International Journal of Food Microbiology 167 (2013) 303–309

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Foci of contamination of Listeria monocytogenes in different cheese processing plants G. Almeida, R. Magalhães, L. Carneiro, I. Santos, J. Silva, V. Ferreira, T. Hogg, P. Teixeira ⁎ CBQF — Centro de Biotecnologia e Química Fina, Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa/Porto, Rua Dr. António Bernardino Almeida, 4200-072 Porto, Portugal

a r t i c l e

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Article history: Received 14 February 2013 Received in revised form 14 September 2013 Accepted 16 September 2013 Available online 21 September 2013 Keywords: Listeria monocytogenes Cheese Sources of contamination Typing PFGE Persistence

a b s t r a c t Listeria monocytogenes is a ubiquitous bacterium widely distributed in the environment that can cause a severe disease in humans when contaminated foods are ingested. Cheese has been implicated in sporadic cases and in outbreaks of listeriosis worldwide. Environmental contamination, in several occasions by persistent strains, has been considered an important source of finished product contamination. The objectives of this research were to (i) evaluate the presence of L. monocytogenes within the factory environments and cheeses of three processing plants, artisanal producer of raw ewe's milk cheeses (APC), small-scale industrial cheese producer (SSI) and industrial cheese producer (ICP) each producing a distinct style of cheese, all with history of contamination by L. monocytogenes (ii) and identify possible sources of contamination using different typing methods (arsenic and cadmium susceptibility, geno-serotyping, PFGE). The presence of markers specific for 3 epidemic clones (ECI– ECIII) of L. monocytogenes was also investigated. Samples were collected from raw milk (n = 179), whey (n = 3), cheese brining solution (n = 7), cheese brine sludge (n = 505), finished product (n = 3016), and environment (n = 2560) during, at least, a four-year period. Listeria monocytogenes was detected in environmental, raw milk and cheese samples, respectively, at 15.4%, 1.1% and 13.6% in APC; at 8.9%, 2.9% and 3.4% in SSI; and at 0%, 21.1% and 0.2% in ICP. Typing of isolates revealed that raw ewe's milk and the dairy plant environment are important sources of contamination, and that some strains persisted for at least four years in the environment. Although cheeses produced in the three plants investigated were never associated with any case or outbreak of listeriosis, some L. monocytogenes belonging to specific PFGE types that caused disease (including putative epidemic clone strains isolated from final products) were found in this study. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Listeria monocytogenes is a bacterium that causes a rare but severe disease in susceptible individuals with a mortality rate between 20 to 30% (Swaminathan and Gerner-Smidt, 2007). In Portugal, the estimated incidence of the disease was 1.4 and 2.3 cases per million inhabitants in 2003 and 2007, respectively, with a mortality rate between 17% and 37.5% (Almeida et al., 2006, 2010). The consumption of raw milk or raw milk products has caused several listeriosis outbreaks resulting in several hundred cases (Lundén et al., 2004). In an analysis of data from foodborne outbreaks reported internationally between 1988 and 2007, 337 out of 4093 were associated with dairy products and that 6.6% of which were attributed to L. monocytogenes (Greig and Ravel, 2009). Cheese has been implicated in some of the major listeriosis outbreaks reported worldwide (Bille et al., 2006; Kabuki et al., 2004; Koch et al., 2010; Vít et al., 2007; Warriner and Namvar, 2009; Yde et al., 2012).

⁎ Corresponding author. Tel.: +351 22 5580095. E-mail address: [email protected] (P. Teixeira). 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.09.006

Listeria monocytogenes was detected in different types of Portuguese cheese with contamination rates ranging from 1.6% to 26.2% (Almeida et al., 2007; Guerra and Bernardo, 1999; Mena et al., 2004; Santos et al., 1994). Given the ability of L. monocytogenes to grow/survive at temperatures ranging between 0 °C and 45 °C, pH values ranging from 4.5 and 9, and in salt medium with 10 to 20% (w/v) of NaCl (Le Monnier and Leclercq, 2009), prevention of the establishment of L. monocytogenes in a food producing plant is essential. Environmental contamination has been considered an important source of finished product contamination, a fact demonstrated by characterization of L. monocytogenes isolates by molecular subtyping methods with high discriminatory power, such as pulsed-field gel electrophoresis (PFGE, generally considered the “gold-standard” of molecular typing methods), amplified fragment length polymorphism (AFLP), or ribotyping (Di Ciccio et al., 2012; Kabuki et al., 2004; Lomonaco et al., 2009; Mendonça et al., 2012; Parisi et al., 2013). Although food processing environments and equipment are regularly cleaned and sanitized, persistence of specific L. monocytogenes PFGE types, over time periods ranging from a few months to more than a decade, has been documented (Di Ciccio et al., 2012; Ferreira et al., 2011; Lappi et al., 2004; Lomonaco et al., 2009;

