Molecular epidemiology of Listeria ... - Wiley Online Library

Journal of Applied Microbiology 2001, 91, 888±899

Molecular epidemiology of Listeria monocytogenes isolates collected from the environment, raw meat and raw products in two poultry- and pork-processing plants E. Chasseignaux, M.-T. Toquin, C. Ragimbeau, G. Salvat, P. Colin and G. Ermel AFSSA-Ploufragan, UniteÂ d'HygieÁne et QualiteÂ des Produits Avicoles et Porcins, Ploufragan, France 796/02/01: received 23 February 2001, revised 9 May 2001 and accepted 14 May 2001

E . C H A S S E I G N A U X , M . - T . T O Q U I N , C . R A G I M B E A U , G . S A L V A T , P . C O L I N A N D G . E R M E L . 2001.

Aims: In order to study the transmission of Listeria monocytogenes in a poultry and a pork meat plant, we analysed the contamination by this pathogen over several months. Methods and Results: Five hundred and two isolates of L. monocytogenes were collected and characterized by genotyping and serotyping. Thirty-seven genotypes were obtained by ApaIrestriction analysis-pulsed ®eld gel electrophoresis (REA-PFGE) and 35 by SmaI-REA-PFGE and resulted in 50 combined genotypes. The tracing of the contamination in both plants showed that some clones were able to survive for several months. However, some other clones were found only during processing operations, were not detectable after cleaning and seemed to enter continuously into the plant. Conclusions: Some L. monocytogenes strains may persist for a long period in the plant environment. Different genotypes can be associated with poultry as well as pork meat. Signi®cance and Impact of the Study: Listeria monocytogenes contamination can be due to contaminated raw materials, bacterial spread and also ineffective cleaning procedures. INTRODUCTION Listeria monocytogenes has emerged as one of the major human foodborne pathogens. Listeriosis can occur as sporadic disease or as outbreaks. In France, it has been associated with three major outbreaks in humans: in 1992 with pork tongue in jelly, in 1993 with pork `rillettes' and in 1995 with a soft cheese `Brie de Meaux' (Jacquet et al. 1995). As outbreaks are linked with a food product, it is interesting to learn about the epidemiology of L. monocytogenes in food industry plants. Epidemiological studies are able to indicate the potential sources of contamination, trace the contamination in the plant and enhance knowledge about the environmental conditions where L. monocytogenes can survive and develop in a plant. Studies have shown that the ®rst major ampli®cation source of food product contamination with L. monocytogenes might be cross contamination, which occurs in the environment of the processing plants (Graham and Colins 1991; Wendtland and Bergann 1994). This phenomenon can be enhanced by Correspondence to: G. Ermel, AFSSA Ploufragan, BP 53, 22 440 Ploufragan, France (e-mail: [email protected]).

the psychrotrophic nature of L. monocytogenes (Helke and Wong 1994) and its ability to adhere to various surfaces (Blackman and Frank 1996). The colonization of working surfaces represents a potential source of contamination of food products (Cox et al. 1989; Sammarco et al. 1997; Wong 1998). In a dairy plant, Unnerstad et al. (1996) showed contamination lasting several years with one particular clone. Destro et al. (1996) were able to trace contamination by L. monocytogenes in a shrimp-processing plant. Therefore, these studies evaluated the genomic diversity of the different clones of L. monocytogenes which are present in particular environments such as food industries. The characterization of L. monocytogenes isolates can be achieved using phenotypic (serotyping and phage typing) and genotyping methods (DNA restriction analysis (REA) with pulsed ®eld gel electrophoresis (PFGE), ribotyping, etc.). Serotyping is the ®rst step in a typing scheme but is not suf®ciently discriminatory. However, three serovars are associated with the majority of sporadic cases of listeriosis (4b, 1/2a and 1/2b) and serotype 4b is linked to almost all recent outbreaks of listeriosis (Farber and Peterkin 1991; Rocourt and Bille 1997). Therefore, serotyping and phage typing are useful in indicating a ã 2001 The Society for Applied Microbiology

EPIDEMIOLOGY OF LISTERIA MONOCYTOGENES

possible source for an outbreak in preliminary investigations. The REA-PFGE is highly discriminatory and reproducible (Rocourt and Bille 1997). Using different enzymes, this technique has been useful in proving that, in France, isolates collected in 1988 in a small outbreak were identical to isolates responsible for a major outbreak in 1975±77 (Buchrieser et al. 1992). It was also useful in appraising the polymorphism of isolates collected from cheeses and dairy products (Unnerstad et al. 1996; Margolles and de los Reyes-Gavilan 1998). This REA-PFGE method is often used in epidemiological studies in combination with serotyping (Buchrieser et al. 1992; Brosch et al. 1994; Jacquet et al. 1995; Destro et al. 1996; Boerlin et al. 1997). In order to ®nd the origin of L. monocytogenes on food products, we monitored two plants, a poultry meat plant (named A) for 1 year and a pork meat plant (named B) for 4 months for L. monocytogenes. We used serotyping and two genotypic methods (REA-PFGE with ApaI and SmaI) to characterize the isolates and to study the dissemination of L. monocytogenes types.

