring concepts for FPEs in small and medium sized en-terprises (SMEs) are not harmonized in terms of frequency ofsampling, sampling sites, and detection methods. The difficulty forFBOs in constantly growing/evolving establishments is to definecritical control areas (CCAs) (sometimes called high risk zones) forL. monocytogenes contamination throughout new buildings, ex-tensions and reconstructions. A further important fact is that FBOsin small-scale production facilities often do not have the resourcesfor extensive environmental sampling and scientifically basedresearch (Winkler & Freund, 2011).

The aim of the current study was to determine the occurrence ofL. monocytogenes in 12 European meat and dairy small-scale directmarketers and SMEs applying a harmonized methodologicalapproach during a one-year period. FPEs were categorized in un-contaminated (UC) and contaminated (C) FPEs according to prioravailable L. monocytogenes occurrence data of each company (e.g.determined by an internal monitoring system). A further aimwas tovisualize actual L. monocytogenes contaminated areas on a con-struction map of each facility, so that the FBO could clearly identifycontaminated areas and implement corrective actions.

2. Materials and methods

2.1. Selection of FPE facilities and sampling

Twelve FPEs located in six European countries (Austria, Ireland,Spain, Slovakia, Greece and Romania) were sampled from March2012 to March 2013. From previous sampling activities, facilityinspections and consultations with quality managers, theL. monocytogenes contamination history was known, resulting in acategorization in presumed uncontaminated (UC) or contaminated(C)-FPEs. The selection of FPEs with regard to the initial contami-nation status (presumed positive/negative) should enable thedetermination of contamination dynamics in a positive FPE, orwhether a negative FPE could remain negative during the 12months of investigation. The FPEs included four small farmhousecheesemaking facilities, five industrial dairy and three meat pro-ducers (Figs. 1e6).

The industrial cheese (Austria, Greece, and Spain) and Romanianmeat producers processed rawmaterials from at least two different

animal sources. Four farmhouse cheese makers, a poultry slaugh-terhouse and the meat producing plants also prepared RTE foodwithout heat treatment. Six cheese producers located in Ireland,Austria, Greece and Spain manufactured ripened soft/semi-soft-,brined cured-, and hard cheeses with a minimum shelf life of twoto three weeks (Table 1).

Four visits per UC-FPE or C-FPE, every three to four months wereconducted. The number of samples related to the surface area of theFPE (small FPEs, <200 m2, large FPEs, >500m2). Due to the nationalpeculiarities of the different types of food chains, the choice of thesampling points was left to the person taking the samples. How-ever, there was an agreement that sampling should be spread overthe entire FPE, including the areas of raw product delivery, pro-cessing and packaging. A minimum number of 15 samples persampling date were taken on consistent sampled areas so thattrends could be monitored over time (Carpentier & Barre, 2012).

Non-food contact surfaces (NFCS; floors, walls, and drains) andfood contact surfaces (FCS; conveyors, belts, tables, slicers, moulds,and knives) were tested during the processing. FCS and NFCSsampling areas (>100e900 cm2 when possible e.g. on flat and openareas as conveyors and shelves) were sampled with sterile sponges(3M, St. Paul, MN, USA) using meandering movement.

When sampling drains (NFCS), 100 ml of liquid samples (drainwater) were collected aseptically and transported in sterile bottles.All samples were maintained at 4 �C during transportation to thelaboratory and were analyzed for the presence of L. monocytogeneswithin 24 h. Food samples were also tested, and additional datafrom food lot controls (determined by the local district labora-tories) were available during the actual one-year study.

2.2. Harmonized methodological approach

Environmental swabs, liquids and solid food samples wereinvestigated according to the ISO 11290-1 protocol (ISO, 1996,Amendment 2004). Swabs were enriched in 50e100 ml half-Fraserbroth (HFB; Merck KgA, Darmstadt, Germany). Liquid samples(100 ml) were centrifuged for 30 min at 6000� g and the sedimentwas transferred to 100 ml HFB. Twenty-five g of each food sample(intermediate products or product before packaging) was enrichedin 225 ml HFB. After incubation for 24 h at 30 �C, 0.1 ml HFB wastransferred to 10 ml Fraser broth (FB; Merck KgA) and incubated for48 h at 37 �C. HFB and FB enriched cultures were streaked onto theselective plating media Agar Listeria acc. to Ottaviani and Agosti(ALOA; Merck KgA) and in addition on Palcam agar (Biokar Di-agnostics, Beauvais Cedex, France).

Up to five L. monocytogenes colonies were isolated from theselective agars (ALOA, Palcam) and sub-cultivated on Tryptone soyagar supplemented with 0.6% (wt/vol) yeast extract (TSAY; OxoidLtd., Basingstoke, UK). For further confirmation based on the po-lymerase chain reaction (PCR) technique, DNA was isolated bydispersing an isolated colony in 100 ml 0.1 M Tris-HCL buffer (SigmaAldrich, St. Louis, MO, USA). A simple DNA isolation methodincluding Chelex® 100-Resin (BioRad, Hercules, CA, USA), asdescribed by Walsh, Metzger, and Higuchi (1991), or heating at95 �C for 20 min, was performed on bacterial suspensions prior toPCR. One of three different PCR confirmation assays was thenapplied, (i) the Listeria spp. multiplex-PCR protocol targeting theinvasion-associated protein (iap), as described by Bubert et al.,(1999); (ii) the method targeting the actA gene, which encodes aprotein involved in the actin filament assembly (Oravcova et al.,2006); (iii) a PCR amplification of the hemolysin (hly)-, iap gene,and the Listeria innocua specific lin02483 target (Rodriguez-L�azaroet al., 2004).

Fig. 1. Listeria monocytogenes occurrence in Slovakian farmhouse cheesemaking FPEs.

M. Muhterem-Uyar et al. / Food Control 51 (2015) 94e10796

Fig. 2. Listeria monocytogenes occurrence in Irish farmhouse cheesemaking FPEs.

M. Muhterem-Uyar et al. / Food Control 51 (2015) 94e107 97

M. Muhterem-Uyar et al. / Food Control 51 (2015) 94e10798

2.3. Statistical analysis

Fisher's exact test and chi square (c)2 tests were performed inIBM SPSS (version 19.0, SPSS Inc., Chicago, USA) to determine the

Fig. 3. Listeria monocytogenes occurrenc

statistical significance (P < 0.05) of the difference in the distribu-tions between various sample categories and parameters[L. monocytogenes isolation on FCS, NCFS, facility type andcontamination status (UC, C)].

e in Austrian industrial dairy FPEs.

