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

Contents lists available at ScienceDirect

International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

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

a r t i c l e i n f o a b s t r a c t

Article history: Sixteen cheesemaking facilities were sampled during the production season at monthly intervals over a two-
Received 25 June 2010 year period. Thirteen facilities were found to have samples positive for Listeria monocytogenes. Samples were
Received in revised form 28 September 2010 divided into 4 categories; cheese, raw milk, processing environment and external to the processing
Accepted 17 October 2010
environment (samples from the farm such as silage, bedding, and pooled water). In order to attempt to
identify the source, persistence and putative transfer routes of contamination with the L. monocytogenes
Keywords:
Cheese processing facilities
isolates, they were differentiated using PFGE and serotyping. Of the 250 isolates, there were 52 different
Listeria pulsotypes. No pulsotype was found at more than one facility. Two facilities had persistent pulsotypes that
Food safety were isolated on sampling occasions at least 6 months apart. Of the samples tested, 6.3% of milk, 13.1% of
Persistence processing environment and 12.3% of samples external to the processing environment, respectively, were
positive 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 the
pulsotypes isolated from the environment external to the processing facility was found on the surface of
cheese, however, a number of them were found in the processing environment. The results suggest that the
farm environment external to the processing environment may in some cases be the source of processing
environment contamination with L. monocytogenes.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction
Listeria spp. frequently contaminate foods (Lianou and Sofos,
2007) and as Listeria monocytogenes is pathogenic, contamination
with L. monocytogenes is considered a public health risk. Food is
considered the major vehicle of Listeriosis transmission (Farber and
Peterkin, 1991) that can affect at risk populations like the young, old,
immunocompromised and pregnant woman, with a high mortality
rate (Lynch et al., 2006). In Europe, the incidence of listeriosis has
increased from 0.1 cases per 100,000 in 2000 to 0.3 cases per 100,000
in 2006 (Denny and McLauchlin, 2008). Dairy related outbreaks in
California in 1985 (Linnan et al., 1988), Japan in 2001 (Makino et al.,
2005), Canada in 2008 (Public Health Agency of Canada, 2009) and
other places (see Warriner and Namvar, 2009) illustrate the relevance
of dairy products in listeriosis outbreaks.
L. monocytogenes is widespread in the environment. Nineteen
percent of dairy farm samples (Fox et al., 2009), 22% of samples from
fish slaughter- and smokehouses (Wulff et al., 2006), 16% of dairy
⁎ Corresponding author. Moorepark Food Research Centre, Fermoy, Co. Cork, Ireland.
Tel.: + 3532542451; fax: + 3532542340.
E-mail address: kieran.jordan@teagasc.ie (K. Jordan).
processing plants (Pritchard et al., 1994) and 12.8% of smoked fish
processing facilities (Thimothe et al., 2004) were positive. The
presence of L. monocytogenes in processing environments is important
as transfer from the environment to the food can occur (D'Amico and
Donnelly 2008).
Certain foods are considered high risk for contamination with
L. monocytogenes. These include raw milk, soft cheese, ready-to-eat
(RTE) meats and smoked fish. Occurrence on various foods has been
reviewed recently by Lianou and Sofos (2007). Of particular concern
are RTE foods that are able to support the growth of L. monocytogenes
as 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 on
the route of contamination. Environmental testing is a more effective
way to assess hygiene and prevent future contamination events
(Tompkin, 2002). Molecular subtyping of isolates from environmental
monitoring is critical in characterising contamination patterns and
transmission 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'Amico
and Donnelly, 2008).
The aim of this study was to undertake environmental sampling at
16 farmhouse cheesemaking facilities over a two-year period, with a
view to identifying the ecology of the strains and to tracing L.
monocytogenes isolates in the cheese processing facilities.

