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Absence of growth of Listeria monocytogenes in naturally contaminated Cheddar cheese

Published online by Cambridge University Press:  17 December 2013

Marion Dalmasso  and
Kieran Jordan
Marion Dalmasso
Affiliation:
Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland
Kieran Jordan*
Affiliation:
Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland
*
* For correspondence; e-mail: kieran.jordan@teagasc.ie
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Abstract

Each cheese producer is responsible by the legislation for the number of Listeria monocytogenes in cheese and is required to prove that numbers will not exceed 100 cfu/g throughout the shelf-life of the cheese. Even in the case of hard-cheese such as Cheddar cheese, the absence of growth of List. monocytogenes during ripening has to be demonstrated to comply with EU legislation. Studies dedicated to assessing List. monocytogenes growth throughout cheese shelf-life are generally based on artificially contaminated cheeses. Contrary to the majority of works, the current study focused on the growth of List. monocytogenes in naturally contaminated raw milk farmhouse Cheddar cheeses during a five-month ripening period. List. monocytogenes growth was assessed by direct count and its presence was detected by enrichment in two naturally contaminated cheese batches. In order to track routes of contamination, 199 processing environment samples from inside and outside the processing facility were taken, and their analysis for the presence of List. monocytogenes was performed on four occasions over a 9-month period. List. monocytogenes isolates were differentiated using PFGE and serotyping. List. monocytogenes never exceeded 20 cfu/g in the cheeses and could not be detected after five months of ripening. Eleven pulsotypes were identified. One pulsotype was found in the yard outside the processing facility, in a vat, on the processing area floor and in a cheese. This indicated that the outside environment constitutes a potential source of contamination of the processing environment and of the cheese. These results demonstrate that this farmhouse Cheddar cheese does not support List. monocytogenes growth and suggests that the efforts to reduce processing environment contamination are worthwhile.

Keywords

Natural contaminationfarmhouse cheesecheese processing facilityListeria monocytogenesfood safety

Information

Type
Research Article
Information
Journal of Dairy Research , Volume 81 , Issue 1 , February 2014 , pp. 46 - 53
Copyright
Copyright © Proprietors of Journal of Dairy Research 2013 

Listeria monocytogenes is a Gram-positive, rod-shaped bacterium. It is known to be the causative agent of listeriosis in humans that can affect susceptible populations, such as newborn children, the elderly and immunocompromised persons, with a mortality rate of 20–30 % (Lorber, Reference Lorber1997; McLauchlin et al. Reference McLauchlin, Mitchell, Smerdon and Jewell2004; Sleator et al. Reference Sleator, Watson, Hill and Gahan2009). List. monocytogenes is ubiquitous in the environment and has been isolated from a wide variety of ready-to-eat foods, such as meat, fish and dairy products (Lianou & Sofos, Reference Lianou and Sofos2007). Although List. monocytogenes is effectively inactivated by pasteurisation, post-processing contamination of dairy products has been well established and control of List. monocytogenes in processing facility environments is thus critical. The presence of List. monocytogenes in cheese made with raw or pasteurised milk has been described over the past few years (Wagner et al. Reference Wagner, Eliskases-Lechner, Rieck, Hein and Allerberger2006; Cocolin et al. Reference Cocolin, Nucera, Alessandria, Rantsiou, Dolci, Grassi, Lomonaco and Civera2009; O'Brien et al. Reference O'Brien, Hunt, McSweeney and Jordan2009; Brooks et al. Reference Brooks, Martinez, Stratton, Bianchini, Krokstrom and Hutkins2012; Schoder et al. Reference Schoder, Rossmanith, Glaser and Wagner2012; Torres-Vitela et al. Reference Torres-Vitela, Mendoza-Bernardo, Castro-Rosas, Gomez-Aldapa, Garay-Martinez, Navarro-Hidalgo and Villarruel-Lopez2012). The public health concerns associated with the occurrence of List. monocytogenes in cheese have been demonstrated by listeriosis outbreaks, for example, in France in 1980–2001 (De Buyser et al. Reference De Buyser, Dufour, Maire and Lafarge2001), Japan in 2001 (Makino et al. Reference Makino, Kawamoto, Takeshi, Okada, Yamasaki, Yamamoto and Igimi2005), Switzerland in 2005 (Bille et al. Reference Bille, Blanc, Schmid, Boubaker, Baumgartner, Siegrist, Tritten, Lienhard, Berner, Anderau, Treboux, Ducommun, Malinverni, Genne, Erard and Waespi2006), USA in 2004–2008 and 2010 (Jackson et al. Reference Jackson, Biggerstaff, Tobin-D'Angelo, Sweat, Klos, Nosari, Garrison, Boothe, Saathoff-Huber, Hainstock and Fagan2011; MMWR, 2011), Canada in 2008 (Gaulin et al. Reference Gaulin, Ramsay and Bekal2012) and Austria/Germany in 2009–2010 (Schoder et al. Reference Schoder, Rossmanith, Glaser and Wagner2012). European Union legislation places responsibility for safety on the food producer (EC, 2005), so it is important that the potential of food products to support growth of List. monocytogenes is understood. In many studies, the determination of growth in food is achieved using shelf-life challenges and/or mathematical modelling (Carrasco et al. Reference Carrasco, Valero, Pérez-Rodríguez, García-Gimeno and Zurera2007). These approaches have been used on various food matrices to assess the influence of different factors on the growth of List. monocytogenes. For example, the influence of packing conditions was investigated for shrimp (Mejlholm et al. Reference Mejlholm, Kjeldgaard, Modberg, Vest, Bøknæs, Koort, Björkroth and Dalgaard2008), the storage temperature for smoked fish (Uyttendaele et al. Reference Uyttendaele, Busschaert, Valero, Geeraerd, Vermeulen, Jacxsens, Goh, De Loy, Van Impe and Devlieghere2009) and the type of matrix for ready-to-eat salad (Skalina & Nikolajeva, Reference Skalina and Nikolajeva2010). For cheese, salt content, type of milk (raw or pasteurised), pH, aw, initial level of contamination and ripening temperature have already been investigated to predict the growth of List. monocytogenes in different types of cheeses (Angelidis et al. Reference Angelidis, Boutsiouki and Papageorgiou2010; Finazzi et al. Reference Finazzi, Daminelli, Serraino, Pizzamiglio, Riu, Giacometti, Bertasi, Losio and Boni2011; Schvartzman et al. Reference Schvartzman, Belessi, Butler, Skandamis and Jordan2011a, Reference Schvartzman, Maffre, Tenenhaus-Aziza, Sanaa, Butler and Jordanb; Shrestha et al. Reference Shrestha, Grieder, McMahon and Nummer2011). A database, ComBase (www.combase.cc), for predictive microbiology has also been available since 2004 to estimate microbial responses to various food environments (Baranyi & Tamplin, Reference Baranyi and Tamplin2004). However, the models developed in laboratory conditions are simplified descriptions of growth of List. monocytogenes in complex food matrices (Vermeulen et al. Reference Vermeulen, Devlieghere, De Loy-Hendrickx and Uyttendaele2011; Rosshaug et al. Reference Rosshaug, Detmer, Ingmer and Larsen2012) and they do not always reflect the reality of a food matrix. For example, Schvartzman et al. (Reference Schvartzman, Belessi, Butler, Skandamis and Jordan2011a) showed that in 40 % of cases where ComBase predicted growth, no growth actually occurred in cheese.

