Prevalence and Genetic Diversity of Listeria monocytogenes in Vacuum-Packaged Ready-to-Eat Meat Products at Retail Markets in Latvia

Abstract

Nine groups of different retail ready-to-eat vacuum-packaged meat products from 10 Baltic meat processing plants were analyzed for presence and numbers of Listeria monocytogenes at the end of shelf life. A total of 38 (18%) of 211 samples tested positive for L. monocytogenes serotype 1/2a (88%) or 1/2c (12%). The prevalence of L. monocytogenes in cold-smoked, sliced, vacuum-packaged beef and pork products (42%) was significantly higher than in cooked, sliced, vacuum-packaged meat products (0.8%) (P < 0.001). Enumeration of L. monocytogenes showed that 84% of the positive samples contained <100 CFU/g upon expiry of product shelf life. The numbers of L. monocytogenes exceeded 100 CFU/g only in cold-smoked, sliced, vacuum-packaged beef products. Identical pulsed-field gel electrophoresis types were recovered from different production lots of cold-smoked vacuum-packaged beef and pork products produced by the same meat processing plant, demonstrating L. monocytogenes contamination as a recurrent problem within one meat processing plant. [PUBLICATION ABSTRACT]

Headnote

ABSTRACT

Nine groups of different retail ready-to-eat vacuum-packaged meat products from 10 Baltic meat processing plants were analyzed for presence and numbers of Listeria monocytogenes at the end of shelf life. A total of 38 (18%) of 211 samples tested positive for L. monocytogenes serotype 1/2a (88%) or 1/2c (12%). The prevalence of L. monocytogenes in cold-smoked, sliced, vacuum-packaged beef and pork products (42%) was significantly higher than in cooked, sliced, vacuum-packaged meat products (0.8%) (P < 0.001). Enumeration of L. monocytogenes showed that 84% of the positive samples contained <100 CFU/g upon expiry of product shelf life. The numbers of L. monocytogenes exceeded 100 CFU/g only in cold-smoked, sliced, vacuum-packaged beef products. Identical pulsed-field gel electrophoresis types were recovered from different production lots of cold-smoked vacuum-packaged beef and pork products produced by the same meat processing plant, demonstrating L. monocytogenes contamination as a recurrent problem within one meat processing plant.

Listeria monocytogenes is a widely recognized foodborne pathogen that can cause listeriosis in humans, with high mortality rates in susceptible individuals. Over the last 2 decades, numerous foodborne cases and outbreaks of human listeriosis have been traced to meat, with evidence of transmission through consumption of contaminated meat products (7, 9, 10, 20). Different delicatessen pork products have been implicated in L. monocytogenes outbreaks in several European countries (11, 15).

Previous studies of L. monocytogenes in ready-to-eat (RTE) meat products have shown that the prevalence of L. monocytogenes may vary from 0 to 72% with different levels of CFU per gram upon expiry of shelf life (8). A particularly high prevalence of L. monocytogenes has been found in meat products that have received no heat treatment steps to eliminate L. monocytogenes in the finished product (4, 8, 20). A risk of foodborne disease must be considered when the pathogen is present. To date, limited data on the prevalence of L. monocytogenes in different retail vacuumpackaged RTE meat products produced in Baltic countries have been available.

In this study, we investigated the prevalence, number, and genetic diversity of L. monocytogenes organisms in different RTE vacuum-packaged meat products of Baltic origin available in retail markets in Latvia. A high prevalence of L. monocytogenes was found in cold-smoked, sliced, vacuum-packaged beef and pork products. Moreover, we showed that identical pulsed-field gel electrophoresis (PFGE) types were found in different cold-smoked RTE vacuum-packaged meat products produced by the same meat processing plant, indicating that a common contamination site may exist at the meat processing plant.

