Evaluation of hygiene practices and microbiological quality of cooked meat products during slicing and handling at retail

Meat Science 86 (2010) 479–485

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Meat Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m e a t s c i

Evaluation of hygiene practices and microbiological quality of cooked meat products during slicing and handling at retail F. Pérez-Rodríguez ⁎, R. Castro, G.D. Posada-Izquierdo, A. Valero, E. Carrasco, R.M. García-Gimeno, G. Zurera Departamento de Bromatología y Tecnología de los Alimentos, Universidad de Córdoba, Campus Rabanales, Edif. Darwin-Anexo, 1014 Córdoba, Spain

a r t i c l e

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Article history: Received 16 January 2010 Received in revised form 24 March 2010 Accepted 20 May 2010 Keywords: Checklist Listeria monocytogenes Slicing machine Escherichia coli Microorganism indicators Hygiene practices Delicatessen establishments Cooked meat Ready-to-eat foods (RTE)

a b s t r a c t Cooked meat ready-to-eat products are recognized to be contaminated during slicing which, in the last years, has been associated with several outbreaks. This work aimed to find out possible relation between the hygiene practice taking place at retail point during slicing of cooked meat products in small and mediumsized establishments (SMEs) and large-sized establishments (LEs) and the microbiological quality of sliced cooked meat products. For that, a checklist was drawn up and filled in based on scoring handling practice during slicing in different establishments in Cordoba (Southern Spain). In addition, sliced cooked meats were analyzed for different microbiological indicators and investigated for the presence of Listeria spp. and Listeria monocytogenes. Results indicated that SMEs showed a more deficient handling practices compared to LEs. In spite of these differences, microbiological counts indicated similar microbiological quality in cooked meat samples for both types of establishments. On the other hand, Listeria monocytogenes and Listeria inocua were isolated from 7.35% (5/68) and 8.82% (6/68) of analyzed samples, respectively. Positive samples for Listeria spp. were found in establishments which showed acceptable hygiene levels, though contamination could be associated to the lack of exclusiveness of slicers at retail points. Moreover, Listeria spp presence could not be statistically linked to any microbiological parameters; however, it was observed that seasonality influenced significantly (P b 0.05) L. monocytogenes presence, being all samples found during warm season (5/5). As a conclusion, results suggested that more effort should be made to adequately educate handlers in food hygiene practices, focused specially on SMEs. © 2010 The American Meat Science Association. Published by Elsevier Ltd. All rights reserved.

1. Introduction Sliced cooked meats are one of the most consumed ready-to-eat food (RTE) products around the world. The two major reasons that fuel the demand for such products are convenience and good acceptance by consumers. During preparation and sale, cooked meat products can be contaminated by pathogenic bacteria causing illness in consumers if infective doses are reached at the time of consumption (EFSA, European Food Safety Authority, 2007a; Norrung & Buncic, 2008). Epidemiological and microbiological studies have identified cross-contamination (during preparation and sale) and subsequent bacterial growth (during storage) as the main causes of RTE contamination and illness. RTE cooked meat products are likely to be contaminated after processing due to recontamination phenomena during handling at retail points. Particularly, slicing machines and cutting utensils are recognized as important vehicles of contamination of cooked meat products both at factory and sale points (PérezRodríguez, van Asselt, García-Gimeno, Zurera & Zwietering, 2007; Vorst, Todd, & Ryser, 2006a; Vorst, Todd, & Ryser, 2006b).

⁎ Corresponding author. Tel.: +34 957 21 20 57; fax: +34 957 21 20 00. E-mail address: [email protected] (F. Pérez-Rodríguez).

Hence, several outbreaks have been associated to contaminated slicing machines (PHAC (Public Health Agency of Canada), 2008). Good-hygienic practices during handling arise as an important means to reduce cross-contamination. Improper food handling practices have been most related to distribution and sale of RTE products (Lues & Van Tonder, 2007; Little & de Louvois 1998) and, especially, to small establishments such as delicatessen and butchery premises. Various studies have suggested that foods handled in small and medium-sized establishments (SMEs) are associated with lower microbiological quality than large-sized establishments (LE) but few data have been published to date (Norrung & Buncic, 2008; Violaris, Bridges & Bridges, 2008). Deficient education programs, financial constraints and insufficient food safety awareness can influence significantly poorer hygiene in SMEs (Violaris et al., 2008). The maintenance of the cold-chain is an effective means in order to prevent growth of microorganisms in food products. However, psychrotrophic pathogens such as Listeria monocytogenes can overcome it due to their capacity to grow at refrigerated storage conditions, reaching infective doses at consumption. L. monocytogenes is a food-borne pathogen which has had an increasing interest in the last decades. It is characterized by its ubiquity since it is present in a wide variety of ecosystems including animals, plants, water, food industries or domestic environments. On the other hand,