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Lundén et al., 2003; Miettinen et al., 1999; Norton et al., 2001; Olsen et al., 2005; Unnerstad et al., 1996). Subtype characterization of L. monocytogenes isolates from listeriosis outbreaks that have occurred over the last three decades has suggested that many outbreaks were caused by a small number of L. monocytogenes epidemic clones (ECs) (Kathariou, 2002), i.e. by a closely related group of isolates that evolved clonally. Seven L. monocytogenes ECs have been reported to date, designated ECI (serotype 4b), ECII (serotype 4b), ECIII (serotype 1/2a), ECIa (serotype 4b; ECIa has also been referred to as ECIV), ECV (serotype 1/2a), ECVI (serotype 1/2b), and ECVII (serotype 1/2a) (Chen et al., 2007; Knabel et al., 2012; Lomonaco et al., 2013). Epidemic clones of L. monocytogenes have been implicated in several outbreaks and sporadic cases of listeriosis worldwide (Chenal-Francisque et al., 2011; Cheng et al., 2008; den Bakker et al., 2010; Knabel et al., 2012) and for this reason, although not yet proven, they have been considered more virulent than other strains (Roberts et al., 2009; Tompkin, 2002; Yildirim et al., 2004). As hypothesize by Fugett et al. (2007), it could also be attributed to a better ability to persist in different environments as well as grow in foods and subsequently transmit. We thus hypothesize that this PFGE type represents a stable pandemic clone, which appears to be able to survive successfully in different environments as well as grow in foods and cause human disease. Unquestionable, however, is their widespread presence and in several cases persistence in food and food processing and other environments (Eifert et al., 2005; Ferreira et al., 2011; Fugett et al., 2007; Kathariou, 2003; Kabuki et al., 2004; Olsen et al., 2005; Sauders et al., 2006; Yildirim et al., 2004). There is a long tradition of cheese making and eating in Portugal; total annual per capita cheese consumption stands at 7.2 kg. Despite the current dominance of industrial cheese produced from pasteurized cows' milk, cheese produced from raw ewes' and goats' milk are still parts of the daily diet in rural areas of Portugal as well as fashionable food products in urban centers. Currently, fourteen traditional Portuguese cheeses are protected under the designation PDO — Protected Designation of Origin. The objectives of this study were to (i) evaluate the presence of L. monocytogenes within the factory environments and cheeses of three processing plants (ii) and identify possible sources of contamination using different typing methods. The presence of markers specific for 3 epidemic clones (ECI–ECIII) of L. monocytogenes was also investigated.

Samples were collected from raw milk (n = 179), whey (n = 3), cheese brining solution (n = 7), cheese brine sludge (n = 505), finished product (n = 3016), and environment (n = 2560) during, at least, a four-year period (Table 1). Environmental sampling was carried out on product contact surfaces and product non-contact surfaces at different processing plant areas, including: plant entrance; milk reception; production line; ripening; cheese washing; refrigerated chambers; and shipping (Table 1). Sampling program was designed to include sites of difficult sanitation, thus most likely to harbor L. monocytogenes, and focused on those previously tested positive. 2.2. Sampling procedure Cheese samples of different lots consisting of the whole cheese were transferred aseptically from the refrigerated chamber to a sterile plastic bag. Sampling sites in the production environment and equipment were swabbed with cotton swabs or non-toxic sterilized sponges moistened with sterile ¼ Ringer's solution (Oxoid, Hampshire, UK) and placed in 10 ml and 30 ml of ½ Fraser broth, respectively (bioMérieux, Marcy l'Etoile, France). Milk and brine solution samples were transferred from the tanks into 500 ml sterile plastic bottles. All samples were maintained below 4 °C during transportation to the laboratory and were analyzed for the presence of L. monocytogenes within 24 h. 2.3. Detection and isolation of Listeria monocytogenes All samples were analyzed using the VIDAS LMO2 automated immunoassay system (bioMérieux) (Anonymous, 1996a). For each VIDAS positive result five presumptive colonies, whenever possible, were confirmed by sugar fermentation and CAMP test, according to the ISO 11290-1:1996 standard method (Anonymous, 1996b). Listeria monocytogenes isolates were preserved in tryptone soy broth supplemented with 0.6% yeast extract (TSBYE, Merck, Darmstadt, Germany) with 30% (v/v) glycerol at −80 °C. 2.4. Genoserotyping by multiplex PCR

