Listeria monocytogenes in ready-to-eat vacuum and modified atmosphere packaged meat and fish products of Estonian origin at retail level

Food Control 67 (2016) 48e52

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Listeria monocytogenes in ready-to-eat vacuum and modified atmosphere packaged meat and fish products of Estonian origin at retail level €esaar a, b, Toomas Kramarenko a, b, *, Mati Roasto a, Riikka Keto-Timonen c, Mihkel Ma a b d c € rman , Hannu Korkeala €e , Maiu Kuningas , Ari Ho Kadrin Merema a

Department of Food Hygiene, Institute of Veterinary Medicine and Animal Sciences, Estonian University of Life Sciences, Kreutzwaldi 56/3, 51014, Tartu, Estonia Veterinary and Food Laboratory, Kreutzwaldi 30, 51006, Tartu, Estonia c Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of Helsinki, P.O. Box 66, FI-00014 University of Helsinki, Finland d The Finnish Defence Forces, P.O. Box 919, 00131 Helsinki, Finland b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 January 2016 Received in revised form 19 February 2016 Accepted 20 February 2016 Available online 23 February 2016

The prevalence, counts and genetic diversity of Listeria monocytogenes in ready-to-eat (RTE) vacuum and modified atmosphere packaged meat and fish products was studied in Estonia. Within two consecutive years 370 RTE food samples were collected at retail level from which 11% were found to be positive for L. monocytogenes. Contamination was higher among RTE fish products (17%) than in RTE meat products (6%). Generally, the counts of L. monocytogenes in positive products remained under ten colony forming units (CFU) per gram of product. Only 1.6% of the RTE meat and fish products contained L. monocytogenes in range of 10e100 CFU/g and 0.3% more than 100 CFU/g at the end of shelf-life. The food category containing highest L. monocytogenes prevalence was RTE lightly salted fish products with the prevalence of 32%. Only one (0.3%) RTE food sample exceeded the 100 CFU/g food safety criterion set out in the EU Regulation 2073/2005. Pulsed-field gel electrophoresis (PFGE) characterization of the isolates showed an overall similarity higher than 70%, and nine clusters based on 100% similarity were revealed. PFGE genotyping revealed that the few predominant pulsotypes were associated with particular food plants. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Listeria monocytogenes Prevalence Counts RTE meat products RTE fish products

1. Introduction Listeria monocytogenes is the causative agent of a severe foodborne infection, listeriosis which shows increasing trend during recent years in Europe (EFSA, 2015). The notification rate for human listeriosis in 2013 was 0.44 cases per 100,000 population which indicates an 8.6% increase compared with 2012 (EFSA, 2015). According to the data of the Estonian Health Board (Terviseamet, 2015) the average notification rate of human listeriosis cases was 0.15 per 100,000 inhabitants from 2011 to 2014 in Estonia. The main route of transmission of L. monocytogenes to humans is

* Corresponding author. Veterinary and Food Laboratory, Kreutzwaldi 30, 51006, Tartu, Estonia. E-mail addresses: [email protected] (T. Kramarenko), mati.roasto@ emu.ee (M. Roasto), [email protected] (M. Kuningas), ari.horman@mil.fi € rman), [email protected] (H. Korkeala). (A. Ho http://dx.doi.org/10.1016/j.foodcont.2016.02.034 0956-7135/© 2016 Elsevier Ltd. All rights reserved.

through the consumption of contaminated food (Allerberger & Wagner, 2010). The majority of the listeriosis outbreaks have been linked to certain RTE foods with extended storage time, and is related with the fact that L. monocytogenes can grow to high counts at refrigeration temperatures (Ericsson et al., 1997; Gottlieb et al., €inen et al., 2000; McLauchlin, Hall, Velani, & Gilbert, 2006; Lyytika 1991; Miettinen et al., 1999; Sim et al., 2002; Stephan et al., 2015). RTE meat and fish products with a long shelf-life are associated with the high risk of transmission of L. monocytogenes. In RTE fish and fishery products the prevalence of L. monocytogenes has been 10.8%, and the criterion of 100 CFU/g during the shelf-life of a product was exceeded in 1.6% of the samples in EU year 2013 (EFSA, 2015). Furthermore, L. monocytogenes was detected in 3.4% and 2.3% of pig and bovine RTE meat products, and L. monocytogenes was found at the levels exceeding the 100 CFU/g in 1.0%, 0.0% and 0.4% of the RTE poultry, bovine and pig meat samples, respectively (EFSA, 2015).

