International Journal of Food Microbiology 145 (2011) S39–S45

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International Journal of Food Microbiology

j ourna l homepage: www.e lsev ie r.com/ locate / i j foodmicro

Listeria monocytogenes in Irish Farmhouse cheese processing environments

Edward Fox, Karen Hunt, Martina O'Brien, Kieran Jordan ⁎Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland

⁎ Corresponding author. Moorepark Food Research CeTel.: +3532542451; fax: +3532542340.

E-mail address: kieran.jordan@teagasc.ie (K. Jordan)

0168-1605/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.ijfoodmicro.2010.10.012

a b s t r a c t

a r t i c l e i n f o

Article history:Received 25 June 2010Received in revised form 28 September 2010Accepted 17 October 2010

Keywords:Cheese processing facilitiesListeriaFood safetyPersistence

Sixteen cheesemaking facilities were sampled during the production season at monthly intervals over a two-year period. Thirteen facilities were found to have samples positive for Listeria monocytogenes. Samples weredivided into 4 categories; cheese, raw milk, processing environment and external to the processingenvironment (samples from the farm such as silage, bedding, and pooled water). In order to attempt toidentify the source, persistence and putative transfer routes of contamination with the L. monocytogenesisolates, they were differentiated using PFGE and serotyping. Of the 250 isolates, there were 52 differentpulsotypes. No pulsotype was found at more than one facility. Two facilities had persistent pulsotypes thatwere isolated on sampling occasions at least 6 months apart. Of the samples tested, 6.3% of milk, 13.1% ofprocessing environment and 12.3% of samples external to the processing environment, respectively, werepositive for L. monocytogenes. Pulsotypes found in raw milk were also found in the processing environment,however, one of the pulsotypes from raw milk was found in cheese on only one occasion. One of thepulsotypes isolated from the environment external to the processing facility was found on the surface ofcheese, however, a number of them were found in the processing environment. The results suggest that thefarm environment external to the processing environment may in some cases be the source of processingenvironment contamination with L. monocytogenes.

ntre, Fermoy, Co. Cork, Ireland.

.

l rights reserved.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Listeria spp. frequently contaminate foods (Lianou and Sofos,2007) and as Listeria monocytogenes is pathogenic, contaminationwith L. monocytogenes is considered a public health risk. Food isconsidered the major vehicle of Listeriosis transmission (Farber andPeterkin, 1991) that can affect at risk populations like the young, old,immunocompromised and pregnant woman, with a high mortalityrate (Lynch et al., 2006). In Europe, the incidence of listeriosis hasincreased from 0.1 cases per 100,000 in 2000 to 0.3 cases per 100,000in 2006 (Denny and McLauchlin, 2008). Dairy related outbreaks inCalifornia in 1985 (Linnan et al., 1988), Japan in 2001 (Makino et al.,2005), Canada in 2008 (Public Health Agency of Canada, 2009) andother places (seeWarriner and Namvar, 2009) illustrate the relevanceof dairy products in listeriosis outbreaks.

L. monocytogenes is widespread in the environment. Nineteenpercent of dairy farm samples (Fox et al., 2009), 22% of samples fromfish slaughter- and smokehouses (Wulff et al., 2006), 16% of dairy

processing plants (Pritchard et al., 1994) and 12.8% of smoked fishprocessing facilities (Thimothe et al., 2004) were positive. Thepresence of L. monocytogenes in processing environments is importantas transfer from the environment to the food can occur (D'Amico andDonnelly 2008).

Certain foods are considered high risk for contamination withL. monocytogenes. These include raw milk, soft cheese, ready-to-eat(RTE) meats and smoked fish. Occurrence on various foods has beenreviewed recently by Lianou and Sofos (2007). Of particular concernare RTE foods that are able to support the growth of L. monocytogenesas numbers can reach higher levels, even at refrigeration tempera-tures, and pose a higher risk.

