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 Article history: Received 25 June 2010 Received in revised form 28 September 2010 Accepted 17 October 2010 Keywords: Cheese processing facilities Listeria Food safety Persistence a b s t r a c t Sixteen cheesemaking facilities were sampled during the production season at monthly intervals over a twoyear period. Thirteen facilities were found to have samples positive for Listeria monocytogenes. Samples were divided into 4 categories; cheese, raw milk, processing environment and external to the processing 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 isolates, they were differentiated using PFGE and serotyping. Of the 250 isolates, there were 52 different pulsotypes. No pulsotype was found at more than one facility. Two facilities had persistent pulsotypes that were isolated on sampling occasions at least 6 months apart. Of the samples tested, 6.3% of milk, 13.1% of 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). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.10.012 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 temperatures, 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 subtypes persist over time (reviewed by Tompkin, 2002) and that crosscontamination 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. 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 “SpeciSponge” 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. % Samples positive Pulsotypes Duration of pulsotype detection Frequency of isolation Serotype Persistence 1 2 Number of samples 58 48 0 10.4 56 3.6 4 5 6 62 43 127 1.6 0 3.9 7 8 9 66 32 50 1.5 3.1 18.0 10 473 14.1 11 40 7.5 12 13 21 44 0 9.1 14 56 5.4 15 296 20.9 16 119 16.8 – Jul08–Aug08 Jul08 Jul08 Mar08 Jun08 Dec08 – Oct07 Jun07–Oct07 Aug07 May08 Jun08 Nov07 Jul08 Aug08 Sep08 Jul08–Aug08 Aug08 Apr07–Dec08 Apr08 Apr08 Nov07 Apr08 Apr08 Feb09 Apr08 Jul07 Apr08 Aug08 May08–Aug08 Nov08 – Jul07 Aug08 Oct08 Oct08 Sep08 Mar08 Jul08 Jun07–Nov08 Oct08 Jul07–Sep08 Aug07–Sept08 Jun07 Apr07–Jun07 Apr07 Apr07–Jun07 Apr07 Apr07 Apr07–Jun07 Apr07 Apr07 Apr07 Jun07 – 3 – 2/1 2/2 2/3 3/1 3/2 4/1 – 6/1 6/2 6/3 7/1 8/1 9/1 9/2 9/3 9/4 9/5 9/6 10/1 10/2 10/3 10/4 10/5 10/6 10/7 10/8 10/9 10/10 10/11 11/1 11/2 – 13/1 13/2 13/3 13/4 14/1 14/2 14/3 15/1 15/2 15/3 15/4 16/1 16/2 16/3 16/4 16/5 16/6 16/7 16/8 16/9 1610 16/11 – 1/2a 4b 4b 1/2a 4b 4b – 1/2a 4b 1/2a 1/2a 1/2a 1/2b 4b 1/2b 1/2b 1/2b 4b 1/2a 1/2a 1/2b 1/2a 1/2a 4b 4b 4b 4b 1/2a 1/2b 1/2a 1/2a – 1/2a Untypable 4b 1/2a 1/2a 1/2a 4b 1/2c 1/2b 1/2c 1/2c 4b 4b 4b 3b 1/2b 4b 1/2b 4b 4b 1/2b Untypable – No No No No No No – No No No No No No No No No No No Yes No No No No No No No No No No No No – No No No No No No No Yes No Yes Yes No No No No No No No No No No No 3 1 1 1 1 1 – 1 5 1 2 1 2 1 1 1 3 1 122 1 2 1 1 1 2 1 1 1 1 2 1 – 1 1 1 1 1 1 1 47 1 2 3 2 3 5 3 1 2 5 1 2 1 1 S41 E. Fox et al. / International Journal of Food Microbiology 145 (2011) S39–S45 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 Similarity 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. 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 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. 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. 2.5. Serotyping 4. Discussion 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). 