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Keywords:

  • butchers;
  • E. coli;
  • E. coli O157;
  • minced meat;
  • Scotland;
  • supermarkets

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aims:  To determine if contamination levels of Escherichia coli O157 and generic E. coli in retail-minced meat products are greater in rural shops compared with urban shops in Grampian, NE Scotland. We also investigated whether meat from supermarkets and meat from local butcher shops had a similar bacteriological quality.

Methods and Results:  Minced beef and minced lamb were tested from November 2004 to August 2006. Escheichia coli O157 was found at low levels in four samples out of 530 tested samples (0·75%). Generic E. coli were present in 11% of the samples tested, of which 67% came from supermarkets. We observed no significant difference in the prevalence of generic E. coli between rural and urban areas.

Conclusions:  Low levels of contamination with E. coli O157 and generic E. coli in retail meat suggest that meat is not a major route of infection in NE Scotland.

Significance and Impact of the Study:  The study does not suggest that the high incidence of E. coli O157 human infection in the rural areas of Grampian is because of meat consumption – this provides further evidence of contact with animals or water being the routes of infection. Hence, risk mitigation should be focussed more on environmental pathways of infection.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Verocytotoxigenic Escherichia coli O157 infections can lead to severe symptoms in humans including haemorrhagic colitis, haemolytic uraemic syndrome and in some cases death, making this pathogen a significant public health concern (Willshaw et al. 2001). The natural reservoir is ruminants, in particular cattle and sheep (Kudva et al. 1997; Omisakin et al. 2003; Ogden et al. 2005). The organism can be found in both the surrounding environment (Ogden et al. 2002) and food derived from these animals (Chapman et al. 2001); therefore, E. coli O157 is now recognized worldwide as a food borne and environmentally transmitted pathogen.

The Grampian region is a largely rural area of NE Scotland with Aberdeen as its capital. It exhibits a higher infection rate compared elsewhere in UK according to the public health records (Locking et al. 2003). The infection rate of the rural population from 1997 to 2002 was, on an average, 1·7 times higher than the infection rate in the urban population (Strachan et al. 2006). Figure 1 compares the infection rate for the two populations from 2001 to 2005. The reasons for the high rural rate are unclear although in the latter study the authors reported a positive correlation between farm animal densities and infection rates, risk assessment models which demonstrate relatively high risk from animal contact and a number of outbreaks associated with the environment. Transmission by direct or indirect contact with faeces from carrier animals has also been reported to be important (Locking et al. 2001). However, meat is still recognized as a vehicle of transmission and information on the microbiological safety of meat sold to the Grampian public is lacking, specifically in the status of E. coli O157. Therefore, it is worth considering that variations in the microbiological status of meat sold in the city and in rural outlets could possibly explain the difference in infection rates.

image

Figure 1.  National Health Survey Grampian reports of Escherichia coli O157 cases, 2001–2005 (rural postcode district: <200 people per km2inline image; urban postcode district: >200 people per km2inline image).

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Minced meat is one of the foods commonly eaten by the public that is most likely to contain E. coli O157. A large number of outbreaks have been linked to consumption of contaminated minced beef, principally in the United States where undercooked hamburgers from fast food chains have been implicated with the infection and subsequent illness of a large number of people (Riley et al. 1983; Bell et al. 1994). In the UK, the first report along similar lines was published in 1994 (Willshaw et al. 1994) when a small outbreak in Wales was linked to consumption of beef burgers. In 1996, a large outbreak in central Scotland that involved a retail butcher affected more than 500 people with 17 deaths and triggered further measures on management, distribution and handling of meat products to ensure public safety (Cowden et al. 2001). The Pennington Report gave recommendations to avoid a recurrence of such incidences (Pennington 1997).

In Scotland, Omisakin et al. (2003) found 7·5% of abattoir cattle shedding E. coli O157, of which 9% were shedding E. coli O157 at concentrations of >104 CFU g−1. In England, 12·9% of cattle and 7·4% of sheep at the abattoir were carriers of E. coli O157 (Chapman et al. 2001). However, during the same survey, they observed a low incidence of contamination with E. coli O157 of beef and sheep carcasses (1·4% and 0·7% respectively). Approximately 1 year later, in a UK-wide study, a lower prevalence in both ruminants was reported at slaughter (Paiba et al. 2002). All these studies suggest that contamination of beef or sheep products can occur in the food chain.

