- Email: [email protected]
Characterization of the novel Listeria monocytogenes PCR serogrouping profile IVb-v1
International Journal of Food Microbiology 147 (2011) 74–77
Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Short Communication
Characterization of the novel Listeria monocytogenes PCR serogrouping profile IVb-v1 Alexandre Leclercq a,⁎, Viviane Chenal-Francisque a, Hélène Dieye a, Thomas Cantinelli a, Rezak Drali b, Sylvain Brisse c, Marc Lecuit a,d a
Institut Pasteur, WHO Collaborating Centre and French National Reference Centre for Listeria, Microbes and Host Barriers Group, Paris, France Service des Entérobactéries et Hygiène de l'Environnement, Laboratoire des Listeria, Institut Pasteur d'Algérie, Route du Petit Staouéli - Dély Brahim, Alger, Algeria Institut Pasteur, Genotyping of Pathogens and Public Health, Paris, France d Université Paris Descartes, Department of Infectious Diseases and Tropical Medicine, Necker-Pasteur Centre for Infectious Diseases, Hôpital Necker-Enfants Malades, Assistance Publique-Hôpitaux de Paris, Paris, France b c
a r t i c l e
i n f o
Article history: Received 10 December 2010 Received in revised form 9 February 2011 Accepted 13 March 2011 Keywords: PCR serogroup Listeria monocytogenes 4b MLST PFGE Atypical pattern
a b s t r a c t The World Health Organization Collaborating Centre for Listeria (WHOCCL) has developed in 2004 a multiplex PCR assay that separates the 4 major Listeria monocytogenes serovars (1/2a, 1/2b, 1/2c, and 4b) into distinct PCR serogroups. A new PCR profile has been recently identified, constituted of amplified DNA fragments of prs, ORF2819, ORF2110 and lmo0737. Here we characterize 22 L. monocytogenes isolates of the WHOCCL collection with this PCR IVb variant 1 (IVb-v1) profile. The 22 isolates belong to the clinically predominant serovar 4b, exhibit 6 distinct pulsed-field gel electrophoresis ApaI/AscI combined profiles, and belong to 2 unrelated multilocus sequence types, indicating that the novel profile does not correspond to a recent clonal emergence. We have updated the WHOCCL serogroup-related PCR typing scheme to include this new profile. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Listeria monocytogenes is a foodborne pathogen that leads to gastroenteritis, maternal/neonatal infections, septicaemia, meningitis and encephalitis (Farber and Peterkin, 1991; Schlech et al., 1983). The populations at risk for listeriosis are pregnant women and neonates, the elderly and immunocompromised individuals. Listeriosis high hospitalisation and mortality rates justify that this rare illness is closely monitored by public health authorities in most Western countries (Goulet et al., 2008). Since 2005, listeriosis incidence has increased in several European countries, for so far unknown reasons (Goulet et al., 2008). In this context, an effective surveillance system of listeriosis and a reliable and rapid microbiological characterization of isolates of food and clinical origins are needed. The microbiological characterization of L. monocytogenes is usually performed in two steps. The first is serogroup determination, either by the classical serotyping method (Seeliger and Höhne, 1979) or by a molecular method known as PCRserogrouping (Doumith et al., 2004a, 2005; Kérouanton et al., 2010). The second step relies on a reference and standardized subtyping method with a higher discriminatory power, pulsed-field gel electrophoresis (PFGE) with enzymes AscI and ApaI (Graves and Swaminathan, 2001; Martin et al., 2006). As PFGE is time-consuming and labor-intensive,
⁎ Corresponding author at: Institut Pasteur, WHO Collaborating Centre for Listeria, 25 Rue du Docteur Roux, 75724 Paris cedex 15, France. Tel.: +33 140613190; fax: +33 140613567. E-mail address: [email protected] (A. Leclercq). 0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2011.03.010