Fig. 4. Listeria monocytogenes occurrence in Greek industrial dairy FPEs.

M. Muhterem-Uyar et al. / Food Control 51 (2015) 94e107 99

M. Muhterem-Uyar et al. / Food Control 51 (2015) 94e107100

3. Results

A total of 2242 environmental samples were taken with anoverall L. monocytogenes occurrence rate of 12.6% (n ¼ 282; 8.8% in

Fig. 5. Listeria monocytogenes occurr

dairy and 32% in meat plants). Of this total amount, 3.5% (n ¼ 78)and 9.1% (n ¼ 204) were collected from FCS and NFCS, respectively.The extensive sampling protocol revealed that all FPEs previouslydetermined as uncontaminated were contaminated on at least one

ence in Spanish industrial FPEs.

M. Muhterem-Uyar et al. / Food Control 51 (2015) 94e107 101

sampling occasion. Two UC-FPEs and three C-FPEs were contami-nated on every sampling occasion (Tables 2 and 3).

The L. monocytogenes occurrence at farmhouse cheese makingenvironments ranged between 1.3% and 6.4%. The highest

Fig. 6. Listeria monocytogenes occur

prevalence was found in an environment previously categorized asUC-FPE. A similar L. monocytogenes occurrence was detected inindustrial dairy producers (0.5e7.2%), except for one previouslycontaminated cheese processing environment which was

rence in Romanian meat FPEs.

Table 1Overview of the FPEs sampled including type of product and origin of rawmaterials.

Country Products Heattreatmenta

Ripened Sources

Farmhouse cheese makersSlovakia Fresh cheese No No EweSlovakia Fresh cheese No No EweIreland Semi-soft cheese No, yes Yes CowIreland Hard cheese No Yes Cow

Industrial cheese producersAustria Fresh cheese, brined

curd cheeseYes No, yes Ewe, goat, cow

Austria Soft cheese Yes Yes Ewe, cowGreece Fresh cheese, brined

curd cheeseYes No, yes Ewe, goat

Greece Fresh cheese, brinedcurd cheese

Yes No, yes Ewe, goat

Spain Soft and semi-softcheese, brined curedcheese

Yes Yes Ewe, goat, cow

Meat producersRomania Smoked or boiled or

boiled and smokedmeat products

Yesb Yes Pig, cow, sheepchicken

Romania Smoked, boiled, boiledand smoked meatproducts

Yesb Yes Pig, cow, sheep,chicken, turkey

Spain Raw and processedpoultry meat

No, yes Yes Chicken

a Heat treatment as pasteurization and boiling.b With the exception of cold smoked products.

M. Muhterem-Uyar et al. / Food Control 51 (2015) 94e107102

contaminated on all four sampling occasions (average contamina-tionwas 26%). At that facility, the contaminationwas widely spreadaround the entire facility. At the three meat processing plants, theL. monocytogenes contaminationwas 18.8%, 26.5% and 50.5%, for theUC-FPE in Romania, the C-FPE in Romania and the C-FPE in Spain,respectively. The highest contamination rate (50.5%) was detectedwhen slaughter and meat processing was combined at the samebuilding (Tables 2 and 3). The NFCS of one C- FPE (Austrian cheeseproducer) and the FCS and NFCS of C-FPE meat producing facilities(located in Romania and Spain) were significantly higher contam-inated with L. monocytogenes (P � 0.001; P ¼ 0.13).

Based on the results (Tables 2 and 3) and the visualization of theL. monocytogenes positive areas on the facility construction plans(Figs. 1e6), three situations could be observed:

- (i) sporadic contamination in the interface of unclean (receptionof raw materials) and clean areas: UC-FPE Slovakia, C-FPESlovakia, UC-FPE Greece, and UC-FPE Austria, where thecontamination was found once and often located at the begin-ning of the processing chain. A major impact onL. monocytogenes cross-contamination from inner and outerenvironments was evident due to the lack of hygiene barriers inthe interface of unclean (reception of raw materials) and cleanareas at farmhouse cheesemakers and two dairy SMEs.

- (ii) hotspot contamination in the hygienic processing areas: C-FPE Ireland, UC-FPE Spain, C-FPE Greece, UC-FPE Romania,where contamination was located at a distinct niche proved tobe repeatedly L. monocytogenes positive. L. monocytogenes hot-spots could be found in four processing facilities at differentstages of the processing chain. In two facilities, two hotspotswere identified. These were located in the fresh cheese pro-duction area, cheese ripening room (both Greek C-FPE), moldingroom and dispatch room (both Irish C-FPE). One facility showeda hotspot in the brine (Spanish UC-FPE). In a Romanian meatproducer, a hotspot occurred in the room where raw meat wasminced.

- (iii) widely disseminated contamination in the entire FPE: UC-FPE Ireland, C-FPE Romania, C-FPE Austria, and C-FPE Spain,where the contamination was widely spread throughout thefacility. Additional hygiene failures as not including storage andripening rooms in a daily-based cleaning regime, the lack ofone-way personnel traffic routes in the main processing areas,and food soil on equipment, floors and drains could have trig-gered widely disseminated contamination.

Accessible cleaning and disinfection protocols were recordedand main compounds of sanitizers are shown in Table 2. All pro-ducers applied chlorine-based disinfectants, in alternation withperacetic acid or/and quaternary ammonium compounds (QACs).Two farmhouse cheesemakers and a dairy producer used solelychlorine-based formulas.

To demonstrate a possible L. monocytogenes transmission fromFPEs to food, data from intermediate or final products wereincluded in the study.

Out of 1446 food samples tested, 62 (4.3%) proved to be positivefor L. monocytogenes (2.9% in dairy and 26.2% in meat plants). Thehighest L. monocytogenes detection rate was found in meat samplestaken at the slaughterhouse (87.5%), followed by farmhousecheeses and meat products (range: 20e25%). Cheese samples takenat a farmhouse cheesemaking and a SME dairy producer, bothformerly categorized as UC-FPE, were positive for L. monocytogenes(25% and 10.9%). The widely disseminated L. monocytogenescontamination on NFCS of one dairy (26%) and one meat processingSME (18.9%) did not result in higher contamination rates in foodsamples (2.2% and 0% confirmed positive results, respectively)(Table 2).

