Prevalence of Listeria monocytogenes in 13 dried sausage processing plants and their products

International Journal of Food Microbiology 102 (2005) 85 – 94 www.elsevier.com/locate/ijfoodmicro

Prevalence of Listeria monocytogenes in 13 dried sausage processing plants and their products D. The´venot a,T, M.L. Delignette-Muller b, S. Christieans a, C. Vernozy-Rozand b b

a Association pour le De´veloppement de l’Institut de la Viande, 2 rue Chappe, 63039 Clermont-Ferrand cedex 2, France Unite´ de Microbiologie Alimentaire et Pre´visionnelle, Ecole Nationale Ve´te´rinaire de Lyon, B.P. 83, 69280 Marcy l’e´toile, France

Received in revised form 14 June 2004; accepted 11 December 2004

Abstract The aims of the present study were: (i) to investigate the occurrence of Listeria monocytogenes in dried sausage processing plants on surfaces before and during processing, (ii) to study the contamination in meat and sausages at different stages of maturation, (iii) to assess the distribution of L. monocytogenes in the different plants and products studied. Thirteen dried sausage processing plants were sampled at two different times of the working day. The studies were repeated twice to evaluate the persistence of the pathogen. A total of 1029 samples were collected. Among swabbed samples, 15% were positive before the beginning of the working day and 47.3% during working day. Results showed that effectiveness of cleaning and disinfecting operations could be linked with the complexity of processing lines and machines used. The presence of L. monocytogenes in mixed meat amounted to 71.6% of the collected samples. A decrease of the contamination rate in dry sausage was noted, particularly during the drying stage. Nevertheless 3 sausages studied presented a low contamination rate (b3 cfu/g) when ready for consumption. A total of 996 strains of L. monocytogenes were characterised by biochemical tests and serotyping. A majority of isolates were 1/2a (49.5%), 1/2c (19.5%) and 1/2b (13%) strains. A high heterogeneity of serotypes was observed in all plants, raw meat and in sausages during maturation. D 2005 Elsevier B.V. All rights reserved. Keywords: Listeria monocytogenes; Contamination; Sausages; Dried sausage processing plants; Serotypes

1. Introduction

T Corresponding author. Current address: Unite´ de Microbiologie Alinmentaire et Pre´visionnelle. Ecole Nationale Ve´te´rinaire de Lyon. 1 avenue Bourgelat. BP 83. 69 280 Marcy l’Etoile. Tel.: +33 04 73 98 53 80; fax: +33 04 73 98 53 85. E-mail address: [email protected] (D. The´venot). 0168-1605/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2004.12.008

Several listeriosis outbreaks have been reported in North America and Europe (Ryser et al., 2001; Frye et al., 2002). Major pork-related listeriosis outbreaks occurred in France. Pork tongue in jelly (1992), pork brillettesQ (1993), and sausage mixture (2002) were the vehicles of transmission of Listeria monocytogenes