MATERIALS AND METHODS

Sampling. Vacuum-packaged sliced meat products, sampled from January to April 2005, were taken from two to six production lots for each product group from two supermarket chains in Riga, Latvia (Table 1). All available RTE vacuum-packaged meat products of Baltic origin presented in meat counters were represented in the sampling. Samples included cold-smoked beef (n = 54), dried beef "Basturma" (a highly seasoned, air-dried, cured beef) (n = 10), cold-smoked pork (n = 25), salami-type sausages (n = 28), cooked smoked ham (n = 44), cooked sausages (n = 10), cooked smoked beef (n = 11), liver paté (? = 24), and cooked smoked turkey (n = 5). A total of 211 samples originating from 10 meat processing plants were obtained. The samples were transported to the laboratory in transport ice bags and kept at 6°C before being analyzed at 0 to 5 days before the end of their shelf life. Vacuum packages were opened aseptically, and a 25 -g sample was taken and pummeled with 225 ml of half-strength Fraser broth (Oxoid, Basingstoke, UK) in a stomacher. After the sampling, all product packages were aseptically vacuum packaged again and kept at 6°C to await enumeration analyses.

Isolation and enumeration of L. monocytogenes. Isolation and enumeration of L. monocytogenes were performed according to the International Organization for Standardization methods (1, 2), with the modifications suggested by Johansson (14). Examination for L. monocytogenes included a two-step enrichment. The samples were incubated in half-strength Fraser broth at 30°C for 24 h. After incubation, 0.1 ml was transferred to full-strength Fraser broth and incubated at 37°C for 48 h. The half- and fullstrength Fraser broths were plated on PALCAM listeria selective agar (Oxoid) and L. monocytogenes blood agar (LMBA) containing Trypticase soy agar base (Difco, Becton Dickinson, Sparks, MD), 5% sterile sheep blood and, per liter, 10 g of lithium chloride (Merck KgaA, Darmstadt, Germany) and 10 mg of ceftazidime (Abtek Biologicals Ltd., Liverpool, UK). Selective agar plates were incubated at 37°C for 24 to 48 h. Five typical colonies were streaked from both PALCAM and LMBA onto 5% sheep blood Columbia agar (Difco, Becton Dickinson) and then incubated at 37°C for 24 h. Isolates that were beta-hemolytic, catalase positive, and gram positive were presumed to be L. monocytogenes. Presumptive L. monocytogenes isolates were confirmed using the API Listeria kit (bioMérieux, Marcy l'Etoile, France). All confirmed L. monocytogenes isolates were stored at -70°C.

Enumeration was performed upon expiry of shelf life of each of the L. monocytogenes-positive products. The lower limit of enumeration was 10 CFU/g. The procedure included a 1-h resuscitation in buffered peptone water at 20°C and surface plating on PALCAM and LMBA of 1.0 ml of the IO"1 dilution and 0.1 ml of each of the 10^sup -2^ and 10^sup -3^ dilutions. After incubation, presumptive L monocytogenes colonies were counted. Typical colonies were selected and plated on 5% sheep blood agar. L monocytogenes was confirmed as described above.

DNA isolation and PFGE. DNA isolation was performed with modifications as described by Björkroth et al. (5). After overnight incubation in Trypticase soy broth (Difco, Becton Dickinson) at 37°C, 2 ml of the culture was diluted in 5 ml of PIV buffer (10 mM Tris, 1 M NaCl) and concentrated by centrifugation at 1,100 × g for 15 min at 4°C. Plugs for PFGE were prepared with concentrated cell suspension in PIV and 2% (wt/vol) low-meltingpoint agarose (InCert; FMC Bioproducts, Rockland, ME). Cells in prepared plugs were lysed in a solution containing, per ml, 20 µg of RNase, 1 mg of lysozyme, and 10 U of mutanolysin in lysis buffer at 37°C for 3 h. Lysis was continued with a 1-h wash with ESP solution containing 100 µg/ml proteinase K (Sigma), 0.5 M EDTA, and 10% sodium lauroyl sarcosine at 500C. Afterward, the plugs were washed in buffer containing 10 mM Tris and 0.1 mM EDTA at 50°C for 1 h. A cutting enzyme, 20 U/µl Ascl (New England BioLabs, Beverly, MA), was used for digestion at 37°C for 16 h. PFGE was performed with 1.0% (wt/vol) agarose gel (SeaKem Gold, FMC Bioproducts, Rockland, ME) in buffer containing 45 mM Tris, 4.5 mM boric acid, and 1 mM sodium EDTA at 8°C in a Gene Navigator system (Pharmacia, Uppsala, Sweden) operated at 200 V. Pulse times ranged from 1 to 35 s for 18 h. The size of the fragments was determined with a low-range PFG marker (New England BioLabs). Gels were stained with ethidium bromide. Photo images were obtained with the Alpha Imager 2000 photo documentation system (Alpha Innotech, San Leandro, CA).

