Transfer of foodborne pathogenic bacteria to non-inoculated beef fillets through meat mincing machine

Meat Science 90 (2012) 865–869

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Transfer of foodborne pathogenic bacteria to non-inoculated beef fillets through meat mincing machine O.S. Papadopoulou a, b, N.G. Chorianopoulos a,⁎, E.N. Gkana a, A.V. Grounta a, K.P. Koutsoumanis c, G.-J.E. Nychas a a b c

Laboratory of Microbiology and Biotechnology of Foods, Department of Food Science and Technology, Agricultural University of Athens, Iera Odos 75, Athens 11855, Greece National Agricultural Research Foundation, Institute of Technology of Agricultural Products, Sofokli Venizelou 1, Lycovrissi 14123, Greece Laboratory of Food Microbiology and Hygiene, Department of Food Science and Technology, Faculty of Agriculture, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece

a r t i c l e

i n f o

Article history: Received 22 February 2011 Received in revised form 8 August 2011 Accepted 2 November 2011 Keywords: Beef fillets Cross-contamination Meat mincing machine Listeria monocytogenes Salmonella ser. Typhimurium Escherichia coli O157:H7

a b s t r a c t The aim of this study was to evaluate the transfer of pathogens population to non-inoculated beef fillets through meat mincing machine. In this regard, cocktails of mixed strain cultures of each Listeria monocytogenes, Salmonella enterica ser. Typhimurium and Escherichia coli O157:H7 were used for the inoculation of beef fillets. Three different initial inoculum sizes (3, 5, or 7 log CFU/g) were tested. The inoculated beef fillets passed through meat mincing machine and then, six non-inoculated beef fillets passed in sequence through the same mincing machine without sanitation. The population of each pathogen was measured. It was evident that, all noninoculated beef fillets were contaminated through mincing with all pathogens, regardless the inoculum levels used. This observation can be used to cover knowledge gaps in risk assessments since indicates the potential of pathogen contamination and provides significant insights for the risk estimation related to crosscontamination, aiming thus to food safety enhancement. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Cross-contamination contributes to foodborne illnesses due to the potential transfer of pathogens to food products. The common routes for cross-contamination are summarized to the indirect contact from air, the direct contact from hands to foods and the direct contact from equipment and utensils to food (den Aantrekker, Boom, Zwietering, & van Schothorst, 2003). Nowadays, ready-to-eat food products need more attention, since cross-contamination during handling at food processing points and retail has been recognized as a causative agent of human illnesses (Aarnisalo, Sheen, Raaska, & Tamplin, 2007; Perez-Rodrıguez et al., 2007, 2010; Sheen & Hwang, 2010; Vorst, Todd, & Ryser, 2006). Furthermore, bad hygiene practices and improper food handling might result to cross-contamination in domestic kitchens too, leading some times to infections, in cases of contamination with foodborne pathogens. Redmond and Griffith (2003) reported that foodborne diseases are three times more frequent from private kitchens than those occurring from food serving points. Pathogenic bacteria such as Escherichia coli, Salmonella enterica, and Listeria monocytogenes are highly associated with outbreaks

⁎ Corresponding author at: National Agricultural Research Foundation, Institute of Veterinary Research, Neapoleos 25, Agia Paraskevi, 15310, Attiki, Greece. Tel.: + 30 210 529 4939; fax: + 30 210 529 4938. E-mail address: [email protected] (N.G. Chorianopoulos). 0309-1740/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2011.11.008