0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2010.10.012
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 manufacturing
facilities across Ireland over a two-year period from 2007 to 2008. The
distance between facilities ranged from 20 to 400 km. Each facility had
its own individual staff. All facilities have associated farms, 75% source
milk from their own herd, while 25% source milk from local herds.
Sampling was targeted at areas where L. monocytogenes was likely to be
found. The sample type was divided into four groups; 1) cheese, 2) raw
milk, 3) processing environment and 4) external to the processing
environment. Samples external to the processing environment included
straw, faeces, pooled water, soil and dust and were collected aseptically
and placed in sterile containers. The sample sites were 10 to 200 m from
the cheesemaking facilities. Processing environment samples included
drains, 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 and
using a sterile forceps to hold the sponge, the area (1 m2) was swabbed
and the sponge placed back into the bag. Liquid samples were collected
using 100 ml sterile dippers. All samples were collected during cheese
production, wearing gloves and appropriate protective clothing,
individually packaged to prevent cross-contamination, placed in a cool
box with ice packs and transported directly to the laboratory and
analysed immediately. Cheese samples were placed in plastic wrapping
and stored in an insulated container with ice packs for transport to the
laboratory, where they were analysed immediately or stored at less than
4 °C and analysed the following day.

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

Facility no. Number of samples % Samples positive Pulsotypes Duration of pulsotype detection Frequency of isolation Serotype Persistence

1 58 0 – – – – –
2 48 10.4 2/1 Jul08–Aug08 3 1/2a No
2/2 Jul08 1 4b No
2/3 Jul08 1 4b No
3 56 3.6 3/1 Mar08 1 1/2a No
3/2 Jun08 1 4b No
4 62 1.6 4/1 Dec08 1 4b No
5 43 0 – – – – –
6 127 3.9 6/1 Oct07 1 1/2a No
6/2 Jun07–Oct07 5 4b No
6/3 Aug07 1 1/2a No
7 66 1.5 7/1 May08 2 1/2a No
8 32 3.1 8/1 Jun08 1 1/2a No
9 50 18.0 9/1 Nov07 2 1/2b No
9/2 Jul08 1 4b No
9/3 Aug08 1 1/2b No
9/4 Sep08 1 1/2b No
9/5 Jul08–Aug08 3 1/2b No
9/6 Aug08 1 4b No
10 473 14.1 10/1 Apr07–Dec08 122 1/2a Yes
10/2 Apr08 1 1/2a No
10/3 Apr08 2 1/2b No
10/4 Nov07 1 1/2a No
10/5 Apr08 1 1/2a No
10/6 Apr08 1 4b No
10/7 Feb09 2 4b No
10/8 Apr08 1 4b No
10/9 Jul07 1 4b No
10/10 Apr08 1 1/2a No
10/11 Aug08 1 1/2b No
11 40 7.5 11/1 May08–Aug08 2 1/2a No
11/2 Nov08 1 1/2a No
12 21 0 – – – – –
13 44 9.1 13/1 Jul07 1 1/2a No
13/2 Aug08 1 Untypable No
13/3 Oct08 1 4b No
13/4 Oct08 1 1/2a No
14 56 5.4 14/1 Sep08 1 1/2a No
14/2 Mar08 1 1/2a No
14/3 Jul08 1 4b No
15 296 20.9 15/1 Jun07–Nov08 47 1/2c Yes
15/2 Oct08 1 1/2b No
15/3 Jul07–Sep08 2 1/2c Yes
15/4 Aug07–Sept08 3 1/2c Yes
16 119 16.8 16/1 Jun07 2 4b No
16/2 Apr07–Jun07 3 4b No
16/3 Apr07 5 4b No
16/4 Apr07–Jun07 3 3b No
16/5 Apr07 1 1/2b No
16/6 Apr07 2 4b No
16/7 Apr07–Jun07 5 1/2b No
16/8 Apr07 1 4b No
16/9 Apr07 2 4b No
1610 Apr07 1 1/2b No
16/11 Jun07 1 Untypable No
 E. Fox et al. / International Journal of Food Microbiology 145 (2011) S39–S45 S41

2.2. Microbiological analyses
Samples were analysed by the ISO 11290-1 (ISO 2002) for presence/
absence of L. monocytogenes. After this enrichment step, 50 μl was
spread on an ALOA (Agosti & Ottaviani Listeria Agar; LabM, Lancashire,
UK, HAL010) agar plate which was then incubated at 37 °C for 24 to 48 h.
Typical L. monocytogenes colonies (which are blue-green with a
surrounding halo) were isolated and purified by re-streaking on ALOA
agar, followed by streaking on tryptone soy agar (TSA). Single pure
isolated colonies were grown overnight in tryptone soy broth (TSB) and
frozen in cryovials in a glycerol/TSB mixture at −20 °C. For the
environmental sponge samples, 90 ml half Frazer broth was added to
the ‘Speci-Sponge’ bag which was incubated at 37 °C for 24 h. After this
pre-enrichment the samples were treated as detailed in ISO 11290.