2. Materials and methods 2.1. Processing plants and sample collection Three cheese manufacturing plants operated from different companies, and located in different cities in Portugal, were surveyed for L. monocytogenes contamination: artisanal (APC), small scale (SSI), and industrial (ICP) cheese producers (Table 1).

Genoserotyping was determined by PCR grouping using a multiplex PCR assay previously described by Doumith et al. (2004), which detects serotype-specific marker genes. PCR was performed in an Eppendorf thermocycler (Eppendorf, Hamburg, Germany) and PCR products were resolved on a 2% (w/v) agarose gel containing 0.5 μg/ml of ethidium bromide (Eurobio, Courtaboeuf, France) and visualized and photographed under a UV transilluminator (Bio-Rad Gel Doc 2000 TM imaging system, Bio-Rad Laboratories, Milan, Italy). This assay

Table 1 Surveyed cheese processing plants, sampling period and collected products. ⁎Manufacturer

Region

Cheese type

Sampling N° of visits

APC

Setubal, South Portugal

Semi hard raw ovine milk

18

SSI

Braga, North Portugal

Several cheese varieties. pasteurized cow, ewe or goat milk or a mixture of milks

56

ICP

Aveiro, Center Portugal

Semi hard pasteurized cow milk

148

Total number of samples

Period

February 2004 to December 2007 May 2003 to December 2007

January 2004 to December 2007

Cheese

Milk

Other

Surfaces swabs ⁎⁎Product contact

⁎⁎⁎Product non-contact

81

90

Milk whey 3

72

58

Cow 24 ewe 2 goat 91 Mix 91 2727

Goat 50 Cow 19 Ewe 1

Brine 7

224

192

19

Brine sludge 505

1373

641

3016

179

515

1669

891

⁎ APC, artisanal cheese producer; SSI, small-scale industrial cheese producer; ICP, industrial cheese producer. ⁎⁎ General equipment, working tables, trays, molds, cutters, conveyor belts, cheese press, personnel hands, sinks. ⁎⁎⁎ Walls, drains, floors.