Table 1 Distribution of samples and Listeria monocytogenes isolates obtained from contaminated samples in plants A and B

889

MATERIALS AND METHODS Detection and collection of Listeria monocytogenes isolates Two types of sample were taken: swabs from the processing plants (232 in plant A and 116 in plant B) and meat products (raw and processed; 81 in plant A and 39 in plant B) (Table 1). In both plants, the ®nished products were uncooked and had to be well cooked before eating. The swabbing procedure was performed in three different workrooms of plants A and B: reception of raw materials, meat processing and product processing. The sampling surfaces were either in direct contact or without any contact with the raw meat or products and were assessed in two classes: environment (¯oors, walls and drain) and equipment (working tables, boxes, transport belts, machines, knives, cutter and churn). The swabbing was performed either during processing or after the cleaning operations. The meat products were divided into two classes: raw materials (pork and poultry raw meat, rind and water supply) and products (pork and poultry products at the end of the production and

Plant A

Plant B

Types of samples

Samples*

Isolates

Samples*

Isolates

Swabbings Environment Floors Walls Drain

15/54 3/32 1/2

99 22 1

1/24 0/20 0/0

11 0 0

Equipment Working tables Boxes Transport belts Machines Knives Cutter Curn

3/14 8/27 4/67 2/14 1/2 4/9 1/11

17 28 4 9 1 16 2

6/16 1/7 5/28 4/13 2/4 0/0 0/4

65 21 43 38 20 0 0

Meat product samples Raw materials Pork meat Poultry meat Rind Water supply

0/0 7/44 0/0 2/2

0 17 0 5

8/24 0/0 1/1 0/0

30 0 7 0

Products Poultry products Shelf-life poultry products Pork products Shelf-life pork products

6/17 9/18 0/0 0/0

4 0 0 0

0/0 0/0 1/6 4/8

0 0 10 32

*Number of L. monocytogenes positive samples/number of total samples. Number of L. monocytogenes isolates collected. ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 91, 888±899

890 E . C H A S S E I G N A U X ET AL.

pork products at the end of the shelf-life at 4°C). All workrooms were studied at different times, for 1 year for plant A and 4 months for plant B. Meat products (10 g) were analysed using the VIDAS (bioMeÂrieux, Marcy l'Etoile, France) method for L. monocytogenes detection according to the manufacturer's recommendations (Pradel et al. 1995). Swabbing was performed using a tissue swab moistened with 5 ml tryptone salt (AES Laboratoires, Combourg, France) containing 100 g l±1 IsobioÒ (Laboratoire LCB, Lugny, France). They were then treated according to the modi®ed VIDAS method of detection for L. monocytogenes (Chasseignaux et al. 1999), i.e. the 48-h Palcam plates obtained after incubation of samples in half Fraser broth were soaked with 5 ml tryptone salt and the resulting bacterial suspension used for the VIDAS assay. For each positive sample, isolates were then collected. On each plate, typical Listeria colonies were subcultured onto tryptone soy agar supplemented with yeast extract (TSAYe; (Oxoid, Unipath, Dardilly, France). Colonies with a bluish tinge on TSAYe agar were subcultured onto tryptone soy broth (AES Laboratoires) and identi®ed using a microtiter plate method by testing catalase, haemolysis, nitrate reductase and fermentation of xylose, mannitol and rhamnose. An average of six isolates per sample was collected (Table 1); nevertheless, the collecting of isolates depended on the level of L. monocytogenes contamination, at low levels only one isolate could be recovered and, at high levels, up to 21 isolates were collected. Preparation of plugs for pulsed ®eld gel electrophoresis Bacteria were grown on TSAYe at 30°C for 24 h. Cells were recovered by ¯ooding the plates with 2á5 ml TN buffer (0á01 mol l±1 Tris HCl, pH 7á6 and 1 mol l±1 NaCl) and centrifuging (10 min, 5000 g). Bacterial cells were washed twice with TN buffer and then resuspended in 1 ml TN buffer. Washed cells were adjusted to an O.D. of 4 (at 600 nm) in TN buffer and mixed with an equal volume of 1% agarose (Eurobio, Les Ulis, France) solution in TN buffer. The mixture was distributed into insert moulds (100 ll in each mould; Bio-Rad, Ivry sur Seine, France) and then cooled for 10 min at 4°C. The plugs were incubated for at least 5 h at 37°C in a solution of lysozyme (0á01 mol l±1 Tris HCl, pH 8, 0á001 mol l±1 EDTA, 1 mol l±1 NaCl, 0á5 g l±1 N-lauryol-sarcosine and 10 g l±1 lysozyme) and then transferred in a proteinase K solution (0á5 mol l±1 EDTA, pH 9, 10 g l±1 N-lauryol-sarcosine and 1 g l±1 proteinase K) and incubated at 50°C for at least 40 h. They were then washed twice with TE buffer (0á01 mol l±1 Tris HCl and 0á001 mol l±1 EDTA) for 30 min at room temperature. The proteinase K was then inactivated with a