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Table 1 L. monocytogenes in RTE meat and fish products of Estonian origin. Samples

No. of samples

No. of positive samples/positive %

CI95% of positive %

RTE meat RTE fish Total

185 185 370

11/5.9 31/16.8 42/11.4

3.4e10.3 12.1e22.8 8.5e15.0

Present study aimed to determine the prevalence, counts and genetic similarity of L. monocytogenes in Estonian origin RTE fish and meat products. Data will further be used for quantitative risk assessment of L. monocytogenes. 2. Materials and methods 2.1. Sampling A total of 185 RTE meat and 185 RTE fish product samples were collected monthly during two years period at Estonian retail level from the most consumed RTE fish and meat products in the biggest supermarkets. Samples originated from 13 fish and 13 meat enterprises. RTE fish and meat VP and MAP products of Estonian origin having a long shelf-life, more than four weeks, were collected. Among the RTE fish products cold-smoked, hot-smoked, salted (including gravad fish) and matured fish products were sampled. Most of the salted fish products were lightly salted with final sodium chloride content of 1.3e1.5%. Among the RTE meat products cold-smoked, hot-smoked, cooked and fermented meat products were sampled. At retail outlets before sampling the storage temperature was recorded and all the products were kept at proper refrigerated temperature conditions not exceeding 4
C, and transported to the laboratory immediately. The samples were stored under the refrigerated temperatures in accordance with product labeling information at maximum allowed storage temperature. All analyses of the products were performed at use-by-date. 2.2. Isolation and enumeration of L. monocytogenes

broth (Oxoid, Basingstoke, Hampshire, England) at 30
C for 24 h. After the incubation, 0.1 ml was transferred to a tube containing 10 ml of Fraser broth and incubated at 37
C for 48 h. After the incubation, the half- and full-strength Fraser broths were platedout on ALOA agar (Lab M Ltd., Bury, Lancashire, UK) and PALCAM agar (Oxoid). Selective agar plates were incubated at 37
C for 24e48 h. Typical colonies (n ¼ 5) presumed to be Listeria spp. were streaked from both PALCAM and ALOA agars onto the tryptone soya yeast extract agar (TSYEA) made in house from single components (Oxoid) and plates were incubated at 37
C for 24 h. The confirmation tests were performed using the pure culture obtained from TSYEA. Isolates that were catalase and Gram positive and with characteristic tumbling motility were inoculated on 5% sheep blood agar (Oxoid) plates to determine the haemolytic reaction, b-haemolysis for L. monocytogenes. For further confirmation, carbohydrate utilization and CAMP tests were performed. All confirmed L. monocytogenes isolates were stored at e 80
C in bacterial protect tubes (TSC Ltd, Heywood, Lancashire, UK). Enumeration of L. monocytogenes was carried out according to the method described by EVS-EN ISO 11290-2:2000/A1:2004. In all cases enumeration was performed at the use-by-date. For making initial suspensions and dilutions (101 to 103) buffered peptone water (ISO, Oxoid) was used. The procedure included 1-h resuscitation in buffered peptone water at room temperature, and surface plating on ALOA agar of 1.0 ml of 101, and 0.1 ml of each of the 102 and 103 dilutions to duplicates of the ALOA plates. The plates were incubated at 37
C for 24e48 h. Typical colonies were selected and plated on 5% sheep blood agar. L. monocytogenes was confirmed as described above. 2.3. Serological typing

