Article pp.293-303 du Vol.23 n°2 (2003)

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SCIENCES DES ALIMENTS, 23(2003) 293-303

ARTICLE ORIGINAL ORIGINAL PAPER

Phenotypic and molecular characterization of Listeria spp. strains isolated from meat products

in Morocco

M. R. Kriem¹*, J. Rocourt²

RÉSUMÉ

Caractérisation phénotypique et moléculaire des souches de Listeria spp. isolées des produits carnés au Maroc. L’analyse microbiologique de 229 échantillons de viande et produits carnés prélevés au niveau des boucheries et d’abattoir, a permis d’isoler 143 souches de Listeria spp. (Listeria monocytogenes : 33, Listeria innocua : 105, Listeria ivanovii : 4, Listeria seeli- geri : 1). L’incidence totale de Listeria monocytogenes dans la viande et les produits carnés est de 14.4%. Listeria monocytogenes a été isolée dans 10 des 61 échantillons de viande hachée bovine, dans 8 des 59 échantillons de viande hachée chevaline, dans 7 des 22 échantillons des saucisses crues bovine et dans 3 des 29 échantillons des saucisses crues chevalines. Pour les échantillons prélevés au niveau de l’abattoir, Listeria monocytogenes a été trouvée dans 2 des 21 échantillons des poumons bovins, dans 2 des 18 échantillons des diaphragmes bovins et dans un seul des 19 échantillons de la rate chevaline. La caractérisation phénotypique des souches de Liste- ria spp. montre différents sérovars et phagovars. Deux sérovars ont été identifiés pour Listeria monocytogenes, 4b (22 souches, 66,66 %) et 1/2b (11 souches, 33,33 %). Seules trois souches de Listeria monocytogenes (9,09 %) sont lysotypables. Pour les souches de Listeria monocytogenes identiques phénotypiquement, un typage moléculaire par la méthode de macrorestriction d’ADN en champ pulsé a été utilisé. Sept différents profils de restriction d’ADN ont été obtenus en utilisant l’enzyme de restriction ApaI et AscI.

Mots clés :

Listeria monocytogenes ; produit carné ; sérotypage ; lysotypage ; macrores- triction d’ADN en champ pulsé

1. APESA , Institut Agronomique et Vétérinaire Hassan II. B.P. 6202, Rabat-Instituts, Rabat, Morocco.

2. Institut Pasteur, Laboratoire de Listeria, 28, rue du Docteur-Roux, 75724 Paris Cedex 15, France.

* Correspondance : M.R. Kriem.

9-Kriem (293-303) Page 293 Mercredi, 11. juin 2003 1:00 13

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294 Sci. Aliments 23(2), 2003 M. R. Kriem, J. Rocourt

SUMMARY

One hundred forty three Listeria strains (Listeria monocytogenes: 33, Listeria innocua: 105, Listeria ivanovii: 4, Listeria seeligeri: 1) were isolated from 229 samples of meat and meat products collected from butcher's shops and slaughterhouse. The overall incidence of L.monocytogenes in meat and meat products was 14.4%. L. monocytogenes was detected in 10 of 61 ground bovine meats, in 8 of 59 ground equine meats, in 7 of 22 raw bovine sausages, in 3 of 29 raw equine sausages, in 2 of 21 bovine lungs, in 2 of 18 bovine diaphragms and in 1 of 19 equine spleens. The characterization of the isolated Listeria spp. strains by serotyping and phage typing showed different serovars and phagovars. Two serovars of Listeria monocytogenes were recovered, 4b (22 strains, 66.66%) and 1/2b (11 strains, 33.33%). Only 3 strains of Listeria monocytogenes (9.09%) were phage-typable among the thirty-three strains tested. Strains were also studied by DNA macrorestriction patterns obtained after cleavage with ApaI and AscI, and separation by pulsed field gel electrophoresis. Seven different clones of L.monocytogenes could be identified from some strains.

Key words:

Listeria monocytogenes; meat product; serotyping; phage-typing; pulsed field gel electrophoresis

1 – INTRODUCTION

A total of 229 samples of meat and meat products were collected on two sites: butcher's shops and slaughterhouse to determine the frequency of Liste- ria contamination in these products. These results have been previously pub- lished (KRIEM et al., 1998).