Similarity AscI Serotype Pulsotype

20 30 40 50 60 70 80 90 100
1/2a 14/1
4b 15/3
1/2b 9/1
4b 16/1
1/2a 10/4
1/2a 10/5
1/2a 14/2
1/2a 6/3
1/2a 10/1
1/2a 7/1
1/2a 8/1
1/2a 10/10
1/2a 2/1
1/2a 3/1
1/2a 6/1
1/2c 15/1
1/2a 13/1
1/2a 13/4
4b 16/3
1/2a 10/2
4b 16/9
1/2b 15/2
4b 2/2
1/2b 16/10
1/2b 16/5
4b 10/9
4b 16/2
NT 16/11
1/2a 11/2
NT 16/4
1/2b 15/4
1/2b 16/7
1/2b 10/3
1/2b 10/11
4b 6/2
4b 14/3
4b 3/2
4b 10/7
4b 16/6
4b 4/1
4b 16/8
4b 10/6
4b 9/6
4b 10/8
4b 2/3
1/2a 11/1
1/2b 9/5
4b 9/2
1/2b 9/3
1/2b 9/4
4b 13/3
NT 13/2

NT: Not typable

Fig. 1. Cluster analysis (using BioNumerics) for the 52 pulsotypes from 16 farmhouse cheese facilities, 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 using
Real-Time PCR (Rodríguez-Lázaro et al., 2004), as described by O'Brien
et al. (2009).
2.4. Pulsed-field gel electrophoresis
Pulsed-Field Gel Electrophoresis (PFGE) of all L. monocytogenes
isolates was performed primarily with the enzyme AscI using the
standard PulseNet protocol as described by Graves and Swaminathan
(2001). Persistent strains isolated at different sampling times were
also 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 at
least one sample taken from 13 of the facilities. No isolates
were obtained from the remaining 3 facilities. Of the 13 facilities,
L. monocytogenes was isolated on only one sampling date from 3
facilities. For the remaining 10 facilities, it was isolated more than
once. In total, 1591 samples were collected and 250 (15.7%) were
positive for L. monocytogenes. All isolates were confirmed as L.
monocytogenes by PCR. Serotyping of all isolates showed a number of
different serotypes (Table 1). The isolates were mainly serotypes 4b
and 1/2a (72%). At facility 16, most of the isolates were from samples
taken external to the processing facility. Only 3 isolates were
untypable. All isolates were further sub-typed using PFGE. A total of
52 pulsotypes were identified (Fig. 1). These were named by the
facility number from which they were isolated and the number of the
isolate from that facility. No common pulsotype was found between
the facilities. More than one pulsotype was obtained at 10 of the 13
facilities.
Positive milk samples were found at 7 of the 13 facilities. Three of
these PFGE patterns (i.e. 2/1, 11/1, 13/1) were also identified in other
samples at the same facility. At the remaining 6 facilities, the PFGE
patterns of isolates found in the processing facilities were not
identified in samples taken externally. At 2 of the facilities,
indistinguishable patterns were seen in raw milk and the processing
environment. At 1 of the facilities, an indistinguishable pulsotype was
found 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 AscI
patterns.
At each facility, all pulsotypes identified were unique to that
facility. However, two sets of strains showed N95% similarity (10/9
and 16/2; 15/4 and 16/7; Fig. 1). Within these facilities, the putative
Fig. 2. PFGE pulsotypes of the persistent strain 10/1 isolated from different samples at facility 10 since Jan 1999.
transfer patterns of strains were compared using PFGE pulsotypes
(Table 2). Diagrammatic representation of the transfer of strains
within 1 of the facilities is shown in Fig. 3. The data suggest that
transfer between the area external to the processing facility and the
processing environment may have occurred.
From the 13 facilities where isolates were obtained, persistent
isolates were identified at two facilities (10 and 15; see Table 1). For
the purposes of this study, persistence was defined as isolation of the
same pulsotype from a facility over a period of more than 6 months.
Four of the 52 pulsotypes (7.7%) were persistent in the environment
from which they were isolated. The persistent pulsotypes were
isolated from the two facilities where a greater number of samples
were obtained.
4. Discussion
PFGE is a valuable tool in tracing the strain similarities and putative
transfer routes of L. monocytogenes in food and food processing
facilities. 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 (Autio
et al., 2003; Keto-Timonen et al., 2007) have been used to distinguish
L. monocytogenes isolates. However, PFGE (which was used in this
study) has the greatest power of discrimination and is currently the
accepted ‘gold standard’ for such studies.
Using PFGE, the environmental samples from the 16 facilities
resulted in 52 different pulsotypes. Previous studies on fish (Thimothe
et 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 of
cheese processing facilities has involved only one or two facilities
(Jacquet et al., 1993; Silva et al., 2003), or sampling over a short time
period (D'Amico and Donnelly, 2008).
In the current extensive study involving 16 cheesemaking facilities
over a two-year period, L. monocytogenes was not isolated from 3
(19%) of the 16 cheese processing facilities tested, even though
sampling was targeted at locations likely to harbour L. monocytogenes.
On the basis of workflows, sanitation and access practices, these
facilities were similar to facilities where L. monocytogenes was
detected. L. monocytogenes was isolated from N10% of the samples
at 5 (31%) of the facilities. The prevalence of L. monocytogenes
detected in cheese processing facilities in the current study (13 of 16
facilities sampled) was considerably higher than the 50 of 181
facilities found positive in Austria (Wagner et al., 2006). However, in
the Austrian study, most of the samples were from cheese or smear
(89%), effectively making it a survey of prevalence in cheese. The
current study was more extensive in terms of non-food contact
samples.
Strain diversity in both serotype and pulsotype was seen in the
isolates obtained at the facilities. Facilities 10 and 16 showed the highest
degree of diversity with 11 pulsotypes identified at each one. Serotypes
1/2a and 4b, and serotypes 1/2b and 4b, were the most prevalent at
facilities 10 and 16, respectively. In an independent study, 298 samples
 E. Fox et al. / International Journal of Food Microbiology 145 (2011) S39–S45 S43