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(Jacquet et al., 1995; Goulet et al., 1998). Three serotypes are associated with many sporadic cases of listeriosis (1/2a, 1/2b and 4b) and serotype 4b is linked to almost all outbreaks of listeriosis in Europe (Jay, 1996; Kathariou, 2002). L. monocytogenes presents a severe systemic illness in persons with impaired cell mediated immunity such as infants, elderly individuals, immunocompromised persons and pregnant women (Anonymous, 2002; Rocourt et al., 2000). L. monocytogenes has been found in every part along the pork processing industry (Nesbakken et al., 1996). The incidence of the microorganism increases when going from the slaughterhouse to the cutting room (Nesbakken et al., 1996). The delicatessen industry also acts as a source of contamination (Salvat et al., 1995; Jay, 1996). In addition, several studies have shown that L. monocytogenes is capable of contaminating food processing machines with bacterial attachment and biofilm formation, representing a source of potential contamination of food products (Autio et al., 1999). Once attached to a surface, microorganisms appear to be more difficult to remove (Aase et al., 2000). Dry sausage is made of cut pork meat and pork fat, together with sugar, NaCl, saltpeter and starter cultures. During fermentation and drying of sausages, the number of L. monocytogenes tends to decrease substantially because of inhibiting environment created by a set of hurdles (low pH and low water activity (a w) and high salt concentration) (Buncˇic´ et al., 1991). However, contamination of raw meat and inherent ability of microorganisms to adapt to their environment may result in production of unsafe sausages. Even if the contamination rate is usually low (b10 to 100 cfu/g), manufacturers have to abide by a regulation requiring the absence of L. monocytogenes in 25 g of foods with a tolerance of below 100 cfu/g for some foods at the best-before date. There is a lack of data about prevalence of L. monocytogenes in French dried sausage processing plants and the aim of this study was to evaluate the occurrence of L. monocytogenes in surfaces after the cleaning and disinfecting operations, during plant operations, in meat and meat products at different processing stages. Isolates were serotyped to assess the distribution of L. monocytogenes strains in the different plants.

2. Materials and methods 2.1. Samples Thirteen French dried sausage processing plants having no apparent relationship with each other and located in different areas of France were examinated for L. monocytogenes. There were both large manufacturers and small factories and were representative of the French production. The combination of ingredients and processes may vary among companies and regions of production (some add wine, rum, spices for chorizo or specific surface fungi). In addition, fermentable sugar, starter cultures, curing temperature and drying conditions change with the type and diameter of product. This diversity is illustrated in Table 1 (Dabin and Jussiaux, 1994). Yet, all sausages are prepared with the same ingredients (pork meat and fat, sugar, starter, spices) and manufactured with the same technology (fermentation and drying). For each plant, different data were collected: annual tonnage, processing lines complexity, products studied, raw material used (fresh or frozen), general plant state, hygiene of staff, equipment visual status (new, old, presence of rust, good state), cleaning and disinfecting procedures, moisture (water, moist, dry) and visual detection of organic residues (minced meat or fat on surfaces). In order to evaluate the persistence of L. monocytogenes in plants, sampling operations were

Table 1 Characteristics of the different types of sausages studied (Dabin and Jussiaux, 1994) Products

Diameter Time of Time of (mm) curinga dryingb (days) (days)

bSaucisseQ

40

2 to 3

21

bSaucissonQ

60

3 to 5

bRosetteQ

100

5 to 6

bChorizoQ

40

2 to 3

21 to 30 45 to 60 21

a

Final pH

Final a w

5.2 to 5.4 0.78 to 0.85 5.1 to 5.3 0.83 to 0.88 4.8 to 5.4 0.90 4.7 to 5.4 0.80

During the curing stage, sausages are placed in steaming rooms (22–24 8C) in order to accelerate the growth of starter cultures. b During the curing stage, sausages are placed in steaming rooms (22–24 8C) in order to accelerate the growth of starter cultures.

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Two types of tissue swab were used: surfaces after the cleaning and disinfecting operations were sampled by swabbing with 10 cm by 10 cm sterile gauze pads moistened with cleaning and disinfecting neutralizing products (Humeau, La chapelle-sur-Edre, France). Surfaces during the processing were sampled with simple sterile gauze pads (Humeau). All tissue swabs were then transferred into sterile boxes with 20 ml of peptone salt (di-sodium phosphate 3.5 M, peptone 10.0 M, sodium chloride 5.0 M, potassium dihydrogen phosphate 1.5 M) (Oxoid, Basingstoke, Hamphire, UK). The numbers of samples collected depended on manufactories size, products manufactured, complexity of processing lines (Table 2) and equipment used.

repeated twice with a time difference of at least 2 weeks. Two dried sausages processing plants were only sampled once (G and N) because of technological problems. Plant A was sampled three times because factory was close to the laboratory. Plant L was sampled twice but with a time interval of only 1 day because of geographic distance. Time difference between sampling varied because of organizational parameters. All factories except L received microbiological results of the first sampling before the realization of the second one. Therefore, corrective actions (more particularly in cleaning and disinfecting procedures) could have been applied in these plants. A total of 1029 samples were collected and among them 413 samples from surfaces with and without contact with meat after the cleaning and disinfections procedures, 372 from the same surfaces during activity and 244 from meat and manufactured meat.