4. Discussion

This study adds a comprehensive view to the L. monocytogenesFPE contamination problem present in dairy and meat small-scaledirect marketers and SMEs in six European countries.L. monocytogenes occurrence in FPEs and food samples was deter-mined with a harmonized methodological approach applied ineach European partner lab. Records on hygiene measurements andthe visualization of actual L. monocytogenes positive areas in theconstruction map of each facility supplemented the data set.

Many food enterprises have evolved from small units to con-glomerates of premises and areas, presenting challenges for theprevention and control of L. monocytogenes contamination. Recentstudies focused on the L. monocytogenes occurrence in FPEs havebeen undertaken in single food processing facilities (Alessandria,Rantsiou, Dolci, & Cocolin, 2010; Blatter, Giezendanner, Stephan,& Zweifel, 2010; Campdepadr�os, Stchigel, Romeu, Quilez, & Sol�a,2012; Latorre et al., 2009; Ortiz et al., 2010; Pagadala et al., 2012;Pappelbaum et al., 2008), at different facilities of one productionchain (Almeida et al., 2013; Barancelli et al., 2011; Fox et al., 2011;Meloni et al. 2013; Parisi et al., 2013; Rotariu, Thomas, Goodburn,Hutchison, & Strachan, 2014; Williams et al., 2011), or highlightedthe phenomenon of strain persistence (Ferreira et al, 2011, 2014;Lomonaco et al., 2009; Stessl et al., 2014; Vongkamjan, Roof,Stasiewicz, & Wiedmann, 2013).

In the present study the recorded L. monocytogenes occurrencein FPEs had a broad range from 0.5 to 50.5% (mean 12.6%). The FPEcontamination during the processing of fresh -, brined cured e andsoft cheeses in SMEs ranged from 0.5 to 7.2% with one outlier (26%).A potential source of L. monocytogenes FPE contamination infarmhouse cheesemaking facilities (varied from 1.3 to 6.4%) couldbe the external farm environment (Almeida et al., 2013; Fox et al.,2011; Ho, Lappi, & Wiedmann, 2007; Latorre et al., 2009; Schoderet al., 2011). Schoder et al. (2011) and Fox et al. (2009) reported a

Table

2Occurren

ceof

L.mon

ocytog

enes

inFP

Esalon

gfoursamplin

gev

ents,F

PEco

ntaminationstatusbe

fore

andaftersamplin

g,an

dap

plie

dhy

gien

eregimes

(clean

ingan

ddisinfection).

Proc

essing

catego

ry/cou

ntry

Con

tamination

statusIa

Con

tamination

statusIIa

Occurence

(%)

FPE(pos/total)

Food

(pos/total)

Hyg

iene

lock

Clean

ing

(allroom

s-daily)

Verified

sanitation

plans

QACg /Biguan

ide

Chlorine

Peracetic

acid

12

34

Farm

house

chee

semak

ers

Slov

akia

CbSP

Cd

1.27

0/30

1/43

0/43

1/41

1/4

No

No

Yes

No

Yes

No

Slov

akia

UCc

SPC

2.2

0/45

1/45

0/45

3/47

0/10

No

No

Yes

No

Yes

Yes

Irelan

dC

HSP

Ce

2.14

0/43

2/50

1/50

1/44

2/9

No

No

Yes

No

Yes

Yes

Irelan

dUC

WDCf

6.42

1/46

2/47

6/47

3/47

5/20

No

No

Yes

No

Yes

No

Industrial

chee

sepro

duce

rsSp

ain

UC

HSP

C2.63

1/36

1/39

1/38

1/39

5/46

No

No

Yes

Yes

Yes

Yes

Greece

CHSP

C7.22

11/45

2/46

0/44

0/45

0/17

No

No

Yes

No

Yes

No

Greece

UC

SPC

1.74

0/30

1/44

2/50

0/48

0/15

Yes

Yes

Yes

Yes

Yes

Yes

Austria

CW

DC

2621

/81

23/80

13/85

66/227

27/122

3Yes

Yes

Yes

Yes

Yes

Yes

Austria

UC

SPC

0.53

0/50

0/38

1/51

0/51

0/18

Yes

No

Yes

No

Yes

Yes

Mea

tpro

duce

rsSp

ain

CW

DC

50.45

14/27

15/28

17/28

10/28

14/16

Yes

No

Yes

No

Yes

Yes

Rom

ania

CW

DC

26.51

8/40

12/41

11/42

13/43

8/40

Yes

No

Yes

Yes

Yes

Yes

Rom

ania

UC

HSP

C18

.82

0/13

2/37

14/35

n.d

.h0/28

Yes

No

Yes

Yes

Yes

Yes

aCon

taminationstatusof

FPEbe

fore

(I)an

daftersamplin

g(II).

bCco

ntaminated

.cUCunco

ntaminated

.dSP

Csp

orad

ically

contaminated

FPE.

eHSP

Chotsp

otco

ntaminated

FPE.

fW

DCwidelyco

ntaminated

FPE.

gQACQuartern

yAmmon

ium

compou

nds.

hn.d

.not

determined

.

M. Muhterem-Uyar et al. / Food Control 51 (2015) 94e107 103

L. monocytogenes occurrence in the dairy farm environmentranging from 0.9 to 15.4% and 19%, respectively. For industrialcheese producers, where the farming of animals is not located inthe same building complex, Almeida et al. (2013) showed that the“outer” environmental contamination was nearly zero. Barancelliet al. (2011) reported a L. monocytogenes contamination on NFCSof Brazilian fresh cheese producers of 13.3%. Furthermore, Parisiet al. (2013) studied 34 dairy facilities in Southern Italy showing18.8% contamination of NFCS.

The data from this study corroborated the general assumption ofhigher L. monocytogenes prevalence in meat plants (18.8e50.5%)compared to most dairies (0.5e7.2%) (Chiarini, Tyler, Farber,Pagotto, & Destro, 2009; Kovacevic, McIntyre, Henderson, &Kosatsky, 2012; Martin, Garriga, & Aymerich, 2011; Prencipeet al., 2012). This can be explained by the high possibility ofL. monocytogenes cross-contamination of meat carcasses and cleancutting and packaging rooms due to unhygienic design of bleeding,plucking and evisceration equipment (Chiarini et al., 2009; Martinet al. 2011). Sakaridis et al. (2011) detected 80% L. monocytogenespositive environmental samples in a Greek chicken slaughterhousecompared to 50.5% prevalence in the Spanish FPE included in thisstudy. The Romanian meat processing FPEs were contaminated athigher rates of L. monocytogenes (18.8 and 26.5%) in contrast to 6.1%and 11.8% environmental contamination in small-scale US meat - orSpanish fermented sausage processors (Martin et al., 2011;Williams et al., 2011).