While product testing is important, it does not give information onthe route of contamination. Environmental testing is a more effectiveway to assess hygiene and prevent future contamination events(Tompkin, 2002). Molecular subtyping of isolates from environmentalmonitoring is critical in characterising contamination patterns andtransmission of L. monocytogenes in processing plants (Lappi et al.,2004; Ho et al., 2007). These techniques have shown that some sub-types persist over time (reviewed by Tompkin, 2002) and that cross-contamination from the environment to cheese can occur (D'Amicoand Donnelly, 2008).

The aim of this study was to undertake environmental sampling at16 farmhouse cheesemaking facilities over a two-year period, with aview to identifying the ecology of the strains and to tracing L.monocytogenes isolates in the cheese processing facilities.

S40 E. Fox et al. / International Journal of Food Microbiology 145 (2011) S39–S45

2. Materials and methods

2.1. Sample collection

Samples were collected from 16 farmhouse cheese manufacturingfacilities across Ireland over a two-year period from 2007 to 2008. Thedistance between facilities ranged from 20 to 400 km. Each facility hadits own individual staff. All facilities have associated farms, 75% sourcemilk from their own herd, while 25% source milk from local herds.Sampling was targeted at areas where L. monocytogeneswas likely to befound. The sample type was divided into four groups; 1) cheese, 2) rawmilk, 3) processing environment and 4) external to the processingenvironment. Samples external to the processing environment includedstraw, faeces, pooledwater, soil and dust and were collected asepticallyand placed in sterile containers. The sample siteswere 10 to 200 m from

Table 1Summary of the facilities sampled, the sub-types and persistence of the isolates obtained.

Facility no. Number of samples % Samples positive Pulsotypes Duratio

1 58 0 – –

2 48 10.4 2/1 Jul08–A2/2 Jul082/3 Jul08

3 56 3.6 3/1 Mar083/2 Jun08

4 62 1.6 4/1 Dec085 43 0 – –

6 127 3.9 6/1 Oct076/2 Jun07–O6/3 Aug07

7 66 1.5 7/1 May088 32 3.1 8/1 Jun089 50 18.0 9/1 Nov07

9/2 Jul089/3 Aug089/4 Sep089/5 Jul08–A9/6 Aug08

10 473 14.1 10/1 Apr07–10/2 Apr0810/3 Apr0810/4 Nov0710/5 Apr0810/6 Apr0810/7 Feb0910/8 Apr0810/9 Jul0710/10 Apr0810/11 Aug08

11 40 7.5 11/1 May08–11/2 Nov08

12 21 0 – –

13 44 9.1 13/1 Jul0713/2 Aug0813/3 Oct0813/4 Oct08

14 56 5.4 14/1 Sep0814/2 Mar0814/3 Jul08

15 296 20.9 15/1 Jun07–N15/2 Oct0815/3 Jul07–S15/4 Aug07–

16 119 16.8 16/1 Jun0716/2 Apr07–16/3 Apr0716/4 Apr07–16/5 Apr0716/6 Apr0716/7 Apr07–16/8 Apr0716/9 Apr071610 Apr0716/11 Jun07

the cheesemaking facilities. Processing environment samples includeddrains, floors, walls, doors, food contact surfaces and brine.

Swab samples were taken and collected using Whirl-Pak “Speci-Sponge” Bags (Nasco, Modesto, California). To moisten the sponge,10 ml of half Frazer broth was added to the bag with the sponge andusing a sterile forceps to hold the sponge, the area (1 m2) was swabbedand the sponge placed back into the bag. Liquid samples were collectedusing 100 ml sterile dippers. All samples were collected during cheeseproduction, wearing gloves and appropriate protective clothing,individually packaged to prevent cross-contamination, placed in a coolbox with ice packs and transported directly to the laboratory andanalysed immediately. Cheese samples were placed in plastic wrappingand stored in an insulated container with ice packs for transport to thelaboratory,where theywere analysed immediately or stored at less than4 °C and analysed the following day.

n of pulsotype detection Frequency of isolation Serotype Persistence

– – –

ug08 3 1/2a No1 4b No1 4b No1 1/2a No1 4b No1 4b No

– – –

1 1/2a Noct07 5 4b No

1 1/2a No2 1/2a No1 1/2a No2 1/2b No1 4b No1 1/2b No1 1/2b No

ug08 3 1/2b No1 4b No

Dec08 122 1/2a Yes1 1/2a No2 1/2b No1 1/2a No1 1/2a No1 4b No2 4b No1 4b No1 4b No1 1/2a No1 1/2b No