2.3. Meat and meat products Pork cut items were sampled by excising surface. Sausage mixing and sausages from the same batch were aseptically sampled and placed in sterile bags. Sausages were studied at the ends of the curing (sausages are placed in steaming rooms, at 22/24 8C, in order to accelerate the growth of starter cultures) and the drying phases (at 12 8C in drying rooms). All samples were kept in ice boxes at 3 8C during transport and were immediately analyzed upon arrival at the laboratory.

2.2. Surfaces samples by swabbing All surfaces sampled were accessible by cleaning and disinfecting products. Sampled surfaces were grouped into two classes: the surface without any contact with raw meat or sausage (floor, wall, steaming and drying rooms) and the surfaces in direct contact with meat and meat products (cutter, mincing, dicing, mixing and stuffing machines).

Table 2 Some characteristics of the 13 dried sausages processing plants Plants

A B C D E F G H I/J K L M N

Tonnage/year

4000 to b1000 4000 to 1000 to 2000 to b1000 1000 to 2000 to 2000 to b1000 b1000 b1000 b1000

5000 5000 2000 3000 2000 3000 3000

Processing lines complexity +++ ++ +++ + ++ ++ + +++ ++ ++ ++ ++ +

Types of products bSaucisseQ 2

bSaucissonQ

Raw meat bRosetteQ

bChorizoQ

3 2 2 2

1 1

Fresh

Frozen

 

   

   

2 2 4

1

1 2

2 3

2 1

    

       

Processing lines complexity: +(few simples machines)/++(more machines (more complex))/+++(lot of machines (with a complex design)). 1, 2, 3, 4 design the number of different types of bsaucisseQ, bsaucissonQ, brosetteQ and bchorizoQ produced in the plant.

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2.4. Detection and isolation of L. monocytogenes All samples were analyzed to test the presence of L. monocytogenes using the two enrichments procedure recommended by V08-055 AFNOR Norm (Association Franc¸aise de la Normalisation). More precisely, a portion (25 g) aseptically taken from raw meat, sausage mixing or from the central portion of each sausage was added to 225 ml of Fraser-demi (Oxoid). Tissue swabs were resuspended in 9/10 of their weight in the same broth. Samples were homogenized in a stomacher (90 s) and incubated at 37 8C for 18 to 24 h. A portion (0.1 ml) of the homogenate was inoculated into 9.9 ml of Fraser (Oxoid) broth for a second enrichment at 37 8C for 18 to 24 h twice. Each enrichment broth was streaked to ALOA agar (AES Laboratories, Combourg, France), and plates were examined for typical L. monocytogenes colonies after 48 h incubation at 37 8C. Four characteristic colonies (round, regular and blue/green colonies with opaque halo) from positive sampling plates were tested with a biochemical identification with bmonoconfirm testQ (ref AEB 193000, AES Laboratories, Combourg, France). This is a micro method based on 4 biochemicals characters to identify Listeria spp. and L. monocytogenes. Each L. monocytogenes isolate was grown into Brain Heart Infusion (BHI) (AES Laboratories) agar or broth and stored respectively into bcryo beadsQ system or in BHI broth supplemented with 15% glycerol and maintained at 82 8C. 2.5. Serotyping Serotyping was performed using commercial Listeria antisera according to the instructions given by the manufacturer (Eurobio Les Ulis, France). This method differentiates L. monocytogenes into 13 different serotypes based on the association of somatic (O) and flagellar (H) antigens with a series of polyvalent and monovalent antisera. For the O antigen, isolates were grown on BHI 10 g l 1 agar for 24 h at 37 8C. A positive reaction consisted of an agglutination of the isolate after at least 1 min of mixing in one drop serum. The polyvalent sera (OI/OII and OV/OVI) were tested first. In the case of a positive reaction with OI/OII, OI