related to meat consumption, leading to human diseases and deaths worldwide (Rhoades, Duffy, & Koutsoumanis, 2009). According to data collected from European countries for zoonotic diseases in humans (EFSA, 2006), Salm. ser. Typhimurium and Salm. ser. Enteritidis were the most frequent serovars related to human illnesses, whereas Salm. ser. Typhimurium was more often associated with the consumption of contaminated poultry, pork and bovine meat. For the year 2005, 177963 outbreaks of salmonellosis reported for 26 European countries. According to Global Salmonella Surveillance Progress Report (WHO, 2005), Salmonella ser. Typhimurium was found to be the second most common human serotype emerging in Europe. Likewise, verotoxigenic E. coli O157 has been linked with severe outbreaks and is broadly recognized as an important and threatening pathogen since 1980's (Davis & Brogan, 1995; Duffy, Cummins, Nally, O'Brien, & Butler, 2006). 3314 outbreaks were reported for E. coli O157 on 2005 (EFSA, 2006) and is one of the main threatening bacteria of beef, that could be potentially transferred from gut or hide during slaughtering (Duffy et al., 2006). Additionally, E. coli O157 can survive for hours or days on hands, cloths or utensils, leading to a potential cross-contamination if bad hygiene practices are followed (Chen, Jackson, Chea, & Schaffner, 2001; Kusumaningrum, Riboldi, Hazeleger, & Beumer, 2003). In the case of listeriosis, recorded data showed 0.3 confirmed cases per 100000 populations (EFSA, 2006). The high mortality and high hospitalization rates caused by L. monocytogenes in tandem with its ability to grow on refrigerated temperatures have increased the interest in this bacterium as a serious post processing contaminant pathogen

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(Perez-Rodrıguez et al., 2010; Rhoades et al., 2009; Vorst et al., 2006). Several studies indicated that L. monocytogenes might contaminate ready-to-eat food products at post processing points (Aarnisalo et al., 2007; Lin et al., 2006; Vorst et al., 2006; Wilks, Michels, & Keevil, 2006). Nowadays, the consumption of minced meat is increasing worldwide, so and the risk for contamination. This risk becomes even more serious because the consumption of raw minced meat (like Tartar) or undercooked meat products is now very frequent (Rhoades et al., 2009). In addition, contaminated minced meat could lead indirectly to cross-contamination because of the transfer of pathogenic bacteria to food through the equipment in household or/and food serving kitchens, such as mincing machine. To our knowledge, there is limited (if any) information related to cross-contamination caused by meat mincing machine. However, knowledge gaps related to the transfer of foodborne pathogens caused by meat mincing machine need to be addressed for risk assessments. The aim of the current study was to evaluate the transfer of Listeria monocytogenes, Salmonella ser. Typhimurium and Escherichia coli O157: H7 from inoculated beef fillets to subsequently non-inoculated fillets, through their passage from mincing machine. Such research provides significant insights in order to estimate the risk related to crosscontamination and thus enhancing food safety.

1000 mg/L of free available chlorine determined by titration with sodium thiosulfate) was applied for 6 min. Then, the parts were washed with detergent and hot water. Subsequently, the parts were rinsed with pure ethanol, burned to let ethanol evaporated, rinsed well with sterile distilled water and let dry. 2.3. Inoculation and treatment of the samples Fresh beef was purchased from the central meat market of Athens and transported under refrigeration to the laboratory within 30 min. A factorial experiment 3X3 (3 inoculum levels by 3 pathogenic bacteria) was designed and performed. The meat was divided in portions of 100 g in a laminar flow cabinet. Beef fillets were inoculated with 3, 5, or 7 log CFU/g population level of each of L. monocytogenes, Salm. ser. Typhimurium or E. coli. In detail, 1 ml from 105, 107, or 109 CFU/ml dilution was added to the fillets (fillet sample) providing a population approximately of 3, 5, or 7 log CFU/g, respectively. 30 min after inoculation, mincing of each inoculated fillet was performed using mincing machine (sample 1). Then, six additional non-inoculated fillets (samples 2 to 7) were minced in sequence using the same mincing machine without sanitation. 2.4. Microbiological analysis

