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Prevalence of Salmonella Spp. and Listeria Monocytogenes at Small-Scale Spanish Factories Producing Traditional Fermented Sausages

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Prevalence of Salmonella Spp. and Listeria Monocytogenes at Small-Scale Spanish Factories Producing Traditional Fermented Sausages 812 Journal of Food Protection, Vol. 74, No. 5, 2011, Pages 812–815 doi:10.4315/0362-028X.JFP-10-437 Copyright G, International Association for Food Protection Research Note Prevalence of Salmonella spp. and Listeria monocytogenes at Small-Scale Spanish Factories Producing Traditional Fermented Sausages BELEN MARTIN, MARGARITA GARRIGA, AND TERESA AYMERICH* Downloaded from http://meridian.allenpress.com/jfp/article-pdf/74/5/812/1684169/0362-028x_jfp-10-437.pdf by guest on 25 September 2021 IRTA, Food Safety Program, Finca Camps i Armet, 17121 Monells, Spain MS 10-437: Received 6 October 2010/Accepted 20 January 2011 ABSTRACT The manufacturing of fermented sausages is subject to natural contamination processes that can potentially carry foodborne pathogens along the process chain and result in contamination of the final product. The aim of this study was to evaluate the occurrence of Salmonella spp. and Listeria monocytogenes at different sampling points during the manufacturing process of fuet, a type of traditional fermented sausage, at 10 small-scale Spanish factories. The presence of both pathogens was studied in the raw materials (19 casings and 19 meat batters), the final products (19 fermented sausages), and the factory equipment (12 mincing, 12 mixing, and 19 stuffing machines, 19 cutting tables, 11 knives, and 12 cold rooms) by using classical microbiological techniques and real-time PCR. Salmonella was not detected in the equipment analyzed or in the final products, but it was detected in the raw materials (23.7% of samples). L. monocytogenes showed higher incidence than Salmonella and was detected in the equipment (11.8% of samples), the raw materials (28.9%), and the final products (15.8%), confirming its ubiquity throughout the manufacturing process of fermented sausages. Five factories were further investigated to study the changes in the distribution of pathogens in the fuet production process over a period of either 2 or 3 years. There was considerable variation in the incidence of both pathogens at different sampling periods, and there was no relation between seasonal variations or geographic location of the factories. The presence of bacterial foodborne pathogens in ing foodborne listeriosis that can result in meningitis, ready-to-eat (RTE) meat products is one of the major encephalitis, septicemia, and abortion in pregnant women. concerns for the meat industry and consumers today. The knowledge of microbial ecosystems of the food Among the known foodborne pathogens, Salmonella spp. environment is crucial for the improvement of hygiene and Listeria monocytogenes are the most relevant in the control systems in small-scale meat factories (20). Fuet is a production of RTE meat products. Salmonella is the second traditional fermented sausage from Catalonia (an autonomous most commonly reported cause of zoonotic disease in region located in the northeast of Spain) made from a batter of humans in the European Union (8) and the cause of recent pork meat, pork fat, and salt along with sugar, nitrate and/or foodborne outbreaks in Europe and the United States (1, 8). nitrite, and pepper. This kind of fermented sausage is Salmonella spp. frequently colonize the gastrointestinal tract characterized by a mild fermentation process (final pH . of many species of livestock and constitute a potential 5.3) that relies on the natural contamination of the raw source of contamination in meat and meat products. L. materials (2) by useful microorganisms (lactic acid bacteria, monocytogenes, although less frequent, is the causative gram-positive catalase-positive cocci) as well as by spoilage agent of human listeriosis, a serious illness with a high and even pathogenic microorganisms (12, 20). The aim of mortality rate (20 to 30%) among individuals with this study was to evaluate the occurrence of Salmonella spp. suppressed cellular immunity such as the young, the elderly, and L. monocytogenes at different sampling points during the individuals experiencing immunosupression (due to chemo- manufacturing process of fuet at 10 local small-scale factories therapeutic treatment, diabetes, alcoholism, or cardiovascu- and the prevalence of these pathogens during a 3-year period lar disease), and pregnant women (15). This bacterium is at five selected factories. These data will contribute to a better widely distributed through the environment and is often identification of hazards and control points in the production found in food processing plants (11, 17, 20), where it can chain of fermented sausages, which could be the basis of an contaminate food products (22) including RTE meat appropriate risk assessment. products. Consumption of contaminated RTE foods by immunocompromised individuals poses a risk for contract- MATERIALS AND METHODS * Author for correspondence. Tel: (z34) 972-630-052; Fax: (z34) 972- Sampling procedures. Nine different