To investigate whether human E. coli O157 infection via retail meat can account for the rural/urban difference in infection rates in NE Scotland, we carried out a retail survey of minced beef and minced lamb products sold in butcher shops and supermarkets in Grampian. Prevalence and concentration of generic E. coli were also determined as an indicator of faecal contamination.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Food samples

Minced beef and minced lamb were purchased from retail butchers and supermarkets throughout Grampian between December 2004 and August 2006. A total of 530 samples were tested. From butcher shops, 100 g of loose mince was purchased, while from supermarkets either small volumes of prepacked mince or 100 g of loose mince were obtained when the supermarket displayed a butcher section. For ease of interpretation, these samples are referred as supermarket samples. During the collection prior to delivery to the laboratory, samples were stored in a cool box at 4°C. Sampling took place during three periods (December/January, May and August) over 2 years to assess a seasonal effect.

Premises sampled

Sampling was structured to collect retail meats from all available butcher shops and supermarkets (one in each chain) in Aberdeen city and rural Grampian. This represents a total of 16 urban butcher shops, five urban supermarkets, 46 rural butcher shops and 17 rural supermarkets. When possible, all premises identified were visited for testing at each sampling round (some butcher shops had ceased trading between sampling rounds).

Isolation of E. coli O157

The organism was isolated using enrichment and immunomagnetic separation (IMS) followed by culture onto selective agar. The mince (25 g) was homogenized with 225 ml of buffered peptone water (Oxoid CM509, Basingstoke, UK) supplemented with vancomycin (8 mg l−1). The samples were then incubated for 6–7 h at 42°C (Ogden et al. 2001). After enrichment, 1 ml was processed by automated IMS (KingFisher, Thermoelectron, Basingstoke, UK). Recovered magnetic beads (0·1 ml) were spread equally on sorbitol MacConkey agar (SMAC; Oxoid CM813) supplemented with cefixime (0·05 mg l−1) and potassium tellurite (2·5 mg l−1) and on Harlequin™ SMAC BCIG agar (LabM, IDG, Bury, UK) supplemented with cefixime and tellurite (as above). Plates were incubated at 37°C for 15–18 h. Escherichia coli O157 presumptive colonies (nonsorbitol fermenting and lacking β-glucuronidase activity) were confirmed by latex agglutination (Oxoid DR620). A maximum of five presumptive colonies from each sample were stored at −20°C for further analyses.

Enumeration of E. coli O157

Enumeration of target organisms in samples that tested positive by IMS was determined by serial dilution. Ten grams of original sample was mixed with 90 ml of PBS. Each dilution was spread (0·1 ml) on duplicate Harlequin™ CT-BCIG agar. Plates were incubated at 37°C for 18 h and target numbers recorded.

Generic E. coli count

Generic counts of E. coli were performed only from the second sampling round. The total number of sample tested was 476. Two dilutions (1 : 10 and 1 : 20) of the sample were plated separately onto Chromocult® TBX agar (Merck, Darmstadt, Germany), and presumptive E. coli colonies (blue) were confirmed by indole production in tryptone water (Oxoid CM87) prior to recording.

Virulence markers

The toxigenic pattern of each E. coli O157 isolate (two to five isolates from each sample) was determined by the presence of the verotoxin genes (vt1, vt2) and the gene coding for one of the proteins involved in the attaching/effacing lesions, intimin (eaeA). The detection of virulence markers was determined by PCR. The amplification products were separated on a 2% agarose gel in 0·5 mol l−1 Tris–borate–EDTA buffer and visualized under UV by using a 100-bp ladder as a standard (Promega G2101, Madison, WI, USA). The expected product sizes were as follows: vt1, 282 bp; vt2, 164 bp; and eaeA, 410 bp (Lin et al. 1993).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

E. coli O157 prevalence

The details of samples tested are listed in Table 1. Escherichia coli O157 was found in three beef samples and in one lamb sample that gave an overall incidence of 0·75%. Less than six E. coli O157 colonies were observed in all four samples post-IMS and no target colonies were isolated after direct plating of a 1 : 10 dilution of samples on CT-BCIG-SMAC. The concentration of E. coli O157 in those meat samples was therefore estimated to be low (<102 CFU g−1).