2. Materials and methods 2.1. Inoculum preparation Listeria monocytogenes, Salmonella ser. Typhimurium and Escherichia coli O157:H7 were the tested bacteria of the study. For each bacterium, a cocktail of strains was prepared. More specifically, six strains of L. monocytogenes (NCTC 10527, serotype 4b, isolated from spinal fluid of child with meningitis, Germany, kindly provided by Dr. E. Drosinos; ScottA, serotype 4b, epidemic strain, human isolate, kindly provided by Dr. E. Smid, ATO-DLO, Netherlands; FMCC B-126, isolated from meat, Food Microbiology Culture Collection of Agricultural University of Athens; FMCC 21085, isolated from soft cheese, Food Microbiology Culture Collection of Agricultural University of Athens; FMCC 21350, isolated from ready-to-eat frozen meal — minced meat based, Food Microbiology Culture Collection of Agricultural University of Athens; FMCC 21411, serotype 4b, isolated from conveyor belt of ready-to-eat frozen foods, Food Microbiology Culture Collection of Agricultural University of Athens), three strains of Salm. ser. Typhimurium (DT 193, human isolate-epidemic; 4/74, isolated from calf bowel, kindly provided by Dr. P. Skandamis; JH3298, a mutant derived from Salmonella enterica subsp. enterica serovar Typhimurium strain 4/74, kindly provided by Dr. P. Skandamis;) and three strains of E. coli O157:H7 (NCTC 12079, serotype O157:H7 / Produces Vero cytotoxins VT1 and VT2, isolated from human faeces, kindly provided by Dr. E. Drosinos; NCTC 13125, serotype O157:H7 / Vero cytotoxins negative; NCTC 13127, serotype O157: H7 / Vero cytotoxins negative) were activated from a stock culture stored at −80 °C, subcultured into 10 ml Tryptone Soy Broth (TSB, LabM, LAB004) and incubated overnight at the appropriate temperature for each bacterium (30 °C for L. monocytogenes and 37 °C for E. coli and Salm. ser. Typhimurium). A second subculture was prepared in fresh 10 ml TSB and incubated for 18 h at appropriate temperatures for each strain. Cells were then harvested by centrifugation (5000 g, 10 min, 4 °C, in Multifuge 1S-R, Thermo-Electron Corporation), washed twice with sterile 10 ml Ringer solution (LabM, 100Z), resuspended in Ringer solution and combined to provide a population of approximately 109 CFU/ml.

To estimate the number of viable cells transferred during the mincing process, 25 g of meat was placed in stomacher bag with 50 ml Ringer solution (1:2; Sample weight: Volume Ringer) and homogenized in the Stomacher (Lab Blender 400, Seward Medical, London, UK) for 60 sec at room temperature. In addition, control samples (fillets without pathogen inoculation) were tested to confirm the absence of pathogens in the raw meat. Serial dilutions were prepared with the Ringer solution and duplicate 0.1 or 1 ml samples of the appropriate dilutions were spread or mixed on the following media: Palcam Listeria Agar Base (Biolife, 4016042) for Listeria monocytogenes, incubated at 30 °C for 48 h; Harlequin Tryptone Bile Glycuronide Agar (LabM, HAL 003) for Escherichia coli, incubated at 37 °C for 4 h and then transferred to 44 °C for 18–24 h; Xylose Lysine Deoxycholate Agar (Merck, 1.052.87.0500) for Salmonella spp, incubated at 37 °C for 24 h; Plate Count Agar (Biolife, 4021452) for total viable counts, incubated at 30 °C for 48 h; Pseudomonas Agar Base selective supplement (Biolife, 401961) for Pseudomonas spp., incubated at 25 °C for 48–72 h; Streptomycin Thallous AcetateActidione Agar (Biolife, 402079) for Brochothrix thermosphacta, incubated at 25 °C for 72 h; Violet Red Bile Glucose Agar (Biolife, 402185) for Enterobacteriaceae counts, incubated at 37 °C for 18–24 h; de Man-Rogosa-Sharpe medium with pH adjusted at 5.7 (Biolife, 4017282) for lactic acid bacteria, incubated at 30 °C for 48–72 h. The detection limit of the enumeration method was 0.48 log CFU/g. 2.5. Data analysis Each experiment was replicated two times (two different batches of meat for each pathogen–totally six batches) with three samples analyzed each time for each pathogen (six replicates). A multifactor analysis of variance (ANOVA) was performed to evaluate the effect of different meat samples (fillet, 1st, 2nd, 3rd, 4th, 5th, 6th, and 7th) on pathogen counts and on total bacterial counts. The multiple range test (MRT) was applied to determine which level of each factor was perceptibly different (p b 0.05). In the MRT, the F-distribution (LSD) was used to check equality of variances. All the statistical analyses were done with XLSTAT. ® v2006.06 (Addinsoft, Paris, France).