The isolation of L. monocytogenes was carried out in Estonian Veterinary and Food Laboratory and all the analyses and confirmation tests were performed in accordance with instructions of the detection methods described by EVS-EN ISO 11290-1:2000/ A1:2004. Examination for L. monocytogenes included a primary and secondary enrichment. All samples were incubated in half Fraser

In house method of EU Reference Laboratory for L. monocytogenes (AFSSA, 2009) was followed for conventional serotyping. Somatic (O) and flagellar (H) antigenes were identified by using specific antisera produced by Denka Seiken (Tokyo, Japan). 2.4. In situ DNA isolation and pulsed-field gel electrophoresis (PFGE)

Table 2 L. monocytogenes presence and counts in different RTE fish and meat products. Product category

RTE meat Cold-smoked Hot-smoked Cooked Fermented All RTE meat RTE fish Cold-smoked Hot-smoked Matured Salted All RTE fish Total (%) a b

Absence in 25 g. Presence in 25 g.

No. of samples Negativea

Positiveb

10 … 100

>100

66 70 18 20 174

6 3 0 0 11

1 0 0 1 2

0 0 0 0 0

62 2 38 52 154 328 (88.6%)

4 1 4 17 31 42 (11.4%)

0 0 0 4 4 6 (1.6%)

0 0 0 1 1 1 (0.3%)

In situ DNA isolation and PFGE were performed as described earlier by Autio et al. (2002). Agarose-embedded DNA was digested with restriction endonuclease AscI (New England Biolabs, Ipswich, USA). PFGE patterns were analyzed using BioNumerics software, version 5.10 (Applied Maths, Sint-Martens-Latem, Belgium), and similarity between PFGE patterns was expressed as a Dice coefficient (position tolerance 1.0%). Clustering and construction of dendrograms were performed by using the unweighted pair-group method with arithmetic averages. 2.5. Statistical analyses All individual results were recorded using MS Excel 2010 software (Microsoft Corporation, Redmond, Wash.), and statistical analysis was performed with the Statistical Package R in order to determine if there were statistically significant differences at 95%

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Fig. 1. Dendrogram of AscI macrorestriction patterns of Listeria monocytogenes isolates. Similarity analysis was performed by using the Dice coefficient and clustering was performed by using the unweighted pair-group method with arithmetic averages. In the figure L. monocytogenes isolates from fish and meat plants are represented as F and M, respectively.

and 99% confidence levels in the prevalence and counts of L. monocytogenes in RTE fish and meat products of different categories, producers and retail outlets using the KruskaleWallis rank sum test and Chi-square test. 3. Results and discussion The overall prevalence of L. monocytogenes in RTE meat and fish products is shown in Table 1. The contamination of RTE fish products (16.8%) with L. monocytogenes was significantly higher than of RTE meat products (5.9%). Similarly, the European baseline survey carried out in 2010 and 2011 concluded, that the EU wide prevalence of L. monocytogenes was highest in fish products (10%) and lower in meat products 2.1% at the end of the products shelf-life (EFSA, 2013). Similar prevalence of RTE fish has been reported in

different studies performed in Bulgaria (Gyurova, KrumovaVulcheva, Daskalov, & Gogov, 2015), Finland (Lyhs, Hatakka, Maki-Petays, Hyytia, & Korkeala, 1998), Iran (Fallah, SaeiDehkordi, & Mahzounieh, 2013) and Northern Spain (Garrido,  n, 2009). Another Spanish study by Gonz Vitas, & García-Jalo alez, n (2013) found only 4.8% of Vitas, Díez Leturia, and García-Jalo smoked salmon products to be L. monocytogenes positive with low levels. In a previous Estonian study by Kramarenko et al. (2013) performed in 2008e2010 the prevalence of L. monocytogenes in RTE fish was 5.7% and in RTE meat products 2.0%. According to the EFSA (2015) the overall L. monocytogenes prevalence in RTE fish and fishery products in EU was higher than in RTE meat products. L. monocytogenes prevalence found in current study showed higher contamination percentages compared to the EU average. However, in the present study the analyses focused only to the VP and MAP