Aug08 2 1/2a No1 1/2a No

– – –

1 1/2a No1 Untypable No1 4b No1 1/2a No1 1/2a No1 1/2a No1 4b No

ov08 47 1/2c Yes1 1/2b No

ep08 2 1/2c YesSept08 3 1/2c Yes

2 4b NoJun07 3 4b No

5 4b NoJun07 3 3b No

1 1/2b No2 4b No

Jun07 5 1/2b No1 4b No2 4b No1 1/2b No1 Untypable No

S41E. Fox et al. / International Journal of Food Microbiology 145 (2011) S39–S45

2.2. Microbiological analyses

Sampleswere analysed by the ISO 11290-1 (ISO 2002) for presence/absence of L. monocytogenes. After this enrichment step, 50 μl wasspread on an ALOA (Agosti & Ottaviani Listeria Agar; LabM, Lancashire,UK,HAL010) agarplatewhichwas then incubatedat37 °C for 24 to48 h.Typical L. monocytogenes colonies (which are blue-green with a

Similarity10090807060403020 50

NT: Not typable

Fig. 1. Cluster analysis (using BioNumerics) for the 52 pulsotypes from 16 farmhouse cheese f

surrounding halo) were isolated and purified by re-streaking on ALOAagar, followed by streaking on tryptone soy agar (TSA). Single pureisolated colonieswere grown overnight in tryptone soy broth (TSB) andfrozen in cryovials in a glycerol/TSB mixture at −20 °C. For theenvironmental sponge samples, 90 ml half Frazer broth was added tothe ‘Speci-Sponge’ bag whichwas incubated at 37 °C for 24 h. After thispre-enrichment the samples were treated as detailed in ISO 11290.

1/2a

4b

1/2b

4b

1/2a

1/2a

1/2a

1/2a

1/2a

1/2a

1/2a

1/2a

1/2a

1/2a

1/2a

1/2c

1/2a

1/2a

4b

1/2a

4b

1/2b

4b

1/2b

1/2b

4b

4b

NT

1/2a

NT

1/2b

1/2b

1/2b

1/2b

4b

4b

4b

4b

4b

4b

4b

4b

4b

4b

4b

1/2a

1/2b

4b

1/2b

1/2b

4b

NT

14/1

15/3

9/1

16/1

10/4

10/5

14/2

6/3

10/1

7/1

8/1

10/10

2/1

3/1

6/1

15/1

13/1

13/4

16/3

10/2

16/9

15/2

2/2

16/10

16/5

10/9

16/2

16/11

11/2

16/4

15/4

16/7

10/3

10/11

6/2

14/3

3/2

10/7

16/6

4/1

16/8

10/6

9/6

10/8

2/3

11/1

9/5

9/2

9/3

9/4

13/3

13/2

AscI Serotype Pulsotype

acilities, showing the serotype and the pulsotype identification (using the enzyme AscI).

S42 E. Fox et al. / International Journal of Food Microbiology 145 (2011) S39–S45

2.3. Confirmation by PCR

All purified isolates were confirmed as L. monocytogenes usingReal-Time PCR (Rodríguez-Lázaro et al., 2004), as described by O'Brienet al. (2009).

2.4. Pulsed-field gel electrophoresis

Pulsed-Field Gel Electrophoresis (PFGE) of all L. monocytogenesisolates was performed primarily with the enzyme AscI using thestandard PulseNet protocol as described by Graves and Swaminathan(2001). Persistent strains isolated at different sampling times werealso typed with ApaI to confirm strain similarities.

2.5. Serotyping

Serotyping of the isolates was performed as described in Fox et al.(2009), using a combination of antisera and serotype-specific PCR(Doumith et al., 2004).