solution of 0á002 mol l±1 PefablocÒ (Boehringer Mannheim, Meylan, France) in TE buffer for 2 h at 37°C (Benjamin and Datta 1995). Finally, the plugs were washed twice with TE buffer for 30 min at room temperature. The plugs were divided into four slices and stored in TE buffer at 4°C. DNA restriction enzyme analysis-pulsed ®eld gel electrophoresis One quarter of a plug was used for endonuclease digestions in separate reactions with 60 units ApaI (Roche Diagnostics, Meylon, France) or 40 units SmaI under the conditions recommended by the manufacturer, in a ®nal volume of 100 ll and for at least 5 h of incubation at 25°C. The plugs were then cast in 1á2% agarose prepared with 0á5 ´ TBE buffer (0á045 mol l±1 Tris, 0á045 mol l±1 boric acid and 0á001 mol l±1 EDTA; Eurobio). The resulting DNA fragments were separated by PFGE using a CHEF DR III system (Bio-Rad). The electrophoresis for ApaI was performed at 14°C, 7á6 V cm±1, a ®rst pulsed time ramp from 15 to 35 s for 7 h and then a second from 2 to 20 s for 13 h and for SmaI at 14°C, 7á6 V cm±1, with one ramp from 2 to 20 s for 20 h. The resulting DNA patterns were visualized after staining in ethidium bromide solution (0á5 mg l±1) under short wave u.v. light. The images were captured by a video system (gel DOC 1000 system; Bio-Rad). The estimation of the size of fragments and the comparison of patterns belonging to different isolates were performed using Molecular Analyst Software Fingerprinting (Bio-Rad). Similarities between the pro®les, based on band positions, were derived by the Dice correlation coef®cient with a maximum position tolerance of 1á2% (Struelens 1996). Dendrograms were constructed to re¯ect the similarities between isolates in the matrix. Isolates were clustered by the Unweighted Pair Group Method using Arithmetic average (Struelens 1996). Serotyping Serotyping was performed using sera (Eurobio) according to the method described by Seeliger and HoÈhner (1979). For the O antigen, isolates were grown on brain heart infusion agar (BHI; 10 g l±1 agar) for 24 h at 37°C. A positive reaction consisted of an agglutination of the isolate after at least 1 min of mixing in one drop of serum. The polyvalent sera (OI/OII and OV/OVI) were tested ®rst. 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, OVI, OVII, OVIII and OVIX serums were tested. For the H antigen, isolates were grown in BHI half-agar (4á5 g l±1 agar) for 48 h at room temperature by sticking into the agar. Isolates at the far end of the growth were then grown on BHI broth for 24 h at room temperature. A volume

ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 91, 888±899


(1á2 ml) of the culture was then mixed with 1á2 ml 9 g l±1 NaCl. In separate tubes, two drops of sera (A, AB, C, D and nothing) were mixed with 0á5 ml mixed culture. The ®ve tubes were incubated at 50°C for 2±3 h. A positive result showed an agglutination at the bottom of the tube. The serotyping was then determined according to the table of Larpent (1995). RESULTS Status of Listeria monocytogenes contamination in both plants Among the 468 samples, 65 of 313 in plant A and 33 of 155 in plant B were L. monocytogenes positive (Table 1). The environment and the equipment were not very contaminated (18á3 and 16á4% in plants A and B, respectively). Nevertheless, the ®nished products were more frequently contaminated (40 and 35á7% for plants A and B, respectively). There was an increase in contamination in plant A with regard to the level of contamination of the raw materials (19á5%) while the contamination of raw materials (36%) in plant B was similar to that determined in the ®nished products. Analysis of the patterns obtained by ApaI-restriction analysis-pulsed ®eld gel electrophoresis Five hundred and two isolates were genotyped by ApaIREA-PFGE. The results showed 37 different ApaI genotypes (named A1±A37) but three isolates were resistant to this restriction enzyme. Figure 1 represents the genetic relationships and the distribution of the 37 pro®les obtained. Four major ApaI genotypes, A12, A20, A5 and A11, were predominant with 33á3, 11, 8 and 8% of the genotyped isolates, respectively. A large diversity of the ApaI genotypes is obvious. However, some pro®les showed great similarity (at least 0á85) and, therefore, nine clusters (named AA±AI) could be de®ned (Fig. 1). The main cluster AH (55á3% of the collection) was composed of seven ApaI genotypes (A11±A17) and included the major ApaI genotype A12 (33á3% of the collection); these pro®les were quite similar and the main differences were due to the absence or presence of bands of 70±140 kb in length. Cluster AE contained three ApaI genotypes (A19, A20 and A28) which were very similar; it represented 11á4% of the genotyped isolates. Cluster AB also gathered two similar ApaI genotypes (A5 and A6) representing 8á4% of the isolates. Cluster AD contained three ApaI genotypes (A8, A18 and A26); the main differences were due to bands of 50±97 kb in length. Clusters AA, AC, AG and AI represented less than 5% of the collection.