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differentiates isolates in five major serogroups: IIa (grouping serotypes 1/2a and 3a), IIb (grouping serotypes 1/2b, 3b, and 7), IIc (grouping serotypes 1/2c and 3c), IVb (grouping serotypes 4b, 4d, and 4e), and IVa (grouping serotypes 4a and 4c). Each amplification reaction included four L. monocytogenes control strains: NCTC 11994 (serotype 4b), CECT 911 (serotype 1/2c), CECT 936 (serotype 1/2b) and CIP 104794 (serotype 1/2a). 2.5. Resistance to arsenic and cadmium The characterization of the differential resistance of the isolates to arsenic and cadmium was performed in Isosensitest agar plates (Oxoid), containing 500 μg/ml sodium arsenite (Merck) or 75 μg/ml cadmium chloride (Merck) as previously described (McLauchlin et al., 1997). Isolates were classified as resistant (R) or sensitive (S) to each compound, and results were combined to generate a unique resistance profile. 2.6. Pulsed Field Gel Electrophoresis (PFGE) PFGE was performed according to the PulseNet protocol using the restriction enzymes AscI and ApaI (Graves and Swaminathan, 2001). Bacterial cultures were embedded in agarose, lysed, washed, digested with the restriction enzyme, and electrophoresed on a Chef III (BioRad Laboratories, Hercules, CA). XbaI-digested DNA of Salmonella enterica serotype Braenderup H9812 was used as a size reference standard. The PFGE profiles obtained were scanned, and the computerized data were analyzed using the Gelcompare II software (Applied Maths, Kortrijk, Belgium). Bands automatically assigned by the computer were checked visually and corrected manually when necessary. A band position tolerance of 1.5 was selected for each restriction enzyme. Cluster analysis of the individual or combined PFGE profiles was done by the unweighted pair group method with average linkages (UPGMA), using the Dice coefficient to analyze the similarities of the banding PFGE profiles. Classification of isolates into different ApaI and AscI PFGE profiles was visually validated. A capital letter was ascribed to each indistinguishable AscI PFGE profile; strains that were similar by AscI but not similar by ApaI, were denoted, in addition to the capital letters, by lower case letters. The PFGE types obtained for the selected isolates were compared to the PFGE types of 95 L. monocytogenes strains isolated from human cases of listeriosis that occurred in Portugal between 1994 and 2007, and integrated in the L. monocytogenes PFGE types database of Escola Superior de Biotecnologia-Universidade Católica Portuguesa. 2.7. Detection of epidemic-clones associated genetic markers for ECI, ECII and ECIII The screening for genetic markers for epidemic clones ECI, ECII and ECIII was performed by multiplex PCR as previously described by Chen and Knabel (2007). Only L. monocytogenes isolates recovered from finished products were selected for this analysis. Fourteen isolates belonging to molecular serotype IVb (nine collected from APC and five from ICP) were screened for ECI and ECII genetic markers (as these ECs are associated with serotype 4b), while two isolates (collected from APC) belonging to molecular serotype IIa were screened for the ECIII genetic markers (as this EC is associated with serotype 1/2a). ECI, ECII, ECIII strains (CDC, 1989, 1999; Linnan et al., 1988) and 1/2a and 4b strains not belonging to ECs were used as reference controls. 3. Results and discussion The prevalence of L. monocytogenes in raw milk, cheese and general factory environment is presented in Tables 2 to 4 for APC, SSI and ICP, respectively.

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Table 2 Positive samples for Listeria monocytogenes collected at an Artisanal Cheese Producer (APC). No. of positive samples Date

Cheese

February 04 February 05 June 05 July 05 August 05 October 05 November 05 December 05 January 06 February 06 March 06 June 06 September 06 Total (%)

1 1 3

Milk

Whey

Contact

Non-contact

1 1

5

1 1 1 1 1 1

1 3 1

2 2 1 4

1 11(13.6)

1 (1.1)

1 (33.3)

6 (8.3)

1 15 (25.6)

The percentage of samples positive for L. monocytogenes decreased with the increase in the size of the cheese plant. EFSA reported a higher occurrence of L. monocytogenes in soft and semi-soft cheeses made from pasteurized milk than in cheese made from raw milk (EFSA, 2009). However, this was not observed in present study (Table 1). All isolates of L. monocytogenes (n = 221) recovered from the positive samples were characterized. With the exception of a sample taken from a drain in the cheese washing area at SSI (isolates 2116/1 and 2116/2a both recovered from the same sample) all the other samples harbored isolates belonging to the same serogroup and presenting the same resistance pattern to arsenic and cadmium (data not shown). Therefore, with the exception of isolates 2116/1 and 2116/2, for all the other samples one isolate per sample was selected was selected for PFGE typing. The combination of PFGE profiles obtained from both ApaI and AscI yielded a total of 22 PFGE types (Fig. 1).

Table 3 Positive samples for Listeria monocytogenes collected at a Small Scale Cheese Producer (SSI). No. of positive samples Date

Cheese

May 03 June 03 July-03 August-03 September 03 November-03 January 04 February 04 April-04 May 04 June-04 October 04 November-04 February-05 April 05 May 05 July-05 August-05 September-05 October 05 November 05 August-06 February-07 June-07 July-07 September-07 October-07 Total (%)

1 1

Milk

Contact

Non-contact

1 1 1

1

1 1 1

1 1 1

2 2 1 2

1 1 1 2 2

1

1 2 2

1

7 (3.4)

2 (2.9)

15 (6.7)

1 1 1 1 1 1

1 1 1 1 1 1 1 22 (11.5)

