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RAPD analysis, serotyping, and esterase typing indicate that the population of Listeria monocytogenes strains recovered from cheese and from patients with listeriosis in Belgium are different
M. Malak, A. Vivier, P. Andr, J. Decallonne, and P. Gilot

Abstract: Populations of Listeria monocytogenes strains isolated in Belgium from cheese and from patients with listeriosis were characterised by randomly amplified polymorphic DNA (RAPD) analysis using two 10-mers primers (OPA-04 and OPA-13). High discrimination levels were obtained with each of these primers alone (discrimination indices (DI) of 0.899 and 0.935 for OPA13 and OPA04, respectively) or in combination (DI of 0.960). The clustering of strains obtained by RAPD was compared with a clustering previously made using serotyping and esterase typing. RAPD allowed the subdivision of each serovar cluster and of most of the clusters determined by the polymorphism of the bacterial esterases. Our analysis indicates that the population of strains of L. monocytogenes found in cheese differs from the one isolated from patients with listeriosis during the same period. Key words: Listeria, typing, RAPD, esterase, serovar. Rsum : Des populations de souches de Listeria monocytogenes isoles en Belgique partir de fromages ainsi que de Notes patients atteints de listriose ont t caractrises daprs le polymorphisme de lADN amplifi au hasard (RAPD) laide de deux amorces 10-mers (OPA04 et OPA13). Une discrimination leve a t obtenue avec chacune des deux amorces utilises sparment (DI= 0,899 pour OPA-13 et DI= 0,935 pour OPA-04) ou ensemble (DI= 0,960). Le regroupement des souches obtenu par RAPD a t compar celui obtenu pralablement par srotypage et par typage des estrases. La technique RAPD a permis de subdiviser chacun des srovars et la plupart des groupes constitus sur la base du polymorphisme des estrases bactriennes. Notre analyse indique que la population des souches de L. monocytogenes isoles du fromage est diffrente de celle isole de patients atteints de listriose durant la mme priode. Mots cles : Listeria, typage, RAPD, estrase, srovar. 887

Listeria monocytogenes is a facultative intracellular bacterium responsible for opportunistic infections leading to abortion, meningitis, and septicaemia in humans and animals. This bacterium is widely distributed in the environment and has been isolated from numerous foodstuffs, such as raw and pasteurized milk, soft cheeses, ice cream, raw meat, egg products, vegetables, and seafood (Doyle 1988; Andr et al. 1990; Massa et al. 1991; Beuchat 1996; Gilot et al. 1997).
Received May 2, 2001. Revision received July 18, 2001. Accepted July 25, 2001. Published on the NRC Research Press Web site at http://cjm.nrc.ca on September 19, 2001. M. Malak, A. Vivier, and J. Decallonne. Laboratory of Microbiology (MBLA) Faculty of Agronomic Sciences, Catholic University of Louvain, Louvain-la-Neuve, Belgium. P. Andr and P. Gilot.1,2 Belgian Reference Centre for Listeriosis, Institute of Hygiene and Epidemiology, Brussels.
1 2

Corresponding author (e-mail: gilot@tours.inra.fr). Present address: Laboratoire de pathologie infectieuse et immunologie, Institut national de la recherche agronomique, Tours, Domaine de lOrfrasire, 37380 Nouzilly, France.