L. monocytogenes cross-contamination from FPEs to foodmatrices was high at the chicken slaughterhouse (WDC FPE) (87.5%positive samples), indicating ineffective hygiene measurements inthe food contact environment. The same observation was given inthe previously categorized C-FPE in a meat plant (26.5%L. monocytogenes environmental contamination resulting in 20%positive food samples). By contrast, in one dairy (26%; C-FPE,Austria; WDC FPE) and one meat processing SME (18.9%; UC-FPE,HSPC, Romania), the L. monocytogenes environmental contamina-tion was not leading to higher contamination rates in food samples(2.2% and 0% confirmed positive results, respectively). Therefore,the hygiene measurements on the FCS could have sufficientlyimpeded the higher contamination of food, or the food matrix itselfdid not support the growth of L. monocytogenes (pH � 4.4 oraW � 0.92; pH � 5.0 and aW � 0.94) (Anonymous, 2005).

It is becoming increasingly clear, that the probability ofL. monocytogenes FPE contamination in certain food processingchains (meat, fish) is not yet be accurately predicted from the in-house monitoring data collection. Not all FBOs undertake suffi-ciently rigorous sampling regimes to detect L. monocytogenes. Theabsence/presence of hygiene barriers, daily sanitation regimes,personnel work flow, and good compartmentalization of uncleanareas from clean processing or packaging rooms can influence theFPE contamination as highlighted by this study. After visualizingthe L. monocytogenes positive areas on the construction plans of thefacilities, three situations were observed as shown in Table 2 andFigs. 1e6. The following conclusions could be drawn on improve-ments needed:

Sporadic FPE contaminations (i) were mainly located in theinterface of unclean (reception of rawmaterials) and clean areas. Inthis case the implementation of basic hygiene rules such as efficienthand -, shoe disinfection, and the installation of hygiene locks(sanitizing barriers) could clearly contribute to a decrease ofL. monocytogenes contamination from the outer environment(Dalmasso& Jordan, 2013; Schoder et al., 2011). A further impact onminimizing the risk of sporadic L. monocytogenes contamination isto raise the awareness of possible biofilm formation onmilking anddairy equipment. A high variety of L. monocytogenes strains couldbe introducedwhich are able tomultiply during processing (Latorre

Table 3Listeria monocytogenes positive FPEs (FCS, NFCS) in European cheese processing facilities.

Processing facility Sampling event Sample category Sample details (number of positive samples)

Farmhouse cheese makersSlovakia (Ca; SPCc) 1 FCSf Equipment-packing machine (1)

4 NFCSg Equipment (1)Slovakia (UCb; SPC) 4 FCS Equipment-milking machine (1)

2 NFCS Fly-paper (1)4 NFCS Rag for shoes (1), feed for sheep (1)

Ireland (C; HSPCd) 2 NFCS Floor-processing room (1), dispatch room (1)3 NFCS Floor-dispatch room (1)4 NFCS Floor-dispatch room (1)

Ireland (UC; WDCe) 1 FCS Equipment-inside milk vat (1)2 NFCS Floor-entrance milk parlor (1), processing room (1)3 NFCS Floor-processing room (4), packing room (2)4 NFCS Floor-processing room (1), packing room (2)

Industrial cheese producersSpain (UC; HSPC) 1 NFCS Drain-salting room (1)

2 NFCS Floor-salting room (1)3 NFCS Floor-packing room (1)4 NFCS Floor-slicing room (1)

Greece (C; HSPC) 1 FCS Equipment-wooden rack (1)1 NFCS Equipment-milk heater (2), floor (3), drain (2), door (2), wall (1)2 NFCS Floor (1), wall (1)

Greece (UC; SPC) 2 NFCS Floor (1)3 NFCS Floor (1), drain (1)

Austria (C; WDC) 1 FCS Cheese processing I-collection tray (1), fresh cheese filling -moulds (1)1 NFCS Preparation (raw material)-transport trolley, drain (2); cooling chamber-drain (1);

wash system- pallet, drain (2); production I, II-drain (3); cheese processing I, II, fresh cheesefilling, yoghurt-drain (4); corridor-floor (2); shoe (2)

2 NFCS Preparation (raw material)-drain (2); cooling chamber-drain (1); wash system- pallet, drain (4);production I, III-drain (3); cheese processing I, II, fresh cheese filling, yoghurt-drain (9);corridor-floor (2); shoe (6)

3 NFCS Preparation (raw material)-drain (1); wash system-drain (2); fresh cheese filling and processing,yoghurt-floor, drain (4); production IV, V-drain, floor (3); shoe (2)

4 NFCS Preparation (raw material)-floor, drain (10); cooling chamber-floor, drain (4); wash system- floor,drain (10); production I, II, III-floor, drain (14); cheese processing I, II, fresh cheese filling,production IV, yoghurt-floor, drain (27); shoe (1)

Austria (UC; SPC) 3 NFCS Milk tank-drain (1)

Listeria monocytogenes positive FPEs (FCS, NFCS) in European meat processing facilities.

Processingfacility

Samplingevent

Samplecategory

Sample details (number of positive samples)

Meat producersSpain

(Ca; WDC)1 FCSf Deboning area: equipment-cutting table (1), conveyor belt (1), knife (1); staff-hands (1);

packaging area: equipment-packaging table (1); transport boxes (1); staff-hands (1)1 NFCSg Evisceration: drain (1); air chilling: floor (1); deboning: floor, drain (2); processing: floor (1); packaging area: floor, drain (2)2 FCS Deboning area: equipment-cutting table (1), conveyor belt (1), knife (1); staff-hands (1); processing area: mincing

machine (1), staff-hands (1); packaging area: equipment-packaging table (1); transport boxes (1); staff-hands (1)2 NFCS Deboning: floor, drain, wall (3); processing: floor (1); packaging area: floor, drain (2)3 FCS Deboning area: equipment-cutting table (1), conveyor belt (1), knife (1); staff-hands (1);

processing area: mincing machine (1), staff-hands (1); packaging area: equipment-packaging table (1);transport boxes (1); staff-hands (1)