and OIV sera were tested. In the case of a positive reaction with OV/OVI, sera OVI, OVII, OVIII and OIX were tested. For the H antigen, isolates were grown as stab cultures in BHI half-agar (4.5 g l 1 agar) for 48 h at room temperature. Isolates at the deepest part of the growth were then grown on BHI broth for 24 h at room temperature. A volume (1.2 ml) of the culture was then mixed with 1.2 ml 9 g l 1 NaCl. In separate tubes, two drops of sera (A, AB, C, D and nothing) were mixed with 0.5 ml of broth culture. The five tubes were incubated at 50 8C for 2–3 h. A positive result showed an agglutination at the bottom of the tube. The serotypes were determined according to the table of Larpent (1995). 2.6. Statistical analysis Contamination rates were compared using the Pearson’s chi-squared test with continuity correction when necessary. These statistical calculations were computed using R Software version 1.6.1 (Ihaka and Gentleman, 1996).

3. Results 3.1. Global contamination by L. monocytogenes A total of 1029 samples were tested. Among the samples, 785 were from swabbed surfaces: 15% (62/ 413) were positive for L. monocytogenes before the beginning of the working day. More precisely, 15.1% (58/383) of the surfaces in the direct contact with meat (machines) and 13.3% (4/30) of the surfaces without any contact with meat were contaminated by this pathogen. During processing, 25.9% (14/54) of these surfaces and 50.9% (162/318) of machines gave positive swabbing for L. monocytogenes (Table 3). 3.1.1. Surfaces contamination before the beginning of the working day All surfaces after the cleaning and disinfecting operations were sampled by swabbing with sterile gauze pads moistened with cleaning and disinfecting neutralizing products. No inhibitory effects of such

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Table 3 Contamination of equipment surfaces, raw meat and products at the end of fermentation in the 13 plants studied Plants

Machines surfaces

Raw meats

Before operations

A

B C D E F G H I/J K L M N Grand total

S1 S2 S3 S1 S2 S1 S2 S1 S2 S1 S2 S1 S2 S1 S1 S2 S1 S2 S1 S2 S1 S2 S1 S2 S1

End of fermentation

During operations

+/Total

%

Total%

+/Total

%

Total%

+/Total

%

4/16 6/18 7/13 0/10 1/10 6/15 3/15 0/10 0/10 2/15 0/15 3/14 1/17 0/11 6/24 5/24 1/21 0/21 1/16 0/16 1/18 1/18 4/12 3/12 0/12 58/383

25.0 33.3 53.8 0.0 10.0 40.0 20.0 0.0 0.0 13.3 0.0 21.4 5.8 0.0 25.0 20.8 4.7 0.0 6.25 0.0 5.5 5.5 33.3 25.0 0.0 15.1

36.1

3/5 5/5 6/7 3/13 4/13 9/12 10/12 8/11 6/11 3/12 2/13 10/14 3/13 6/12 9/16 12/19 10/23 13/23 7/14 8/14 6/19 7/13 4/11 4/11 1/9 162/318

60.0 100.0 85.0 23.0 30.7 75.0 83.3 72.7 54.5 25.0 15.3 71.4 23.0 50.0 56.2 63.1 43.4 56.5 50.0 61.5 46.1 53.8 36.3 63.6 11.1 50.9

82.3

1/6 3/6 0/5 0/3 1/3 2/2 3/3 2/3 1/3 1/7 1/7 2/7 3/5 1/5 2/5 3/4 3/3 2/4 2/4 1/4 3/7 2/7 0/7 1/6 1/6 41/121

20.0 50.0 0.0 0.0 33.3 100.0 100.0 66.6 33.3 14.2 14.2 28.5 60.0 20.0 40.0 75.0 100.0 50.0 50.0 25.0 42.8 28.5 0.0 16.6 16.6 33.9