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L. monocytogenes has an ability to target high risk population presenting a high death rate (20–30%). According to data released by the European Food Safety Authority (EFSA), a range of 0.1 to 1.1 cases of listeriosis per 100,000 population are presented in EU per year (EFSA (European Food Safety Authority), 2007b) and data showed an upward trend of this disease, which could be related to the increase in immune-compromised populations (Goulet, Hedberg & Monnier, 2008). Several worldwide outbreaks have been recently associated to consumption of contaminated RTE foods that involved L. monocytogenes (Vit et al., 2007; Swaminathan & Gerner-Smidt, 2007; Dawson et al., 2006; Makino et al., 2005, Sim et al., 2002). It can be highlighted the recent outbreak in Canada with 20 death associated to consumption of sliced meat products (PHAC (Public Health Agency of Canada), 2008) or the outbreak reported in Chile, with a total of 91 cases including 5 deaths, due to brie cheese consumption (Instituto de Salud Pública de Chile, ISPC, 2008). Cross-contaminations of foods are plausible causes of the occurrence of L. monocytogenes in RTE products. Because cooked meat products are subjected to handling and slicing, there is a chance of contamination of the products by pathogen, as reported by many authors (Garrido, Vitas & García-Jalón, 2009; Norrung & Buncic, 2008; Gudbjornsdottir et al., 2004; Gombas, Chen, Clavero & Scott, 2003). Although viable cells cannot be detected in final products, it is recognized that L. monocytogenes could grow in cooked meat products, since the designed formulations (levels of pH, water activity, atmosphere and preservatives), together with storage temperatures, do not fully guarantee the microbiological safety regarding this food-borne pathogen. This allows L. monocytogenes reach high doses at domestic refrigeration temperatures, especially after long storage periods (Pérez-Rodríguez, Valero, Todd, Carrasco, Carcía-Gimeno & Zurera, 2007; FDA, Food and Drugs Administration, 2003). The aims of this study were to evaluate the hygienic level in vending areas in Southern Spain in order to know the incidence level of L. monocytogenes in cooked meat slices. In addition, potential relationships between handling practices and microbiological quality of sliced cooked meats were studied by identifying the most relevant hygiene aspects.

2. Material and methods 2.1. Sampling Nineteen retail points in Cordoba (Spain) were selected randomly, 10 small and medium-sized establishments (SMEs) and other 9 large-sized establishments (LEs) which were coded by means of letters from A to R. Establishment classification was based on both, number of workers and establishment dimension. Establishments with less than 10 workers and 500 m2 were considered SME, while establishments with higher number of workers and greater dimensions were classified into LE. Cooked ham and chopped pork were selected in this study to be sampled because they are RTE products often sliced at retail point. Cooked ham is a piece of meat from a hog's hind leg, generally from the middle of the shank bone to the aitch bone which is submitted to a heating treatment, and then packaged. On the other hand, chopped pork is a cooked sausage made of a mixture of meats which is based mainly on pork. A total of 68 samples were collected in two different periods corresponding with warm (June–July) and cool (October–November) seasons in Córdoba. Every establishment was visited at least 3 times. Sampling consisted of purchasing 150 g of cooked ham and chopped pork from each establishment, being sliced at the sale moment. Various slices comprised the sample, which was subsequently kept and transported in a cool box (b2 °C) to laboratory for microbiological analysis.

2.2. Checklist A checklist was designed to assess the hygiene level in the establishments studied. Twelve attributes were selected, all of them especially focused on slicing procedure (hygienic state of the slicing machine, use of gloves, etc.). Before the experiment, interviewers were submitted to a training period in order to score attributes with a uniform criterion. Each time when samples were taken, the checklist was fulfilled by the interviewers. The checklist was constructed based on the inclusion of principles and rules of European Union regulations (No. 852/2004). The checklist was divided into two blocks: handler hygiene level (which comprised 7 attributes) and equipment hygiene level (which consisted of 5 attributes). The hygiene level of surfaces was determined by visually inspecting critical zones where handling occurred. If organic residues and/or stains were presented on surfaces, they were considered poor cleaned or dirty. All checklist attributes and their corresponding scores are showed in Table 1. Score was defined as a discrete and scalar variable which means that no decimals were utilized to attribute scores. The attributes were weighted as a function of the impact on hygiene of cooked meat slices. Those attributes entailing more impact (Nos. 1, 3, 7, 10, 11 and 12) were assigned with a maximum score of 2, while the others scored with a maximum of 1. The total score for an establishment was calculated by adding the scores obtained in all attributes. High total score meant high hygiene level in establishments, being 18 as the maximum score. In accordance to the total score, the establishments were classified in three different hygiene levels, considered as the most suitable for explaining the checklists results. This categorization was done by dividing the total score (18) as follows: poor hygiene was assigned when less than 50% of total punctuation was obtained (from 1 to 8). In case the obtained scores were between 9 and 13, the establishments were catalogued as acceptable, while good hygiene was considered when the score was equal or higher than 14.