3 NFCS Slaughter room: drain, wall (2); evisceration: drain (1); deboning: floor, drain (2); packaging area: floor, drain (2)4 FCS Deboning area: equipment-cutting table (1), conveyor belt (1), knife (1); staff-hands (1); processing area:

mincing machine (1); packaging area: equipment-packaging table (1); transport boxes (1); staff-hands (1)4 NFCS Deboning: floor, drain (2); processing: floor (1)

Romania(C; WDCe)

1 FCS Equipment-table (1); cutting board (1); derinding machine (1); slicer (1); vacuum packaging (1)1 NFCS Floor (2); wall (1)2 FCS Equipment-table (1); cutting board (1); knife (1); derinding machine (1); meat cutter (1); balance (1);

slicer (1); vacuum packaging (1); RTE-storage: meat sticks (1)2 NFCS Floor (3)3 FCS Equipment-table (2); derinding machine (1); meat cutter (1); balance (1); slicer (1); collection tray (1)3 NFCS Floor (3), drain (1)4 FCS Equipment-table (2); cutting board (1); knife (1); derinding machine (1); slicer (1); vacuum packaging (1)4 NFCS Equipment: area for hands washing (1); floor (3), drain (1); wall (1)

Romania(UCb; HSPCd)

2 FCS Equippment-meat grinder (2)3 FCS Equippment-meat grinder (2), table (2), tumbler (1), cutter (1); collection tray (2); transport box (1);

processing area: colloid mill (1)3 NFCS Equipment: stainless steel bin (1), area for hands washing (1), floor (1); drain (1)

Contamination status of FPE before (I) sampling.a C contaminated.b UC uncontaminated; aContamination status of FPE after sampling (II).c SPC sporadically contaminated FPE.d HSPC hotspot contaminated FPE.e WDC widely contaminated FPE.f FCS Food-contact-surfaces.g NFCS Non-food-contact surfaces.

M. Muhterem-Uyar et al. / Food Control 51 (2015) 94e107104

M. Muhterem-Uyar et al. / Food Control 51 (2015) 94e107 105

et al., 2009). The source attribution of the zoonotic agent in thatsituation is very difficult (Chambel et al. 2007). Two farmhousecheese makers (Slovakian C-FPE and Irish UC-FPE) included in thestudy applied only chlorine-based disinfectants. In cases of resis-tance, sole use of one class of disinfectant could trigger wide dis-tribution of L. monocytogenes. Therefore, an alteration of acidic andalkaline disinfectants, along with a correct application of workingconcentrations and exposure time should be controlled throughoutthe production chain.

Hotspot contamination (ii) referred in the study to single sam-pling sites that proved to be repeatedly positive. No clear trans-mission route or pattern was found to cause the nichecontamination (Dalmasso & Jordan, 2014). The hygiene manage-ment system may have confined L. monocytogenes FPE contami-nations to certain niches (Blatter et al., 2010; Gounadaki,Skandamis, Drosinos, & Nychas, 2008; Parisi et al., 2013; Stesslet al., 2014). Infrequent or inadequate sampling could easily misshotspots. It also explains why in many outbreaks the route ofcontamination cannot be identified.

L. monocytogenes contamination throughout the facility seem tobe amajor problem of fast growing companies with high number oforders, permanent restructuring processes, high staff turnovers,and complex traffic patterns. In widely contaminated FPEs (iii) thehigher L. monocytogenes floor, drain andwall contamination led to across-border spread to the entire hygiene area including mobilefood transport elements (e.g. trolleys, boxes, conveyors and fork lifttrucks) (Dalmasso & Jordan, 2013; Rückerl et al., 2014). TheL. monocytogenes cross-contamination pressure is likely to be highand therefore assessed to be ‘difficult to manage’. In all widelydistributed contaminated FPEs disinfection seemed to be imple-mented on a daily basis as reported by the quality managementteam (QMT), but L. monocytogenes FPE contamination was noteliminated. Closer inspections showed that the assembly-line workwas not reduced in L. monocytogenes positive FPEs, cleaning pro-cedures and processing of food lots took place simultaneously insome cases, and the storage and ripening rooms were not includedin the cleaning plans. In such cases the QMT should be encouragedto develop a sanitation plan that, step by step, reducing thecontamination pressure. Such a plan should strictly include theestablishment of the CCAs concept since those areas would be thefirst that need to be reliably decontaminated. None of the FBOsexhibiting wide-spread contamination had a CCA concept in place.A CCA should encompass all rooms after the last effective decon-tamination step in the processing chain (e.g. after pasteurization orother thermal treatments) until the area where the food item isfinally packaged. The CCA section of a FPE should be marked andthe access should be restricted to specially trained staff. Disinfec-tion plans in a CCA should be most carefully and comprehensivelydeveloped including the latest state of knowledge with a specialfocus on the avoidance of dilution failures occurring on wet sur-faces. Protein residues and biofilms should be continuously moni-tored after sanitation to avoid the efficiency loss of disinfectantsand saponification process on floor surfaces (Gram, Bagge-Ravn,Ng, Gymoese, & Vogel, 2007; Hoelzer et al., 2012; van der Veen &Abee, 2011). Furthermore, drying of the surfaces after cleaning(physically, during the night or over a weekend) is highly recom-mended to facilitate a L. monocytogenes reduction in “cleaningponds” and airborne spread via condensates and aerosols (Berrang& Frank, 2012; Campdepadros et al., 2012; Lehto, Kuisma, M€a€att€a,Kym€al€ainen, & M€aki, 2011; Williams et al., 2011). The regular effi-cient desiccation of equipment and floors after sanitation mayexplain the low occurrence of L. monocytogenes at the UC-Austrianfacility.

EU regulations neither define details on the choice of samplingsite nor the sampling frequency for L. monocytogenes detection inFPEs. Therefore, FBOs and QMTs often neglect the importance ofdetermining the “in-house” L. monocytogenes FPE contaminationstatus to estimate the probability and risk of cross-contaminationto food lots. Sampling multiple times before (as sanitation con-trol), during processing or vector swabbing as suggested by Malleyet al. (2013) are not often observed. Infrequent sampling and notdefined CCAs can still be encountered during the inspection of foodprocessing facilities (Wagner & Stessl, 2014).