2.2. Mincing machine 3. Results A domestic meat mincing machine was used. For the disinfection of the machine's parts that were coming in contact with the meat, chlorine at concentration of 1000 ppm (sodium hypochlorite at

Beef fillets were inoculated with ca 3, 5, or 7 log CFU/g for each pathogen and passed through meat mincing machine. Then, six non-

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inoculated beef fillets passed in sequence through the mincing machine and the potential transfer of pathogens was evaluated in the minced product. Fillet samples (fillet sample, Table 1) before mincing were analyzed too. Results showed that all non-inoculated beef fillets were contaminated with pathogenic bacteria during the procedure of mincing for all inoculum levels and pathogens tested (Table 1). From Table 1 is evident that, the transfer of the pathogens to beef samples was highest for the first inoculated sample and was decreased progressively with the passage of the sequential non-inoculated meat samples through the mincing machine. Higher pathogen population level at the fillet sample led to higher transfer of bacteria to non-inoculated samples (Table 1). The results regarding the transfer of contamination showed a similar trend of approximately 2-log decrease for the population of L. monocytogenes and Salm. ser Typhimurium from the 1st to the 7th sample for all inoculum levels (Table 1). More specifically for L. monocytogenes at the high inoculum level, the population of the first minced sample (sample 1, Table 1) was 7.12 log CFU/g whereas the population for the last sample (sample 7, Table 1) was 5.07 log CFU/g. The same trend in log-decrease was observed for the medium level of inoculation for the aforementioned pathogen, where the first and the last minced sample was 5.47 log CFU/g and 3.25 log CFU/g, respectively (Table 1). Finally, for the low inoculum level, the population of L. monocytogenes was 3.23 log CFU/g for the first minced sample, while for the last sample was 1.17 log CFU/g (Table 1). Similar trends, regarding all inoculation levels were observed for Salm. ser. Typhimulium (Table 1). In case of E. coli O157:H7, similar trends were observed for high and medium inoculum level. However, for the low level of inoculation, a slight log-decrease from the first till the last sample was observed (Table 1). In particular, the population of the first minced sample was 2.89 log CFU/g while the last minced sample was found to be 1.68 log CFU/g (Table 1), whereas 1st to 5th sample populations were similar (p > 0.05). In terms of total viable counts (TVC), TVCs were a reliable parameter in the case of the low pathogens inoculation only (Table 1), since this level was the only one clearly below the actual meat microbial load,