3. Results

The results of the sampling and analysis are summarised in Table 1.Of the 16 facilities sampled, L. monocytogenes was detected from atleast one sample taken from 13 of the facilities. No isolateswere obtained from the remaining 3 facilities. Of the 13 facilities,L. monocytogenes was isolated on only one sampling date from 3facilities. For the remaining 10 facilities, it was isolated more thanonce. In total, 1591 samples were collected and 250 (15.7%) werepositive for L. monocytogenes. All isolates were confirmed as L.monocytogenes by PCR. Serotyping of all isolates showed a number ofdifferent serotypes (Table 1). The isolates were mainly serotypes 4band 1/2a (72%). At facility 16, most of the isolates were from samplestaken external to the processing facility. Only 3 isolates wereuntypable. All isolates were further sub-typed using PFGE. A total of52 pulsotypes were identified (Fig. 1). These were named by thefacility number from which they were isolated and the number of theisolate from that facility. No common pulsotype was found betweenthe facilities. More than one pulsotype was obtained at 10 of the 13facilities.

Positive milk samples were found at 7 of the 13 facilities. Three ofthese PFGE patterns (i.e. 2/1, 11/1, 13/1) were also identified in othersamples at the same facility. At the remaining 6 facilities, the PFGEpatterns of isolates found in the processing facilities were notidentified in samples taken externally. At 2 of the facilities,indistinguishable patterns were seen in raw milk and the processingenvironment. At 1 of the facilities, an indistinguishable pulsotype wasfound in raw milk and cheese.

Fig. 2 shows the ApaI and AscI digest for a persistent strain (10/1),isolated over a 10-year period, with indistinguishable ApaI and AscIpatterns.

At each facility, all pulsotypes identified were unique to thatfacility. However, two sets of strains showed N95% similarity (10/9and 16/2; 15/4 and 16/7; Fig. 1). Within these facilities, the putative

Fig. 2. PFGE pulsotypes of the persistent strain 10/1 isolate

transfer patterns of strains were compared using PFGE pulsotypes(Table 2). Diagrammatic representation of the transfer of strainswithin 1 of the facilities is shown in Fig. 3. The data suggest thattransfer between the area external to the processing facility and theprocessing environment may have occurred.

From the 13 facilities where isolates were obtained, persistentisolates were identified at two facilities (10 and 15; see Table 1). Forthe purposes of this study, persistence was defined as isolation of thesame pulsotype from a facility over a period of more than 6 months.Four of the 52 pulsotypes (7.7%) were persistent in the environmentfrom which they were isolated. The persistent pulsotypes wereisolated from the two facilities where a greater number of sampleswere obtained.

4. Discussion

PFGE is a valuable tool in tracing the strain similarities and putativetransfer routes of L. monocytogenes in food and food processingfacilities. Other typing methods like serotyping (Gianfranceschi et al.,2009), phage typing (Jacquet et al., 1993), Ribotyping (Norton et al.,2001; Meloni et al., 2009), RAPD (Wulff et al., 2006) and AFLP (Autioet al., 2003; Keto-Timonen et al., 2007) have been used to distinguishL. monocytogenes isolates. However, PFGE (which was used in thisstudy) has the greatest power of discrimination and is currently theaccepted ‘gold standard’ for such studies.

Using PFGE, the environmental samples from the 16 facilitiesresulted in 52 different pulsotypes. Previous studies on fish (Thimotheet al., 2004; Gudmundsdóottir et al., 2005), meat (Felício et al., 2007;Peccio et al., 2003) and dairy (Waak et al., 2002; Wagner et al., 2006;Lomonaco et al., 2009) processing facilities have been undertaken.Apart from the Austrian study, such environmental sampling ofcheese processing facilities has involved only one or two facilities(Jacquet et al., 1993; Silva et al., 2003), or sampling over a short timeperiod (D'Amico and Donnelly, 2008).