5.0 30.0 0.0 6.6 12.9

22.9 2.3 3.1 5.5 29.1

26.9 79.1 63.6 20.0 48.1

60.0 50.0 53.5 50.0 50.0

Total% 25.0

16.6 100.0 50.0 14.2 41.6

55.5 71.4 37.5 35.7 7.7

+/Total 0/2 0/1 1/2 0/1 0/1 ND ND 0/1 1/1 0/1 0/2 0/1 0/1 0/2 0/2 0/2 0/2 1/1 0/1 0/2 0/1 0/1 0/1 0/1 3/30

% 0.0 0.0 50.0 0.0 0.0

Total% 20.0

0.0 ND 0.0

0.0 100.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 100.0 0.0 0.0 0.0 0.0 0.0 0.0 10.0

50.0 0.0

0.0 0.0 50.0 0.0 0.0

S1, S2 and S3: samplings 1, 2 and 3. +: number of samples contaminated with Listeria monocytogenes. Total: total samples. ND: not done.

products were detected on growth of the L. monocytogenes (data not shown). Regarding the contamination rates before processing in the 13 plants studied at each sampling day, surfaces without contact with meat or products were not taken into account because of their low number (30 before the working day and 54 during the working day) and their poor statistical impact. The examination of the machines contamination revealed that different plants could be grouped into 3 categories. The first one brought together 4 plants (A, C, H and M) which presented high levels of contamination at each sampling (Table 3). The second category was represented by only one plant (F) with a contamination rate of 21.4% during the first sampling and 5.8% during the second. The last category grouped 8 plants (B, D,

E, G, I/J, K, L and N) which always presented low rates of contamination by L. monocytogenes (Table 3). A decrease of the contamination rate was observed in most of the plants sampled two times except in plants A and B. In plants D and L the contamination rate remained constant. Large plants (A, C, H and M) showed relatively high contamination rate by L. monocytogenes (29.5% for all samplings) whereas smaller plants (B, D, E, G, I/J, K, L and N) were significantly less contaminated (2.9%) ( pb0.0001). It is noteworthy that in each plant, organic residues wre noticed on the surface in case of L. monocytogenes detection (Table 4). Cleaning and disinfection procedures applied in plants varied (Table 5). Most of the factories used a

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Table 4 Organic residues and presence of Listeria monocytogenes on surfaces after the cleaning and disinfecting operations Plants

A B C D E F G H I/J K L M N

Sampling 1

Sampling 2

Surfaces with organic residues

Presence of L. monocytogenes on these surfaces

Surfaces with organic residues

Presence of L. monocytogenes on these surfaces

Floor and equipment no one Floor and equipment No one Mixing trough/separating machines Cutter No one Floor and equipment Stuffing floor Transport belt Mixing machine Floor and equipment Floor

Yes No Yes No Yes

Floor and equipment (1) Mixing machine Dicing machine Mixing machine No one

Yes (1) Yes Yes Yes No

Yes No Yes Yes Yes Yes Yes Yes

Knifes/transport belt ND Floor and equipment Fabrication floor No one Mixing machine Floor and equipment No one

Yes ND Yes Yes No Yes Yes No

ND: not done. (1): and sampling 3.

two-step procedure using both detergent and disinfectant products. But some plants (F, H, K and M) applied just a one-step procedure with only a disinfecting detergent, often an alkaline chlorinated product. Plants N, I/J and C applied the two procedures in different rooms or different days. Whatever the plants studied, no rotations of products have been applied to prevent the development of resistant strains. 3.1.2. Equipment samples during processing During processing, the contamination rate increased from 15.1% before initiation of processing to 50.9% during processing with high heterogeneity between the different plants with a rate of 11.1% in plant N and a rate of 100% in plant A during the second sampling) (Table 3). 3.1.3. Meat and meat products The contamination rate of raw meat in all plants studied was 33.9% (Table 3). No obvious differences were observed between contamination of frozen or fresh meat (data not shown). Contamination of minced meat and stuffed sausage increased to 71.6%. The contamination rate decreased at the end of the curing stage (results that shown) but mainly after the drying step (10%). In dried sausages, the pH values are low (pH 5), and because of deshydratation