2.3. Microbiological analysis, identification and serotype The microbiological methods followed were performed according to ISO (International Standard Organization) and AOAC (Association of Official Analytical Chemists) analytical standard. All culture media used for microbiological analyses belonged to Oxoid (Oxoid Ltd., Hampshire, England). Every sample was blended in a bowl in order to obtain a homogenous sample. Next, 25-gram blended sample was placed into a stomacher bag containing 225 mL of 1% buffered peptone water to be subsequently homogenized in a Stomacher 400 Circulator Table 1 Hygiene attributes evaluated in the checklist and corresponding minimal and maximal scores. Number Items

Score range

Handler hygiene level 1 Proper use of gloves 2 Adequate hand washing facilities 3 Washing hands before handling 4 Use of tongs or small shovel to collect slices 5 Apron clean 6 Hygiene of handler (nails/hair/jewellery/hairmet) 7 Hygienic practices (shaking hands, coughing, sneezing nose, etc.) Equipment hygiene level 8 RTE cooked meat products separated from cured and raw products 9 RTE products maintained in the original package 10 Slicing machine exclusive for RTE cooked meat products 11 Slicing machine and other work surfaces clean 12 Sanitation schedule

0–10 0/1/2 0/1 0/2 0/1 0/1 0/1 0/1/2 0–8 0/1 0/1 0/2 0/1/2 0/1/2

0.18 ± 0.57 0.49 ± 0.63

0.93 ± 0.26

Subtotals in the same column with no letters in common are significantly (P b 0.05) different for type of establishment. a Maximum score. b Average ± SD.

0.21±0.41

0.85 ± 0.36

0.85 ± 0.36

0.97 ± 0.69

4.47 ± 3.28

0.88 ± 0.32

0.84 ± 0.37

0.03 ± 0.24

0.90 ± 0.65

1.31±0.47

3.95±2.05

8.43 ± 5.33

0.27 ± 0.69a 0.20 ± 0.41a 0.80 ± 0.41a 0.70 ± 0.47b 0.73 ± 0.64b 3.80± 1.85b 0.73 ± 0.45b 0.97 ± 0.18b 0.00 ± 0.00a 0.83 ± 0.59a 1.17 ± 0.38b 3.70 ± 0.70b 7.50 ± 5.18b 0.27±0.58b 0.83 ± 0.38b

7.63 ± 4.44 3.75 ± 0.96 1.25 ± 0.45 0.50 ± 0.52 0.00 ± 0.00 1.00 ± 0.00 1.00 ± 0.00 3.88 ± 3.47 0.63 ± 0.62 0.81± 0.40 0.81±0.40

1.00 ± 0.00 0.64 ± 0.63

0.25 ± 0.68 0.88 ± 0.34 0.19 ± 0.54

0.31± 0.48

7.36 ± 5.13 3.64±1.47 1.07 ± 0.27 1.21±0.43 0.00 ± 0.00 0.93 ± 0.27 0.43 ± 0.51 3.71± 3.66 0.86 ± 0.66 0.57 ± 0.51 0.79 ± 0.43 0.07 ± 0.27 0.29 ± 0.73 0.79 ± 0.43 0.36 ± 0.63

0.21± 0.41a 0.89 ± 0.31a 0.97 ± 0.16a 1.16 ± 0.68a 5.00±2.65a 1.00 ± 0.00a 0.74 ± 0.45a 0.05 ± 0.32a 0.95 ± 0.70a 1.43 ± 0.50a 4.17±1.97a 9.17 ± 4.61a 0.11±0.45a 0.66± 0.63a 1.00 ± 0.00a

8.42± 4.32 4.13±1.72 1.27 ± 0.47 1.07 ± 0.83 0.00 ± 0.00 0.79 ± 0.43 1.00 ± 0.00 4.29± 2.60 0.79 ± 0.80 0.93 ± 0.27 0.71±0.47 0.21± 0.43

1.00 ± 0.00 0.67±0.64

Large (n = 38) Warm (n= 24) Cool (n=14) Subtotal (n = 38) Warm Small and (n = 14) Medium (n = 30) Cool (n = 16) Subtotal (n = 30) Total