List. monocytogenes can occur on dairy farms and in associated cheesemaking facility environments. In Ireland, Fox et al. (Reference Fox, O'Mahony, Clancy, Dempsey, O'Brien and Jordan2009, Reference Fox, Hunt, O'Brien and Jordan2011) reported that 19 % of dairy farm samples and 13 % of processing area samples were positive for List. monocytogenes. Its occurrence in the environment may pose the problem of List. monocytogenes transfer from the environment to the cheese, even though the routes of contamination are not always clearly identified. Molecular subtyping of List. monocytogenes strains by methods such as Pulsed-Field Gel Electrophoresis (PFGE) have provided clear evidence that different List. monocytogenes subtypes can persist in dairy farms and processing facilities (Ho et al. Reference Ho, Lappi and Wiedmann2007). Environmental sampling is an effective way to assess hygiene and prevent future contamination events (Tompkin, Reference Tompkin2002). A stringent Listeria control programme, even in small processing facilities, and measures to prevent and control persistent Listeria contamination in niches in processing facilities are essential.

Owing to its elaborate physiological adaptation mechanisms, List. monocytogenes can survive and even proliferate in a variety of foods under adverse environmental conditions such as low pH, high salinity and low temperature (Khelef et al. Reference Khelef, Lecuit, Buchrieser, Cabanes, Dussurget, Cossart, Dworkin, Falkow, Rosenberg, Schleifer and Stackebrandt2006). In Europe, regulations permit up to 100 cfu/g in certain ready-to-eat foods that cannot support growth of List. monocytogenes (EC, 2005). In ready-to-eat foods that can support growth, absence in five 25 g samples is required, unless the manufacturer can show that numbers will not exceed 100 cfu/g throughout the stated shelf-life of the product. Thus, the responsibility for ensuring that the permitted concentration of List. monocytogenes is not exceeded is placed with the manufacturer. In addition, it is necessary to determine whether or not a food can support the growth of List. monocytogenes; this will determine the regulation that applies.

The aim of this study was to investigate potential routes of contamination of a raw milk farmhouse Cheddar cheese from the processing environment and to assess the ability of naturally contaminated cheese to support the growth of List. monocytogenes.

Materials and methods

Environmental sample collection

Samples from an Irish farmhouse cheesemaking facility with an associated farm providing milk for the cheesemaking were collected in April, June and September 2012 and in January 2013. At each sampling time, approximately fifty samples were collected and analysed. Samples were collected from non food contact surfaces (NFCS) and food contact surfaces (FCS) in the processing area, and from outside the processing environment (yard, entrance of milking parlour). Raw milk, curd and whey were also sampled. Swab samples were collected using pre-moistened with neutralising broth sponge-sticks (3M, St Paul, Minnesota, USA). Liquid samples were collected using 100-ml sterile dippers. All samples were collected during cheese production, wearing gloves and appropriate protective clothing, were individually packaged to prevent cross-contamination, placed in a cool box with ice packs and transported directly to the laboratory where they were analysed immediately.