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ca. 4.10 log CFU/g (Table 2). In both medium and high inoculum levels, TVC actually reflected the pathogens populations (Table 1). Furthermore, the batches used for Salmonella experiment were of slightly higher microbial load, compared to the microbial load of the batches used for E. coli and L. monocytogenes experiments (Table 1). Moreover, counts were 3.59 log CFU/g, 3.25 log CFU/g, 3.26 log CFU/g, 2.81 log CFU/g and 2.94 log CFU/g for Pseudomonas spp., Brochothrix thermosphacta, yeasts and moulds, lactic acid bacteria and Enterobacteriaceae, respectively (Table 2). No pathogens detected for all batches, for no inoculation (Table 2). 4. Discussion Safety is the main issue for the consumers in the EU, and as such a holistic approach e.g. “farm to fork” has been designed for the meat industry. In particular cross-contamination of bacterial pathogens is considered as a major contributing factor for sporadic and epidemic foodborne illnesses (Chen et al., 2001). Food equipments have been already recognized as important vehicles of contamination throughout the food (meat) chain (Gounadaki, Skandamis, Drosinos, & Nychas, 2008; Kennedy et al., 2005; Perez-Rodrıguez et al., 2010; Vorst et al., 2006). Several studies have been conducted with the assessment of various pathogenic bacteria transfers (L. monocytogenes, Salm. enterica, E. coli O157:H7, Staph. aureus) between different processing equipments or utensils. Published results have showed that the tested equipments might contaminate sequential food products during handling, after the initial contamination with bacteria at various inoculum levels (Aarnisalo et al., 2007; Perez-Rodrıguez et al., 2010; Vorst et al., 2006). In the current study, the results confirmed the aforementioned concern, since pathogens were transferred to all the non-inoculated samples for all inoculum levels during mincing of meat. Nowadays, it is well established that many foodborne outbreaks start from domestic kitchens (Redmond & Griffith, 2003). Consumers usually follow unsafe practices during the handling of fresh products, such as the use of cutting boards or knifes after handling raw meat or

Table 1 Transfer (log CFU/g) of E. coli O157:H7, Salm. ser. Typhimurium or L. monocytogenes and Total Viable Counts from a surface inoculated beef fillet (fillet Sample) after the mincing (Sample 1) to 6 non-inoculated fillets through their sequential passage from the meat mincing machine (Samples 2–7), for three different levels of pathogens inoculation (107-high, 105-medium or 103 CFU/g-low). Data reported are means ± standard deviation of two samples (two different batches of meat) with three replications each. Within a group of the different meat samples for the same pathogen and inoculum level, means with different letter differ significantly (p b 0.05). Population (log CFU/g) Inoculum level (CFU/g)

Sample

High (107)

Fillet 1 2 3 4 5 6 7 Fillet 1 2 3 4 5 6 7 Fillet 1 2 3 4 5 6 7

Medium (105)

Low (103)

E. coli

L. monocytogenes

Salm. ser. Typhimurium

Pathogen

TVC

Pathogen

TVC

Pathogen

TVC

7.05 ± 0.11 a 6.54 ± 0.12 b 6.17 ± 0.07c 5.49 ± 0.13 d 5.15 ± 0.16 e 4.53 ± 0.15 f 4.41 ± 0.30 f 4.03 ± 0.27 g 4.67 ± 0.04 a 4.75 ± 0.40 a 3.98 ± 0.58 ab 3.23 ± 0.30 bc 2.91 ± 0.55 bc 3.08 ± 0.26 c 2.45 ± 0.81 c 2.30 ± 0.73 c 2.86 ± 0.13 a 2.89 ± 0.05 ab 2.39 ± 0.42 abc 2.42 ± 0.28 abc 2.20 ± 0.35 abc 2.65 ± 0.27 bc 2.42 ± 0.36 c 1.68 ± 0.14 d

6.81 ± 0.05 a 6.63 ± 0.15 a 6.31 ± 0.04 b 5.61 ± 0.02 c 5.15 ± 0.02 d 4.75 ± 0.01 d 4.73 ± 0.21 e 4.25 ± 0.10 f 4.74 ± 0.05 a 5.14 ± 0.26 ab 4.34 ± 0.27 bc 4.09 ± 0.30 c 4.06 ± 0.21 c 4.01 ± 0.18 c 3.96 ± 0.25 c 4.04 ± 0.22 c 4.11 ± 0.34 a 4.02 ± 0.32 a 3.98 ± 0.30 a 4.03 ± 0.38 a 4.01 ± 0.42 a 3.96 ± 0.35 a 4.06 ± 0.34 a 3.91 ± 0.39 a