The purpose of the present study was to characterize and compare isolates of Listeria spp. by using serotyping, phage typing (methods used in routine) and Pulsed-Field Gel Electrophoresis (PFGE) to determine whether the strains of Listeriamonocytogenes belonged to one or several clones.

However, the phenotypic characterization has proven insufficient, serotyping not being discriminating enough and phage-typing being available for less than 70% of the strains (ROCOURT et al., 1985).

To overcome these difficulties, molecular typing has been used successfully to characterize Listeriamonocytogenes (BROSCH et al., 1991; BUCHRIESER et al., 1993; FARBER and ADDISON 1994; MAZURIER et al., 1992). By use of PFGE, large fragments are generated by digesting DNA with low-frequency cutting restriction enzymes. This method has shown good discriminatory power, is easy to interpret, and has permitted subtyping of Listeria monocytogenes strains.

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Phenotypic and molecular characterization of Listeria spp. 295

2 – MATERIALS AND METHODS

2.1 Genus and species identification

All isolates were biochemically characterized by recommended procedures (WEAVER,1989). Five or more black colonies (aesculin positive) from plates of Palcam medium (Merck), were tested for catalase activity, and tumbling motility at room temperature. Small, coccoid, Gram-positive rods, with blue grayish reflection when using oblique lighting, were inoculated in Columbia agar (Biokar) for further tests.

Species identification of Listeria isolates was carried out using the Api Liste- ria strips (BioMérieux, Marcy l’Etoile, France) with haemolysis on blood agar.

2.2 Phenotypic typing

Serovar determination was performed as described by SEELIGER and HÖHNE, (1979) and phage typing was done using the international set of phages (ROCOURT et al., 1985).

2.3 Molecular characterization

Preparation of genomic DNA was done according to BROSCH etal., (1996).

DNA of in situ-lysed bacterial cells in chilled agarose-plugs was cleaved by the rare cutting restriction endonucleases ApaI (Boehringer) and AscI (Biolabs), and the resulting fragments were subjected to pulsed-field gel electrophoresis (field inversion) at 250 v under various conditions as described by BROSCH et al., (1996).

3 – RESULTS AND DISCUSSION

3.1 Identification

The identification of species of Listeria using the Api Listeria strips, reveled that Listeria innocua was the most often identified species present in butcher's samples, 98 of 131 Listeria spp. (74.8%). Twenty-eight of 131 Listeria spp.

(21.37%) contained Listeria monocytogenes.Listeria ivanovii was found on four instances (3.05%) and one as Listeria seeligeri (0.76%). Listeria monocytogenes was identified along with Listeriainnocua on three occasions in ground equine meat samples (Table I).

With regard to Listeria species isolated from samples of slaughterhouse, Lis- teria innocua was found from 7 of 12 Listeria spp. (58.33%), while Listeria monocytogenes was identified on five occasions (41.66%) Table II.

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Table I

Phenotypic characterization of Listeria spp. strains isolated from meat products.

(Samples from butcher's shops).

Tableau I

Caractérisation phénotypique des souches de Listeria monocytogenes isolées des produits carnés (Échantillons des boucheries).

product species (Nb. of strains) serovars (Nb. of strains)

phagovars (Nb. of strains)

Ground bovine meat

L.monocytogenes (10) 4b (9) NT (9)

1/2b (1) 1967: 4477 (1)

L.innocua (37) 6a (24)* NT (24)

6b (16)* 4211:1090:4276:4277 (3)**

16:4211:4292:187 (2) 1090:42 (2) 4286:5337:4277:3665 (2)**

1090:4277 (3)**

NT (8)**

US (1)* NT (1)

L.ivanovii (2) 5 (2) ND (2)

Raw bovine sausage

1/2b (1) NT (1)

L.innocua (15) 6a (12)* NT (12)

6b (5)* 16:4211:4286:1090:5337:4292 (2)**

4286:5337:4277:3665 (2)**

16:4211:4292:187 (2)**

NT (1)**

L.seeligeri (1) 1/2b (1) 1967:4477 (1)

Ground equine meat

L.monocytogenes (8) 4b (4) NT (4)

1/2b (1) 1967:4477 (1)

1/2b (3) NT (3)

L.innocua (29) 6a (20) NT (20)