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Table 4 Positive samples for Listeria monocytogenes collected at an Industrial Cheese Producer (ICP). No. of positive samples Date July 03 May 04 August 05 November 05 January 06 February 06 Total (%)

Cheese

Milk 1 2

1 1 4 5 (0.2)

4 (21.1)

In APC, six PFGE types were identified. Of these, three were only isolated on one occasion while the other three (Da, Db and Dc), were recurrently isolated during several months from cheeses, different locations in the factory and once from a raw milk sample (Fig. 1). These PFGE types presented similar AscI profiles and less than three bands difference for ApaI profiles; and can therefore be considered as closely related according to the “3-band rule” suggested by Tenover et al. (1995). A higher diversity was observed in SSI compared to the other two plants. Twelve PFGE types were identified, PFGE types Ka and Kb being dominant (Fig. 1). In addition to a non-efficient cleaning and sanitation plan, this higher diversity may be explained by the higher diversity of raw material entering the plant. While SSI produces cheese from ewe's, goat's and cow's milk, the APC and ICP only use ewe's and cow's milk, respectively (Table 1). In ICP five PFGE types were identified, four different PFGE types in different raw cow's milk samples and one (Da) in five cheeses (one collected in one visit and four collected in another visit; Fig. 1). The lower rate of cheese contamination and the fact that L. monocytogenes was never detected in environmental samples suggest that the source of contamination may be in an unusual environmental site which was not sampled. Only PFGE type Da was found in isolates from two processing plants (APC and ICP; Fig. 1). The distance between the two factories is more than 250 km, and the use of common raw materials is unlikely as the type of process and cheeses produced are different. Therefore, it was not possible to identify a common source of contamination. PFGE type Da recovered from six APC cheeses was also recovered from raw ewe's milk and from environmental samples collected at the milk reception and the production zones. It is therefore possible to speculate that the source of these cheese isolates was the raw milk itself, and also contributed to the contamination detected on the floor at milk reception. The suggestion that milk carries the microorganism into cheese manufacture was also made by Leite et al. (2006) who found the same genotype in milking and cheese making environments in two farmhouses. Mendonça et al. (2012) attributed the introduction of L. monocytogenes in the environment of a chicken slaughterhouse to the raw material. In a longitudinal study over a period of six years, Di Ciccio et al. (2012) concluded that even if raw fish can be a contamination source of the working environment, contamination of smoked salmon occurs mainly during processing. Although results obtained in APC premises have shown that the microorganism was not confined to a single site, it is possible to speculate that the feet of personnel were responsible for the spread of the organism in the premises; studies with Gorgonzola cheese detected L. monocytogenes in locker rooms and toilets (Lomonaco et al., 2009), supporting the hypothesis proposed. In the case of cheese made from pasteurized milk, the contamination is probably due to post-process contamination. In fact, isolates recovered from raw milk differ from isolates recovered from cheese in SSI and ICP and from the majority of environmental isolates concerning their serogroup or PFGE types in SSI. At SSI the highest number of positive samples was recovered from the cheese washing area and the shipping area. The cheese washing area was also the location that yielded the highest number of positive