Microbiological analyses

The two-step enrichment method ISO 11290-1 was used for detection of List. monocytogenes (ISO, 2004a), except that only chromogenic (agaALOA agar, Oxoid, Hampshire, UK) was used. After each enrichment step, 20 μl was spread on an ALOA plate and incubated for 48 h at 37 °C. At least two presumptive-positive List. monocytogenes colonies (blue-green with a surrounding halo) were isolated per positive sample and frozen at −20 °C after purification, pending further analysis. Isolates were purified by streaking a single colony from the ALOA agar onto Brilliance Listeria Agar (BLA, a chromogenic agar selective for Listeria obtained from Oxoid, Hampshire, UK). From BLA a single colony was streaked onto BHI agar and a single colony selected for freezing at −20 °C in cryovials (VWR, Leuven, Belgium).

The enumeration of List. monocytogenes in cheese was assessed by a direct count method according to ISO 11290-2 (ISO, 2004b). Briefly, 25 g of cheese were mixed in 225 ml half Fraser broth base (Merck, Darmstadt, Germany) using a Colworth Stomacher 400 (Seward, Worthing, UK) and 1 ml (2×0·5 ml) was immediately spread onto ALOA plates. Direct counts were performed in duplicate for each cheese sample. The rest of the cheese dilution was analysed as already mentioned for the detection of List. monocytogenes.

Confirmation by PCR

All purified isolates were confirmed as List. monocytogenes using Real-Time PCR (Rodríguez-Lázaro et al. Reference Rodríguez-Lázaro, Hernández, Scortti, Esteve, Vázquez-Boland and Pla2004), as described by O'Brien et al. (Reference O'Brien, Hunt, McSweeney and Jordan2009). Samples with a threshold value (Ct value) >35 were considered negative.

Serotyping

Serotyping of the isolates was performed as described by Fox et al. (Reference Fox, O'Mahony, Clancy, Dempsey, O'Brien and Jordan2009), using a combination of antisera and serotype-specific PCR (Doumith et al. Reference Doumith, Buchrieser, Glaser, Jacquet and Martin2004).

Pulsed-field gel electrophoresis (PFGE)

PFGE of all List. monocytogenes isolates was carried out using the International Standard PulseNet protocol (PulseNet USA, 2009). Two restriction enzymes, AscI and ApaI (Thermo Scientific, Dublin, Ireland), were used and isolate similarity dendrograms were obtained using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) method in Bionumerics version 5.10 software (Applied Maths, Belgium) as previously described (Fox et al. Reference Fox, deLappe, Garvey, McKeown, Cormican, Leonard and Jordan2012).

Cheese analysis

Cheese samples from batches I and II (from the same manufacturer but made on different days) were collected after 1, 2, 3 and 5 months of ripening (May to September 2012). Cheese samples were placed in sterile plastic bags, transported to the lab in a cool box and analysed immediately. Each cheese analysis was performed in duplicate. The presence/enumeration of List. monocytogenes and isolate identification and characterisation were performed as already described.

Cheeses samples were also analysed for pH, salt and moisture after 3 months of ripening. For pH measurement, 20 g cheese were blended with 12 ml distiled water and the pH was then measured using a pH211 microprocessor pH-meter (Hanna instruments, Bedfordshire, UK). The moisture content in cheese was determined by drying 3 g cheese at 102 °C for 5 h according to IDF Standard 4:2004/ISO 5534:2004 (ISO, 2004c). The salt content in cheese was determined by silver nitrate titration according to AOAC method No. 975·20 (AOAC, Reference Helrich1990). The salt-to-moisture (S : M) ratio was then calculated.

Results

Growth of List. monocytogenes in naturally contaminated cheese

Growth of List. monocytogenes in the two independent batches of naturally contaminated Cheddar cheese was monitored. In cheese batch I, List. monocytogenes was detected by enrichment during the first three months of ripening, but the level was always under the enumeration threshold (10 cfu/g) of the direct count method (Fig. 1). In cheese batch II, the number of List. monocytogenes never exceeded 20 cfu/g (Fig. 1) for the first two months of ripening. At three months of ripening, the number dropped under the enumeration threshold of the direct count method, but List. monocytogenes was still detected by enrichment. After five months of ripening, List. monocytogenes could not be detected in either cheese batches, either by direct count or by enrichment.

Fig. 1.

Growth of List. monocytogenes in Cheddar cheese monitored by direct counts and enrichment during a five-month ripening period. ○ Number of List. monocytogenes in cheese batch I; ● Number of List. monocytogenes in cheese batch II; + List. monocytogenes detected by enrichment in cheese batch I; * List. monocytogenes detected by enrichment in cheese batch II. † Maximum authorised number in food that do not support List. monocytogenes growth (EC, 2005).

Composition of cheeses

The pH, salt and moisture contents of both cheese batches were analysed during ripening. The pH values of the cheeses were 5·54 and 5·45 in batch I and batch II, respectively. The salt contents were 2·28±0·33 % and 1·89±0·03 % in batch I and in batch II, respectively. The moisture contents were 24·9±0·4 % and 28·6±1·2 %, respectively. The S : M ratios were 9·14 and 6·61 in cheese I and II, respectively.