The three described L. monocytogenes FPE “contamination sce-narios” were important in increasing the FBOs safety awareness,but must be seen as dynamic. Previous studies have shown thatextensive sanitation can lead from widely distributed FPEcontamination to hotspot contamination (Campdepadr�os et al.,2012; Dalmasso & Jordan, 2013; Ferreira et al., 2011; Holah, Bird,& Hall, 2004; Ortiz et al., 2010). Though extensiveL. monocytogenes contamination could be restricted to hotspots, orreduced below the methodological limits of detection, the path-ogen is still present in the FPE and needs to be addressed (Dalmasso& Jordan, 2014; Garrido, Vitas, & García-Jal�on, 2009; Kovacevicet al., 2012; Pappelbaum et al., 2008; Rotariu et al., 2014;Tenenhaus-Aziza, Daudin, Maffre, & Sanaa, 2014).

Hygiene barriers, when present, did not prevent contamina-tion from being widespread or hotspot in nature. Furtherresearch on the type of hygiene barrier, the maintenance of suchbarriers and their efficacy would be required to define this moreclearly.

5. Conclusions

These data demonstrate that L. monocytogenes were commoncolonizers of FPEs in the European processing facilities sampled andthat a consistent cross-contamination risk existed either in farm-house based or larger food producing facilities.

The L. monocytogenes contamination rate determined in thecurrent study was not in accordance with the previous contami-nation status estimated by the project team, FBOs and QMTs.L. monocytogenes could be detected in each included FPE with ahigh variability in the occurrence rate between the plants. Knowl-edge on the type of contamination in a FPE, as described in thisstudy, could facilitate a better targeting of sanitation activities andimplement corrective action. Improving the awareness of FBOs andtheir staff on good hygiene practices should be mandatory andestimated by questionnaires as published by Lehto et al. (2011) andRotariu et al. (2014).

Verification of the sampling plan throughmonitoring the FPE forL. monocytogenes before and after sanitation should be an integralpart of the hygiene plan, particularly for food categories that areknown to support growth of L. monocytogenes. Close monitoring ofCCAs could increase processing environment hygiene with respectto L. monocytogenes contamination, thus reinforcing the progressthat has been achieved in the recent years (Carpentier & Barre,2012; Luber et al., 2011).

Acknowledgments

This work was supported by the project ‘Protection of con-sumers by mitigation of segregation of expertise’ (PROMISE,project number 265877, EU 7th Framework program). The authorswish to acknowledge the cooperation of the food business ownerswho participated in this work. We would like to thank Dr.

M. Muhterem-Uyar et al. / Food Control 51 (2015) 94e107106

Alexander Tichy (Vetmeduni, Vienna) for assistance with themanuscript.

References

Alali, W. Q., & Schaffner, D. W. (2013). Relationship between Listeria monocytogenesand Listeria spp. in seafood processing plants. Journal of Food Protection, 76,1279e1282.

Alessandria, V., Rantsiou, K., Dolci, P., & Cocolin, L. (2010). Molecular methods toassess Listeria monocytogenes route of contamination in a dairy processingplant. International Journal of Food Microbiology, 141, S156eS162.

Almeida, G., Magalh~aes, R., Carneiro, L., Santos, I., Silva, J., Ferreira, V., et al. (2013).Foci of contamination of Listeria monocytogenes in different cheese processingplants. International Journal of Food Microbiology, 167, 303e309.

Anonymous. (2005). Commission Regulation (EC) No 2073/2005 of 15 November2005 on microbiological criteria for foodstuffs. Official Journal of the EuropeanUnion L, 338, 1e29.

Barancelli, G. V., Camargo, T. M., Reis, C. M., Porto, E., Hofer, E., & Oliveira, C. A.(2011). Incidence of Listeria monocytogenes in cheese manufacturing plantsfrom the northeast region of S~ao Paulo, Brazil. Journal of Food Protection, 74,816e819.

Berrang, M. E., & Frank, J. F. (2012). Generation of airborne Listeria innocua frommodel floor drains. Journal of Food Protection, 75, 1328e1331.

Blatter, S., Giezendanner, N., Stephan, R., & Zweifel, C. (2010). Phenotypic and mo-lecular typing of Listeria monocytogenes isolated from the processing environ-ment and products of a sandwich-producing plant. Food Control, 21, 1519e1523.

Bubert, A., Hein, I., Rauch, M., Lehner, A., Yoon, B., Goebel, W., et al. (1999). Detectionand differentiation of Listeria spp. by a single reaction based on multiplex PCR.Applied and Environmental Microbiology, 65, 4688e4692.

Campdepadr�os, M., Stchigel, A. M., Romeu, M., Quilez, J., & Sol�a, R. (2012). Effec-tiveness of two sanitation procedures for decreasing the microbial contami-nation levels (including Listeria monocytogenes) on food contact and non-foodcontact surfaces in a dessert-processing factory. Food Control, 23, 26e31.

Carpentier, B., & Barre, L. (2012). Guidelines on sampling the food processing area andequipment for the detection of Listeria monocytogenes, Version 3 e 20/08/2012,EURL for Listeria monocytogenes, Maisons-Alfort Laboratory for Food Safety (pp.1e15). France: ANSES. https://pro.anses.fr/eurl-listeria/index.htm; (Accessed on17.11.14).

Carpentier, B., & Cerf, O. (2011). ReviewePersistence of Listeria monocytogenes infood industry equipment and premises. International Journal of Food Microbi-ology, 145, 1e8.

Chambel, L., Sol, M., Fernandes, I., Barbosa, M., Zilhao, I., Barata, B., et al. (2007).Occurrence and persistence of Listeria spp. in the environment of ewe and cow'smilk cheese dairies in Portugal unveiled by an integrated analysis of identifi-cation, typing and spatial-temporal mapping along production cycle. Interna-tional Journal of Food Microbiology, 116, 52e63.

Chiarini, E., Tyler, K., Farber, J., Pagotto, F., & Destro, M. (2009). Listeria mono-cytogenes in two different poultry facilities: manual and automatic evisceration.Poultry Science, 88, 791e797.

Dalmasso, M., & Jordan, K. (2013). Process environment sampling can help to reducethe occurrence of Listeria monocytogenes in food processing facilities. IrishJournal of Agricultural and Food Research, 52, 93e100.

Dalmasso, M., & Jordan, K. (2014). Absence of growth of Listeria monocytogenes innaturally contaminated Cheddar cheese. J. Dairy Research, 81, 46e53.

Di Ciccio, P., Meloni, D., Festino, A. R., Conter, M., Zanardi, E., Ghidini, S., et al. (2012).Longitudinal study on the sources of Listeria monocytogenes contamination incold-smoked salmon and its processing environment in Italy. InternationalJournal of Food Microbiology, 158, 79e84.

