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Keywords:

  • Listeria monocytogenes;
  • beef slaughterhouse;
  • epidemiological characterization;
  • pork processing;
  • REA –PFGE

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Sampling
  6. Isolation and identification of L. monocytogenes
  7. DNA analysis by PFGE
  8. Results
  9. Discussion
  10. References

Aims: The prevalence, level of contamination and epidemiological profile of Listeria monocytogenes were investigated in two meat-producing plants during a 20-month period.

Methods and Results: Sampling for L. monocytogenes was carried out in a cattle slaughterhouse (n = 72) and a swine meat-processing plant (n = 68) during a 20-month period. Swabs and food samples were analysed with the most probable number (MPN) technique for L. monocytogenes and the isolated strains were characterized by AscI-restriction analysis pulsed-field gel electrophoresis (REA–PFGE). Contamination of meat and meat products was always at low level (below 50 MPN per gram). The seven L. monocytogenes positive samples isolated in the bovine slaughterhouse yielded strains with the same REA–PFGE profile. However, the seven strains isolated in the swine meat processing plant showed six different profiles. Two of them showed indistinguishable profiles with L. monocytogenes strains collected from other meat processing facilities located in the same area.

Significance and Impact of the Study: The genotyping method is a valuable tool to investigate contamination sources. The study of REA–PFGE profiles indicated that environmental contamination was probably responsible for the persistence of over 16 months of one strain of L. monocytogenes in the cattle slaughterhouse. Several meat suppliers could be responsible for the contamination in the pig meat processing facility, and this is confirmed by the finding of some identical strain in other meat processing facilities located in the same area.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Sampling
  6. Isolation and identification of L. monocytogenes
  7. DNA analysis by PFGE
  8. Results
  9. Discussion
  10. References

Occurrence of Listeria monocytogenes within slaughterhouses and meat processing facilities has been associated with environmental colonization, because of its ability to adapt and survive even on ‘clean’ equipment and rooms (Lundén et al. 2000). However, L. monocytogenes can enter through infected animals and raw meat or intermediate products processed by suppliers (Boerlin and Piffaretti 1991; Gill and Jones 1995; Fenlon et al. 1996; Nesbakken et al. 1996; Sammarco et al. 1997). This poses a microbiological risk in products, which requires enforcement of specific control measures. Strategies and control measures could be improved if data on the prevalence and location (hot spot) of Listeria within processing facilities is analysed. Characterization of isolated strains is an essential tool in tracing contamination sources and genotyping of L. monocytogenes can be achieved by using pulsed-field gel electrophoresis (PFGE) (Destro et al. 1996; Autio et al. 1999; Giovannacci et al. 1999; Miettinen et al. 1999; Autio et al. 2000; Chasseignaux et al. 2001). The aim of this study was to determine the prevalence and the epidemiological profile of L. monocytogenes in two EC-approved meat-producing plants to find the correlation between environmental contamination and occurrence of this food-borne pathogen in the final products.

Sampling

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Sampling
  6. Isolation and identification of L. monocytogenes
  7. DNA analysis by PFGE
  8. Results
  9. Discussion
  10. References

The samples were collected from two meat processing facilities, a cattle slaughterhouse (plant A) and a pork processing plant (plant B) from September 2000 to April 2002 (Tables 1 and 2). Both plants were located in the same producing area in north-east Italy. Environment (fridge rooms and drains), equipment (tables, saws, kneaders and mincers) and utensils (knives) were sampled by swabbing a 20 cm2 area (46 from plant A and 51 from plant B). The swabs were taken both during processing and/or before starting the process, after the cleaning operations. Swabs (n = 14) were also taken to detect Listeria on cattle carcass (i.e. from shoulder and abdominal incision), swine large meat cuts (n = 6) and from batches of minced meat (n = 4). Swabbing was performed using several sterile cotton swabs moistened with NaCl 0·85%. After sampling, the swabs were soaked in 2 ml of saline solution. Meat samples (12 from plant A and seven from plant B) were taken at different phases of the processing. They included raw meat cuts, minced meat prior to salting and unseasoned and uncooked sausages that had to be cooked before eating. Meat samples and swabs were stored at 0–8°C and were examined within 72 h of arrival.

Table 1.  Level, prevalence and characterization of Listeria monocytogenes contamination in raw meat, carcasses, environmental and equipment samples in a bovine slaughterhouse plant A
SourceNo. of L. monocytogenes positive/no. of samplesContamination level (MPN)AscI REDP
During processAfter cleaning
  1. *During process.