0.0 ±0.00

9.58 ± 4.11 4.17±2.00 1.50 ± 0.51 0.88 ± 0.61 0.08 ± 0.41 0.71±0.46 1.00 ± 0.00 5.42± 2.11 1.38 ± 0.49 1.00 ± 0.00 1.00 ± 0.00 0.21± 0.41

(2) (1) (1) (1) (2) (1)

0.17 ± 0.56

(1)

(2)

(2)

(2)

Total equipment (8) 12 11 10 9

Scores equipment Total 8 handler (10) (1) 7 6 5 4

(2)a

Checklist results for all establishments are presented in Table 2. LEs showed an average score of 9.17 (acceptable hygiene level), even though a poor hygiene classification (b9) was surveyed in 50% of visits. In the case of SMEs, the average score was below 9 (7.50), thus being classified as poor hygiene level. It is also remarkable that, regarding SMEs, only in 27% of visits, an acceptable hygiene level was observed, and no establishment obtained a good hygiene level classification. According to statistical analysis, scores obtained for handler hygiene and equipment hygiene level in SMEs were significantly lower than in LEs, being this difference more pronounced in aspects related to handler hygiene. Regarding season effect, there were not significant differences between warm and cool seasons in

b

3.1. Checklist results

Establishment Season

3. Results

3

Descriptive statistics for microbiological data and checklist scores were applied with Microsoft Excel (Microsoft, Redmond, Wash.). Statistical analysis was performed by means of SPSS 8.0 software (SPSS Inc. Chicago, Illinois, USA). Correlation coefficient, t-student test, and variance analysis (significance at P b0.05) were applied on microbiological counts. On the other hand, checklist scores and prevalence data were analyzed by Mann–Whitney and Chi-square test, respectively.

481

Scores handler 1 2

2.4. Statistical analysis

Table 2 Average score for different hygiene aspect evaluated through checklist during handling and slicing of cooked meat products in Small and medium-sized establishments and large-sized establishments.

(Seward, UK) blender for 2 min. When necessary, decimal dilutions were prepared in 0.1 % water peptone. Mesophilic aerobic bacteria were analyzed in Plate Count agar, incubating at 30 °C for 48 h (ISO 4833:1991). For coliforms, Violet Red Bile Lactose agar was used followed by incubation at 30 °C for 24 h (ISO 4832:1991). Lactic acid bacteria were pour-plated onto De Man Rogosa and Sharpe agar and incubating at 30 °C–48 h with 10% CO2 (ISO 15214:1998). Psychrotrophic bacteria were surface-plated on Plate Count agar, and then incubated at 7 °C during 10 days (ISO 17410:2001). As microbial indicators, enumeration of coagulase-positive Staphylococci was carried out using Baird Parker agar with Rabbit Plasma Fibrinogen supplement (RPF) incubating at 37 °C–24 h (ISO 68882:1999). Escherichia coli β-glucuronidase was enumerated by using a Most Probable Number (MPN) method in 9 mL tubes of Lauryl Tryptose Broth supplemented with MUG (4-methylumbelliferyl-β-Dglucuronide) as established by the AOAC Official Method 992.30. Enumeration and investigation of Listeria spp. and L. monocytogenes were also performed. Counts of L. monocytogenes were obtained by following the EN/ISO 11290-2 enumeration method, while investigation of the pathogen was based on EN/ISO 11290-1 detection method. For identification of Listeria spp., from Oxford and Palcam Listeria Selective agar (Oxoid), five presumptive colonies were subcultured on Tryptone-Soya Agar at 37 °C for 24 h. The following tests were performed on colonies isolated in TSA: catalase test (3% H2O2 solution was prepared), motility test in SIM medium and Gram stain test, following the directions of the EN/ISO 11290-1 detection method. Afterwards, the isolated colonies of Listeria were identified by means of API Listeria identification galleries (Biomèrieux, Marcyl'Etoile, France). The manufacturer's instructions were followed. The software APILAB PLUS was used. Serotyping of Listeria was carried out using commercial specific antisera (Denka Seiken Co., Ltd., Tokyo, Japan), following the manufacturer's instructions. Both polyclonal anti-O antisera (O-I/II, O-V/VI, O-I, OII, O-VI, OVII, O-VIII, Y O-IX) and anti-H antisera (H-A, H-AB, H-C, H-D) were used in the determination of somatic and flagelar antigens, respectively. Interpretation of the results was carried out according to the serotyping scheme established by Seeliger and Höhne (1979).