PFGE pattern analysis. Macrorestriction patterns of Asci were analyzed with BioNumerics software version 4.01 (Applied Maths, Kortrijk, Belgium), and Dice coefficient correlation was applied to identify similarities among PFGE types. A dendrogram was constructed with the unweighted pair group method with arithmetic mean. In addition, macrorestriction patterns of L. monocytogenes isolates from meat processing plant A were compared with those of isolates from products of the same plant (A) in our previous study carried out in 2003 and 2004 (4).

Serotyping. At least two L monocytogenes isolates of each PFGE type were serotyped with Listeria antisera (Denka Seiken, Tokyo, Japan) following the manufacturer's instructions.

Statistical analysis. Statistical analyses were performed using a Microsoft Excel-based software program. The chi-square test or the Fisher's exact test was performed to compare the numbers of L monocytogenes-positive products produced by different meat processing plants and the prevalence data in different groups of meat products.

RESULTS

Altogether, 38 samples (18%) were positive for L monocytogenes. The prevalence of L. monocytogenes in different RTE, sliced, vacuum-packaged meat products is shown in Table 1. Cold-smoked beef, dried beef "Basturma," and cold-smoked pork products were positive for L monocytogenes, with overall prevalences of 57, 10, and 20%, respectively. None of the cooked or hot-smoked sliced meat products and only one liver paté sample of the 24 samples tested were positive for L. monocytogenes. The prevalence of L. monocytogenes in cold-smoked meat products (42%) was significantly higher than in cooked meat products (0.8%) (P < 0.001). Thirty-six (95%) of the 38 L. monocytogenes-positive samples were obtained from meat processing plant A, while the remaining 2 L. monocytogenes-positive samples came from meat processing plants B and C. The prevalence of L. monocytogenes in cold-smoked, sliced, vacuum-packaged pork products of meat processing plant A was significantly higher (P < 0.05) than in those of plants B, C, and D. Thirty-two (84%) of the 38 L. monocytogenes-positive samples contained <100 CFU/g (Table 2). None of the positive samples contained more than 1,000 CFU/g; however, 6 (16%) of the 38 samples contained 100 to 1,000 CFU/g. All L. monocytogenes-positive samples from cold-smoked pork products contained < 100 CFU/g. Only in cold-smoked beef products and dried beef "Basturma" was the number of L. monocytogenes between 100 and 1,000 CFU/g.

Based on genetic similarity, seven different PFGE types were detected in 34 L. monocytogenes isolates from cold-smoked pork and beef and dried beef "Basturma" products produced in meat processing plant A. A dendrogram demonstrating genetic similarity among PFGE (Asci) restriction profiles of L. monocytogenes isolates from different production lots originating from meat processing plant A over a 15 -month period is shown in Figure 1. PFGE type 6 contained 71% of all L. monocytogenes isolates belonging to serotype 1/2a, whereas the remaining six PFGE types were either serotype l/2a or l/2c. Moreover, PFGE type 6 was found in six different production lots of coldsmoked beef and pork products from meat processing plant A over a 2-month period. L monocytogenes isolates belonging to PFGE type 5 (serotype l/2c) were found repeatedly within 1 month in two different production lots of dried beef "Basturma" and cold-smoked beef.