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

Site Cheese type, Pulsotype Putative Positive samples

no. milk type transfer routea collected

1 Soft white, cow – – –

MILK
2 Semi-hard wax 2/1 M; PE Milk, drain
coated, sheep 2/2 – Milk
2/3 – Dairy
3 Soft white mould, 3/1 – Drain
DAIRY BUILDING
goat 3/2 – Drain
4 Soft blue, sheep 4/1 – Milk OUTSIDE BUILDING
5 Hard cheese, goat – – –
6 Semi-soft smear 6/1 – Door
EXTERNAL TO PROCESSING ENVIRONMENT
ripened, cow 6/2 PE; C Door, cheese
6/3 – Cheese PROCESSING ENVIRONMENT
7 Semi-soft, wax 7/1 – Cheese
coated, cow
8 Gouda, cow 8/1 – Milk
9 Soft Blue mould, cow 9/1 – Cheese
9/2 – Milk
9/3 – Drain
9/4 – Prep table
9/5 EPE; PE Floor, drain, soil
9/6 Cheese CHEESE
10 Semi-soft smear 10/1 PE; C Cheese, process
ripened, cow environment
10/2 – Cheese FOOD CONTACT SURFACES
10/3 – Cheese
10/4 – Cheese RIPENING ROOM
10/5 – Cheese
10/6 – Drain
PROCESSING HALL
10/7 – Milk
10/8 – Cheese
10/9 – Cheese 10/1 10/3 10/5 10/7 10/9
10/10 – Cheese
10/11 – Milk sock 10/2 10/4 10/6 10/8 10/10
11 Fresh, cow 11/1 M; PE Milk, drain
11/2 – Drain
10/11
12 Hard, cow – – –
Fig. 3. Spread of various pulsotypes isolated at facility 10.
13 Semi-hard, cow 13/1 M; C Cheese, milk
13/2 – Drain
13/3 – Milk
13/4 – Pooled water remaining 5 sets of similar strains could be sub-types that were
14 Gouda, goat 14/1 – Drain isolated at different sites. This suggests the possibility of some link
14/2 – Floor between these sites at some time. In Gorgonzola cheese, indistin-
14/3 – Drain
guishable pulsotypes have been isolated from different cheesemaking
15 Soft blue mould, cow 15/1 EPE; PE; C Straw, process
environment, cheese facilities that are apparently unrelated (Lomonaco et al., 2009).
15/2 – Walls Persistence of particular sub-types in an environment was
15/3 C; PE Cheese, processing monitored by Harvey and Gilmour (1993). In a review by Tompkin
environment (2002), persistence of L. monocytogenes in various food processing
15/4 – Ripening room racks
16 Hard cheese, cow 16/1 C; PE Cheese, sink
facilities was discussed. Many different serotypes showed persistence,
16/2 PE Sieve drain, floor many of the persistent strains were implicated in illness and the
16/3 EPE; PE Straw, cheese press, persistence time ranged from a few months to 10 years, including a
drain study by Unnerstad et al. (1996) showing persistence in a dairy
16/4 – Pooled water
environment for more than 7 years. Wulff et al. (2006) and Lomonaco
16/5 – Straw
16/6 – Drain et al. (2009) also reported persistent strains in fish and cheese
16/7 – Bovine manure processing facilities, respectively. In the current study, persistence
16/8 – Straw was defined as repeated isolation from samples taken at least
16/9 – Floor 6 months apart; four of the 52 sub-types were persistent. These
16/10 – Processing environment
16/11 – Processing environment
were isolated at two of the 16 facilities. One of these persistent strains
a
(10/1) was isolated from the same facility over a 10-year period
EPE = external to processing environment; PE = processing environment; M =
(Fig. 2) while the remaining three persistent strains were isolated
milk, and C = cheese.