891

Analysis of the patterns obtained by SmaI-restriction analysis-pulsed ®eld gel electrophoresis Following typing by ApaI-REA-PFGE, isolates were selected for the second genotyping method. At least one isolate per sample and per ApaI genotype was chosen for analysis by SmaI-REA-PFGE, thus, if isolates recovered from the same sample showed the same pattern by ApaIREA-PFGE, only one was randomly selected. Therefore, 145 isolates were analysed, showing 35 different SmaI genotypes (named S1±S35). Figure 2 represents the genetic relationships and distribution of the 35 pro®les obtained. Three major different SmaI genotypes were observed, S9, S4 and S14 with, respectively, 35á9, 8á3 and 5á5% of the isolates. As for the ApaI genotypes, a great diversity was present and, as some SmaI genotypes showed great similarity (at least 0á85), eight clusters (named SA±SH) were de®ned (Fig. 2). The main cluster SF (42á6% of the genotyped isolates) was composed of ®ve SmaI genotypes (S9±S13) with one major genotype S9 (35á9% of the isolates); the major differences were due to the absence or presence of three bands (120, 145á5 and 162 kb). Cluster SB contained only two closely related SmaI genotypes (S4 and S5) representing 9% of the isolates. Cluster SE (7á6% of the isolates) grouped three SmaI genotypes (S14, S25 and S26) with differences in lower DNA fragments (length below 100 kb). The other clusters represented less than 5% of the genotyped isolates. Combination of the two genotypic methods In order to discriminate the L. monocytogenes isolates more precisely, the pro®les obtained using the two genotyping methods were associated. The combination of both typing methods resulted in 50 different combined genotypes (named T1±T50) (Table 2). One ApaI pro®le can result in two or three different SmaI pro®les, e.g. A13 is divided into S9 and S10 to give the combined genotypes T17 and T18 (Table 2). Correspondingly, one SmaI pro®le can result in different ApaI pro®les, e.g. S9 is divided into A11, A12, A13 and A14 to give the combined genotypes T14, T16, T17 and T19 (Table 2). Comparing the clusters obtained for ApaI and SmaI, there were three notable occurrences. The ®rst concerned isolates that were grouped in one cluster by ApaI as well as by SmaI: cluster AF matched with cluster SH and cluster AH with cluster SF. In the second, isolates that belonged to the same genotype by one method were divided in different genotypes by the other typing method: SmaI genotype S15 was distributed into cluster AC and ApaI genotype A32 into cluster SA. The last concerned



0·4 0·5 0·6 Similarity

0·7 0·8 0·9 1·0

M

485 436·5 388 339·5 291 242·5 194 145·5

97

48·5

AE

AD

1/2a-3a-1/2c-NT

AC

AB

1/2b-3b

AA

0·6

AF

A1

AG

0·2 2·4 0·4 8 0·4 0·2 0·4 0·6 0·2 2·4 4·2 0·6 0·2 11 0·2 2·2 1 0·2

0·2 1·8 5·8 2 8 33·3 2·4 3·6 0·2 1·4 0·6 2·4 0·2 0·4 0·8 0·2 0·2 0·8 4a-4ab-NT

AH

A2 A3 A4 A5 A6 A7 A9 A10 A19 A35 A8 A18 A26 A20 A28 A24 A25 A22

A23 A21 A13 A14 A11 A12 A16 A17 A15 A37 A27 A30 A36 A33 A31 A34 A29 A32 AI

Profile Percent of isolates Cluster

Serotypes

Fig. 1 Schematic representation, genetic relationship and distribution of the 37 ApaI genotypes of the Listeria monocytogenes isolates. The matrix was subjected to cluster analysis by the unweighted pair group average linkage analysis clustering method using Dice's index (maximum tolerance 1á2%). The serotypes of the isolates are also indicated. M, Lambda ladder marker in kb

isolates that were gathered in one cluster by one method but corresponded to different clusters and genotypes by the other method. Two examples can illustrate this occurrence. The ®rst concerned cluster AC which is distributed by SmaI genotyping into clusters SD and SG. The second corresponded to cluster SC which included cluster AA and ApaI genotype A7.

Serotyping Isolates were selected for serotyping as for typing by SmaIREA-PFGE (at least one isolate per sample and per combined genotype; 133 isolates were serotyped). One major serotype was observed, 64% of the isolates being from 1/2a serotype. Serotypes 1/2b, 4a, 1/2c, 3b, 3a and 4ab



893

0·4 0·5 0·6 Similarity

0·7 0·8 0·9 1·0

M 388 339·5 291 242·5 194

145·5

97

48·5 S32 S20 0·7 4·1

0·7 0·7 0·7

1/2b - 3b

S35 S23 S19

SB

0·7 0·7 2·1

1/2a - 3a - 1/2c - NT

SC

S24 S29 S34

SD

0·7 0·7 0·7 8·3 1·4 3·5

0·7 2·7 2·1 0·7 0·7 5·5

0·7

SE

S21 S18 S5 S4 S6 S7

S8 S1 S2 S33 S25 S14

35·9 2·1 0·7 SF

S26

S9 S10 S13

2·1 2·1 2·7

4·2

0·7 0·7 1·4 4·2

2 1·4 1·4

SG

S11 S12 S15

S16

S17 S3 S28 S27

S31 S30 S22

SH

Profiles

Percent of isolates

Cluster

SA

4a - 4ab -NT

Serovar

Fig. 2 Schematic representation, genetic relationship and distribution of the 35 SmaI genotypes of the Listeria monocytogenes isolates. The matrix was subjected to cluster analysis by the unweighted pair group average linkage analysis clustering method using Dice's index (maximum tolerance 1á2%). The serotypes of the isolates are also indicated. M, Lambda ladder marker in kb

represented, respectively, 14, 7á5, 4, 2, 1á5 and 1% of the chosen isolates. However, 6% of the isolates were not serotypable either because they did not react with the O or H factor antisera or with either of them.