Table 1.   Microbiological (generic Escherichia coli and Escherichia coli O157) status in minced beef and minced lamb from butcher shops and supermarkets in Grampian (NE Scotland) from November 2004 to August 2006
 No. of samples tested for E. coli O157No. positive for E. coli O157 (%)No. of samples tested for E. coliNo. positive for E. coli* (%)
  1. *Generic E. coli counts are for a total of 476 samples; 54 samples were not tested in the first sampling round.

Beef samples from supermarkets
 Urban440385 (13·16)
 Rural1013 (2·97)10124 (23·76)
Beef samples from butcher shops
 Urban870778 (10·39)
 Rural249021110 (4·74)
Lamb samples from supermarkets
 Urban14014 4 (28·57)
 Rural321 (3·13)32 3 (9·38)
Lamb samples from butcher shops
 Urban101 0
 Rural202 0
Total rural384434637
Total urban146013017
Total5304 (0·75)47654 (11·34)

The first two positive samples were identified within a 1 week interval in January 2006, and the last two positive samples were identified on the same day in August 2006. All four positive samples came from the same supermarket chain but all from different rural towns. No generic E. coli were detected in these samples.

Virulence markers

Table 2 shows the toxigenic pattern of the different E. coli O157 strains isolated during this survey. All samples contained at least one isolate that was potentially pathogenic.

Table 2.   Incidence of vt1, vt2 and eaeA genes in E. coli O157 isolated from retail-minced beef and minced lamb
SampleIsolatevt1vt2eaeA
1 Beef 12/1/06A++
B++
C++
2 Beef 19/1/06A++
B+
3 Lamb 10/8/06A++
B++
C++
D++
E++
4 Beef 10/8/06A++
B++
C++

Prevalence and concentration of generic E. coli

The overall prevalence of generic E. coli in mince was 11·3% (11·0% for beef and 14·3% for lamb). The prevalence of generic E. coli in supermarkets and butcher shops was 19·5% and 6·2% respectively (independent chi-squared test demonstrated greater prevalence in supermarkets at the P < 0·001 level). Frequency and concentration of E. coli in mince samples from supermarkets and butcher shops are compared in Table 3.

Table 3. Escherichia coli concentration in minced beef and minced lamb from supermarkets and butcher shops
E. coli concentration (CFU g−1)E. coli not detected (<102 CFU g−1) 102 to <103 103 to <104 104 to <105Total
Supermarkets1493213185
Butchers2731530291
Total4224743476

The overall prevalence of minced meat samples containing generic E. coli from urban (13·1%) and rural (10·7%) outlets was found not to be significantly different (chi-squared test, P > 0·05).

Table 4 shows the seasonal prevalence and concentration of E. coli in mince. The prevalence in May (8·6% and 7·8%) and December (8·2%) varied little but was higher in August (18·4% and 13·7%).

Table 4.   Seasonality of Escherichia coli prevalence and concentration in minced meat (beef and lamb) from supermarkets and butcher shops
DateE. coli not detected (<102 CFU g−1) 102 to <103 103 to <104 104 to <105Total
May 200585893
August 20057114287
December/January 2006786185
May 200695512103
August 200693141108
Total4224743476

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The four positive mince samples containing E. coli O157 were purchased from rural supermarkets where most of the meat displayed was of UK origin. The positive samples came from England (data supplied by the company) and were part of a batch widely distributed. It is feasible therefore that those contaminated minced meat packs may have been found in other premises from the same supermarket chain including those located in the city of Aberdeen. As it takes 3 days from collection to obtain the results (purchase on day 1, enrichment/IMS on day 2, reading plates on day 3), further samplings of the same batches from those urban premises to prove this assumption were not possible. No butcher shop samples, either rural or urban, were contaminated by E. coli O157. Hence, the data reported in this paper suggest that the meat infection route is unlikely to explain why the incidence is higher in rural dwellers compared with urban inhabitants. However, further sampling over a longer time period is required to eliminate meat as being responsible for the discrepancy in human infection rates in rural/urban populations.