more than 6 months apart. Persistence may be related to a range of
physiological characteristics including attachment and biofilm for-
mation (Mafu et al., 1990; Lunden et al., 2000; Møretrø and Langsrud,
from 16 farms found serotypes 1/2a, 1/2b and 4b were most prevalent 2004), resistance to sanitisers (Pan et al., 2006; Chavant et al., 2002)
(Fox et al., 2009). Serotypes 1/2a, 1/2b and 4b are also the most and adaptive responses (Lundén et al., 2003). The basis for persistence
prevalent in clinical cases (Tompkin, 2002; Zhang et al., 2007). of the strains isolated needs to be investigated.
Each facility had unique pulsotypes. However, there were 6 pairs In most studies of this kind, sampling is limited to food and the
of strains with N90% similarity, 5 at different sites and one within site processing environment, although Ho et al. (2007) also found that
16. (Fig. 1). Strains 16/2 and 16/11 were isolated at the same site and farm and processing facility strains were distinct. In the current study,
although 16/11 was untypable, it could be a sub-type of 16/2. The 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 processing
facilities. In the case of strain 15/1, this strain was also found on the
final product, indicating a putative transfer between the external
environment and the food product. There is a possibility that the
transfer of the strains was from the processing facility to the external
environment, but even if such an event occurred, the opportunity for
recontamination 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 processing
facility was observed over the two-year period. This implies that
cross-contamination within the processing facility could be occurring
(Reij et al., 2003). Preventing cross-contamination between dairy
production and processing facilities and preventing contamination
cycles within processing facilities are critical to controlling L.
monocytogenes thus assuring the microbial safety of farmhouse
dairy 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-milk
samples at the same facilities, indicating that milk is a possible vector
for contamination of cheesemaking facilities. This highlights the
importance of prevention of milk contamination.
5. Conclusions
In order to control L. monocytogenes contamination of final product,
and possible infection of consumers, care must also be taken to ensure
that recontamination of the processing environment is not originating
from an external source. The results of this study indicate that
contamination of food processing facilities with L. monocytogenes
appears to be sporadic with the majority of strains not persisting.
With 75% of cheesemaking facilities showing contamination with
L. monocytogenes in the processing environment, strict monitoring and
implementation 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 Programme
under the project BIOTRACER, project number 036272 and by the Irish
Government under the FIRM Programme, project number
06RDTMFRC434. The authors wish to acknowledge the assistance of
CAIS – the Irish Farmhouse Cheesemakers Association – and the
cooperation of all 16 cheesemakers who participated in this work.
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