Occurrence of List. monocytogenes at the farmhouse cheese facility

Overall, 199 samples were analysed and 185 (93 %) were negative for List. monocytogenes. The number of positive samples increased with each sampling occasion up to September 2012 with 12 % of the total samples positive (Table 1). Sampling in January 2013, following implementation of corrective actions regarding work-flows and cleaning and disinfection, resulted in 6 % total samples positive (Table 1). NFCS were responsible for the majority of positive samples, increasing from 0 to 15 % between April and September 2012 and reducing to 8 % following implementation of corrective actions. The contaminated NFCS included floors and drains in the processing area, and floors in the packing area and in the cold room and samples from the yard outside the processing facility. In the processing facility, one FCS, a vat, was positive only at one sampling time. On one sampling occasion, two products (some curd from a vat and cheese waste from the packing room) were positive for List. monocytogenes. All the tested raw materials were negative for List. monocytogenes (Table 1).

Table 1.

Number of samples taken at a farmhouse cheesemaking facility indicating the number and percentage that were positive for Listeria monocytogenes

† Non-food contact surface

‡ Food contact surface

§ Other: raw materials (raw milk, brine), products (whey, curd, cheese)

Characterisation of List. monocytogenes isolates and routes of contamination

From each positive sample found between April 2012 and January 2013, two isolates were selected from each of the first and second enrichments, where possible. This resulted in thirty-nine isolates which were confirmed as List. monocytogenes by PCR. The source of these isolates is shown in Fig. 2. Eleven isolates were taken from cheese. Thirty-four isolates were serotype 1/2a and five isolates were serotype 1/2b.

Fig. 2.

Location of isolation of the 11 pulsotypes from inside and outside the cheesemaking facility.

Combining PFGE patterns from both enzymes, ApaI and AscI, resulted in 11 distinguishable types (T1 to T11, Fig. 3). Strains of types T1, T5 and T9 were found in 2012 and 2013. All the other types were only found at one sampling time. The type T5 strains were all isolated at the same spot on the floor in the processing room (Figs. 2 and 3). The type T9 strains were found in cheese waste in the packing room in 2012 and on the floor in the processing area in 2013 (Figs. 2 and 3). Except for type T1, the PFGE results also indicated that no type was common to NFCS and FCS samples inside the processing area.

Fig. 3.

Dendrogram of PFGE profiles combining ApaI and AscI enzymes from isolates in this study. Eleven pulsotypes (T1 to T11) were identified and marked with symbols that are used in Fig. 2 to indicate their location in the cheesemaking facility.

The List. monocytogenes strains isolated from each batch of cheese were of two different types in each cheese. The strains from cheese batch I were identified as types T3 and T4 whereas strains from cheese batch II were types T1 and T10 (Fig. 3). In cheese batch I, the type T3 and T4 strains were isolated during the first three months. In cheese batch II, the type T10 strains could only be detected during the first month of ripening and were not isolated afterwards. The type T1 strains were found in cheese batch II in the first months of ripening and were also found in the yard and in a vat in April 2012, and also in the processing area and on the floor at the entrance to the packing room in January 2013 (Fig. 2).

Discussion

List. monocytogenes did not grow in two different and independent batches of naturally contaminated Cheddar cheese. Under these conditions, ComBase predicts List. monocytogenes growth reaching 100 cfu/g after 4 d and 108 cfu/g after 5 months of ripening whereas no List. monocytogenes growth actually occurred in the Cheddar cheeses studied. Performing challenge studies always raises questions such as, what strain to use, what should the pre-growth conditions be and what should the inoculation level be. The present study presented the opportunity to assess the growth of List. monocytogenes in the case on a natural contamination of cheese. The List. monocytogenes level never exceeded the legal threshold, even in the first months of ripening. List. monocytogenes could not be detected after five months of ripening. This is consistent with previous observations in long-time ripening cheese such as Cheddar which create a hostile environment for pathogenic bacteria (Pearson & Marth, Reference Pearson and Marth1990). The pH values around 5·5 and the S : M ratio higher than 6 % in cheese could explain the inability of List. monocytogenes to grow during ripening (Bishop & Smukowski, Reference Bishop and Smukowski2006). The presence of competitive flora like lactic acid starters could have significant potential to prevent List. monocytogenes growth in long-time ripening cheese (Buyong et al. Reference Buyong, Kok and Luchansky1998). All this implies that the ripening conditions of this farmhouse cheese do not support growth of List. monocytogenes resulting in a mature cheese suitable for consumption from a microbial safety perspective.

Deciphering the potential routes of contamination of the farmhouse cheese was also undertaken. Over a 9-month period, List. monocytogenes was isolated from 7 % of the samples collected. The prevalence of List. monocytogenes in this processing facility is consistent with results obtained in Irish farmhouse cheese processing environments (Fox et al. Reference Fox, Hunt, O'Brien and Jordan2011). These authors described List. monocytogenes prevalence ranging from 0 to 20·9 % depending on the processing facility. The present study was mainly focused on the occurrence of List. monocytogenes on NFCS and FCS, but also included some raw materials and products. It appeared that List. monocytogenes was found preferentially on NFCS rather than on FCS as it was also the case in previous studies (Barancelli et al. Reference Barancelli, Camargo, Reis, Porto, Hofer and Oliveira2011). From a food safety perspective, this means there is less chance of food contamination. The contamination of NFCS increased from April to September 2012. Advice regarding control strategies to prevent the dispersion of List. monocytogenes in the environment of the processing facility using adequate cleaning and disinfection procedures, and altered workflows was given. Once these recommendations were applied, the occurrence in January 2013 showed a decrease in the number of positive samples for List. monocytogenes. This study shows the effective contribution of environment sampling plans to the implementation of appropriate corrective actions and consequently to the improvement of food safety in cheesemaking facilities as also stated by Tompkin (Reference Tompkin2002).