Serotypes and pro®les were then linked (Table 2). The analysis of the distribution of ApaI genotypes and serotypes resulted in three genomic divisions (Fig. 1). The ®rst division contained clusters AA and AB and ApaI genotypes A4 and A7.



Table 2 Characteristics of the different combined genotypes (ApaI and SmaI) and associated serotypes Categories of combined genotypes*

Combined genotypes ApaI

SmaI

I

T14

A11

S9

T16 T17

A12 A13

S9 S9

T18 T28

A13 A21

S10 S16

T19 T23 T3 T4 T21 T24 T25 T45 T38 T5

A14 A16 A3 A4 A15 A17 A18 A35 A31 A5

S9 S12 S7 S6 S9 S9 S1 S31 S23 S4

T27 T37

A20 A30

S14 S20

T13 T31 T32 T1

A10 A24 A25 A1

S15 S27 S28 S7

T2 T6 T7 T8 T9 T10 T11 T12 T15 T20 T22 T26 T29 T30 T33 T34 T35 T36 T39 T41

A2 A5 A6 A7 A8 A8 A8 A9 A11 A14 A16 A19 A22 A23 A26 A27 A28 A29 A31 A32

S7 S5 S4 S8 S1 S2 S16 S15 S10 S13 S11 S3 S16 S17 S2 S22 S25 S18 S24 S34

T42 T43

A33 A33

S26 S29

II III

IV

V

VI

Genotypes using

Percentage Serotypes of isolates 1/2a 3a 1/2a 1/2a 1/2b 1/2a 1/2c NT 1/2a 1/2a 3b 1/2b ND 1/2a 1/2a ND ND 1/2b 1/2a 1/2a 4a 4ab NT 1/2a 1/2a 1/2a 3b 1/2b 1/2b 1/2b 1/2b 1/2b 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2c NT 1/2a NT ND 4a NT 4a NT 1/2b ND

4 1á3 18á8 8á1 0á7 1á3 2 1á3 2 2 1á3 1á3 0á7 0á7 1á3 2 0á7 6á7 0á7 5á4 2á7 0á7 0á7 1á3 4 2 0á7 0á7 0á7 0á7 1á3 0á7 1á3 1á3 0á7 1á3 0á7 0á7 2 0á7 0á7 0á7 0á7 1á3 0á7 0á7 1á3 0á7 0á7 0á7 0á7

Table 2 (Continued) Categories of combined genotypes*

Genotypes using Combined genotypes ApaI SmaI

Percentage Serotypes of isolates

T46 T47 T48 T40 T44 T49 T50

4a 1/2a ND 4a NT 1/2c 4a

A36 A37 NT A32 A34 NT NT

S35 S30 S4 S21 S19 S32 S33

0á7 1á3 0á7 0á7 0á7 0á7 0á7

*Categories: I, genotypes present in both plants; II, genotypes present only in plant B in different workrooms; III, genotypes restricted to one workroom and one sample in plant B; IV, genotypespresent only in plant A in different workrooms; V, genotypes present in plant A in one workroom and on different samples; VI, genotypes restricted to one sample in plant A. NT, Untypable isolates; ND, not determined.

These genotypes were either of serotype 1/2b or 3b. The second division gathered together clusters AC±AH and ApaI genotypes A19, A35 and A37, all of serotypes 1/2a, 3a, 1/2c or untypable. The last division grouped cluster AI and ApaI genotypes A29, A31, A32 and A34. These genotypes were either of serotype 4a, 4ab or untypable. The combination of SmaI genotypes and serotypes also resulted in three genomic divisions (Fig. 2). The ®rst division gathered together cluster SA and SmaI genotypes S19, S20, S23, S24, S29, S32 and S35. These genotypes were either of serotype 4a, 4ab or untypable. The second division included clusters SB and SC and the SmaI genotype S18 and showed either serotype 1/2b or 3b. The last division grouped clusters SD±SH and SmaI genotypes S3, S15, S22, S30, S31 and S33. They were all of serotypes 1/2a, 3a, 1/2c or untypable. When each combined genotype was considered, it was also observed that, generally, one combined pro®le corresponded to one serotype (Table 2). However, some pro®les could have different serotypes. For example, the combined genotype T14 presented two serotypes, 1/2a and 3a. For a few pro®les, isolates could either have a serotype or did not possess one. This was the case for the combined genotypes T28, T37 and T41. In the same way, three combined genotypes (T30, T34 and T44) were represented by only one isolate which did not show any serotype. Analysis of the distribution of the combined types in the two different plants Forty-one combined genotypes were observed in plant A, 37 during processing operations and eight after the cleaning and disinfecting procedures. Four of these latter genotypes (T40, T44, T49 and T50) were only detected after the