Listeriosis is considered mainly as a foodborne disease. Through contaminated food, people with a weak or suppressed immune system may contract a severe disease that could be potentially fatal (Farber and Peterkin 1991). We previously reported the identification and biochemical characterization of five L. monocytogenes esterases (Gilot and Andr 1995). These enzymes, separated by electrophoresis, showed a number of electrophoretic variants. A typing scheme based on the polymorphism of four of them was developed. This esterase-typing method, in conjunction with serotyping, was used to analyse L. monocytogenes populations recovered in Belgium from cheeses and from human patients with listeriosis (Gilot et al. 1996). That study indicated that whereas the populations could be divided into 23 different subtypes, most of the strains were grouped in only a few of them. The population of strains found in cheeses was also found to be rather different from the one recovered in human patients with listeriosis. The most striking differences were found in the serovar 1/2a-esterase type IB group, which accounted for 44.2% of the strains recovered from cheese, but for 4.2% of the strains isolated from human patients with listeriosis. It is not known if all strains of the
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Can. J. Microbiol. Vol. 47, 2001 Table 1. Distribution of 176 strains of Listeria monocytogenes isolated from cheese and from human patients with listeriosis in Belgium after analysis by serotyping, esterase typing (ET), and RAPD. RAPD Subtype 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 Serovar 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2b 1/2b 1/2b 1/2b 1/2b 1/2b 1/2b 1/2b 1/2b 1/2b 1/2b 1/2c 1/2c 1/2c 3a 3a 3a 4b ET IA IA IA IA IB IB IB IB IB IB IC IC IC IC IC IC IC IC ID IE IE IF IG IH Ii Ii Ii IJ IIA IIA IIA IIA IIA IIA IIA IIG IIC IIC IIC IIC IIC IIC IIC IIC IIF IIF IIF IIE IIE IIIB IIA IIIB IIIB IIB OPA13 1 1 1 2 2 4 21 22 23 29 5 5 11 12 18 18 19 30 1 4 6 5 13 14 25 26 27 24 1 3 15 15 16 17 17 31 7 7 7 7 7 7 7 8 7 8 8 20 31 1 1 1 15 10 OPA04 3 6 40 41 41 39 40 40 40 40 1 2 5 17 6 40 12 12 6 4 4 2 9 18 19 19 43 10 7 20 11 13 8 8 14 19 22 24 25 26 27 28 29 25 32 30 31 36 19 15 11 15 16 34 Origin of strains* Human 2 0 0 0 0 1 0 0 0 3 0 2 1 1 0 0 0 2 0 1 2 2 3 1 1 1 1 2 0 1 0 0 1 1 1 1 4 1 7 2 1 1 1 0 1 0 0 0 3 0 0 0 0 0 Cheese 0 4 3 1 1 0 1 5 18 0 1 0 0 0 1 1 1 0 1 0 0 0 0 0 0 0 0 0 1 0 5 1 0 0 0 0 0 0 0 0 0 0 0 2 0 2 5 2 0 1 1 1 1 12
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Table 1 (concluded). RAPD Subtype 55 56 57 58 59 60 61 62 63 64 65 66 67 Serovar 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b ET IIB IIB IIB IIC IIC IIC IIC IIC IIC IID IID IIH IIIA OPA13 10 28 28 7 7 7 7 8 8 7 8 28 9 OPA04 35 21 23 24 29 37 38 33 34 24 25 37 42 Origin of strains* Human 0 16 1 13 4 2 1 0 0 5 0 1 0 Cheese 3 0 0 0 0 0 0 1 3 0 1 0 1

*Numbers indicate the quantity of isolates found in each subtype. This strain (ATCC 51781) was tentatively typed as 4b, but could not be serotyped with assurance further than serogroup 4.

serovar 1/2a-esterase type IB group have a clonal origin or if only some strains form a subtype with a higher pathogenicity level. Following this hypothesis, the few strains of serovar 1/2a-esterase type IB isolated from human patients with listeriosis should be of the same above-mentioned subtype. On the other hand, serovar 4b-esterase type IIB and serovar 4b-esterase type IIC strains accounted for only 10.6% of the strains recovered from cheeses, but accounted for 40% of the strains of human origin (Gilot et al. 1996). In this work, the heterogeneity of L. monocytogenes strains forming the previously analyzed populations and the clonal origin of strains belonging to the above defined major groups were further analyzed, with a more highly discriminatory method. For that aim, strains were typed by the random amplification of polymorphic DNA (RAPD) method, and relationships between serovars, esterase types, and RAPD patterns were examined. All analysed strains were collected in Belgium during the period 19901992 and were described previously (Gilot et al. 1996). In brief, all the 95 strains isolated in sporatic cases of listerosis, reported to the Belgian Reference Center for listerosis, and 81 of the 178 strains collected from cheeses (45 strains chosen at random among the serovar 1/2a strains and all the strains belonging to the other serovars) during the same period were analysed. Strains were cultured overnight without agitation at 30C in 10 mL of tryptic soy broth (Merck, Darmstadt, Germany) enriched with 0.6% of yeast extract (Merck). The nucleic acids were then extracted following the method of Cancilla et al. (1992), slightly modified as follows. Bacteria from 1.0 mL of the overnight culture were collected by centrifugation (17 400 g for 2 min), resuspended in 500 L of lysis buffer (250 mmolL1 NaCl, 10 mmolL1 Na2EDTA, 10 mmolL1 TrisHCl (pH 8), 105 UmL1 of lysozyme (Fluka, BioChemika, Buchs, Switzerland)), and incubated for 30 min at 37C. Sodium dodecyl sulfate (70 L of a 5% (w/v) solution) was then added, and the tube was incubated at 80C for 5 min. The obtained lysate was mixed with 700 L of phenolchloroform (1:10 (v/v)) by gentle inversion and centrifuged