PFGE is recognised as an ideal method for tracking List. monocytogenes isolates and for identifying routes of contamination in food processing facilities (Fox et al. Reference Fox, Hunt, O'Brien and Jordan2011). In the present study, use of PFGE resulted in 11 different pulsotypes. Three pulsotypes were identified in 2012 and 2013, indicating the presence of persistent strains in the processing environment. List. monocytogenes strain persistence over time is a widely described matter in several types of food processing plants. For example, Peccio et al. (Reference Peccio, Autio, Korkeala, Rosmini and Trevisani2003), Gudmundsdottir et al. (Reference Gudmundsdottir, Gudbjornsdottir, Einarsson, Kristinsson and Kristjansson2006), Keto-Timonen et al. (Reference Keto-Timonen, Tolvanen, Lundén and Korkeala2007), Lomonaco et al. (Reference Lomonaco, Decastelli, Nucera, Gallina, Manila Bianchi and Civera2009) and Chen et al. (Reference Chen, Pyla, Kim, Silva and Jung2010) reported persistent strains in meat, shrimps, ready-to-eat meals, Gorgonzola cheese and fish processing environments, respectively. In the current work, serotypes 1/2a and 1/2b were found for 34 strains and 5 strains, respectively. This is congruent with previous results showing that serotypes 1/2a and 1/2b along with serotypes 1/2c and 4b are the most prevalent in processing environments, clinical and food samples (Fox et al. Reference Fox, Hunt, O'Brien and Jordan2011; Pontello et al. Reference Pontello, Guaita, Sala, Cipolla, Gattuso, Sonnessa and Gianfranceschi2012).

In the current study, the same pulsotype was found outside the processing area (in the yard), in a vat and in a cheese in 2012, and on the processing area floor in 2013. This identified a possible route of contamination from the outside to the inside of the processing facility and finally to the cheese, although the transfer of List. monocytogenes from inside to outside the cheesemaking facility is also plausible. Nevertheless, the most likely hypothesis is that this List. monocytogenes strain could have been carried from the yard to the processing facility by the processing plant staff. The dairy farm environment is known to be a potential source of List. monocytogenes contamination (Ho et al. Reference Ho, Lappi and Wiedmann2007; Fox et al. Reference Fox, O'Mahony, Clancy, Dempsey, O'Brien and Jordan2009). Boots and footbaths with inappropriate sanitiser concentration have also been identified as potential vectors of contamination in farmhouse cheesemaking facilities (Ho et al. Reference Ho, Lappi and Wiedmann2007; Schoder et al. Reference Schoder, Melzner, Schmalwieser, Zangana, Winter and Wagner2011). In the current survey, other routes of contamination could not be clearly identified as the other pulsotypes found in cheeses, in the processing facility or on the farm were distinguishable. List. monocytogenes found in cheese and in the processing area were likely to also come from the dairy farm environment. Thus, preventing cross contamination between dairy production and processing facilities is critical to assuring the microbial safety of farmhouse cheese (Ho et al. Reference Ho, Lappi and Wiedmann2007). The presence of List. monocytogenes in cheese could also be due to other sources of contamination as pathogens may access the food-processing environment through raw material like raw milk (Hunt et al. Reference Hunt, Drummond, Murphy, Butler, Buckley and Jordan2012) or brine (Alessandria et al. Reference Alessandria, Rantsiou, Dolci and Cocolin2010). The raw material samples tested in the present work were not positive for List. monocytogenes. An increased number of raw material samples may have permitted identification of other routes of contamination, but focus was given to FCS and NFCS. Staff members, mobile equipment, leaks and openings in buildings (doors, windows) or even pests may also be the source for List. monocytogenes contamination in processing facilities (Reij et al. Reference Reij and Den Aantrekker2004).

The results of this study suggest that NFCS in cheesemaking facilities and outside the processing area may pose a risk to food contamination with List. monocytogenes and indicate that efforts to reduce processing environment contamination are worthwhile. Indeed, a potential route of contamination of cheese was identified from outside to inside of the processing area and finally to the cheese. This case of naturally contaminated cheeses offered the opportunity to assess List. monocytogenes growth in this type of farmhouse cheese. It shows that List. monocytogenes cannot grow in this cheese which is valuable information to the food business operator regarding the safety of the food produced.

This work was supported by the EU 7th Framework Programme under the project PROMISE (project number 265877). The authors wish to acknowledge the cooperation of the cheesemaker who participated in this work.