895

Table 3 Distribution of the combined genotypes in the different workrooms of plants A and B in relation to the different samples Workrooms reception Samples Plant A Environment

Equipment

T5, T9, T11, T35, T39, T41, T42, T46, T48, T22, T27, T34, T37. T40, T44, T49, T50 T18, T37

Raw meat Processed meat Product D0 Product DSL

T15, T17, T33, T43, T47 ND ND ND

Plant B Environment Equipment

T14, T16, T24 No L. monocytogenes

Raw meat Processed meat Product D0 Product DSL

T19 ND ND ND

Meat processing

Product processing

T17

T5, T13, T17, T20, T26, T27, T28, T31, T32, T37

T1, T2, T5, T7, T8, T16, T17, T28, T29 T30 No recovered isolate ND ND

T5, T6, T10, T12, T13, T14, T31, T36

No L. monocytogenes T3, T14, T16, T17, T18, T19, T21, T23, T24, T25, T28, T45 T16 No L. monocytogenes ND ND

ND T17, T31 T5, T32 No recovered isolate No L. monocytogenes T38, T16, T19, T23 ND T16 T16 T4, T14

D0, Product at the end of the production day; DSL, product at the end of the shelf life; ND, not done. Normal type, Genotypes found during plant operations; bold type, genotypes present in both plants A and B; italic type, genotypes detected only after cleaning and disinfecting procedures; underlined type, genotypes present both during plant operations and after cleaning and disinfecting procedures.

cleaning and disinfecting procedures. Fourteen combined genotypes were found in plant B during processing, three of which were also recovered after the cleaning procedures. In contrast to plant A, no genotype was found only after the cleaning operations. Five combined genotypes (T14, T16, T17, T18 and T28) were isolated in both plants A and B during plant operation. Two of these were also isolated after the cleaning procedures in plant B. The categories of the different combined genotypes were de®ned according to their distribution in plants A and B (Table 2). The ®rst category corresponded to the ®ve combined genotypes present in both plants A and B, which were found in at least one workroom and on at least one sample. Categories II and III represented combined genotypes present only in plant B, largely spread in different workrooms (category II, genotypes T19 and T23) or only detected in one workroom and on one sample (category III, genotypes T3, T4, T21, T24, T25, T38 and T45). Categories IV, V and VI concerned those found in plant A in different workrooms, in one workroom but on many samples and restricted to only one sample, respectively. Table 3 presents the distribution of the combined genotypes observed in the different workrooms for plants A and B. The combined genotypes of category I were generally spread

over all the workrooms (environment of reception, equipment and raw meat in the processing area and transformed meat and product at the end of the production), except T18, which was not found in meat- and product-processing areas in plant A, and T28, which was detected in the meatprocessing areas in both plants and in the product-processing area in plant A. Contrasting situations appeared in plant A when the distribution of the 41 combined genotypes was analysed. A great diversity was observed in the environment of the reception and product-processing areas and also on the equipment of the meat-processing area. Moreover, the diversity diminished along the processing operation. From the three combined genotypes of category IV (genotypes present in different workrooms), genotype T5 was present in the three workrooms and on different samples, the environment in the reception area, the equipment in the meatprocessing area and the environment and equipment in the product-processing area. For genotype T31 belonging to category V (genotypes restricted to one workroom), the contamination was limited to the product-processing area (environment, equipment and transformed meat). Some combined genotypes (T40, T44, T49 and T50) were found only after the cleaning and disinfecting procedures, in the



reception area. Other genotypes (T22, T27, T34 and T37) were recovered both during processing and after the cleaning operations in the same workroom. In plant B, diversity was observed on the equipment of the meat-processing area especially during plant operation. The two genotypes T14 and T16 were also detected in the different meat products and were largely spread over all the plant (environment of the reception area and equipment of the meat- and product-processing areas). Genotype T16 was also isolated, after the cleaning operations, on the equipment in the product-processing workroom. Three combined genotypes (T19, T23 and T24) were present in at least two workrooms. The two combined genotypes T19 and T23, belonging to category II, were detected in the meatprocessing area during plant operation and also in the product-processing workroom after the cleaning procedures. Genotype T24 was ®rst found in the environment of the reception area and also detected on the equipment of the meat-processing area. The other genotypes (category III) were generally found on the equipment of the meatprocessing area. The majority (74%) of samples in both plants A and B only presented one combined genotype. Nevertheless, 17á6% of the samples showed two genotypes and 6% contained three genotypes. However, some samples contained up to four different combined genotypes. Two samples presented four different genotypes: a ¯oor in the reception workroom of plant A consisted of genotypes T5, T9, T42 and T48 and a working table in the meatprocessing area of plant B consisted of genotypes T14, T16, T23 and T25. DISCUSSION Two different plants (A and B) were followed: one poultry-processing plant for 1 year and one pork-processing plant for 4 months. In both cases, plants were divided into different workrooms, studied separately and visited at least twice (during plant operation and after the cleaning and disinfecting operations). The 502 isolates of L. monocytogenes were characterized with the enzymes ApaI and SmaI into 50 combined genotypes. We have, therefore, observed great diversity in our collection. We found a higher number of PFGE pro®les than in previous reports: 37 ApaI and 35 SmaI pro®les instead of the 15 ApaI and 13 SmaI genotypes on 115 isolates tested by Destro et al. (1996), 17 ApaI genotypes on 42 isolates (Brosch et al. 1991) and 17 ApaI genotypes on 35 isolates (Buchrieser et al. 1991). The last two studies also combined the pro®les obtained with three enzymes, resulting in only 24 different types whereas our combination of only two enzymes resulted in 50 different types. However, Buchrieser et al. (1991) obtained a greater