6b (9) 4211:1090:4276:4277 (2) 4286:5337:4277:3665 (1) 1090:4277 (1) NT (5) Raw equine

sausage

1/2b (1) 1967:4477 (1)

L.innocua (17) 6a (10) NT (10)

6b (7) 1090 (1)

4286:5337:4277:3665 (1) 16:4211:4286:1090:5337:4292 (2) 16:4211:4292:187 (1) NT (2)

L.ivanovii (2) 5 (2) ND (2)

Total (131) (137) (143)

NT: Non-phage-typable US: undesignated serovar ND: not done

* Six positive samples contained both serovars 6b and 6a/US ** Three positive samples, everyone contained three different phagovars 9-Kriem (293-303) Page 296 Mercredi, 11. juin 2003 1:00 13

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Phenotypic and molecular characterization of Listeria spp. 297

3.2 Phenotypic typing

The distribution of Listeria spp. according to their serovars and phagovars is given in Tables I and II.

Only two serovars of Listeria monocytogenes were recovered among the 13 described by SEELIGER and HÖNE (1979), 4b (22 strains 66.66%) and 1/2b (11 strains 33.33%).

Most of serovar 4b (21 strains) was found from samples collected at butcher's shops, whereas serovar 1/2b was present in those samples for seven instances. Among samples from slaughterhouse, four strains belonged to serovar 1/2b and one to serovar 4b. Therefore, it is interesting to note that serovar 4b, which is frequently responsible for human infections, was detected in a large variety of samples and with high frequency (66.66%). This contrast with previous studies where 4b strains were rarely isolated from meat products (COMI et al., 1992; NICOLAS et al., 1989; TRÜSSEL, 1989).

The Listeria innocua isolates belonged to serovars 6a, 6b and undesignated one. Serovar 6a (62.83%) was more frequent among Listeria innocua isolates than serovar 6b (36.28%). On ninety-nine occasions, either serovar 6a or 6b were identified as the only serovar present in the Listeria positive samples. On seven occasions, both serovars (6a and 6b) were found (ground bovine meat:

3 samples, raw bovine sausage: 2 samples and bovine lung: 2 samples), and on one occasion serovar 6b was isolated with undesignated serovar (ground bovine meat: 1 sample).

Table II

Phenotypic characterization of Listeria spp. strains isolated from meat products.

(Samples from Slaughterhouse).

Tableau II

Caractérisation phénotypique des souches de Listeria monocytogenes isolées des produits carnés (Échantillons de l’abattoir).

product species (Nb. of strains) serovars (Nb. of strains)

phagovars (Nb. of strains)

Bovine lung L.monocytogenes (2) 4b (1) NT (1)

1/2b (1) NT (1)

L.innocua (4) 6a (3)* NT (3)

6b (3)* 16:4286:5337:3665 (1)

16:1090:4292:3665:2528:1807 (1) NT (1) Bovine

diaphragm

L.monocytogenes (2) 1/2b (2) NT (2)

L.innocua (2) 6a (1) NT (1)

6b (1) 4211 (1)

Equine spleen L.monocytogenes (1) 1/2b (1) NT (1)

L.innocua (1) 6a (1) NT (1)

Total (12) (14) (14)

NT: Non-phage-typable US: undesignated serovar ND: not done

* Two positive samples contained both serovars 6b and 6a.

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Strains resistant to the phages employed in this set belonged to serovar 6a of Listeria innocua and serovar 4b of Listeria monocytogenes.

Only three strains of Listeria monocytogenes (9.09%) were phage typable among the 33 strains tested. The serovar of the three strains was 1/2b and we were able to distinguish only one phagovar (Table I and II). Overall typability of Listeria innocua was 25.21%; nine phagovars were detected among the 48 strains of serovar 6b tested. Strains of serovars 6a and undesignated serovar were resistant to phages. Among three samples (ground bovine meat: 2 samples and raw bovine sausage: 1 samples), three different phagovars of serovars 6b were detected from each sample (Table I).

The percentages of phage typable of Listeria monocytogenes and Listeria innocua are lower than those reported by other workers, from 52%

(ROCOURT et al., 1985) to 78% (AUDURIER and MARTIN, 1989) for sero- groups 4b and 1/2b, and 61.7% for Listeria innocua (ROCOURT et al., 1982).