Total Checklist (18)

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both types of establishments for hygiene level (total score), as well as for handler and equipment hygiene subcategories. Table 2 also shows the average scores obtained for each hygiene aspect evaluated through checklist. Hand washing facilities were more deficient in SMEs in accordance to the obtained scores and statistical analysis. However, hand washing practices were absent or not correctly performed in both types of establishments. Hand washing was not frequent after handling raw products and/or before slicing cooked meat products. On the other hand, in LEs, the use of gloves was more correct than in SMEs, obtaining scores of 0.66 ± 0.63 and 0.27 ± 0.58, respectively (P b 0.05). In LEs, the use of tongs and shovels to collect the cooked meat slices was more frequent, which were observed in 4/9 establishments (data not shown). Only in 2/10 SMEs, tong were used (data not shown). However, statistical analysis did not show significant differences for use of tongs and shovels (P = 0.052). Scores for hygiene practices were significantly higher in LEs (1.16 ± 0.68) compared to SMEs (0.73 ± 0.64). Likewise, separation between cooked meat products and cured and raw products was statistically less frequent in the case of SMEs compared to LEs, which had adequate separation between both categories of products for all sampled establishments. An important deficiency found for both types of establishments was the lack of exclusiveness of the slicing machine for RTE products (with the exception of one LEs), even when different products, i.e. cooked meat products and cured and raw products, were separated in the vending area. 3.2. Microbiological analysis Microbiological parameters and results are presented in Table 3. Microbiological analysis revealed counts of more than 5 log cfu/g for the bacterial groups studied. The levels obtained for mesophilic aerobic bacteria, psychrotrophic bacteria, and Lactic-Acid Bacteria (LAB) averaged 5.50, 5.79, and 5.24 log cfu/g respectively. Coliforms were found in 65% of analyzed samples, and counts were significantly lower than the other groups of microorganisms (P = 0.01). The average value was 1.88 log cfu/g, though it was obtained a maximum value of 4.90 log cfu/g. E. coli was detected in 8 samples (b10 cfu/g): 7 samples coming from SMEs and only one from LEs. Moreover, the statistical analysis confirmed that prevalence of E. coli was significantly higher in SMEs. Most of samples were positive for coagulase-positive Staphylococci (67/68), with a mean of 3.88 log cfu/g, although levels as high as 6.36 log cfu/g were reached. According to variance analysis, microbiological levels were statistically similar between both types of product analyzed (i.e., cooked ham and chopped pork). Levels obtained for mesophilic

aerobic bacteria, psychrotrophic bacteria, LAB and coagulasepositive Staphylococci were not correlated to the type of establishment and season. In turn, levels of coliforms were significantly higher on warm season (2.35 and 1.32 log cfu/g in warm and cold season, respectively). Despite this fact observed in coliforms, most of positive samples for E. coli (6/8) were found in cool season, which could be explained by the survival of E. coli on surfaces, which is highly influenced by hydric stress, decreasing when temperatures are high, thus meaning more likely cross-contamination of E. coli during cool season, i.e. when temperature is low (Wilks, Michels & Keevil, 2005). Finally, no significant correlation (i.e., linear relationship) was found between checklist scores and microbiological results.

3.3. Prevalence of Listeria spp. and L. monocytogenes in cooked meat products Listeria spp. was detected in 10 samples (b10 cfu/g) from which 5 samples were contaminated by L. innocua, 4 samples by L. monocytogenes and one sample by both L. innocua and L. monocytogenes. Therefore, the percentage of positive sample for L. monocytogenes and L. innocua corresponded to 7.35% and 8.82%, respectively. The pathogen was isolated from one sample coming from SMEs, being the rest found in LEs. On the contrary, in the case of L. innocua, 4 samples came from LEs and 2 samples from SMEs (see Table 4). Serovar analysis indicated that L. monocytogenes found in the positive samples belonged to the serovars 1/2c (2/6), 1/2b (1/6), 1/2a (2/6), and 3b (1/6) (see Table 4). It should be mentioned that one LE showed contamination for L. monocytogenes in two different inspection visits (serovars 1/2a, 3b and 1/2b). The statistical analysis indicated that no significant relationship was found between the type of establishment and the presence of L. monocytogenes. Nevertheless, significant influence of season (pvalue = 0.03) was observed on L. monocytogenes prevalence, as all contaminated samples were found during warm period. Based on the results obtained, it can be stated that the presence of Listeria spp. may be associated to eventual cross-contamination events, being irrespective of the hygiene level, since the establishments with positive samples of Listeria spp. had on average an acceptable hygiene level. In this line, checklist results revealed no exclusiveness in the use of slicing machine for cooked meat products, which could be an important factor causing cross-contamination. Also, hand washing was not sufficiently practiced by workers before slicing, which could result in contamination of the product when handled.