7.33 ± 0.00 a 7.12 ± 0.22 a 6.75 ± 0.09 b 6.24 ± 0.28 c 5.76 ± 0.16 d 5.60 ± 0.21 d 5.14 ± 0.10 e 5.07 ± 0.09 e 5.34 ± 0.05 a 5.47 ± 0.19 a 4.94 ± 0.20 b 4.38 ± 0.22 c 3.65 ± 0.08 d 3.47 ± 0.16 de 3.47 ± 0.19 de 3.25 ± 0.14 e 3.50 ± 0.20 a 3.23 ± 0.11 ab 3.09 ± 0.10 b 2.60 ± 0.12 c 1.96 ± 0.17 d 1.70 ± 0.13 de 1.48 ± 0.04 e 1.17 ± 0.27 f

6.81 ± 0.05 a 6.63 ± 0.15 a 6.60 ± 0.11 ab 6.28 ± 0.22 b 5.67 ± 0.47 c 5.29 ± 0.15 c 5.15 ± 0.29 cd 5.30 ± 0.22 d 5.44 ± 0.01 a 5.49 ± 0.13 a 4.47 ± 0.18 b 3.59 ± 0.16 c 3.51 ± 0.18 c 3.55 ± 0.14 c 3.62 ± 0.20 c 3.60 ± 0.15 c 4.18 ± 0.19 a 3.51 ± 0.14 b 3.49 ± 0.12 b 3.55 ± 0.16 b 3.58 ± 0.18 b 3.55 ± 0.20 b 3.59 ± 0.19 b 3.53 ± 0.17 b

6.73 ± 0.02 a 6.50 ± 0.03 ab 5.99 ± 0.24 b 5.22 ± 0.12 c 4.82 ± 0.12 cd 4.78 ± 0.78 cd 4.54 ± 0.41 d 4.32 ± 0.28 d 4.86 ± 0.17 a 4.45 ± 0.21 ab 4.18 ± 0.17 b 3.43 ± 0.15 c 3.47 ± 0.15 cd 3.05 ± 0.22 d 2.86 ± 0.16 d 2.69 ± 0.21 d 2.46 ± 0.17 a 2.36 ± 0.07 a 2.16 ± 0.01 a 1.43 ± 0.22 b 0.96 ± 0.44 c 0.84 ± 0.39 cd 0.58 ± 0.17 cd 0.48 ± 0.00 d

6.81 ± 0.05 a 6.63 ± 0.11 ab 6.13 ± 0.09 b 5.34 ± 0.28 c 4.58 ± 0.16 cd 4.89 ± 0.75 d 4.58 ± 0.43 d 4.30 ± 0.18 d 4.81 ± 0.09 a 4.64 ± 0.20 ab 4.24 ± 0.05 b 4.48 ± 0.22 b 4.58 ± 0.20 b 4.52 ± 0.17 b 4.55 ± 0.24 b 4.51 ± 0.25 b 4.01 ± 0.40 a 4.77 ± 0.18 b 4.70 ± 0.22 b 4.71 ± 0.25 b 4.68 ± 0.28 b 4.75 ± 0.20 b 4.67 ± 0.19 b 4.73 ± 0.24 b

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Table 2 Microbial counts (log CFU/g) of meat for no inoculation. Data reported are means±standard deviation of six samples (six different batches of meat) with three replications each. Microbial counts

Log CFU/g

Total viable counts Pseudomonas spp. Brochothrix thermosphacta Yeasts and moulds Lactic acid bacteria Enterobacteriaceae E. coli L. monocytogenes Salm. ser. Typhimurium

4.10 ± 0.63 3.59 ± 0.59 3.59 ± 0.59 3.25 ± 0.42 3.26 ± 0.32 2.81 ± 0.15 2.94 ± 0.28 Not detected Not detected Not detected