As previously observed, the lytic spectrum of each phage was restricted to strains of the parent serogroup of each species of Listeria spp. (ROCOURT et al., 1985; ROCOURT et al., 1982). An exception for one strain of Listeria seel- igeri which shared the same serovar and the same lytic phage with Listeria monocytogenes (Table I).

Some authors reported very high specificities of the phages, with respect to the species and serovars of the lytic action (AUDURIER and MARTIN, 1989;

ROCOURT et al.,1985; and ORTEL, 1978).

Listeria monocytogenes strains of the same serovar 1/2b and phagovar (1967:

4477) were recovered from ground equine meat and raw equine sausage purchased from the same butcher's shops at the same time, suggesting an unique source of contamination. Listeria innocua strains serovar 6b and phagovar (4286: 5337: 4277:

3665) were also isolated both from ground equine meat and raw equine sausage.

Despite its usefulness, phage typing is hampered by the non-typeability of some strains, the percentage of non-typeable strains may vary according to the origin of the strain (LŒSSNER, 1991). Isolates from meat and poultry have revealed higher degrees of resistance than did strains stemming from cheese and other dairy products with another set of phages (LŒSSNER and BUSSE, 1990).

3.3 Molecular characterization

The distribution of Listeria monocytogenes according to their serovars, phagov- ars and profiles of DNA restriction with ApaI and AscI is given in Table III.

Considering the phenotypic results showing a low number of serovars and a large number of non-typable strains, for a better characterization PFGE was used. The results of DNA macrorestriction patterns are described in Fig 1 and 2.

According to the number and size of the fragments generated, 2 profiles could be differentiated for Listeria monocytogenes serovar 4b strains with ApaI and 2 profiles with AscI.

Within the 7 strains of Listeria monocytogenes serovar 1/2b and non-typa- ble, we could determine 4 different profiles with ApaI and 3 with AscI. Only one clone was detected among Listeria monocytogenes serovar 1/2b phagovar 1967: 4477 with ApaI and AscI (CLIP 77195 and 77201, Fig 1 and 2).

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The two 4b strains isolated from ground equine meat and raw equine sausage samples collected at the same butcher’s shops, shared indistinguishable DNA patterns. Since the two samples were of the same site at the same day, thus a common source of contamination is reasonable (CLIP 72288 and 72294).

Two strains isolated from ground equine meat and raw equine sausage in the same butcher’s shops considered to be similar as inferred from their serovar (4b) and non-typable lysovar, were characterized by different DNA restriction patterns, suggesting two different sources of contamination (CLIP 77300 and 77171).

Molecular typing showed a greater heterogeneity among the 15 strains of Listeria monocytogenes. While serotyping divided the strains in two serovars, and phage typing allowed to detect 1 phagovar, DNA macrorestriction patterns allowed to distinguish 7 different clones.

These findings emphasize the need to characterize several isolates from the food sample, because it could harbor different clones of Listeria monocy- togenes (DANIELSSON-THAM et al., 1993). It is well documented that mixed populations of Listeria species (BERGANN, 1990; BREER and SCHOP- FER,1988; OZARI and STOLLE,1990) as well as mixed populations of Listeria monocytogenes serovars may contaminate a single food sample (JOHNSON et al., 1988; PINI and GILBERT,1988).

Table III

Molecular characterization of Listeria monocytogenes strains isolated from meat products.

Tableau III

Caractérisation moléculaire des souches de Listeria monocytogenes isolées des produits carnés.

CLIP product Sérovars phagovars Profiles of

ApaI

DNA restriction AscI 72281

72288 72294 77300 77171 77185 77195 77201 77466 77467 77479 77480 77481 77482 72286

GEM GEM RES GEM RES BL GEM RES GEM GEM BL BD BD ES GEM

4b 4b 4b 4b 4b 4b 1/2b 1/2b 1/2b 1/2b 1/2b 1/2b 1/2b 1/2b 1/2b

NT NT NT NT NT NT 1967: 4477 1967: 4477

NT NT NT NT NT NT NT

1 1 1 1 2 2 3 3 4 4 4 4 5 6 7

1 1 1 1 2 2 3 3 4 4 4 4 4 5 6 CLIP: Collection Listeria Institut Pasteur; GEM: Ground Equine Meat; RES: Raw Equine Sausage;

BD: Bovine Diaphragm; ES: Equine Spleen; BL: Bovine Lungs; NT: Non-phageTypable.