DISCUSSION

Cold-smoked, sliced, vacuum-packaged beef and pork products showed the highest levels of contamination with L. monocytogenes, with prevalences of 57 and 20%, respectively. Cold-smoked beef and pork products have similar manufacturing methods involving no processing steps to eliminate L. monocytogenes from the finished products, which may explain the high prevalence of L. monocytogenes. The prevalence of L. monocytogenes in products from plant A was significantly higher than in products from other plants, where it varied from 0 to 10%. The high prevalence of L. monocytogenes (57%) in cold-smoked, sliced, vacuum-packaged beef products from plant A is in agreement with our previous study, where we found a similarly high prevalence of L. monocytogenes (41%) in coldsmoked pork products from the same plant (4). Thus, since 2003, various types of RTE cold-smoked meat products have been continuously contaminated with L. monocytogenes in this meat processing plant.

The majority of the L. monocyto genes-positive samples (84%) contained <100 CFU/g at the end of product shelf life. The relatively low numbers of L. monocytogenes in cold-smoked, sliced, vacuum-packaged pork and beef samples can be explained by the extensive use of starter cultures, which is consistent with our previous findings (4). Starter cultures in production of different RTE meat products have been recognized as one of the most important factors for controlling growth of L. monocytogenes (9, 22, 23). The naturally occurring microbial population of coldsmoked meat products is also, to some extent, able to control growth of L. monocytogenes. However, contamination of L. monocytogenes in cold-smoked, sliced, vacuum-packaged meat products is of concern to manufacturers and retailers alike.

Identical PFGE types were recovered from different production lots of RTE vacuum-packaged cold-smoked pork and beef produced by the same meat processing plant over a 2-month period. Two of the seven PFGE types (types 5 and 6) were found repeatedly in cold-smoked pork, dried beef "Basturma," and cold-smoked beef from meat processing plant A. This may indicate that continuous L. monocytogenes contamination existed in the production line of both pork and beef products. After our visit to this meat processing plant during our study, we found that one production line with the same equipment was used for the production of both products, demonstrating that common contamination sites may exist. L. monocytogenes PFGE type 6 was recovered from six production lots of cold-smoked beef and pork products, revealing L. monocytogenes contamination as a continuous and recurrent contamination problem in this meat processing plant. No PFGE types found in the L. monocytogenes isolates obtained from meat processing plant A were identical to those of isolates of the same meat processing plant in our previous study (4).

Two distinct L. monocytogenes serotypes were detected: 88% of the isolates were serotype 1/2a, and 12% were serotype 1/2c. Several studies have shown that L. monocytogenes strains isolated from meat processing plants are predominantly of serotypes 1/2a, l/2b, and 1/2c (18, 22). Moreover, serotype 1/2c especially has been demonstrated to be able to adhere to, and persist on, food processing surfaces (16, 18, 21). Lundén et al. (16, 17) subsequently showed that serotype l/2c strains also possess the highest adherence properties to stainless steel surfaces, thus facilitating development of persistent microbial population in meat processing plants.

Due to its wide environmental stress tolerance and ubiquitous nature, L. monocytogenes has adapted to different food processing environments, in which RTE products may easily become contaminated through raw foodstuffs or contact surfaces of the processing plants (3, 13, 17). The enhanced ability of L. monocytogenes to survive on different surfaces of meat processing plants, such as conveyor belts, slicers, dicers, and other equipment, has been described previously (6, 16). However, recent studies have indicated that marked differences in environmental stress tolerance between L. monocytogenes strains exist, and thus, specific preventive measures should be designed to be effective against tolerant strains (19). Greer et al. (12) and Bêrzins et al. (4) showed that some factors, such as brining injections and long cold-smoking times, may contribute to the level of L. monocytogenes contamination in the finished cold-smoked pork products; certain production steps should therefore be monitored closely. The high prevalence of L monocytogenes in cold-smoked meat products indicates that special control measures should be implemented by the meat industry to restrict occurrence of this bacterium in the finished products to levels meeting international food safety standards.

ACKNOWLEDGMENTS

We thank Maria Stark and Jari Ano for excellent technical assistance. The Latvian Ministry of Education and Science and the Finnish Veterinary Foundation are acknowledged for financial support. This work was performed in Centre of Excellence on Microbial Food Safety Research, Academy of Finland (118602).

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