Table 3 Microbiological results of cooked meat samples sliced at retail for small and medium-size establishments (SMEs) and large-size establishments (LEs). Season

Type of establishment

No samples

Mesophilic aerobic bacteria

Lactic Acid Bacteria

Coliforms

Staphylococcus coagulasa +

Psychrotrophic bacteria

Listeria monocytogenesc

Listeria innocuac

Esherichia colic

Warm

LEs

24

a

5.42±1.06 (2.70–7.26) 5.37±1.29 (2.57–7.49) 5.40±1.14a 5.13±1.06 (3.35–7.21) 4.97±1.28 (2.22–7.55) 5.04±1.17a 5.24±1.19

2.23±1.63 (0.00–4.38) 2.54±1.51 (0.00–3.91) 2.35±1.57a 1.72±1.86 (0.00–4.90) 0.97±1.31 (0.00–3.62) 1.32±1.61b 1.88±1.62

4.04 ± 0.64 (2.78–5.73) 3.96±1.72 (0.00–6.36) 4.01±1.14a 3.39 ± 0.94 (2.18–5.22) 4.01±0.91 (2.58–6.10) 3.72±0.96a 3.88±1.06

5.89±1.01 (3.57–7.05) 6.14 ± 0.73 (4.86–7.09) 5.98 ± 0.91a 5.45 ± 0.94 (3.44–7.08) 5.73±1.19 (2.90–7.09) 5.60±1.07a 5.79±1.01

16.70% (4/24)

4.20% (1/24)

0% (0/24)a

7.14% (1/14)

14.29% (2/14)

14.29% (2/14)b

13.16% (5/38)a 0% (0/14)

7.89% (3/38)a 21.43% (3/14)

5.26% (2/38) 7.14% (1/14)a

0% (0/16)

0% (0/16)

31.25% (5/16)b

0% (0/30)b 7.35% (5/68)

10.00% (3/30)a 8.82% (6/68)

20.00% (6/30) 11.76% (8/68)

5.65 ± 0.98 (4.10–7.50) 5.71±1.25 (2.62–7.49) 5.67±1.07a 5.46 ± 0.90 (3.90–7.19) 5.16±1.47 (1.72–7.49) 5.30±1.22a 5.50±1.22 b

Cool

Total

SMEs

14

Subtotal LEs

38 14

SMEs

16

Subtotal

30 68

Data in the same column with no letters in common are significantly (P b 0.05) different. Letters were referred to the season effect on different microbiological parameters, excepting for E. coli, in which letters were referred to type of establishment. a Average ± SD: b(minimum–maximum);c(% of positive samples).

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483

4.2. Escherichia coli

Table 4 Details on Listeria monocytogenes and Listeria innocua isolates.

4. Discussion

Prevalence of E. coli was high, especially in SMEs (Table 3), in comparison with the usual low incidence found in cooked meat. Looking at the checklist results, some reasons could be put forward. The main explanation could be the poor hygiene level of the SMEs from which positive samples were collected. However, no statistical relationship could be observed between checklist scores and prevalence of E. coli. Improper hand washing and glove use were associated to SMEs, which could increase the probability of crosscontamination by E. coli (Lues & Van Tonder, 2007). In addition, although SMEs presented separation between RTE cooked meat products and raw or/and cured products, in practice, space limitation in vending area made cross-contamination possible through working surfaces. Pérez-Rodríguez, van Asselt, García-Gimeno, Zurera and Zwietering (2007) and Gormley et al. (2009) highlighted the relevance of slicing at retail as a cross-contamination source for microorganisms such us E. coli.