diversity with fewer isolates than in the present study with regard to the ratio of genotypes vs studied isolates. Only one study (Brosch et al. 1994) showed a greater diversity, with 87 types for 176 tested isolates, but these isolates came from various collections and sources (human, food and animal) whereas our isolates were only collected from two plants in the raw meat industry. These results also showed that three isolates were not cut by ApaI, despite several attempts, while they were digested by SmaI. Daniellson-Tham et al. (1993) and Brosch et al. (1994) observed the same phenomenon with the same enzyme. Considering the genotype obtained by SmaI for these three isolates, two different observations can be made: (i) for genotype T48, the SmaI genotype was already observed in two different combined types (T5 and T7) and (ii) for combined types T49 and T50, however, the SmaI genotypes (S32 and S33) were only observed for these two isolates. This shows that a resistance to ApaI is rare and probably due to a system of modi®cation present in these isolates. The analysis of the genetic relationship of either ApaI or SmaI genotypes resulted in great diversity. However, some types showed great similarity and a genotype which was de®ned with one enzyme can match a cluster de®ned by the other enzyme. In contrast, one ApaI pro®le can result in different SmaI genotypes and, conversely, one SmaI pro®le can result in several ApaI genotypes. This could be due to a mutation only noticed by one of the two enzymes and the relationship between these isolates is, therefore, very important. These facts were observed in the two plants. It is interesting to note that genotypes T1 and T2 were found in plant A whereas the close genotype T3 was detected in plant B. These three genotypes possessed the same SmaI genotype (S7) and three related ApaI genotypes (A1, A2 and A3) with similarities greater than 85%. Genotypes T9, T10 and T11 also illustrated these observations; they corresponded to the combination of the ApaI genotype A8 and the SmaI genotypes S1, S2 and S16. If genotypes S1 and S2 were closely related, genotype S16 only showed 65% of similarity with S1 and S2. Therefore, genotypes T9 and T10 were closely related. Moreover, they were not found in the same plant (plant A, genotype T9 and plant B, genotype T10). In both cases, these observations supported the hypothesis of clonal origins for genotypes T1 and T2 and T9 and T10, respectively. Therefore, the use of two restriction enzymes showed greater discrimination than the use of a single enzyme. The other observation regarding the genetic relationship was that isolates could be grouped in the same cluster by both methods. For example, cluster AH was the major cluster by the ApaI method and corresponded to the major cluster SF by SmaI. In both clusters, we observed one major genotype: (i) A12 representing 33á3% of the



genotyped isolates in cluster AH and (ii) S9 representing 35á9% of the genotyped isolates in cluster SF. In cluster AH, we also observed two secondary genotypes, A11 and A13, representing, respectively, 8 and 5á8% of the genotyped isolates. The other genotypes were less important. Moreover, genotypes A11 and A13 were highly similar to genotype A12 (respectively 97 and 92%). In cluster SF, except for genotype S9, all the genotypes were minor. All these isolates were closely related. These results suggested that the secondary and minor genotypes derived from the major genotype by different mutations and may reveal a major clone of L. monocytogenes which could be characterized by the ApaI pro®le AH and the SmaI pro®le SF. The serotyping results showed that different types were present: 1/2a, 1/2b, 1/2c, 3a, 3b, 4a and 4ab, the major serotype being 1/2a. Different studies have shown that only three serotypes (4b, 1/2a and 1/2b) were associated with the majority of sporadic cases of listeriosis but were also present in outbreaks. However, serotype 4b is predominant in most European outbreaks whereas serotypes 4b, 1/2a and 1/2b are equally responsible for epidemic cases in Canada and the USA (Farber and Peterkin 1991; Rocourt and Bille 1997). No serotype 4b isolates were found in this study. Indeed, L. monocytogenes serotype 4b have caused almost all recent outbreaks of foodborne listeriosis. Accordingly, these results indicate that the presence and predominance of both serotypes 1/2a and 1/2b in these pork and poultry industries might be a source for sporadic cases in France. Moreover, Buchrieser et al. (1992) showed that there was no direct correlation between the restriction pro®les using three different enzymes and the different serotypes. Our results con®rmed this observation that six different combined pro®les (T1, T5, T14, T28, T37 and T41) each displayed more than one serotype. Nevertheless, the majority of combined types presented only one serotype. Furthermore, the pro®les which were obtained after cleavage of genomic DNA by ApaI as well as by SmaI resulted in different genomic divisions that were more discriminating: the ®rst included serotypes 4a, 4ab and unserotypable isolates; the second included serotypes 1/2a, 1/2c, 3a and unserotypable isolates and the last included serotypes 1/2b and 3b. Our results con®rm those of Brosch et al. (1994) who observed two major genomic divisions correlated with the serotype. The ®rst genomic division included serotypes 1/2a, 3a, 1/2c and 3c and the second included serotypes 1/2b, 3b, 4b, 4d and 4e. These two genomic divisions were therefore linked with the ¯agellar antigen type. Following this analysis of genotypes, the L. monocytogenes isolates obtained can be divided into four different classes. The ®rst category (I) is composed of isolates collected during processing but also after the cleaning operations (e.g. genotype T16) after an interval of 9 months for plant A