Ferreira, V., Barbosa, J., Stasiewicz, M., Vongkamjan, K., Moreno Switt, A., Hogg, T.,et al. (2011). Diverse geno- and phenotypes of persistent Listeria monocytogenesisolates from fermented meat sausage production facilities in Portugal. Appliedand Environmental Microbiology, 77, 2701e2715.

Ferreira, V., Wiedmann, M., Teixeira, P., & Stasiewicz, M. (2014). Listeria mono-cytogenes persistence in food-associated environments: epidemiology, straincharacteristics, and implications for public health. Journal of Food Protection, 77,150e170.

Fox, E., Hunt, K., O'Brien, M., & Jordan, K. (2011). Listeria monocytogenes in IrishFarmhouse cheese processing environments. International Journal of FoodMicrobiology, 145(Suppl. 1), S39eS45.

Fox, E., O'Brien, M., Dempsey, R., O'Mahony, T., Hunt, T., & Jordan, K. (2009). Theoccurrence if Listeria monocytogenes in the Irish farm environment. Journal ofFood Protection, 72, 1450e1456.

Garrido, V., Vitas, A., & García-Jal�on, I. (2009). Survey of Listeria monocytogenes inready-to-eat products: prevalence by brands and retail establishments forexposure assessment of listeriosis in Northern Spain. Food Control, 20, 986e991.

Gounadaki, A. S., Skandamis, P. N., Drosinos, E. H., & Nychas, G. E. (2008). Microbialecology of food contact surfaces and products of small-scale facilities producingtraditional sausages. Food Microbiology, 25, 313e323.

Gram, L., Bagge-Ravn, D., Ng, Y. Y., Gymoese, P., & Vogel, B. F. (2007). Influence offood soiling matrix on cleaning and disinfection efficiency on surface attachedListeria monocytogenes. Food Control, 18, 1165e1171.

Ho, A. J., Lappi, V. R., & Wiedmann, M. (2007). Longitudinal monitoring of Listeriamonocytogenes contamination patterns in a farmstead dairy processing facility.Journal of Dairy Science, 90, 2517e2524.

Hoelzer, K., Pouillot, R., Gallagher, D., Silverman, M. B., Kause, J., & Dennis, S. (2012).Estimation of Listeria monocytogenes transfer coefficients and efficacy of bac-terial removal through cleaning and sanitation. International Journal of FoodMicrobiology, 157, 267e277.

Holah, J. T., Bird, J., & Hall, K. E. (2004). The microbial ecology of high-risk, chilledfood factories; evidence for persistent Listeria spp. and Escherichia coli strains.Journal of Applied Microbiology, 97, 68e77.

Ibba, M., Cossu, F., Spanu, V., Virdis, S., Spanu, C., Scarano, C., et al. (2013). Listeriamonocytogenes contamination in dairy plants: evaluation of Listeria mono-cytogenes environmental contamination in two cheese-making plants usingsheeps milk. Italian Journal of Food Safety, 2, 109e112.

Keto-Timonen, R., Tolvanen, R., Lunden, J., & Korkeala, H. (2007). An 8-year sur-veillance of the diversity and persistence of Listeria monocytogenes in a chilledfood processing plant analyzed by amplified fragment length polymorphism.Journal of Food Protection, 70, 1866e1873.

Kovacevic, J., McIntyre, L. F., Henderson, S. B., & Kosatsky, T. (2012). Occurrence anddistribution of Listeria species in facilities producing ready-to-eat foods inBritish Columbia, Canada. Journal of Food Protection, 75, 216e224.

Lambertz, S. T., Ivarsson, S., Lopez-Valladares, G., Sidstedt, M., & Lindqvist, R. (2013).Subtyping of Listeria monocytogenes isolates recovered from retail ready-to-eatfoods, processing plants and listeriosis patients in Sweden 2010. InternationalJournal of Food Microbiology, 166, 186e192.

Latorre, A. A., Van Kessel, J. A., Karns, J. S., Zurakowski, M. J., Pradhan, A. K.,Zadoks, R. N., et al. (2009). Molecular ecology of Listeria monocytogenes: evi-dence for a reservoir in milking equipment on a dairy farm. Applied and Envi-ronmental Microbiology, 75, 1315e1323.

Lehto, M., Kuisma, R., M€a€att€a, J., Kym€al€ainen, H., & M€aki, M. (2011). Hygienic leveland surface contamination in fresh-cut vegetable production plants. FoodControl, 22, 469e475.

Lomonaco, S., Decastelli, L., Nucera, D., Gallina, S., Manila Bianchi, D., & Civera, T.(2009). Listeria monocytogenes in Gorgonzola: subtypes, diversity and persis-tence over time. International Journal of Food Microbiology, 128, 516e520.

Lomonaco, S., Verghese, B., Gerner-Smidt, P., Tarr, C., Gladney, L., Joseph, L., et al.(2013). Novel epidemic clones of Listeria monocytogenes, United States, 2011.Emerging Infectious Diseases, 19, 147e150.

Luber, P., Crerar, S., Dufour, C., Farber, J., Datta, A., & Todd, E. C. (2011). ControllingListeria monocytogenes in ready-to-eat foods: working towards global scientificconsensus and harmonizationerecommendations for improved prevention andcontrol. Food Control, 22, 1535e1549.

Malley, T. J., Stasiewicz, M. J., Grohn, Y. T., Roof, S., Warchocki, S., Nightingale, K.,et al. (2013). Implementation of statistical tools to support identification andmanagement of persistent Listeria monocytogenes contamination in smoked fishprocessing plants. Journal of Food Protection, 76, 796e811.

Martin, B., Garriga, M., & Aymerich, T. (2011). Prevalence of Salmonella spp. andListeria monocytogenes at small-scale Spanish factories producing traditionalfermented sausages. Journal of Food Protection, 74, 812e815.

Meloni, D., Piras, F., Mureddu, A., Fois, F., Consolati, S. G., Lamon, S., et al. (2013).Listeria monocytogenes in five sardinian swine slaughterhouses: prevalence,serotype, and genotype characterization. Journal of Food Protection, 76,1863e1867.

Oravcova, K., Kaclikova, E., Krascsenicsova, K., Pangallo, D., Brezna, B., Siekel, P., et al.(2006). Detection and quantification of Listeria monocytogenes by 5'-nucleasepolymerase chain reaction targeting the actA gene. Letters in Applied Microbi-ology, 42, 15e18.