4.1. Hygiene practices

4.3. Listeria spp and L. monocytogenes

This work found significant differences in hygiene practices between LEs and SMEs, being more deficient in SMEs. In spite of this fact, high levels of microorganisms were found in both types of establishments. Lower levels in sliced cooked meat have been found by other authors (Elson, Burgess, Little & Mitchell, 2004; Gillespie, Little & Mitchell, 2000; Little, Monsey, Nichols & de Louvois, 1998). As an example, Little et al. (1998) reported that most samples (97%) had an acceptable microbiological quality, reporting levels for Staphylococcus aureus below the detection limit in most cases. In turn, our study shows much higher counts for coagulase-positive Staphylococci (3.88±1.06 log cfu/g). Several causes could explain such a high microbiological count on the basis of checklist data. For example, the incorrect or absent hand washing practices observed in both types of establishments (being more frequent in SMEs) could have led to cross-contamination between different products and handlers. These deficiencies were also observed by Angelillo, Viggiani, Rizzo and Bianco (2000) when food safety attitudes were evaluated in Italy, and by Veiros, Proenç, Santos, Kent-Smith and Rocha (2009) in Portuguese canteens by checklist examination. Little & de Louvois (1998) reported that in the majority of premises, raw and unwrapped cooked meat products were physically separated in displays (94% cases) and refrigerators (81% cases ), and dedicated equipment/ utensils (69–89% cases) were used for raw meat and unwrapped cooked meat products and other RTE foods indistinctly. In our study, although separation between different types of products was observed in most cases, slicer and other utensils were not exclusive for cooked meat products, which could have been an important cross-contamination source. As mentioned before in the previous section, deficient handling practices may have led to an increasing probability of the occurrence of Listeria contamination in cooked meat slices. At this respect, it is noteworthy that, though most sampled cooked meat slabs were maintained in their original package (57/68), 4 out of 5 products in which L. monocytogenes were found, were not maintained in original package and a new plastic film was used to wrap the whole slab every time slabs were sliced. This procedure allows a major contact between hands, work surfaces and the cooked meat slab, which could increase the likelihood of cross-contamination during preparation. Our study corroborated what other authors have previously observed. Elson et al. (2004) reported more deficient microbiological quality in cooked meat samples which were covered at the sampling moment than in uncovered samples. On the contrary, Gormley, Little, Grant, de Pinna and McLauchlin (2009) did not find significant differences in the microbiological quality between covered and open sliced cooked meat samples.

Although prevalence of L. monocytogenes reported in this study was relatively high (7.35%), it was not unexpected since other Spanish studies reported similar levels. Garrido et al. (2009) reported a prevalence of 8.5% in cooked meat products sliced at retail. Also, Vitas, Aguado and Garcia-Jalon (2004) and de Simón and Ferrer (1998) reported 8.8 and 5.2% of contaminated sliced cooked meat and cooked meat products, respectively. Other countries also reported similar prevalence levels for L. monocytogenes in sliced cooked meat products at retail (Yucel, Cıtak & Onder, 2005; Beak, Lim, Lee, Min & Kim, 2000). However, higher values between 14 and 16% (Van Coillie, Werbrouck, Heyndrickx, Herman & Rijpens, 2004; Lake, Hudson, Cressey & Nortje, 2002) and lower levels between 0.8 and 1.1% (Uyttendaele et al., 2009; Gombas et al., 2003) have been reported. On the other hand, the same authors reported in a previous study (Uyttendaele, De Troy & Debevere, 1999) higher prevalence (6.5% positive samples, n = 1,156), pointing out that prevalence increased in cooked meat product after slicing (from 1.5 to 6.5%), demonstrating crosscontamination as a probable event occurring during slicing or/and handling at the point of sale. Findings from Garrido et al. (2009) and Gombas et al. (2003) also support cross-contamination during slicing and handling as likely causes of the relatively high prevalence of L. monocytogenes detected in our study. For all positive samples for Listeria spp., concentration levels were below 10 cfu/g, only detectable by investigation methods. Most studies are focused only on presence/absence data. Enumeration data reported show levels usually below the detection limit of the enumeration method used (e.g. b10 cfu/g). However, some studies have reported higher counts (Garrido et al., 2009), with values exceeding 100 cfu/g; in 33.3 and 41.2% of vacuum-packaged cooked meat and in-store-packaged cooked meat samples, respectively. In general, when high levels are reported, they are present in very few samples (Elson et al., 2004; de Simón and Ferrer, 1998). The samples analyzed in our study did not present high levels of Listeria spp., probably due to the prevention of growth of the microorganism, as the time elapsed from collection of samples at retail to sample analysis was very short, together with the low temperature registered during transport to laboratory (b2 °C). In our study, L. monocytogenes serovars frequently linked to listeriosis have been identified as 1/2a and 1/2b. The serovars most often causing disease are 4b, 1/2a and 1/2b (Low & Donuche, 1997), though are not usually the most common organisms recovered from food or the environment (McLauchlin, 1997). In our study, the serovars mostly associated with food environments were 3b and 1/2c, have been isolated. These serovars have been also detected in human listeriosis cases. At this respect, some authors have reported serovar

Establishment Size

Product

Establishment Specie

Serotype

Large Small–medium Small–medium Small–medium Large Large Large Large Large Large Large Large

Cooked ham Chopped Pork Chopped pork Cooked ham Cooked ham Cooked ham Chopped pork Chopped pork Chopped pork Cooked ham Cooked ham Cooked ham