(15 min at 17 400 g, at 4C). The nucleic acids in the aqueous phase were precipitated by the addition of 700 L cold (20C) isopropanol, incubation for 2 h at 20C, and centrifugation for 20 min at 17 400 g, at 4C. The pellet was finally washed with 300 L of 70% (v/v) ethanol, dried under vacuum for 10 min, and dissolved in 50 L of sterile distilled water. The nucleic acids concentration of the resulting extracts were measured by spectrophotometry (GeneQuant RNA/DNA calculator, Pharmacia-Biotech, Uppsala, Sweden), and all extracts were diluted to stock solutions of 100 gmL1 in sterile distilled water and stored at 20C. For the amplification of L. monocytogenes DNA, two 10mers OPA-04 (5-AATCGGGCTG-3) and OPA-13 (5CAGCACCCAC-3) from kit A of Operon Technologies (Alameda, Calif., U.S.A.) were used separately. These primers were chosen among decamer primers of the kit based on their ability to generate distinguishable and reproducible electrophoresis patterns for L. monocytogenes. nucleic acids amplification was performed in 25 L of a solution containing 100 ng of nucleic acids, 0.2 molL1 primer, 100 molL1 of each deoxynucleotide triphosphate, 50 mmolL1 KCl, 10 mmolL1 TrisHCl (pH 8.8), 1.5 mmolL1 MgCl2, 0.1% (v/v) Triton X-100, and 1 U of DNA polymerase (DyNAZyme II, Finnzymes Oy, Espoo, Finland). The reactions were carried out in a thermocycler (GeneAmpR PCR System 2400, Perkin Elmer, Foster City, Calif.) through the following temperature program: 94C for 4 min, 35C for 1.45 min, and 72C for 1.45 min. This first cycle was followed by 43 cycles of the following profile: 94C for 50 s, 35C for 1.45 min, and 72C for 1.45 min. A cycle of 94C for 50 s, 35C for 1.45 min, and 72C for 10 min was finally used. All amplification products (20 L) were then subjected to electrophoresis for 4 h at 120 V, 200 mA in 1.4% (w/v) agarose gels, containing 0.25 gmL1 ethidium bromide in a TBE buffer (45 mmolL1 TrisHCl, 45 mmolL1 H3BO3, 2 mmolL1 Na2EDTA). The agarose gels were photographed under UV light by a Charge Coupled Device camera: Kappa CCD CF 8/1 (Pharmacia-Biotech), digitalized on a Tagged Image File Format (tiff) picture by the software CF 8/1 DX
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Table 2. Discrimination indices (DI) and number of types distinguished by serotyping (ST), esterase typing (ET), and RAPD in combination with other techniques or not. Food strains Method ST ET ST + ET RAPDOPA13 RAPDOPA13 + ST RAPDOPA13 + ET RAPDOPA13 + ST + ET RAPDOPA04 RAPDOPA04 + ST RAPDOPA04 + ET RAPDOPA04 + ST + ET RAPD(OPA13+OPA04) RAPD(OPA13+OPA04) + ST RAPD(OPA13+OPA04) + ET RAPD(OPA13+OPA04) + ST + ET DI 0.617 0.844 0.849 0.861 0.886 0.890 0.895 0.836 0.868 0.882 0.883 0.916 0.916 0.917 0.918 No. of types 5 12 13 13 17 19 21 18 21 26 27 27 27 28 29 Clinical strains DI 0.638 0.809 0.885 0.758 0.859 0.815 0.891 0.914 0.920 0.933 0.938 0.917 0.924 0.939 0.942 No. of types 3 17 18 20 21 26 27 28 29 32 34 31 34 37 38 Food and clinical strains DI 0.650 0.868 0.899 0.899 0.934 0.922 0.946 0.935 0.937 0.952 0.954 0.960 0.963 0.965 0.965 No. of types 5 20 23 31 36 43 47 43 48 56 59 57 62 64 67