897

and 3 months for plant B. These isolates probably show an adaptation to survival in a food industry plant. This has previously been observed by Giovannacci et al. (1999) in pork slaughtering and cutting plants, Boerlin et al. (1997) in ®sh products and Unnerstad et al. (1996) in a dairy plant. The study of Giovannacci et al. (1999) pointed out the persistence of L. monocytogenes strains over a 1-year period in the environments of two pork plants. Boerlin et al. (1997) noted survival for several months whereas Unnerstad et al. (1996) showed survival over 7 years. Two hypotheses can be expressed: either the cleaning operations are not suf®ciently ef®cient to eliminate these clones or these clones are adapted to animals and continuously enter the plants and ®nd appropriate conditions to survive and eventually grow in the plants. The second category (II and IV) is composed of isolates found only during plant processing operations in different workrooms, at different times. These isolates probably entered continuously into the plant but were eliminated by the cleaning and disinfecting procedures. The third category (V) gathers together isolates found only during processing, in one workroom but on different samples. For example, genotype T31 was recovered in three sample types (environment, equipment and raw meat) during the product processing in plant A. These isolates were probably brought in by the raw meat and contaminated the equipment and environment of the workrooms. The last category (III and VI) is composed of isolates found only once in one workroom. The majority of these isolates can be considered as transitory as they do not survive to the cleaning and disinfecting procedures. However, four genotypes, T40, T44, T49 and T50, were only detected after the cleaning operations but never during processing. These isolates, which were not eliminated by the cleaning and disinfecting procedures, were still present during processing but not recovered, probably because of their low levels. Some pro®les were also common to both plants, which are from two different geographical locations (combined pro®les gathered in category I, Table 2). This was previously observed by Buchrieser et al. (1991) and Boerlin et al. (1997). We can, therefore, postulate that some clones are widely spread. Moreover, when these genotypes were compared with the genotypes obtained by Brosch et al. (1994), isolate 1570 (CDC/G-4484) was very similar to our major genotype A12. This isolate came from the collection of the Center for Disease Control and Prevention (CDC, Atlanta, GA, USA) and had been isolated from food. Its serotype was 1/2a as with the majority of our isolates showing ApaI genotype A12. When our ApaI genotypes were then compared with the genotypes obtained by Giovannacci et al. (1999), their major cluster was quite similar to cluster AH. Furthermore, plants A and B



processed different kinds of meat, poultry meat in plant A and pork meat in plant B. This strengthened the idea that some genotypes might be widely spread in the food industry. The results of the present study allow us to trace the contamination in the two plants, in a similar way to Destro et al. (1996). Some isolates were found during the plant processing operations and after the cleaning and disinfecting procedures. For plant A, contamination was present in 12 different points of the reception area, only in the environment and never on the equipment. A deep cleaning of this area might be necessary. However, two genotypes (T22 and T34) found after cleaning and disinfecting procedures and during plant operation were limited to the reception area. Four genotypes (T40, T44, T49 and T50) were found only after cleaning and disinfecting procedures and were not recovered during plant operation. These genotypes might not be predominant and might be masked by the major genotypes collected. For plant B, the contamination of the entire plant with genotypes T14 and T16 was obvious; these genotypes were found in different places and on different processed meat products. The contamination with three different combined types (T16, T19 and T23) in the product-processing area after the cleaning and disinfecting procedures highlights the problem of the ef®ciency of these procedures. In both plants, some samples were contaminated with up to four different types. However, a majority of samples (74%) contained only one genotype. These types were either closely related or very different. This has already been observed by Margolles and de los Reyes-Gavilan (1998) who found four samples contaminated with two different L. monocytogenes isolates (about 85% of similarity). It is, therefore, of interest to have more than one isolate per sample, when possible, to give better understanding of the spreading. In conclusion, the molecular genotyping methods (i.e. digestions using a restriction enzyme followed by PFGE) helped us to trace contamination by L. monocytogenes in both plants. Some clones were recovered in plants for several months while others were eliminated during the cleaning and disinfecting procedures. Finally, some clones were found only during processing operations but in different workrooms and seemed to enter continuously into the plant. Different genotypes can be associated with poultry as well as pork meat. Therefore, these clones might be associated with meat production. Further studies are required to prove whether these clones are found in other meat plants and meat products. ACKNOWLEDGEMENTS The authors acknowledge Europe (FEDER) for ®nancial support of the molecular assays and the Ultra-propre

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