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Table 3 Genotypes of Listeria monocytogenes isolates originating from RTE fish and meat products of distinct Estonian enterprises. Producer Fish F1 F2 F3 F4 F5 F6 F7 F8 e F13 Meat M1 M2 M3 M4 e M13

No of samples

No of positive samples (%)

Pulsotype (no of isolates)

36 2 15 24 21 45 2 40

3 2 10 10 3 1 2 0

(8.3) (100.0) (66.7) (41.7) (14.3) (2.2) (100.0) (0.0)

P3(1), P4(1), P10(1) P6(1), P10(1) P3(8), P5(1), P9(1) P10(3), P9(2), P5(2), P4(1), P2(1), P6(1) P3(2), P1(1) P8(1) P6(2)

15 36 47 87

1 7 3 0

(6.7) (19.4) (6.4) (0.0)

P3(1) P11(6), P2(1), P5(1) P3(1), P7(1), P8(1)

ready-to-eat products with long shelf-life. Additionally, 39% and 76% of the RTE meat and fish products sampled in present study were not heat-treated e.g. were cold-smoked or lightly salted, therefore belonging to the known high risk food categories. Also, Lado and Yousef (2007) reported that L. monocytogenes can grow in vacuum packages (VP) as well as in modified atmosphere packages (MAP) which extend the shelf-life of foods, giving the opportunity for L. monocytogenes multiply to high numbers towards to the end of shelf-life, especially if the recommended temperature during the storage is not maintained. The prevalence of L. monocytogenes in RTE meat products has been generally low but controversially in a recent Greek study by Manios et al. (2014) overall 28% of tested RTE meat products were contaminated. Also in a Belgium study L. monocytogenes was detected in 27.8% (25/90) smoked fish samples (Uyttendaele et al., 2009). Enumeration of L. monocytogenes in RTE meat and fish products of Estonian origin (Table 2) resulted with only one RTE fish product exceeding the limit of 100 CFU/g at the end of the shelf-life. All the RTE meat products of Estonian origin were in compliance with the EU food safety criterion. In comparison, 1.6% of RTE fish and 0.7% of RTE meat products was found to exceed the criterion of 100 CFU/g in the EU wide survey in average (EFSA, 2015). L. monocytogenes was not detected in 328 samples (89%) and only 1.6% of the RTE fish and meat products contained L. monocytogenes in range of 10e100 CFU/g and 0.3% more than 100 CFU/g at the end of shelf-life (Table 2). For a healthy human population, foods where the levels do not exceed the 100 CFU/g limit are considered to pose a very low or negligible risk (EFSA, 2015). L. monocytogenes positive samples were found from the products of three out of thirteen meat plants. One meat plant (coded as M2) had proportion of L. monocytogenes positive RTE meat products 19% while for others it was ranging from 0%e6.7%. Among different RTE meat product categories the highest L. monocytogenes prevalence (3.8%) was found for cold-smoked meat products (Table 2). Also in previous Baltic studies cold-smoked pork and beef products rzin¸s, have been found to have high L. monocytogenes prevalence (Be €rman, Lunde n, & Korkeala, 2007; Be rzin¸s, Terentjeva, & Ho Korkeala, 2009). In other RTE meat categories the L. monocytogenes prevalence was low. PFGE analysis revealed that one pulsotype (P11) predominated in the products of company M2 (Fig. 1; Table 3). In addition, all strains were isolated from cold-smoked meat products of different batches, which suggests a possible persistent L. monocytogenes contamination in the food processing plant. L. monocytogenes can persist in food processing plants even for several years and cause postprocessing contamination of the food products (Keto-Timonen n, Autio, Sjo € berg, & Korkeala, 2003). et al., 2008; Lunde