during drying, a w is low (about 0.80) and salt concentration high. Only 3 final sausages among the 30 studied (produced in the 3 plants A, E and K) were contaminated. L. monocytogenes counts indicated that the contamination was lower than 3 cfu/g. 3.2. Serotypes A total of 996 strains were serotyped. Different serotypes were detected: 1/2a, 1/2b, 1/2c, 4b and 4e. The serotype distribution based on these results was as follow: 49.5% of isolates were of serotype 1/2a, 19.5% of serotype 1/2c, 13% of serotype 1/2b, 8% of serotype 4b and 0.1% of serotype 4e. However 6.5% of the isolates were not serotypable; they did not react with the O, H factors antisera or an autoagglutination was observed (Table 6). Table 7 indicates that a majority of 1/2a, 1/2b and 1/2c serotypes strains were detected in raw meat. The stuffed sausages showed a high heterogeneity of strains. Indeed, some serotypes tended to appear in several plants (E, M and A) whereas in others some serotypes disappeared (plants C, G and N). The two phenomena occurred in most of the plants (B, D, I/J, F, H, K and L). During sausage maturation, the number of serotypes of L. monocytogenes tended to decrease, but no particular serotype remained in the end of the process. In all cases, a very

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Table 5 Hygienic and sanitizing observations of plants Plants General Staff Equipment state Cleaning and disinfecting procedures state hygiene Separated cleaning and Freq disinfection A

New

New

B

Old

C

New

D

Old

E

I

Good state

F

I

Good state

G

Old

H

I

Good state

I/J

I

New

K

I

New

L

I

M

I

N

Old

+

++

++

0

+

Cutter used, rusted Rest more recent New

Good state

Rust

C: alkaline chlorinated D: QACs C: alkaline chlorinated D: glutaraldehyde

All week

C: acid detergent D: glutaraldehyde or QACs

Friday

C: alkaline chlorideD: QACs C: alkaline chlorideD: QACs

All week

Moisture Cleaning and disinfection combined

Freq Water R: morning Moist R: evening

All week

CD: alkaline chlorinated

All week CD: alkaline chlorinated 2 products alterned

C: alkaline chlorinated D: QACs

All week CD: alkaline chlorinated

Stuffing: C: alkaline (1) detergent D: QACs

All week Fabrication: CD: alkaline chlorinated CD: alkaline chlorinated

Rust in mincing C: alkaline chlorinated and stuffing D: QACs machines Heterogeneous

All week water except Friday Dry R: evening Dry R: evening All week Dry R: evening Water R: morning All week Dry R: evening Dry R: evening All week Dry R: evening Dry No rinse

CD: alkaline chlorinated Week 1 Dry R: CD: tensio active detergent Week 2 evening C: surface active detergent (2) All week CD: alkaline chlorinated (3) All week Moist R: D: derived of QACs morning

Old

General state of factory: new/I(intermediate)/old. Staff hygiene/ (bad)/0(mediocre)/+(good)/++ QACs: Quaternary Ammonium Compounds. (1): Friday: acid detergent. (2): often used equipment. (3): less used equipment. (4): and sampling 3. C: cleaning D: disinfecting R: last rinse after cleaning and disinfection.