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 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 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. S43 E. Fox et al. / International Journal of Food Microbiology 145 (2011) S39–S45 Table 2 Source and epidemiology of the L. monocytogenes isolates from 16 cheesemaking facilities. Site Cheese type, no. milk type 1 2 Soft white, cow Semi-hard wax coated, sheep 3 Soft white mould, goat Soft blue, sheep Hard cheese, goat Semi-soft smear ripened, cow 4 5 6 7 8 9 10 11 12 13 14 15 16 Semi-soft, wax coated, cow Gouda, cow Soft Blue mould, cow Semi-soft smear ripened, cow Pulsotype Putative Positive samples transfer routea collected – 2/1 2/2 2/3 3/1 3/2 4/1 – 6/1 6/2 6/3 7/1 8/1 9/1 9/2 9/3 9/4 9/5 9/6 10/1 10/2 10/3 10/4 10/5 10/6 10/7 10/8 10/9 10/10 10/11 Fresh, cow 11/1 11/2 Hard, cow – Semi-hard, cow 13/1 13/2 13/3 13/4 Gouda, goat 14/1 14/2 14/3 Soft blue mould, cow 15/1 Hard cheese, cow – M; PE – – – – – – – PE; C – – – Milk, drain Milk Dairy Drain Drain Milk – Door Door, cheese Cheese Cheese – – – – – EPE; PE Milk Cheese Milk Drain Prep table Floor, drain, soil Cheese Cheese, process environment Cheese Cheese Cheese Cheese Drain Milk Cheese Cheese Cheese Milk sock Milk, drain Drain – Cheese, milk Drain Milk Pooled water Drain Floor Drain Straw, process environment, cheese Walls Cheese, processing environment Ripening room racks Cheese, sink Sieve drain, floor Straw, cheese press, drain Pooled water Straw Drain Bovine manure Straw Floor Processing environment Processing environment PE; C – – – – – – – – – – M; PE – – M; C – – – – – – EPE; PE; C 15/2 15/3 – C; PE 15/4 16/1 16/2 16/3 – C; PE PE EPE; PE 16/4 16/5 16/6 16/7 16/8 16/9 16/10 16/11 – – – – – – – – a EPE = external to processing environment; PE = processing environment; M = milk, and C = cheese. 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 most prevalent in clinical cases (Tompkin, 2002; Zhang et al., 2007). Each facility had unique pulsotypes. However, there were 6 pairs of strains with N90% similarity, 5 at different sites and one within site 16. (Fig. 1). Strains 16/2 and 16/11 were isolated at the same site and although 16/11 was untypable, it could be a sub-type of 16/2. The MILK DAIRY BUILDING OUTSIDE BUILDING EXTERNAL TO PROCESSING ENVIRONMENT PROCESSING ENVIRONMENT CHEESE FOOD CONTACT SURFACES RIPENING ROOM PROCESSING HALL 10/1 10/3 10/5 10/7 10/9 10/2 10/4 10/6 10/8 10/10 10/11 Fig. 3. Spread of various pulsotypes isolated at facility 10. remaining 5 sets of similar strains could be sub-types that were isolated at different sites. This suggests the possibility of some link between these sites at some time. In Gorgonzola cheese, indistinguishable pulsotypes have been isolated from different cheesemaking facilities that are apparently unrelated (Lomonaco et al., 2009). Persistence of particular sub-types in an environment was monitored by Harvey and Gilmour (1993). In a review by Tompkin (2002), persistence of L. monocytogenes in various food processing facilities was discussed. Many different serotypes showed persistence, many of the persistent strains were implicated in illness and the persistence time ranged from a few months to 10 years, including a study by Unnerstad et al. (1996) showing persistence in a dairy environment for more than 7 years. Wulff et al. (2006) and Lomonaco et al. (2009) also reported persistent strains in fish and cheese processing facilities, respectively. In the current study, persistence was defined as repeated isolation from samples taken at least 6 months apart; four of the 52 sub-types were persistent. These were 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 isolated more than 6 months apart. Persistence may be related to a range of physiological characteristics including attachment and biofilm formation (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 persistence of the strains isolated needs to be investigated. In most studies of this kind, sampling is limited to food and the processing environment, although Ho et al. (2007) also found that farm 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 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 workflows, 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. monocytogenes. 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 prevention 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. 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