L. monocytogenes positive samples were found from the products of seven out of thirteen fish plants (Table 2). Among different RTE fish product categories the highest L. monocytogenes prevalence (12%) was found for salted fish products (Table 2), The prevalence was higher than in other fish product categories (pvalue <0.05). In the present study only one RTE product, salted salmon fillet slices, was found to exceed the criterion of 100 CFU/g at the use-by-date. We found one predominant pulsotype (P3) for fish plant F3 (Fig. 1) which was associated with various salmon products at different sampling times. It may indicate the existence of persistent L. monocytogenes in the fish plant. At the same time latter pulsotype was also found in products of other plants (F1, F5, M1, M3) which means that dominating strains in certain food enterprises are not always plant-specific. Similar findings were described by Autio et al. (2002). Serotyping revealed that all L. monocytogenes isolates belonged to the 1/2a serotype which is similar to our previous findings (Kramarenko et al., 2013). 4. Conclusion Study showed that in vacuum and modified atmosphere packaged RTE fish products the prevalence of L. monocytogenes was higher than in similarly packaged RTE meat products. However, for both RTE fish and meat products the counts of L. monocytogenes were in compliance with the food safety criterion of 100 CFU/g during the products self-life, except for one product. Prevalence differences in RTE fish and meat products between food plants were found. Only serotype 1/2a was determined among L. monocytogenes isolates. PFGE genotyping revealed that the few predominant pulsotypes may be associated with particular food companies. Acknowledgements This study was supported by the Estonian Scientific Council (Sihtasutus Eesti Teadusagentuur, ETAg) Grant No. 9315, and by Ministry of Agriculture of Estonia, applied science project T13057VLTH. References AFSSA - Agence Francaise de Securite Sanitaire des Aliments. (2009). In house method: Listeria monocytogenes agglutination serotyping. Method AFSSA LERQAP.CEB.03. Allerberger, F., & Wagner, M. (2010). Listeriosis: a resurgent foodborne infection. Clinical Microbiology and Infection, 16, 16e23. n, J., Fredriksson-Ahomaa, M., Bjo €rkroth, J., Sjo €berg, A.-M., & Autio, T., Lunde

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T. Kramarenko et al. / Food Control 67 (2016) 48e52

Korkeala, H. (2002). Similar Listeria monocytogenes pulsotypes detected in several foods originating from different sources. International Journal of Food Microbiology, 77, 83e90. rzin¸s, A., Ho €rman, A., Lunde n, J., & Korkeala, H. (2007). Factors associated with Be Listeria monocytogenes contamination of cold-smoked pork products produced in Latvia and Lithuania. International Journal of Food Microbiology, 115, 173e179. rzin¸s, A., Terentjeva, M., & Korkeala, H. (2009). Prevalence and genetic diversity of Be Listeria monocytogenes in vacuum-packaged ready-to-eat meat products at retail markets in Latvia. Journal of Food Protection, 72, 1283e1287. EFSA, European Food Safety Authority. (2013). Analysis of the baseline survey on the prevalence of Listeria monocytogenes in certain ready-to-eat foods in the EU, 2010-2011. Part A: Listeria monocytogenes prevalence estimates. EFSA Journal, 11(6), 3241e3275. EFSA, European Food Safety Authority. (2015). The european Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2013. EFSA Journal, 13(1), 3991. €w, A., Danielsson-Tham, M. L., Loncarevic, S., Mentzing, L. O., Ericsson, H., Eklo Persson, I., et al. (1997). An outbreak of listeriosis suspected to have been caused by rainbow trout. Journal of Clinical Microbiology, 35, 2904e2907. Fallah, A. A., Saei-Dehkordi, S. S., & Mahzounieh, M. (2013). Occurrence and antibiotic resistance profiles of Listeria monocytogenes isolated from seafood products and market and processing environments in Iran. Food Control, 34, 630e636.  n, I. (2009). Survey of Listeria monocytogenes in Garrido, V., Vitas, A. I., & García-Jalo ready-to-eat products: prevalence by brands and retail establishments for exposure assessment of listeriosis in Northern Spain. Food Control, 20, 986e991. lez, D., Vitas, A. I., Díez Leturia, M., & García-Jalo n, I. (2013). Listeria monoGonza cytogenes and ready-to-eat seafood in Spain: study of prevalence and temperatures at retail. Food Microbiology, 36, 374e378. Gottlieb, S. L., Newbern, E. C., Griffin, P. M., Graves, L. M., Hoekstra, R. M., Baker, N. L., et al. (2006). Multistate outbreak of listeriosis linked to turkey deli meat and subsequent changes in US regulatory policy. Clinical Infectious Diseases, 42, 29e36. Gyurova, E., Krumova-Vulcheva, G., Daskalov, H., & Gogov, Y. (2015). Prevalence of Listeria monocytogenes in ready-to-eat foods in Bulgaria. Journal of Hygienic Engineering and Design, 112e118. UDC 579.67:579.869.1. n, J., & Korkeala, H. (2007). An 8-year surKeto-Timonen, R., Tolvanen, R., Lunde veillance 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, 1866e1873.