Table 6 Serotypes distribution in the 13 plants sampled (surfaces, raw meat and meat products) Serotypes

1/2a 1/2b 1/2c 4b 4e Non-serotypable strains

Plants A

B

C

D

E

F

G

H

I/J

K

L

M

N

%

84/142 19/142 22/142 7/142 0/142 10/142

21/34 0/34 9/34 0/34 0/34 4/34

21/112 12/112 32/112 14/112 1/112 9/112

15/47 10/47 18/47 0/47 0/47 4/47

20/27 4/27 0/27 0/27 0/27 3/27

11/84 4/84 31/84 35/84 0/84 3/84

2/17 9/17 1/17 2/17 0/17 4/17

92/136 19/136 4/136 4/136 0/136 7/136

103/137 13/137 14/137 0/137 0/137 7/137

36/90 34/90 13/90 6/90 0/90 1/90

56/73 5/73 0/73 11/73 0/73 3/73

28/81 0/81 34/81 0/81 0/81 8/81

4/16 0/16 10/16 0/16 0/16 2/16

49.5 13.0 19.5 8.0 0.1 6.5

X/Y: number of strains positive for the serotype tested/number of serotyped strains.

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Table 7 Serotypes observed in raw meat and in sausages at different stages of maturation Plants A

B C D E F G H I/J K L M N

S1 S2 S3 S1 S2 S1 S2 S1 S2 S1 S2 S1 S2 S1 S1 S2 S1 S2 S1 S2 S1 S2 S1 S2 S1

Raw meat

Sausage mixture and sausage stuffed

Sausage after curing

Sausage after drying

1/2c 1/2a 1/2c 1/2a T 1/2c 1/2a 1/2c 1/2a 1/2b 4b 1/2c 1/2a 1/2a 1/2a 1/2c 4b 1/2a 1/2c 1/2b 1/2a 1/2b 1/2a 1/2a 1/2a 1/2b 1/2c 1/2a 1/2b 1/2a 1/2a 1/2b 4e T T 1/2a 1/2c

1/2c 1/2a 1/2b 1/2c 4b 1/2a 1/2b 1/2c 1/2a 1/2a 1/2a Autoa 1/2a 1/2b 1/2b 1/2c 1/2c 1/2a 1/2b 1/2a 1/2c 4b Autoa T T 1/2a 1/2b 4b 1/2b 1/2c 1/2a 1/2c 1/2a 1/2a 1/2b 4b 1/2a 4b 1/2a 1/2b 4b 1/2a 1/2c 1/2c

T 4b T 1/2a 1/2a ND ND 1/2c 1/2c T T 4b T 4b 1/2a 1/2b T 1/2a 1/2b 1/2a T 1/2b ND ND

T T 1/2a T T ND ND T T 1/2b T T T T T T T T 1/2a T T T

T

T

ND: not done. S1/S2/S3: Sampling 1, 2 and 3. Autoa: autoaglutination. T No Listeria monocytogenes detected.

pronounced serotype and strains heterogeneity was observed.

4. Discussion The aim of this study was to estimate contamination by this pathogen in sausages and sausage manufactories. A decrease of contamination of equipment after the cleaning and disinfecting operations was observed in most of the factories between the two sampling days (except in plants B and A). These results were probably due to corrective actions applied in plants linked to the first bacteriological results. Such results might indicate insufficiencies of cleaning and disinfection procedures. But it also seems to show the effectiveness of regular cleaning and disinfecting operations. The high contamination rate by L. monocytogenes of large plants could be correlated with the complexity

of processing line and complexity of machines used. The eradication of L. monocytogenes from contaminated processing lines and machines has proved to be difficult (Autio et al., 1999; Miettinen et al., 1999). A study led by Lunde´n et al. (2002) showed that such equipment with poor hygienic properties design is susceptible to persistent L. monocytogenes contamination. Moreover, in each plant that was studied, organics residues on the machines surfaces were associated to L. monocytogenes detection. A study led by E Chasseignaux et al. (2001) in raw pork meat processing plants confirms this conclusion. Fifty point nine percent (50.9%) of the equipment samples contained L. monocytogenes during processing. This contamination rate was close to that obtained by Salvat et al. (1995) in a raw pork meat plant, but higher than that observed by Chasseignaux et al. (2002) repeating 37% in pork processing plants. This increase of contamination could be due to the contamination from raw meat, the multiplication of