  2. †Cleaned sites.

  3. MPN: most probable number; REDP, restriction enzyme digestion profiles.

Meat
 Excised samples3/12 3·6; 3·6 and 43 g−11, 1, 1
 Carcass swabs1/14 0.33 cm−21
Environment and equipment
 Knives2/61/50·33* 9·2* and 0·46† cm−21, 1, 1
 Fridge room0/40/4  
 Tables0/40/5  
 Saws0/40/4  
 Floor drains0/5   
 Others0/20/3  
Total6/511/21  
Table 2.  Level, prevalence and characterization of Listeria monocytogenes contamination in meat products, environmental and equipment samples in a pork processing plant B
SourceNo. of L. monocytogenes positive/no. of samplesContamination level (MPN)AscI REDP
During processAfter cleaning
  1. MPN: most probable number; REDP, restriction enzyme digestion profiles.

Meat products
 Swine raw meat1/2  3·6 g−1 5
 Swine raw meat swabs0/6   
 Minced meat for sausage1/1  9·2 g−1 2
 Swine minced meat swabs0/4   
 Cotechino2/2  3·6 and 21 g−1 4, 6
 Country-style sausage1/2 23 g−1 4
Environment and equipment in the sausage processing room
 Kneader1/30/6 0·184 cm−2 7
 Mincer0/21/6 0·184 cm−211
 Stuffer0/30/8  
 Tables0/30/6  
 Others0/70/7  
Total6/351/33  

Isolation and identification of L. monocytogenes

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Sampling
  6. Isolation and identification of L. monocytogenes
  7. DNA analysis by PFGE
  8. Results
  9. Discussion
  10. References

Samples were examined using the most probable number (MPN) technique according to the Italian official method (O.M. Sanità 1993). Ten grams of each food sample was homogenized in a stomacher with 90 ml of buffered peptone water (Oxoid, Basingstoke, UK) for 4 min and two 10-fold serial dilutions in 0·1% tryptone (Oxoid) were made. The saline solution (2 ml) including the swabs was also used to prepare 10-fold serial dilutions. One millilitre of each dilution was aseptically added to a series of three tubes containing 9 ml of Fraser enrichment broth (Oxoid). Tubes were incubated at 32°C for 48 h. The volumes (10 μl each) of the broth were plated on Oxford agar (Oxoid) and incubated at 37°C for 48 h. Five suspected colonies from Oxford agar were picked and streaked out on Tryptone soya agar (Oxoid) plus 0·6% yeast extract (YE; Difco Becton Dickinson, Sparks, MD, USA) and incubated at 37°C for 24 h. Suspected colonies were streaked out on blood agar [blood agar base (Difco), 5% sheep blood] and incubated at 37°C for 24 h. β-haemolytic colonies were further identified as L. monocytogenes by testing motility at 25°C, catalase reaction, carbohydrates acid reaction of rhamnose 0·5% (Sigma), xylose 0·5% (Sigma) and mannitol 1% (Sigma) in purple broth base (Difco). Two isolates for each positive sample were stored at −20°C in Tryptone soya broth (Oxoid) + 20% glycerol (Sigma).

DNA analysis by PFGE

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Sampling
  6. Isolation and identification of L. monocytogenes
  7. DNA analysis by PFGE
  8. Results
  9. Discussion
  10. References

Cultures for DNA isolation were grown overnight in brain heart infusion (BHI) broth (Oxoid) at 37°C. DNA was isolated and digested with AscI restriction enzyme (New England Biolabs, Beverly, MA, USA) in agarose plugs and characterized by PFGE as described by Autio et al. (1999). Isolates which displayed indistinguishable restriction enzyme digestion profiles (REDP) were placed in the same group, whereas isolates that differed by one or more bands were considered different (Miettinen et al. 1999). Patterns were also compared with those of other six strains isolated from different meat processing facilities located in the same area (Fig. 1).

image

Figure 1. AscI restriction enzyme digestion profile (REDP) of Listeria monocytogenes strains. Lanes: M, low range PFGE marker; 1, REDP-4; 2, REDP-9*; 3, REDP-10*; 4, 6, 8 and 9, REDP-1; 5, REDP-4; 7, REDP-11; 10, REDP-8*. *REDPs of L. monocytogenes strains belong to our collection

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Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Sampling
  6. Isolation and identification of L. monocytogenes
  7. DNA analysis by PFGE
  8. Results
  9. Discussion
  10. References

Listeria monocytogenes was detected in four of the 26 cattle carcasses sampled in plant A with contamination level between 3·6 and 43 MPN per gram in meat samples and 0·33 cm−2 on carcass swabs. Presence of L. monocytogenes was also detected in three of the 11 knife swabs, including one that was presumed to be ‘clean’, but was not in the fridge room, drain, tables and saws. The level of contamination on knives was between 0·33 and 9·2 MPN per cm2 (Table 1).