This survey describes an overall low prevalence of E. coli O157 in raw minced beef and lamb sold to the public in Grampian, Scotland (0·75%). These results are in agreement with recent E. coli O157 prevalence surveys in the UK (Chapman et al. 2001, 0·4%; Little and de Louvois 1998, 0·3%); the USA (US Department of Agriculture 2006, 0·19%); Croatia (Uhitil et al. 2001, 0%); France (Vernozy-Rozand et al. 2002, 0·12%) and Italy (Conedera et al. 2004, 0·4%) but lower than those reported from Ireland where a group found 2·8% of contaminated minced beef (Cagney et al. 2004). A second group in Italy found three raw minced beef samples contaminated from the tested 149 samples, all of which came from butcher shops (Stampi et al. 2004). Although results from the present study suggest that the prevalence and concentration of E. coli O157 in meat sold to the Grampian public are low, the organisms isolated were potentially pathogenic to humans. Considering that even a single organism has been estimated to have a 1% chance of causing an infection (Strachan et al. 2001), the presence of any positive sample is of concern.

No significant differences were observed in generic E. coli prevalence from urban and rural sources; however, generic E. coli was found more often and at higher levels in samples from supermarkets (Table 3). While generic E. coli is not harmful itself, it does indicate faecal contamination at slaughter or during processing. These results are lower than that reported by Chapman et al. (2001) where they found a maximum of 23·8% of beef product (mostly mince) and 50·8% of lamb product that had generic E. coli in excess of 104 CFU g−1 in the summer months. In the Italian survey described above, Stampi et al. (2004) found 30·2% of minced beef tested positive for E. coli and the incidence was greater in samples purchased from butcher shops (32·3%) than in those obtained from supermarkets (15·7%). The differences observed might be explained by the methodology, i.e. in the present study, a method with a detection limit of 102 CFU g−1 was considered to be sufficient to prove faecal contamination of meat (by generic E. coli), while the other studies used more sensitive methods that allowed the detection at the <10 CFU g−1 level. However, it is feasible that the differences observed are real and that hygienic practices in the slaughterhouses and butcher shops in the UK might have improved since 1997. This may be applied particularly in Scotland which is influenced by the central Scotland outbreak and the Pennington report recommendations (Pennington 1997).

Two surveys have described no correlation between the presence of E. coli O157 and the concentration of generic E. coli (Chapman et al. 2001; Crowley et al. 2005). Results reported here agreed, but the number of positive samples was too low to be of significance. However, the supermarket chain with E. coli O157-positive samples was frequently found to be contaminated by generic E. coli (30% of total samples bought from this particular chain), and account for the three samples with the highest E. coli count (data not shown) suggesting there could be a systematic hygiene failure in that particular meat processing chain. Purchasing from butchers or supermarkets is a suitable way to analyse meat eaten by the public rather than analysing slaughterhouse products as the latter does not take into account the growth that may occur along the food chain and during processing (often done by the butcher in house, which potentially adds further contamination from, for example, operator, equipment and work areas). A larger number of beef samples were tested (Table 1) because many butchers did not sell minced lamb routinely. We also observed a much lower quantity of minced lamb on display in supermarkets. We assumed that the proportion of both meat types for retail sale and bought for this investigation was a representative of public consumption. Sampling over 2 years allowed for seasonal effects to be tested as infection with E. coli O157 in humans peaks during the summer months (Simmons 1997; Willshaw et al. 2001) and animal studies reported a higher prevalence or concentration during the warmer months (Chapman et al. 2001; Ogden et al. 2004). According to this survey, contamination by generic E. coli occurs throughout the year and contamination of meat with verocytotoxigenic E. coli O157 can occur in the winter as well as in the summer (contaminated samples found in January and August).

The overall low prevalence of E. coli and E. coli O157 demonstrates that precautions and recommendations for meat handling appear to be effective in the majority of meat retailers in Grampian. However, it is important to maintain this level of hygiene to avoid further outbreaks.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors wish to thank the Food Standards Agency Scotland for funding this survey.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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