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microorganisms, cross-contaminations or bad staff hygiene as hands were tested positively in several plants (unpublished information). Regarding the contamination rate of raw meat, our results (33.9%) were similar to those obtained in a study by Chasseignaux et al. (2001). The heterogeneity observed in the different plants could be the result of numerous facts likes methods of meat storage, i.e. frozen or fresh, sample of meat selected for analysis, suppliers or origin of meat. Mixing meat and stuffed sausage were contaminated by L. monocytogenes to an average of 71.6%. This high rate was probably due to combination of cross contaminations, growth of bacteria and bad hygiene of staff. Two previous studies, a Greek (Samelis et al., 1998) and a Spanish one (Encinas et al., 1999) confirmed that Listeria spp. were initially present in all sausage preparations. The contamination rate in sausage decreased slightly after the curing stage. The starter culture decreases the pH of the sausage close to pH-value 5.0 which acts as the first important hurdle (a w remains constant) for several pathogens and spoilage organisms. This slight decrease could be explained by results of Vialette et al. (2003) which indicate an overall weak effect of acid stress alone in exponential phase strains. An important decrease of the contamination rate of dry sausage was observed in this study. It was probably the result of a set of hurdles like low pH, low a w and high salt concentration, which become more important during the maturation of the product. A total of 996 strains were serotyped and different serotypes were detected: 1/2a (49.5%), 1/2b (13%), 1/2c (19.5%), 4b (8%) and 4e (0.1%). Only 3 serotypes (4b, 1/2a and 1/2b) are associated with the majority of sporadic cases of listeriosis or outbreaks (Farber and Peterkin, 1991; Rocourt and Cossart, 1997). In an extensive study of strains contaminating pork slaughtering and cutting plants, Giovannacci et al. (1999) distinguished 4 serotypes: 1/2a, 1/2c, 3a and 3c. Chasseignaux et al. (2001) observed a majority of serotype 1/2a (64%) among isolates collected from two poultry and pork processing plants. In the present study 8% of serotype 4b were found in the dried sausages processing plants. The presence of such a serotype is of major importance for the food industry and could be source

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of listeriosis. Indeed, a previous study (Rocourt and Bille, 1997) showed that most cases of listeriosis associated with the consumption of meat products were caused by strains of serotype 4b. After the cleaning and disinfecting procedures, plants could be clustered into 2 different groups (B, C, E, F, I/J, K and L) and (A, H and M). In the first one, the strain serotypes varied between the 2 days of sampling. These findings confirm that contamination was probably a result of insufficiencies in cleaning and disinfecting programs. In the second group, serotypes that remained were identical during the two samplings. In plants A and M, several machines were contaminated by the same serotypes. These results indicate a possible persistence of strains or the presence of biofilms on machines. That is well linked with the results (Table 4) indicating that the eradication of L. monocytogenes from complex processing lines and machines is difficult. Our results indicate the predominance of 3 serotypes 1/2a, 1/2b and 1/2c of L. monocytogenes in pork meat. This confirms the conclusions of Farber and Peterkin (1991) and Hof and Rocourt (1992). High heterogeneity in serotypes collected in raw meat material and in sausages during maturation was pointed out. This result allowed us to think that no specific serotype survived sausage processing and maturation. As a conclusion, even if sausage maturation tends to yield products without or with low level of L. monocytogenes, the potential presence in at the time of consumption still remains a major concern for the manufacturers. In a further study Pulsed Field Gel Electrophoresis will be applied on the collected strains in order to make an epidemiological study and determine the potential presence of persistent strains, which is a source of contamination in plants and in products. Challenge tests will also be performed to evaluate more precisely L. monocytogenes growth and survival in the different products studied.

Acknowledgements This work was supported by funds from RhoˆneAlpes and Massif Central regions, from OFIVAL (Office Nationale Interprofessionnel des Viandes de

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