References

Alessandria, V, Rantsiou, K, Dolci, P & Cocolin, L 2010 Molecular methods to assess Listeria monocytogenes route of contamination in a dairy processing plant. International Journal of Food Microbiology 141 S156–S162 CrossRefGoogle Scholar
Angelidis, AS, Boutsiouki, P & Papageorgiou, DK 2010 Loss of viability of Listeria monocytogenes in contaminated processed cheese during storage at 4, 12 and 22 °C. Food Microbiology 27 809–818 CrossRefGoogle Scholar
AOAC 1990 Salt in cheese. Official method of analysis, 15th ed., vol. 2 (Ed. Helrich, K, section 971·19). Washington, DC, USA: Association of Official Analytical Chemists Google Scholar
Barancelli, GV, Camargo, TM, Reis, CMF, Porto, E, Hofer, E & Oliveira, CAF 2011 Incidence of Listeria monocytogenes in cheese manufacturing plants from the Northeast region of Sao Paulo, Brazil. Journal of Food Protection 74 816–819 CrossRefGoogle ScholarPubMed
Baranyi, J & Tamplin, ML 2004 ComBase: a common database on microbial responses to food environments. Journal of Food Protection 67 1967–1971 Google Scholar
Bille, J, Blanc, DS, Schmid, H, Boubaker, K, Baumgartner, A, Siegrist, HH, Tritten, ML, Lienhard, R, Berner, D, Anderau, R, Treboux, M, Ducommun, JM, Malinverni, R, Genne, D, Erard, PH & Waespi, U 2006 Outbreak of human listeriosis associated with tomme cheese in northwest Switzerland, 2005. Eurosurveillance 11 91–93 CrossRefGoogle ScholarPubMed
Bishop, JR & Smukowski, M 2006 Storage temperatures necessary to maintain cheese safety. Food Protection Trends 26 714–724 Google Scholar
Brooks, JC, Martinez, B, Stratton, J, Bianchini, A, Krokstrom, R & Hutkins, R 2012 Survey of raw milk cheeses for microbiological quality and prevalence of foodborne pathogens. Food Microbiology 31 154–158 Google Scholar
Buyong, N, Kok, J & Luchansky, JB 1998 Use of a genetically enhanced, pediocin-producing starter culture, Lactococcus lactis subsp. lactis MM217, to control Listeria monocytogenes in Cheddar cheese. Applied and Environmental Microbiology 64 4842–4845 Google Scholar
Carrasco, E, Valero, A, Pérez-Rodríguez, F, García-Gimeno, RM & Zurera, G 2007 Management of microbiological safety of ready-to-eat meat products by mathematical modelling: Listeria monocytogenes as an example. International Journal of Food Microbiology 114 221–226 Google Scholar
Chen, B-Y, Pyla, R, Kim, T-J, Silva, JL & Jung, Y-S 2010 Incidence and persistence of Listeria monocytogenes in the catfish processing environment and fresh fillets. Journal of Food Protection 73 1641–1650 Google Scholar
Cocolin, L, Nucera, D, Alessandria, V, Rantsiou, K, Dolci, P, Grassi, MA, Lomonaco, S & Civera, T 2009 Microbial ecology of Gorgonzola rinds and occurrence of different biotypes of Listeria monocytogenes . International Journal of Food Microbiology 133 200–205 Google Scholar
De Buyser, M-L, Dufour, B, Maire, M & Lafarge, V 2001 Implication of milk and milk products in food-borne diseases in France and in different industrialised countries. International Journal of Food Microbiology 67 1–17 Google Scholar
Doumith, M, Buchrieser, C, Glaser, P, Jacquet, C & Martin, P 2004 Differentiation of the major Listeria monocytogenes serovars by multiplex PCR. Journal of Clinical Microbiology 42 3819–3822 Google Scholar
European Commission 2005 Commission regulation (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs. European Commission. Official Journal of the EU L.338 1–26 Google Scholar
Finazzi, G, Daminelli, P, Serraino, A, Pizzamiglio, V, Riu, R, Giacometti, F, Bertasi, B, Losio, MN & Boni, P 2011 Behaviour of Listeria monocytogenes in packaged water buffalo mozzarella cheese. Letters in Applied Microbiology 53 364–370 Google Scholar
Fox, EM, O'Mahony, T, Clancy, M, Dempsey, R, O'Brien, M & Jordan, K 2009 Listeria monocytogenes in the Irish dairy farm environment. Journal of Food Protection 72 1450–1456 Google Scholar
Fox, EM, Hunt, K, O'Brien, M & Jordan, K 2011 Listeria monocytogenes in Irish farmhouse cheese processing environments. International Journal of Food Microbiology 145 S39–S45 Google Scholar
Fox, EM, deLappe, N, Garvey, P, McKeown, P, Cormican, M, Leonard, N & Jordan, K 2012 PFGE analysis of Listeria monocytogenes isolates of clinical, animal, food and environmental origin from Ireland. Journal of Medical Microbiology 61 540–547 CrossRefGoogle ScholarPubMed
Gaulin, C, Ramsay, D & Bekal, S 2012 Widespread listeriosis outbreak attributable to pasteurized cheese, which led to extensive cross-contamination affecting cheese retailers, Quebec, Canada, 2008. Journal of Food Protection 75 71–78 Google Scholar
Gudmundsdottir, S, Gudbjornsdottir, B, Einarsson, H, Kristinsson, KG & Kristjansson, M 2006 Contamination of cooked peeled shrimp (Pandalus borealis) by Listeria monocytogenes during processing at two processing plants. Journal of Food Protection 69 1304–1311 CrossRefGoogle ScholarPubMed