In the pork processing facility (plant B), L. monocytogenes was isolated from uncooked end products, partially processed (salted minced meat mixes with nitrites and spices), and swine raw meat from suppliers. Finished products like raw sausages (country style and cotechino) were more frequently contaminated. Positive samples showed contamination level between 3·6 and 23 MPN per gram. Contamination by L. monocytogenes was found on kneader and on mincer, but not on stuffers, tables and other surfaces in contact with meat. It was also observed that the contaminated mincer was presumed to be ‘clean’. Contamination level was 0·184 MPN cm−2 in both the positive swabs (Table 2).

The PFGE analysis showed 11 AscI pulsotypes characterized by 9–12 fragments between 23 and 486·5 kb (Fig. 1). All the seven L. monocytogenes strains collected in plant A showed the same pulsotype (REDP-1), while the seven strains collected in plant B had six different profiles. Two strains with the same profile (REDP-4) were isolated from sausages at different times. Two pulsotypes (REDP-2 and REDP-7) were also found among the strains isolated from other meat processing facilities located in the same area.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Sampling
  6. Isolation and identification of L. monocytogenes
  7. DNA analysis by PFGE
  8. Results
  9. Discussion
  10. References

Listeria monocytogenes was isolated with relatively high frequency in meat products, but concentration was low, below the unacceptable limits set in Italy (110 cells per gram) and other European countries (100 cells per gram) for products intended to be consumed after cooking. However, the occurrence of this pathogen in food processing facilities must be investigated. Many authors have proved the widespread occurrence of this pathogen in raw meat (e.g. McLauchlin 1996, Nørrung et al. 1999). Some authors emphasized on environmental conditions for the spread of L. monocytogenes contamination (Nesbakken et al. 1996), others have focused on the equipments used as points of L. monocytogenes cross-contamination (Gill and Jones 1995; Autio et al. 2000). In both plants, L. monocytogenes was detected in equipment and tools occasionally at a very low level. It was isolated from kneader and mincers, used in the pig meat processing facility, and on knives, used in the beef slaughterhouse. However, it was never found in fridge rooms and in floor drains. It was found that L. monocytogenes is able to persist after the cleaning procedures. Indeed, it was detected on a mincer, supposed to be clean, and on a knife cleaned and left in the sterilizing tank. In plant A, all seven strains isolated from different samples (three excised meat samples, one carcass swab and three knife swabs) showed the same AscI restriction profile (REDP-1). The isolates were detected from samples taken at five different times, over 16 months. Twice cross-contamination through the knives used for de-hiding operations was pointed out, because in two cases isolates were collected at the same time from knife and carcass swabs or from the forelegs. Although a common source cannot be identified, these results showed that a unique strain could persist. This result is consistent with studies of other researchers who indicated that slaughtering equipment and environment are probable sites of cross-contamination even for several months (Giovannacci et al. 1999; Miettinen et al. 1999; Autio et al. 2002).

Differently, seven strains with six different AscI restriction profiles were isolated in the pork processing plant. Only strain REDP-4 was detected twice. The results suggest that raw materials, which are supplied by several producers, may play a major role as a source of contamination of the plant. It cannot be neglected that the cleaning operations were not effective in eliminating L. monocytogenes, as shown by an isolate recovered from a cleaned mincer. Among the isolates collected at this plant, two showed a restriction enzyme digestion profile (REDP-2 and REDP-7) identical to other strains of our collection recovered at that time in different plants and retail shop from the same region. This finding may suggest these genotypes could be widespread within a geographical region or food industry environment and should be further investigated.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Sampling
  6. Isolation and identification of L. monocytogenes
  7. DNA analysis by PFGE
  8. Results
  9. Discussion
  10. References
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  • Autio, T., Lundén, J., Fredriksson-Ahomaa, M., Björkroth, J., Sjöberg, A.-M. and Korkeala, H. (2002) Similar Listeria monocytogenes pulsotypes detected in several foods originating from different sources. International Journal of Food Microbiology 77, 8390.
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