Ortiz, S., L�opez, V., Villatoro, D., L�opez, P., D�avila, J. C., & Martínez-Su�arez, J. V. (2010).A 3-year surveillance of the genetic diversity and persistence of Listeria mon-ocytogenes in an Iberian pig slaughterhouse and processing plant. FoodbornePathogens and Disease, 7, 1177e1184.

Pagadala, S., Parveen, S., Rippen, T., Luchansky, J. B., Call, J. E., Tamplin, M. L., et al.(2012). Prevalence, characterization and sources of Listeria monocytogenes inblue crab (Callinectus sapidus) meat and blue crab processing plants. FoodMicrobiology, 31, 263e270.

Pappelbaum, K., Grif, K., Heller, I., Wuirzner, R., Hein, I., Ellerbroek, L., et al. (2008).Monitoring hygiene on- and at-line is critical for controlling Listeria mono-cytogenes during produce processing. Journal of Food Protection, 71, 735e741.

Parisi, A., Latorre, L., Fraccalvieri, R., Miccolupo, A., Normanno, G., Caruso, M., et al.(2013). Occurrence of Listeria spp. in dairy plants in Southern Italy and mo-lecular subtyping of isolates using AFLP. Food Control, 29, 91e97.

Prencipe, V. A., Rizzi, V., Acciari, V., Iannetti, L., Giovannini, A., Serraino, A., et al.(2012). Listeria monocytogenes prevalence, contamination levels and strainscharacterization throughout the Parma ham processing chain. Food Control, 25,150e158.

Reij, M. W., Den Aantrekker, E. D., & ILSI Europe Risk Analysis in Microbiology TaskForce. (2004). Recontamination as a source of pathogens in processed foods.International Journal of Food Microbiology, 91, 1e11.

Rodríguez-L�azaro, D., Hern�andez, M., Scortti, M., Esteve, T., V�azquez-Boland, J. A.,& Pla, M. (2004). Quantitative detection of Listeria monocytogenes and Listeriainnocua by real-time PCR: assessment of hly, iap, and lin02483 targets andAmpliFluor technology. Applied and Environmental Microbiology, 70,1366e1377.

M. Muhterem-Uyar et al. / Food Control 51 (2015) 94e107 107

Rotariu, O., Thomas, D. J. I., Goodburn, K. E., Hutchison, M. L., & Strachan, N. J. (2014).Smoked salmon industry practices and their association with Listeria mono-cytogenes. Food Control, 35, 284e292.

Rückerl, I., Muhterem-Uyar, M., Muri-Klinger, S., Wagner, K., Wagner, M., & Stessl, B.(2014). L. monocytogenes in a cheese processing facility: learning fromcontamination scenarios over three years of sampling. International Journal ofFood Microbiology, 18, 98e105.

Sakaridis, I., Soultos, N., Iossifidou, E., Papa, A., Ambrosiadis, I., & Koidis, P. (2011).Prevalence and antimicrobial resistance of Listeria monocytogenes isolated inchicken slaughterhouses in northern Greece. Journal of Food Protection, 74,1017e1021.

Schoder, D., Melzner, D., Schmalwieser, A., Zangana, A., Winter, P., & Wagner, M.(2011). Important vectors for Listeria monocytogenes transmission at farmdairies manufacturing fresh sheep and goat cheese from raw milk. Journal ofFood Protection, 74, 919e924.

da Silva, E. P., & De Martinis, E. C. (2013). Current knowledge and perspectives onbiofilm formation: the case of Listeria monocytogenes. Applied Microbiology andBiotechnology, 97, 957e968.

Stessl, B., Fricker, M., Fox, E., Karpiskova, R., Demnerova, K., Jordan, K., et al. (2014).Collaborative survey on the colonization of different types of cheese-processingfacilities with Listeria monocytogenes. Foodborne Pathogen and Disease, 11, 8e14.

Tenenhaus-Aziza, F., Daudin, J. J., Maffre, A., & Sanaa, M. (2014). Risk-based approachfor microbiological food safety management in the dairy industry: the case ofListeria monocytogenes in soft cheese made from pasteurized milk. Risk Analysis,34, 56e74.

Todd, E., & Notermans, S. (2011). Surveillance of listeriosis and its causative path-ogen, Listeria monocytogenes. Food Control, 22, 1484e1490.

Van der Veen, S., & Abee, T. (2011). Mixed species biofilms of Listeria mono-cytogenes and Lactobacillus plantarum show enhanced resistance to

benzalkonium chloride and peracetic acid. International Journal of FoodMicrobiology, 144, 421e431.

Viswanath, P., Murugesan, L., Knabel, S. J., Verghese, B., Chikthimmah, N., &Laborde, L. F. (2013). Incidence of Listeria monocytogenes and Listeria spp. in asmall-scale mushroom production facility. Journal of Food Protection, 76,608e615.

Vitas, A. I., Díez-Leturia, M., Tabar, L., & Gonz�alez, D. (2013). Improving the meth-odology for Listeria monocytogenes detection in smoked salmon by using thewet pooling test. International Journal of Food Microbiology, 184, 109e112.

Vongkamjan, K., Roof, S., Stasiewicz, M. J., & Wiedmann, M. (2013). Persistent Lis-teria monocytogenes subtypes isolated from a smoked fish processing facilityincluded both phage susceptible and resistant isolates. Food Microbiology, 35,38e48.

Wagner, M., & Stessl, B. (2014). Sampling the food processing environment: takingup the cudgel for preventive quality management in food processing environ-ments. In Anonymous, Listeria monocytogenes (pp. 275e283). New York:Springer.

Walsh, P. S., Metzger, D. A., & Higuchi, R. (1991). Chelex 100 as a medium for simpleextraction of DNA for PCR-based typing from forensic material. Biotechniques,10(4), 506e513.

Williams, S. K., Roof, S., Boyle, E. A., Burson, D., Thippareddi, H., Geornaras, I., et al.(2011). Molecular ecology of Listeria monocytogenes and other Listeria species insmall and very small ready-to-eat meat processing plants. Journal of Food Pro-tection, 74, 63e77.

Winkler, A., & Freund, M. (2011). The potential for third party standards atmanufacturing and retail to reduce the risk of listeriosis arising from con-sumption of Listeria monocytogenes from ready-to-eat foods. Food Control, 22,1503e1505.