serogrouping is performed as a rapid initial screening in epidemiological investigations. L. monocytogenes serotyping is based on the determination of somatic and flagellar antigens, according to Seeliger and Höhne's scheme (Seeliger and Höhne, 1979). Thirteen serovars have been described, with serotype 4b (phylogenetic lineage I) being more frequent in clinical cases, whereas serovar 1/2a and 1/2c (phylogenetic lineage II) are more frequent in food. Because serotyping is not costeffective, necessitates technical expertise and antisera (Schonberg et al., 1996), it is now often substituted by a quick and reproducible PCR-based method, developed by Doumith et al. (2004a, 2005) and which targets the four DNA fragments prs, ORF2110, ORF2819, lmo1118, lmo0737 (Doumith et al., 2004a; Doumith et al., 2005). All Listeria species but L. rocourtiae possess an amplifiable prs gene fragment (Leclercq et al., 2010). As shown in Fig. 1, PCR serogroup IIa comprises strains of serovars 1/2a and 3a (amplification of prs and lmo0737 DNA fragments); PCR serogroup IIb comprises strains of serovars 1/2b, 3b, and 7 (amplification of prs and ORF2819 DNA fragments); PCR serogroup IIc comprises strains of serovars 1/2c and 3c (amplification of prs, lmo0737 and lmo1118 DNA fragments). PCR serogroup IVb comprises strains of serovars 4b, 4d and 4e (amplification of both prs, ORF2819 and ORF2110 DNA fragments) (Doumith et al., 2004a). Finally, PCR serogroup Listeria species comprises strains of other serovars of L. monocytogenes and other species (amplification of prs DNA fragments), except L. rocourtiae. Interestingly, these groupings are consistent with phylogenetic relationships disclosed among serotypes based on MLST (Doumith et al., 2004b; Ragon et al., 2008).
A. Leclercq et al. / International Journal of Food Microbiology 147 (2011) 74–77
75
Fig. 1. Multiplex PCR assay profiles of the five PCR serogroup of Listeria monocytogenes obtained after agarose gel electrophoresis of amplified DNA fragments. Lane 1, amplified DNA fragments for PCR serogroup IIa obtained from DNA of L. monocytogenes reference strain of serovar 1/2a (CIP 104794); lane 2, amplified DNA fragments for PCR serogroup IIc obtained from DNA of L. monocytogenes reference strain of serovar 1/2c (CIP 105448); lane 3, amplified DNA fragments for PCR serogroup IIb obtained from DNA of L. monocytogenes reference strain of serovar 1/2b (CIP 105449); lane 4, amplified DNA fragments for PCR serogroup IVb obtained from DNA of L. monocytogenes reference strain of serovar 4b (CIP 78.38); lane 5, amplified DNA fragments for PCR serogroup Listeria species (other serovars and Listeria species, except L. rocourtiae) obtained from DNA of L. monocytogenes reference strain of serovar 4a (CIP 74908); lane 6, amplified DNA fragments for PCR serogroup IVb-v1 obtained from DNA of L. monocytogenes strain CLIP 2006/00270 of serovar 4b; lane 7, amplified DNA fragments for PCR serogroup IVb-v1 obtained from DNA of L. monocytogenes strain CLIP 2007/00779 of serovar 4b; lane 8, amplified DNA fragments for PCR serogroup IVb-v1 obtained from DNA of L. monocytogenes strain CLIP 2007/01070 of serovar 4b; lane M, SmartLadder SF molecular weight markers (Eurogentec). Genes corresponding to the amplified fragments are indicated on the right. CIP, Collection of the Institut Pasteur; CLIP, Listeria Culture Collection of the National Reference Centre and the World Health Organization Collaborating Centre for Listeria, Institut Pasteur. Molecular sizes are given in base pairs at the left.
Institut Pasteur (CIP) of serovars 1/2a (CIP 104794), 1/2b (CIP 105449), 1/2c (CIP 105448), 3a (CIP 78.34), 3b (CIP 78.35), 3c (CIP 78.36), 4a (CIP 74908), 4b (CIP 78.38), 4ab (CIP 106065), 4c (CIP 78.39), 4d (CIP 105458), 4e (CIP 105459), and 7 (CIP 78.43).