CLIP: Collection Listeria Institut Pasteur; VHC: Viande hachée chevaline; SCC: Saucisse crue chevaline;

DB: Diaphragme bovin; RT: Rate chevaline; PB: Poumon bovin; NT: Non-Lysotypable.

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The results of this study confirm also, that PFGE should be used with serotyping for a better characterization (at least 7 different genomic clones within the 15 strains of serovars 4b and 1/2b) and allowed to characterize all the isolates, including the 13 non-phage typable strains.

There was no correlation between the restriction profiles and the different phagovars: some strains of Listeria innocua serovar 6b ( phagovar 4211: 1090:

4276: 4277 and non-phage typable), shared the same restriction profiles performed by the two restriction endonucleases (data not shown).

In the same context, some strains of serovars 1/2a, 1/2b and 1/2c shared the same DNA patterns with ApaI, SmaI and NotI (BUCHRIESER et al., 1991).

Regarding the strains from slaughterhouse, three samples of bovine diaphragm, bovine lung and equine spleen shared the same clone. The three samples were of the same site; this suggest that a common source of contamination during slaughtering, handling, cutting, processing or storage could have occurred.

Figure 1

DNA macrorestriction patterns of L.monocytogenes strain (serovars 4b and 1/2b) after cleavage with ApaI and PFGE.: lane 1, profile m4/1 (CLIP 72281); lane 2, profile m4/1 (CLIP 72288); lane 3, profile m4/1 (CLIP 72294) lane 4: lambda DNA- concatamers; lane 5, profile m4/1 (CLIP 77300); lane 6, profile m4/2 (CLIP 77171);

lane 7, profile m4/2 (CLIP 77185); lane 8: lambda DNA-concatamers; lane 9, profile m2/3 (CLIP 77195); lane 10, profile m2/3 (CLIP 77201); lane 11, profile m2/4 (CLIP 77466); lane 12, profile m2/4 (CLIP 77467); lane 13: lambda DNA- concatamers; lane 14, profile m2/4 (CLIP 77479); lane 15, profile m2/4 (CLIP 77480); lane 16, profile m2/5 (CLIP 77481); lane 17, profile m2/6 (CLIP 77482); lane 18, profile m2/7 (CLIP 72286); lane 19: lambda DNA-concatamers.

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4 – CONCLUSION

This study further demonstrates the need to use both phenotypic and molecular typing methods to characterize Listeria strains. No human cases of listeriosis have been reported yet in Morocco, however the presence of Listeria monocytogenes strain of serovar and lysovar known to be responsible for human infections on meat products, could suggest that this illness may be present in this country. These data emphasize the need of a surveillance system for human cases in Morocco.

Figure 2

DNA macrorestriction patterns of L.monocytogenes strain (serovars 4b and 1/2b) after cleavage with AscI and PFGE.: lane 1, profile m4/1 (CLIP 72281); lane 2, profile m4/1 (CLIP 72288); lane 3, profile m4/1 (CLIP 72294) lane 4: lambda DNA- concatamers; lane 5, profile m4/1 (CLIP 77300); lane 6, profile m4/2 (CLIP 77171);

lane 7, profile m4/2 (CLIP 77185); lane 8: lambda DNA-concatamers; lane 9, profile m2/3 (CLIP 77195); lane 10, profile m2/3 (CLIP 77201); lane 11, profile m2/4 (CLIP 77466); lane 12, profile m2/4 (CLIP 77467); lane 13: lambda DNA- concatamers; lane 14, profile m2/4 (CLIP 77479); lane 15, profile m2/4 (CLIP 77480); lane 16, profile m2/4 (CLIP 77481); lane 17, profile m2/5 (CLIP 77482); lane 18, profile m2/6 (CLIP 72286); lane 19: lambda DNA-concatamers.

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ACKNOWLEDGMENTS

This research was supported in a party by grant from the Centre Interna- tional de Hautes Etudes Agronomiques Méditerranéennes (CIHEAM), PRAD N°

98/20, France. We would like to thank B.Catimel ( Centre National de Référence pour la lysotypie et le typage moléculaire de Listeria, Institut Pasteur, Paris) for technical assistance.

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