poultry, prior to any sanitation/disinfection treatment (Ravishankar, Zhu, & Jaroni, 2010). Moreover, consumer's lifestyle has changed and once-a-week shopping became more common (Koutsoumanis, Stamatiou, Drosinos, & Nychas, 2008), leading to the refrigeration of raw minced meat for hours or days before consumption. Following this practice, the growth of psychrotrophic pathogens such as L. monocytogenes becomes a serious concern, since the contamination during processing was feasible, regarding the findings of this study. The current research evaluated the potential transfer of foodborne pathogens through meat mincing machine. It is evident from the results that, the inoculated fillets contaminated sequential pathogens-free meat samples through mincing machine, for all inoculum levels (Table 1). Cells of bacteria might loosely be attached on the contaminated fillet and easily transferred to the mincing machine. Therein, bacteria could be attached to the spare parts of the mincing machine (mincer barrel, worm, blade, mincing plate, screw ring and feed tray) or to the machine (the feed funnel and worm housing sleeve) and then passed to pathogens-free fillets. In addition, pieces of meat tended to be entrapped in the machine and transferred to the next portion of meat, which was being minced. A factor that affects the cross-contamination levels during mincing is the weight of meat which is minced in the machine. Usually, a portion of 0.5 to 1 kg of meat is a normal quantity to be minced. In the current study, fillets of 100 g were used for the purpose of the research. As aforementioned, pieces of meat tended to be entrapped mainly in the mincer barrel, during mincing. Using lower weight pieces of meat (such as the 100 g beef fillets of the study) for mincing, more meat in analog is entrapped in the mincer barrel than that of using higher weight pieces. Besides, when turbid cell suspensions are used for inoculations of stainless steel surfaces, the cells attached onto them are ca. 2 logs lower. In this respect for the current study, following mincing in the machine, levels of 1st (inoculated) and 2nd (non-inoculated) fillet did not differ that much (Table 1). This observation strengthens the hypothesis that pieces of meat tended to be entrapped in the machine and transferred to the next portion of meat. Consequently, it is possible that the study overestimated potential cross contamination levels during meat mincing operations, and thus, the associated safety risks. On the other hand, portions of 100 g of beef fillets lead to approximately 100 g beef burgers which represent a normal quantity of a burger in private kitchens. Furthermore, the total weight of the seven beef fillets that were being minced in the study was 700 g which is a normal portion of meat between sequential cleanings of the domestic mincer. Accordingly, these practices represent a real scenario in private kitchens and reinforce the practical usefulness of the current study. Earlier studies have shown that inoculum size, or/and bacterial species might influence the transfer rates of bacteria (Aarnisalo et al., 2007; Montville & Schaffner, 2003; Sheen & Hwang, 2010). In this concept, results regarding MRT showed differences between the studied pathogens and the various inoculum levels. Evidently, it seems that there were no significant differences (p > 0.05) in the logarithmic reduction from the fourth till the seventh sample for all inoculum levels

of Salm. ser. Typhimurium. On the other hand, E. coli O157:H7 and L. monocytogenes expressed different behaviour for the various inoculum levels (Table 1), with E. coli O157:H7 exhibiting a slight log-decrease from the first till the last sample for the low level of inoculation. Although the levels of pathogens such as Salmonella and Listeria monocytogenes in natural contaminated meats are low, in the current study three different inoculums, a low, a medium and a higher one were tested. The low level of inoculation represents a real scenario when contamination occurs. The other two-inoculum levels represent not only the worst-case scenario of contamination when consumers fail to handle and prepare food in a hygienic and safe manner but also provide us with relative information regarding the quantification of cross-contamination of pathogens in higher levels of inoculation. In this regard, the results provided us with information able to be quantified and to be used in risk (exposure) assessment. Similar approaches have been reported in other studies relevant to cross-contamination through processing equipment (Lin et al., 2006; Perez-Rodrıguez et al., 2007; Vorst et al., 2006). In conclusion, successful completion of such researches will lead to the evaluation of the risk of contamination from food processing equipment to meat. Also, such studies provide data useful in risk assessments in order to cover knowledge gaps related to the transfer of foodborne pathogens caused by bad handling, to estimate the impact of contaminated foods in public health and to contribute to the improvement of decision-making processes within the food industry.

Acknowledgements This work was financially supported by the European Union project ProSafeBeef within the 6th Framework Programme (ref. Food-CT2006-36241).

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