Ho, AJ, Lappi, VR & Wiedmann, M 2007 Longitudinal monitoring of Listeria monocytogenes contamination patterns in a farmstead dairy processing facility. Journal of Dairy Science 90 2517–2524 CrossRefGoogle Scholar
Hunt, K, Drummond, N, Murphy, M, Butler, F, Buckley, J & Jordan, K 2012 A case of bovine raw milk contamination with Listeria monocytogenes . Irish Veterinary Journal 65 13 Google Scholar
International Standarisation Organisation 2004a ISO 11290-1: 1997; Amd 1:2004. Microbiology of food and animal feeding stuffs – Horizontal method for the detection and enumeration of Listeria monocytogenes. Part 1: Detection method. International Organization for Standardization (ISO) Google Scholar
International Standarisation Organisation 2004b ISO 11290-2: 1998; Amd 1:2004. Microbiology of food and animal feeding stuffs – Horizontal method for the detection and enumeration of Listeria monocytogenes – Part 2: Enumeration method. International Organization for Standardization (ISO) Google Scholar
International Standarisation Organisation 2004c ISO 5534:2004 (IDF 4: 2004) – Cheese and processed cheese – Determination of the total solids content (Reference method). International Organization for Standardization (ISO) Google Scholar
Jackson, KA, Biggerstaff, M, Tobin-D'Angelo, M, Sweat, D, Klos, R, Nosari, J, Garrison, O, Boothe, E, Saathoff-Huber, L, Hainstock, L & Fagan, RP 2011 Multistate outbreak of Listeria monocytogenes associated with Mexican-style cheese made from pasteurized milk among pregnant, Hispanic women. Journal of Food Protection 74 949–953 CrossRefGoogle ScholarPubMed
Keto-Timonen, R, Tolvanen, R, Lundén, J & Korkeala, H 2007 An 8-year surveillance of the diversity and persistence of Listeria monocytogenes in a chilled food processing plant analyzed by Amplified Fragment Length Polymorphism. Journal of Food Protection 70 1866–1873 CrossRefGoogle Scholar
Khelef, N, Lecuit, M, Buchrieser, C, Cabanes, D, Dussurget, O & Cossart, P 2006 Listeria monocytogenes and the genus Listeria . In The Prokaryotes, Vol. 4, pp. 404–476 (Eds Dworkin, M, Falkow, S, Rosenberg, E, Schleifer, K-H, Stackebrandt, E). Springer Google Scholar
Lianou, A & Sofos, JN 2007 A Review of the incidence and transmission of Listeria monocytogenes in ready-to-eat products in retail and food service environments. Journal of Food Protection 70 2172–2198 Google Scholar
Lomonaco, S, Decastelli, L, Nucera, D, Gallina, S, Manila Bianchi, D & Civera, T 2009 Listeria monocytogenes in Gorgonzola: subtypes, diversity and persistence over time. International Journal of Food Microbiology 128 516–520 Google Scholar
Lorber, B 1997 Listeriosis. Clinical Infectious Diseases 24 1–9 CrossRefGoogle ScholarPubMed
Makino, SI, Kawamoto, K, Takeshi, K, Okada, Y, Yamasaki, M, Yamamoto, S & Igimi, S 2005 An outbreak of food-borne listeriosis due to cheese in Japan, during 2001. International Journal of Food Microbiology 104 189–196 CrossRefGoogle ScholarPubMed
McLauchlin, J, Mitchell, RT, Smerdon, WJ & Jewell, K 2004 Listeria monocytogenes and listeriosis: a review of hazard characterisation for use in microbiological risk assessment of foods. International Journal of Food Microbiology 92 15–33 CrossRefGoogle ScholarPubMed
Mejlholm, O, Kjeldgaard, J, Modberg, A, Vest, MB, Bøknæs, N, Koort, J, Björkroth, J & Dalgaard, P 2008 Microbial changes and growth of Listeria monocytogenes during chilled storage of brined shrimp (Pandalus borealis). International Journal of Food Microbiology 124 250–259 CrossRefGoogle ScholarPubMed
MMWR 2011 Outbreak of invasive listeriosis associated with the consumption of hog head cheese - Louisiana, 2010. MMWR Morbidity and Mortality Weekly Report 60 401–405 Google Scholar
O'Brien, M, Hunt, K, McSweeney, S & Jordan, K 2009 Occurrence of foodborne pathogens in Irish farmhouse cheese. Food Microbiology 26 910–914 CrossRefGoogle ScholarPubMed
Pearson, LJ & Marth, EH 1990 Listeria monocytogenes – Threat to a safe food supply: a review. Journal of Dairy Science 73 912–928 Google Scholar
Peccio, A, Autio, T, Korkeala, H, Rosmini, R & Trevisani, M 2003 Listeria monocytogenes occurrence and characterization in meat-producing plants. Letters in Applied Microbiology 37 234–238 Google Scholar
Pontello, M, Guaita, A, Sala, G, Cipolla, M, Gattuso, A, Sonnessa, M & Gianfranceschi, MV 2012 Listeria monocytogenes serotypes in human infections (Italy, 2000–2010). Annali Dell Istituto Superiore Di Sanità 48 146–150 Google Scholar
PulseNet USA 2009 One-day (24–28 h) standardized laboratory protocol for molecular subtyping of Listeria monocytogenes by Pulsed Field Gel Electrophoresis (PFGE). Available at: http://www.cdc.gov/pulsenet/pathogens/listeria.html. Accessed July 12th 2013 Google Scholar