points were analyzed 630-373; E-mail: [email protected]. during fuet production at 10 small-scale factories located in J. Food Prot., Vol. 74, No. 5 PREVALENCE OF FOODBORNE PATHOGENS AT MEAT FACTORIES 813 Catalonia: mincing (n ~ 12), mixing (n ~ 12), and stuffing machines (n ~ 19); cutting tables (n ~ 19); knives (n ~ 11); walls of the cold storage rooms (n ~ 12); meat batters (n ~ 19); casings (n ~ 19); and fermented sausages (n ~ 19) after the fermentation and ripening processes. Twenty-five grams of meat product sample was homogenized in 9 volumes of buffered peptone water (BPW; AES Laboratories, Combourg, France) in a Stomacher Lab-Blender model 400 (Cooke Laboratories, Alexan- dria, VA). For the environmental samples, 25 ml of BPW was added to every piece of sterile gauze (40 by 40 cm; Laboratories Humeau, La Chapelle sur Edre, France) that had been used to sample 500 cm2 of each clean surface, except for knives, for which the entire surface was sampled. Five factories were analyzed two or three times during a 3-year period to study the prevalence of the pathogens studied throughout this time period. Each factory was Downloaded from http://meridian.allenpress.com/jfp/article-pdf/74/5/812/1684169/0362-028x_jfp-10-437.pdf by guest on 25 September 2021 sampled in different seasons (winter and spring or summer) during this 3-year period. L. monocytogenes. Quantification of L. monocytogenes was carried out after 1 h of resuscitation at room temperature. Duplicates of 0.5 ml of meat and environmental sample dilutions were plated onto agar Listeria according to Ottaviani and Agosti (ALOA) plates (AES Laboratoires, Combourg, France) and incubated for 48 h at 37 C. Five presumptive colonies (blue-green u FIGURE 1. Prevalence of Salmonella spp. and L. monocytogenes colonies with an opaque halo) per dish (if available) were in each sample type analyzed (A) and in the different groups of confirmed by PCR. For presence or absence determination, samples (B). samples were preenriched in BPW for 24 h at 37uC. A secondary enrichment was prepared by adding 2 ml of the preenriched samples to 18 ml of Fraser broth (Difco Laboratories, BD, Detroit, the annealing temperature of L. monocytogenes PCR was increased MI) and incubating for 48 h at 37uC. The enriched Fraser broths to 64uC in order to avoid unspecific amplification of background were submitted to a pre-PCR treatment to confirm the presence of flora of the fermented sausages. Both PCR protocols included L. monocytogenes by PCR. internal amplification controls for the indication of PCR inhibitors derived from the sample template. A 20-ml aliquot of each PCR Salmonella spp. Detection of Salmonella was carried out product was subjected to 1.5% (wt/vol) agarose gel electrophoresis following two procedures: a culture method based on ISO containing 0.1 mg/ml ethidium bromide (Sigma, St. Louis, MO) for 6579:2002 and a PCR procedure. For both methods, samples 45 min at 100 V. The amplicon visualization was performed using homogenized in BPW were submitted to 24 h of incubation at a UV transilluminator (Pharmacia LKB, Uppsala, Sweden). 37uC. Semisolid Rappaport-Vassiliadis agar plates (Oxoid, Ba- singstoke, Hampshire, England) were spotted with three drops Culture confirmation. Samples of enrichment broth yielding (approximately 100 ml) of the enriched samples and incubated for positive PCR results for L. monocytogenes but with counts that 20 to 22 h at 42uC. When a halo appeared around the inoculation were under the detection limit were streaked onto ALOA plates. spot, a loopful from the edge of the halo was streaked onto brilliant Colonies with typical L. monocytogenes morphology were green agar (Difco, BD). Colonies with typical morphology were confirmed by PCR. confirmed by PCR. An aliquot of the remaining enriched BPW culture was used for PCR detection of Salmonella after the pre- RESULTS AND DISCUSSION PCR treatment. L. monocytogenes was the most commonly isolated Pre-PCR treatment. Enriched samples (1.5 ml) in BPW or pathogen both on work surfaces and in meat products Fraser broth were centrifuged for 5 min at 14,000 | g. The (16.9% of positive samples). Raw material (meat batters and supernatants were carefully discarded; the pellets were resuspended casings) showed more positive samples (28.9%) than the in 300 mlof6% Chelex-100 chelating ion exchange resin (BioRad, equipment (11.8%) and the fermented sausages analyzed Munich, Germany), incubated at 56uC for 20 min, boiled for (15.8%). Figure 1 shows the occurrence of L. monocyto- 10 min, and immediately incubated on ice. After a centrifugation genes in the different samples studied. This pathogen could step at 14,000 | g for 5 min, 50 ml of the supernatants was be detected in all sample types except for the mixing transferred to a new tube and stored at 220uC. Five microliters machines. A high incidence rate of L. monocytogenes was directly used in PCR assays. For colony confirmation, the (47.4%) was noted for meat batter. The presence of L. presumptive pathogen colonies were resuspended in 30 mlof1mM monocytogenes in the pork meat industry has been shown Tris-HCl (pH 8.0) and 0.01 mM EDTA, incubated at 95 C for u previously (17, 21).
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