samples at APC. Jacquet et al. (1993) have already mentioned that cheese contamination can occur during washing of soft cheese. In the present study, the same PFGE type (Kd) was shared by isolates recovered from cheese, from the brush used to wash cheese and from the cheese washing zone. Brushes could be the vehicles transferring the microorganism from one contaminated cheese to another cheese. Apart from the contaminated cheese that could serve as a source in L. monocytogenes transfer, cleaning procedures involving the use of pressurized water may also result in aerosol production and subsequent contamination. The presence of L. monocytogenes in the shipping area at SSI and APC represents a serious threat of post-processing contamination. In fact, isolates recovered from this area and from cheese shared the same PFGE types. All environmental sites tested at ICP were negative for L. monocytogenes and therefore it was not possible to speculate about possible sources of contamination. The approach used in this company to control the establishment of the microorganism in its premises, is based on the analysis of Listeria spp. as an indicator of potential L. monocytogenes contamination. It appears that this strategy in controlling the microorganism is effective. Nevertheless, isolation of putative ECI strains from cheese on two different occasions, five months apart, is of great concern. As previously stated by Chen et al. (2007), “Given the ubiquitous nature of L. monocytogenes in food processing plants, its ability to grow in foods at refrigeration temperatures, and the difficulty in detecting routes of transmission, it seems reasonable that previously or newly identified epidemic clones and outbreak strains will likely be implicated in future listeriosis outbreaks”. In APC, PFGE type Da persisted in the environment for 15 months, since the first isolation, until the end of the study. PFGE type Dc persisted in the environment but was apparently eliminated after 8 months. Although isolation of strains belonging to persistent PFGE types in the environment of SSI was also observed, the global pattern of PFGE types was not constant, suggesting cycles of elimination and recontamination during the production cycle. However, PFGE type Ka was recurrently isolated over a period of four years and PFGE type Kb was isolated on several occasions during approximately a three-year period. The persistence of strains for long periods in cheese plants was previously reported by several authors (Kabuki et al., 2004; Parisi et al., 2013; Unnerstad et al., 1996; Wagner et al., 2006). It is important to note that in the past strains able to persist in food processing plants have been linked to listeriosis outbreaks (McLauchlin et al., 2004; Tompkin, 2002). ECI genetic markers were only detected in isolates recovered from finished products of APC (n = 6) and ICP (n = 5), all sharing the same PFGE type Da (Fig. 1). Outbreaks that occurred in Canada, with coleslaw (1981), in USA with Mexican-style cheese (1985), in Switzerland with soft cheese (1983–87), in Denmark with cheese (1989–90) and in France with jellied pork tongue (1992) were due to this epidemic clone (Liu, 2008). According to Fugett et al. (2007) ECI may represent a stable pandemic clone that has the ability to survive in different environments, to grow in foods and to cause human listeriosis. The ECI genetic marker was found in PFGE type Da isolates but not in Db or Dc PFGE types. Diversification occurring during persistence in food processing plants has been previously reported (Ferreira et al., 2011; Orsi et al., 2008) and attributed to prophage diversification as well plasmid loss or gain (Orsi et al., 2008). Strains belonging to some of the identified PFGE types had been previously implicated in human cases of listeriosis. PFGE type Da matched with the PFGE type of L. monocytogenes strains isolated from two human listeriosis cases occurred in two different dates, July 2003 and September 2006, PFGE type Dc matched with the PFGE type of one clinical strain isolated in September 1997, PFGE type C was identified in one human isolate occurred in December 2006 and PFGE type Ka was found

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in two isolates from human cases of listeriosis occurred in January 2005 and in May 2006. Following this investigation, some measures were implemented by the cheese producers. A more careful selection and greater control of suppliers of raw milk, changes in the entrance of the plant, acquisition of cleaning materials specific for each zone, a greater number of brushes

307

made to eradicate L. monocytogenes in the environment and final products at APC. In SSI several efforts were also made to eradicate it. However, although drains were repaired to avoid backs-up, aprons were used exclusively in the cheese washing area, and brushes were placed in disinfection solution every day and frequently changed, the pathogen persisted. Therefore it may be necessary to increase the number of

Fig. 1. PFGE profiles of selected isolates of Listeria monocytogenes obtained with restriction enzymes AscI and ApaI, and information of cheese producer, site and date of occurrence, serogroup and resistance pattern to arsenium and cadmium, and PFGE type. Isolates showing ECI-specific markers are labeled with an asterisk. Isolates were classified as resistant (R) or sensitive (S) to arsenic (As) and cadmium (Cd).

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Fig. 1 (continued).

environmental sites testes and to launch ‘search and destroy’ operations i.e. to locate the individual niches of persistent strains and to apply effective cleaning/sanitizing to those specific areas.

Acknowledgments This work was supported by National Funds from FCT — Fundação para a Ciência e a Tecnologia through projects PEst-OE/EQB/LA0016/ 2011 and PTDC/AGR-ALI/64662/2006, Pos-doc grants to Joana Silva (SFRH/BPD/35392/2007) and to Vânia Ferreira (SFRH/BPD/72617/ 2010) and PhD grant to Rui Magalhães (SFRH/BD/71704/2010). We thank Dr. Martin Wiedman, Department of Food Science, Cornell University, for providing the L. monocytogenes control strains used in the PCR screen for epidemic-clones associated genetic markers assay. Editing of this manuscript by Dr P.A. Gibbs is gratefully acknowledged.

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