A I I H A B A A C O E P

1/2b 1/2c – – – 1/2c 1/2a 3b – – 1/2a –

Listeria monocytogenes Listeria monocytogenes Listeria innocua Listeia innocua Listeria innocua Listeria monocytogenes Listeria monocytogenes Listeia monocytogenes Listeria innocua Listeria innocua Listeria monocytogenes Listeria innocua

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1/2c as the most frequent serovar found in meats (Hof & Rocourt, 1992), although cooked meat products are frequently contaminated by serovar 1/2a. In the study by Garrido et al. (2009) it was reported that deli meat food isolates belonged mainly to 1/2a and 1/2c serovars (37.5 % and 33.3 %, respectively), while serotypes 1/2b and 4c were detected in lower proportion. The same group reported previously (Vitas et al., 2004) a high percentage of serovar 1/2a isolates (60%), it was also found that the most of L. monocytogenes isolates in cooked meat were 1/2a (60 %) followed by 4b and 1/2b. Similarly, de Simón and Ferrer (1998) reported that the most frequent serovars isolated found in cooked meats were 4b and 1/2a. Unlike other studies (Gombas et al., 2003; Jemmia et al., 2002; Wilson, 1995; MacGowan, Bowker, McLauchlin, Bennett & Reeves, 1994), seasonal differences were found for Listeria spp. being exclusively detected during warm period. Temperatures in Southern Spain during summer can be extremely high, often over 40 °C. Despite temperatures registered in refrigerated cabinet of the visited establishments in warm and cold season were not statistically different, inappropriate temperature in establishment environments frequently observed may explain the fact that Listeria spp. was only detected during warm period (Augustin, 2003; Perez-Rodriguez et al. 2007a). In this sense, Gombas et al. (2003) reported the highest values for Listeria sp. numbers to have been detected during a warm month, i.e. May. The present work has made an attempt to find possible bond between L. monocytogenes and L. innocua. Little information has been found dealing with the relationship between L. monocytogenes and other microorganisms present in foods such as cooked meat products (Garrido et al., 2009). Some studies have suggested that L. innocua or Enterococcus faecalis can be satisfactorily used as indicators or surrogates of L. monocytogenes in inactivation processes, survival, and ripening steps (Liu, Puri & Demirci, 2009; Fairchild & Foegeding, 1993; Inghan & Tautorus; 1991). Our results suggested that no relationship between the studied microbiological parameters and L. monocytogenes' presence was found. In addition, L. innocua was present in samples other than those contaminated by L. monocytogenes, excepting one sample in which both microorganisms were present, indicating different contamination patterns. At this respect, Garrido et al. (2009) and Vitas et al. (2004) reported several samples contaminated with L. monocytogenes and L. innocua, though no more details were given. The study by Little, Sagoo, Gillespie, Grant and McLauchlin (2009) reported higher prevalence of L. innocua than L. monocytogenes in sliced cooked ham, but no more detailed information was provided with regard to possible contamination patterns. Similar results have been presented by other authors (Elson et al., 2004; Yucel et al., 2005). In short, information is still incomplete and in some case inconclusive, hence further specific research should be carried out in order to study the possible relationship between potential indicator microorganisms and L. monocytogenes' presence in different foods. 5. Conclusions Checklist results confirmed that Small and medium-sized establishments are associated with worse hygiene practices compared to Largesized establishments. However, this fact was not equally evidenced by the microbiological levels found, which were similar in both establishment types. These findings indicate that educational programs should be more efficiently implemented with particular attention on proper hand washing and glove use, especially in small and medium-sized establishments. On the other hand, the incidence of L. monocytogenes found in this study was high, although comparable to other studies. This prevalence could be related to cross-contamination during slicing process, though contamination at factory cannot be discarded as possible origin. Hence, effort should be made to improve cooked meat safety by appropriate treatment and hygiene measures both at factory

and retail. In addition, as pointed out by Garrido et al. (2009), other alternatives can be proposed to mitigate post-contamination of Listeria spp. such as the use of biopreservatives or active packaging, since in many cases, as observed in our study, L. monocytogenes absence is not assured despite good hygiene practice has been performed. Finally, the seasonal contamination patterns found in this study for both E. coli and L. monocytogenes suggest that specific measures should be implemented to reduce the impact of temperature in each case, such as more effective disinfection of work surfaces or a better control on establishments' temperatures.

Acknowledgments This work was partly financed by the MCYT AGL2005-119 project (Spain Government) and the AGR-01879 and P08-CTS-3620 Excellences Projects (Andalucía Government) and the AGR-170 Research Group, HIBRO, of Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI) and by the European ERDF funding.

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