In the current extensive study involving 16 cheesemaking facilitiesover a two-year period, L. monocytogenes was not isolated from 3(19%) of the 16 cheese processing facilities tested, even thoughsampling was targeted at locations likely to harbour L. monocytogenes.On the basis of workflows, sanitation and access practices, thesefacilities were similar to facilities where L. monocytogenes wasdetected. L. monocytogenes was isolated from N10% of the samplesat 5 (31%) of the facilities. The prevalence of L. monocytogenesdetected in cheese processing facilities in the current study (13 of 16facilities sampled) was considerably higher than the 50 of 181facilities found positive in Austria (Wagner et al., 2006). However, inthe Austrian study, most of the samples were from cheese or smear(89%), effectively making it a survey of prevalence in cheese. Thecurrent study was more extensive in terms of non-food contactsamples.

Strain diversity in both serotype and pulsotype was seen in theisolates obtained at the facilities. Facilities 10 and 16 showed the highestdegree of diversity with 11 pulsotypes identified at each one. Serotypes1/2a and 4b, and serotypes 1/2b and 4b, were the most prevalent atfacilities 10 and 16, respectively. In an independent study, 298 samples

d from different samples at facility 10 since Jan 1999.

Table 2Source and epidemiology of the L. monocytogenes isolates from 16 cheesemakingfacilities.

Siteno.

Cheese type,milk type

Pulsotype Putativetransfer routea

Positive samplescollected

1 Soft white, cow – – –

2 Semi-hard waxcoated, sheep

2/1 M; PE Milk, drain2/2 – Milk2/3 – Dairy

3 Soft white mould, 3/1 – Draingoat 3/2 – Drain

4 Soft blue, sheep 4/1 – Milk5 Hard cheese, goat – – –

6 Semi-soft smearripened, cow

6/1 – Door6/2 PE; C Door, cheese6/3 – Cheese

7 Semi-soft, waxcoated, cow

7/1 – Cheese

8 Gouda, cow 8/1 – Milk9 Soft Blue mould, cow 9/1 – Cheese

9/2 – Milk9/3 – Drain9/4 – Prep table9/5 EPE; PE Floor, drain, soil9/6 Cheese

10 Semi-soft smearripened, cow

10/1 PE; C Cheese, processenvironment

10/2 – Cheese10/3 – Cheese10/4 – Cheese10/5 – Cheese10/6 – Drain10/7 – Milk10/8 – Cheese10/9 – Cheese10/10 – Cheese10/11 – Milk sock

11 Fresh, cow 11/1 M; PE Milk, drain11/2 – Drain

12 Hard, cow – – –

13 Semi-hard, cow 13/1 M; C Cheese, milk13/2 – Drain13/3 – Milk13/4 – Pooled water

14 Gouda, goat 14/1 – Drain14/2 – Floor14/3 – Drain

15 Soft blue mould, cow 15/1 EPE; PE; C Straw, processenvironment, cheese

15/2 – Walls15/3 C; PE Cheese, processing

environment15/4 – Ripening room racks

16 Hard cheese, cow 16/1 C; PE Cheese, sink16/2 PE Sieve drain, floor16/3 EPE; PE Straw, cheese press,

drain16/4 – Pooled water16/5 – Straw16/6 – Drain16/7 – Bovine manure16/8 – Straw16/9 – Floor16/10 – Processing environment16/11 – Processing environment

a EPE = external to processing environment; PE = processing environment; M =milk, and C = cheese.

10/1

10/2

10/3

10/4

10/5

10/6

10/7

10/8

10/9

10/10

OUTSIDE BUILDING

DAIRY BUILDING

MILK

PROCESSING HALL

RIPENING ROOM

FOOD CONTACT SURFACES

CHEESE

EXTERNAL TO PROCESSING ENVIRONMENT

PROCESSING ENVIRONMENT

10/11

Fig. 3. Spread of various pulsotypes isolated at facility 10.

S43E. Fox et al. / International Journal of Food Microbiology 145 (2011) S39–S45

from 16 farms found serotypes 1/2a, 1/2b and 4b were most prevalent(Fox et al., 2009). Serotypes 1/2a, 1/2b and 4b are also the mostprevalent in clinical cases (Tompkin, 2002; Zhang et al., 2007).