~ ltsama, P., & Elias, T. Kramarenko, T., Roasto, M., Merem€ ae, K., Kuningas, M., Po (2013). Listeria monocytogenes prevalence and serotype diversity in various foods. Food Control, 30, 24e29. Lado, B. H., & Yousef, A. E. (2007). Characteristics of Listeria monocytogenes important to food processors. In E. T. Ryser, & E. H. Marth (Eds.), Listeria, listeriosis, and food safety (3rd ed., pp. 157e213). Boca Raton, FL: CRC Press. n, J., Autio, T., Sjo €berg, A.-M., & Korkeala, H. (2003). Persistent and nonperLunde sistent Listeria monocytogenes contamination in meat and poultry processing plants. Journal of Food Protection, 66, 2062e2069. Lyhs, U., Hatakka, M., Maki-Petays, N., Hyytia, E., & Korkeala, H. (1998). Microbiological quality of Finnish vacuum-packaged fishery products at retail level. Archiv für Lebensmittelhygiene, 49, 146e150. €inen, O., Autio, T., Maijala, R., Ruutu, P., Honkanen-Buzalski, T., Miettinen, M., Lyytika et al. (2000). An outbreak of Listeria monocytogenes serotype 3a infections from butter in Finland. The Journal of Infectious Diseases, 181, 1838e1841. Manios, S. G., Grivokostopoulos, N. C., Bikouli, V. C., Doultsos, A., Zilelidou, E. A., Gialitaki, M. A., et al. (2014). A 3-year hygiene and safety monitoring of a meat processing plant which uses raw materials of global origin. International Journal of Food Microbiology. http://dx.doi.org/10.1016/j.ijfoodmicro.2014.12.028. McLauchlin, J., Hall, S. M., Velani, S. K., & Gilbert, R. J. (1991). Human listeriosis and : a possible association. BMJ, 303, 773e775. pate €rkroth, K. J., & Miettinen, M. K., Siitonen, A., Heiskanen, P., Haajanen, H., Bjo Korkeala, H. (1999). Molecular epidemiology of an outbreak of febrile gastroenteritis caused by Listeria mococytogenes in cold-smoked rainbow trout. Journal of Clinical Microbiology, 37, 2358e2360. Sim, J., Hood, D., Finnie, L., Wilson, M., Graham, C., Brett, M., et al. (2002). Series of incidents of Listeria monocytogenes non-invasive febrile gastroenteritis involving ready-to-eat meats. Letters in Applied Microbiology, 35, 409e413. Stephan, R., Althaus, D., Kiefer, S., Lehner, A., Hatz, C., Schmutz, C., et al. (2015). Foodborne transmission of Listeria monocytogenes via ready-to-eat salad: a nationwide outbreak in Switzerland, 2013e2014. Food Control, 57, 14e17. Terviseamet (Estonian Health Board). (2015). Nakkushaiguste registreerimine Eestis 2011‒2014. http://www.terviseamet.ee/fileadmin/dok/Nakkushaigused/ statistika/NH_Eestis_2011-2014.pdf. Uyttendaele, M., Busschaert, P., Valero, A., Geeraerd, A. H., Vermeulen, A., Jacxsens, L., et al. (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, 94e104.