version 3.15 (KAPPA MesstechnikC 1994, PharmaciaBiotech), and RAPD profiles were standardized by using the Diversity One software (Pharmacia-Biotech). Each sample was analyzed in triplicate, and each experiment included a negative control reaction in which DNA was replaced by sterile distilled water. Thirty-one and forty-three reproducible RAPD patterns were obtained using primers OPA13 and OPA04, respectively (Table 1). RAPD OPA13 and RAPD OPA04 enable an important discrimination between strains of identical serovars (Table 1). Most RAPD patterns were only found among strains belonging to the same serovar; nevertheless, some of them are shared by strains belonging to more than one particular serovar. This is the case for RAPD OPA13 profile 8 and for RAPD OPA04 profiles 24, 25, and 29, which are shared by some strains of serovars 1/2b and 4b, but which were never observed within strains belonging to serovars 1/2a, 1/2c, or 3a. A similar situation was found for RAPD OPA13 profiles 1, 15, and 31 and for RAPD OPA04 profiles 11, 15, and 19, which are only displayed by some strains of serovars 1/2a, 1/2c, or 3a and not by any strains of serovars 1/2b and 4b. Using other typing methods, other authors have also found that some profiles are only displayed by strains of serovars 1/2a, 1/2c, or 3a and not by strains of serovars 1/2b and 4b and vice versa (Comi et al. 1997; Graves et al. 1994). One strain (ATCC 51781, subtype 67, in Table 1) was tentatively serotyped as 4b, but typing results beyond serogroup 4 were doubtful. That strain showed very specific RAPD OPA13 and RAPD OPA04 fingerprints. Whereas esterase typing is a more discriminating typing method than is serotyping (Gilot et al. 1996), RAPD OPA13 and RAPD OPA04 allow a further differentiation among strains belonging to most of the determined esterase types. Only 6 (ID, IF, IG, IH, IJ, and IIG) out of the 20 determined esterase types could not be subdivided by RAPD typing. It is worth noting that these latter esterase types were each only formed by very few strains (from one strain to three strains)