Each facility had unique pulsotypes. However, there were 6 pairsof strains with N90% similarity, 5 at different sites and one within site16. (Fig. 1). Strains 16/2 and 16/11 were isolated at the same site andalthough 16/11 was untypable, it could be a sub-type of 16/2. The

remaining 5 sets of similar strains could be sub-types that wereisolated at different sites. This suggests the possibility of some linkbetween these sites at some time. In Gorgonzola cheese, indistin-guishable pulsotypes have been isolated from different cheesemakingfacilities that are apparently unrelated (Lomonaco et al., 2009).

Persistence of particular sub-types in an environment wasmonitored by Harvey and Gilmour (1993). In a review by Tompkin(2002), persistence of L. monocytogenes in various food processingfacilities was discussed. Many different serotypes showed persistence,many of the persistent strains were implicated in illness and thepersistence time ranged from a few months to 10 years, including astudy by Unnerstad et al. (1996) showing persistence in a dairyenvironment for more than 7 years. Wulff et al. (2006) and Lomonacoet al. (2009) also reported persistent strains in fish and cheeseprocessing facilities, respectively. In the current study, persistencewas defined as repeated isolation from samples taken at least6 months apart; four of the 52 sub-types were persistent. Thesewere isolated at two of the 16 facilities. One of these persistent strains(10/1) was isolated from the same facility over a 10-year period(Fig. 2) while the remaining three persistent strains were isolatedmore than 6 months apart. Persistence may be related to a range ofphysiological characteristics including attachment and biofilm for-mation (Mafu et al., 1990; Lunden et al., 2000; Møretrø and Langsrud,2004), resistance to sanitisers (Pan et al., 2006; Chavant et al., 2002)and adaptive responses (Lundén et al., 2003). The basis for persistenceof the strains isolated needs to be investigated.

In most studies of this kind, sampling is limited to food and theprocessing environment, although Ho et al. (2007) also found thatfarm and processing facility strains were distinct. In the current study,sampling was extended to areas external to the processing facility. At

S44 E. Fox et al. / International Journal of Food Microbiology 145 (2011) S39–S45

two of the facilities, indistinguishable pulsotypes (15/1 and 16/3)were isolated from samples internal and external to the processingfacilities. In the case of strain 15/1, this strain was also found on thefinal product, indicating a putative transfer between the externalenvironment and the food product. There is a possibility that thetransfer of the strains was from the processing facility to the externalenvironment, but even if such an event occurred, the opportunity forrecontamination of the processing facility is increased.

Where persistent strains were found (10/1, 15/1, 15/3 and 15/4;Table 1), isolation from a variety of samples within the processingfacility was observed over the two-year period. This implies thatcross-contamination within the processing facility could be occurring(Reij et al., 2003). Preventing cross-contamination between dairyproduction and processing facilities and preventing contaminationcycles within processing facilities are critical to controlling L.monocytogenes thus assuring the microbial safety of farmhousedairy products. This control can be achieved by appropriate work-flows, good quality raw materials and improved hygiene practices(Holah, 2003).

L. monocytogenes was isolated from 6.3% of raw milk samples (8/127). At 7 of the 16 facilities, milk samples contained L. mono-cytogenes. Three of these pulsotypes were also found in non-milksamples at the same facilities, indicating that milk is a possible vectorfor contamination of cheesemaking facilities. This highlights theimportance of prevention of milk contamination.

5. Conclusions

In order to control L. monocytogenes contamination offinal product,and possible infection of consumers, care must also be taken to ensurethat recontamination of the processing environment is not originatingfrom an external source. The results of this study indicate thatcontamination of food processing facilities with L. monocytogenesappears to be sporadic with the majority of strains not persisting.With 75% of cheesemaking facilities showing contamination withL. monocytogenes in the processing environment, strictmonitoring andimplementation of a control regime is an essential part of the pre-vention of contamination of the final product.

Acknowledgements

This work was supported by the EU 6th Framework Programmeunder the project BIOTRACER, project number 036272 and by the IrishGovernment under the FIRM Programme, project number06RDTMFRC434. The authors wish to acknowledge the assistance ofCAIS – the Irish Farmhouse Cheesemakers Association – and thecooperation of all 16 cheesemakers who participated in this work.

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