Reij, MW, Den Aantrekker, ED & ILSI Europe Risk Analysis in Microbiology Task Force 2004 Recontamination as a source of pathogens in processed foods. International Journal of Food Microbiology 91 1–11 Google Scholar
Rodríguez-Lázaro, D, Hernández, M, Scortti, M, Esteve, T, Vázquez-Boland, JA & Pla, M 2004 Quantitative detection of Listeria monocytogenes and Listeria innocua by Real-Time PCR: assessment of hly, iap, and lin02483 targets and AmpliFluor technology. Applied and Environmental Microbiology 70 1366–1377 CrossRefGoogle ScholarPubMed
Rosshaug, PS, Detmer, A, Ingmer, H & Larsen, MH 2012 Modelling the growth of Listeria monocytogenes in soft blue white cheese. Applied and Environmental Microbiology 78 8508–8514 Google Scholar
Schoder, D, Melzner, D, Schmalwieser, A, Zangana, A, Winter, P & Wagner, M 2011 Important vectors for Listeria monocytogenes transmission at farm dairies manufacturing fresh sheep and goat cheese from raw milk. Journal of Food Protection 74 919–924 Google Scholar
Schoder, D, Rossmanith, P, Glaser, K & Wagner, M 2012 Fluctuation in contamination dynamics of List. monocytogenes in quargel (acid curd cheese) lots recalled during the multinational listeriosis outbreak 2009/2010. International Journal of Food Microbiology 157 326–331 CrossRefGoogle Scholar
Schvartzman, MS, Belessi, C, Butler, F, Skandamis, PN & Jordan, K 2011a Effect of pH and water activity on the growth limits of Listeria monocytogenes in a cheese matrix at two contamination levels. Journal of Food Protection 74 1805–1813 CrossRefGoogle Scholar
Schvartzman, MS, Maffre, A, Tenenhaus-Aziza, F, Sanaa, M, Butler, F & Jordan, K 2011b Modelling the fate of Listeria monocytogenes during manufacture and ripening of smeared cheese made with pasteurised or raw milk. International Journal of Food Microbiology 145 S31–S38 CrossRefGoogle ScholarPubMed
Shrestha, S, Grieder, JA, McMahon, DJ & Nummer, BA 2011 Survival of Listeria monocytogenes introduced as a post-aging contaminant during storage of low-salt Cheddar cheese at 4, 10, and 21 °C. Journal of Dairy Science 94 4329–4335 Google Scholar
Skalina, L & Nikolajeva, V 2010 Growth potential of Listeria monocytogenes strains in mixed ready-to-eat salads. International Journal of Food Microbiology 144 317–321 Google Scholar
Sleator, RD, Watson, D, Hill, C & Gahan, CGM 2009 The interaction between Listeria monocytogenes and the host gastrointestinal tract. Microbiology 155 2463–2475 Google Scholar
Tompkin, RB 2002 Control of Listeria monocytogenes in the food-processing environment. Journal of Food Protection 65 709–725 Google Scholar
Torres-Vitela, MR, Mendoza-Bernardo, M, Castro-Rosas, J, Gomez-Aldapa, CA, Garay-Martinez, LE, Navarro-Hidalgo, V & Villarruel-Lopez, A 2012 Incidence of Salmonella, Listeria monocytogenes, Escherichia coli O157:H7, and staphylococcal enterotoxin in two types of Mexican fresh cheeses. Journal of Food Protection 75 79–84 CrossRefGoogle ScholarPubMed
Uyttendaele, M, Busschaert, P, Valero, A, Geeraerd, AH, Vermeulen, A, Jacxsens, L, Goh, KK, De Loy, A, Van Impe, JF & Devlieghere, F 2009 Prevalence and challenge tests of Listeria monocytogenes in Belgian produced and retailed mayonnaise-based deli-salads, cooked meat products and smoked fish between 2005 and 2007. International Journal of Food Microbiology 133 94–104 Google Scholar
Vermeulen, A, Devlieghere, F, De Loy-Hendrickx, A & Uyttendaele, M 2011 Critical evaluation of the EU-technical guidance on shelf-life studies for List. monocytogenes on RTE-foods: a case study for smoked salmon. International Journal of Food Microbiology 145 176–185 Google Scholar
Wagner, M, Eliskases-Lechner, F, Rieck, P, Hein, I & Allerberger, F 2006 Characterization of Listeria monocytogenes isolates from 50 small-scale Austrian cheese factories. Journal of Food Protection 69 1297–1303 CrossRefGoogle ScholarPubMed
Figure 0

Fig. 1. Growth of List. monocytogenes in Cheddar cheese monitored by direct counts and enrichment during a five-month ripening period. ○ Number of List. monocytogenes in cheese batch I; ● Number of List. monocytogenes in cheese batch II; + List. monocytogenes detected by enrichment in cheese batch I; * List. monocytogenes detected by enrichment in cheese batch II. † Maximum authorised number in food that do not support List. monocytogenes growth (EC, 2005).

Figure 1

Table 1. Number of samples taken at a farmhouse cheesemaking facility indicating the number and percentage that were positive for Listeria monocytogenes

Figure 2

Fig. 2. Location of isolation of the 11 pulsotypes from inside and outside the cheesemaking facility.

Figure 3

Fig. 3. Dendrogram of PFGE profiles combining ApaI and AscI enzymes from isolates in this study. Eleven pulsotypes (T1 to T11) were identified and marked with symbols that are used in Fig. 2 to indicate their location in the cheesemaking facility.