(Table 1). Most of the determined RAPD patterns were only found among strains of the same esterase type. This is the case for 22 out of the 31 RAPD OPA13 profiles identified and also for 34 out of the 43 RAPD OPA04 profiles identified (Table 1). The discrimination indices (DI) of RAPD OPA13 and of RAPD OPA04, calculated by the method of Hunter and Gaston (1988), were 0.899 and 0.935, respectively (Table 2). Both RAPD methods were more discriminatory than were serotyping (DI = 0.650) or esterase typing (DI = 0.868). As in our work, all these methods do not permit the differentiation of the same strains, and as no clear relationships between RAPD patterns, serotypes, and esterase types were found, we combined some or all of these methods to further improve the discrimination level. When analyses based on both fingerprints generated by OPA13 and OPA04 were used, the DI value increased up to 0.960. The highest DI value was achieved when all typing methods (RAPD OPA13, RAPD OPA04, esterase typing, and serotyping) were used in conjunction, giving rise to 67 different L. monocytogenes subtypes and to a DI of 0.965. The high discriminatory power of the RAPD method was used to further analyse the L. monocytogenes population. By RAPD OPA13 and RAPD OPA04, we could divide the strains of serovar 1/2a-esterase type IB into six RAPD subtypes. None of the subtypes present in cheeses were isolated from patients suffering from human listeriosis, and none of the subtypes isolated from patients were recovered from cheeses (Table 1). Other large clusters of strains, such as those formed by serovar 4b-esterase type IIB and serovar 4besterase type IIC, could also be divided by RAPD. Again, RAPD types found in cheese were completely different from those isolated from human patients with listeriosis (Table 1). This work reinforces our previous conclusions that the population of strains implicated in sporadic human listeriosis in Belgium and isolated from cheese are different and confirms other epidemiological studies, done in several countries, showing that certain strains found in foodstuffs differ
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887 Doyle, M.P. 1988. Effect of environmental and processing conditions on Listeria monocytogenes. Food Technol. 42: 169171. Farber, J.M., and Addison, C.J. 1994. RAPD typing for distinguishing species and strains in the genus Listeria. J. Appl. Bacteriol. 77: 242250. Farber, J., and Peterkin, P. 1991. Listeria monocytogenes, a foodborne pathogen. Microbiol. Rev. 55: 476511. Gilot, P., and Andr, P. 1995. Characterization of five esterases from Listeria monocytogenes and use of their electrophoretic polymorphism for strain typing. Appl. Environ. Microbiol. 61: 16611665. Gilot, P., Genicot, A., and Andr, P. 1996. Serotyping and esterase typing for analysis of Listeria monocytogenes populations recovered from foodstuffs and from human patients with listeriosis in Belgium. J. Clin. Microbiol. 34: 10071010. Gilot, P., Hermans, C., Yole, M., Gigi, J., Janssens, M., Genicot, A., Andr, P., and Wauters, G. 1997. Sporadic case of listeriosis associated with the consumption of a Listeria monocytogenes Camembert cheese. J. Infect. 35: 195197. Graves, L., Swaminathan, B., Reeves, M., Hunter, S., Weaver, R., Plikaytis, B., and Schuchat, A. 1994. Comparison of ribotyping and multilocus enzyme electrophoresis for subtyping of Listeria monocytogenes isolates. J. Clin. Microbiol. 32: 29362943. Hunter, P.R., and Gaston, M.A. 1988. Numerical index of the discriminatory ability of typing systems: an application of Simpsons index of diversity. J. Clin. Microbiol. 26: 24652466. Jersek, B., Gilot, P., Gubina, M., Klun, N., Melhe, J., Tcherneva, E., Rijpens, N., and Herman, L. 1999. Typing of Listeria monocytogenes strains by repetitive element sequence-based PCR. J. Clin. Microbiol. 37: 103109. Kerr, K.L., Kite, P., Heritage, J., and Hawkey, P.M. 1995. Typing of epidemiologically associated environmental and clinical strains of Listeria monocytogenes by random amplification of polymorphic DNA. J. Food Prot. 58: 609613. Massa, L., Trovatelli, L.D., and Canganella, F. 1991. Survival of Listeria monocytogenes in yogurt during storage at 4C. Lett. Appl. Microbiol. 13: 112114.

from those isolated from humans (Alleberger et al. 1997; Gilot et al. 1996; Farber and Addison 1994; Kerr et al. 1995; Jersek et al. 1999). Furthermore, the high discriminatory power of RAPD with primers OPA04 and OPA13 indicates the usefulness of the method to show causal relationship between epidemiologically related strains of the same serovar or esterase type during foodborne outbreaks of listeriosis.

Acknowledgements
We thank A. Genicot for serotyping L. monocytogenes strains used in this work and Martine Braibant for reading the manuscript. This work has been supported by the Ministre de la Rgion Wallonne. Direction Gnrale des Technologies, de la Recherche et de lEnergie (contract FIRST-9713509), Belgium.

References
Alleberger, F., Dierich, M., Grundmann, H., Hartung, D., Bannerman, E., and Bille, J. 1997. Typing of Listeria monocytogenes isolates by automated laser fluorescence analysis of randomly amplified polymorphic DNA. Zenbl. Bakteriol. 286: 3340. Andr, P., Roose, H., Van Noyen, R., Dejaegher, L., Uyttendaele, I., and de Schrijver, K. 1990. Listeriose neuro-mninge associe la consommation de crme glace. Md. Mal. Infect. 20: 570572. Beuchat, L.R. 1996. Pathogenic microorganisms associated with fresh produce. J. Food Prot. 59: 204216. Cancilla, M., Powell, I., Hillier, A., and Davidson, B. 1992. Rapid genomic fingerprinting of Lactococcus lactis strains by arbitrarily primed polymerase chain reaction with 32P and fluorescent labels. Appl. Environ. Microbiol. 58: 17721775. Comi, G., Cocolin, L., Cantoni, C., and Manzano, M. 1997. A REPCR method to distinguish Listeria monocytogenes serovars. FEMS Immunol. Med. Microbiol. 18: 99104.

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