An atypical and novel PCR profile was identified in 2007 for serogroup PCR IVb L. monocytogenes isolates from France as shown in Table 1 (Le Monnier and Leclercq, 2008), and other countries, including the USA (Graves et al., 2007), Chile (Moledo de Vasconcelos et al., 2008) and Australia (Huang et al., 2010). No outbreak linked to L. monocytogenes strains with this atypical PCR profile has been detected or reported to our knowledge. The aim of this study was to characterize the clonal relatedness of isolates with this atypical PCR serogroup profile, using PFGE with AscI/ApaI endonuclease enzymes and multilocus sequence typing (MLST) (Graves and Swaminathan, 2001; Ragon et al., 2008).
The amplified PCR product of gene lmo0737 was sequenced to confirm its identity, using primers developed previously by Doumith et al. (2004a).
2. Materials and methods
2.5. PFGE analysis
2.1. Bacterial isolates
Molecular typing of strains by PFGE with AscI and ApaI restriction enzymes were performed according to the PulseNet protocol for L. monocytogenes (Graves and Swaminathan, 2001) and analyzed as described by Martin et al. (2006).
A total of 22 Listeria monocytogenes isolates were included, either collected from clinical and food laboratories during French and international listeriosis surveillance activities, or from the culture collections of the National Reference Centre and of the WHO-CC for Listeria (Collections of Listeria from Institut Pasteur, CLIP), and from Seeliger's Listeria Culture Collection (SLCC) (Rocourt and Seeliger, 1985). These isolates have no epidemiological link.
2.4. Sequencing of fragment lmo0737
2.6. Multilocus sequence typing Multilocus sequence typing of strains representing all distinct combined AscI/ApaI profiles (Table 1) was performed as described by Ragon et al. (2008).
2.2. Identification 3. Results and discussion Strains were revived by plating onto Columbia Agar (BioRad, Marnes la Coquette, France) and identified as Listeria monocytogenes according to standard methods (Bille et al., 1992; Rocourt et al., 1983). 2.3. Classical serotyping and multiplex PCR serogrouping The strains were serotyped on the basis of somatic and flagellar antigens, according to Seeliger and Höhne (1979) with the use of commercially available antisera (Denka Seiken Co., Tokyo, Japan), except for serum IX, and WHO-CC for Listeria reference antisera (Seeliger and Höhne, 1979). The PCR-multiplex assay was performed as previously described (Doumith et al., 2004a). The specificity and reliability of this PCR-multiplex method was controlled using the 13 Listeria monocytogenes reference strains from the Collection of the
Since 2004, we have applied the PCR serogrouping method to nearly 9000 strains of L. monocytogenes. Of these, 22 strains from Algeria, Brazil, France, and Switzerland (Table 1, Fig. 1) were found to display a novel atypical profile of PCR serogroup IVb composed of the amplified fragments of prs, ORF2819, ORF2110 and lmo0737 (Le Monnier and Leclercq, 2008; Graves et al., 2007; Huang et al., 2010; Moledo de Vasconcelos et al., 2008). The 22 strains were isolated from human listeriosis cases including septicaemia, maternal/neonatal and central nervous system infections, and from milk and meat products including red meat and poultry (Table 1). This atypical profile combined the three PCR products observed in group IVb, with PCR product lmo0737 observed so far only in groups IIa and IIc. The unexpected amplification of DNA fragment lmo0737 (691 bp) was confirmed by multiplex-PCR
76
A. Leclercq et al. / International Journal of Food Microbiology 147 (2011) 74–77
Table 1 Strains of Listeria monocytogenes with PCR serogroup IVb-v1 analyzed in this study. Country
France
Straina
CLIP 2006/00270 CLIP 2007/00779 CLIP 2007/01070 Brazil CLIP 16673 CLIP 16678 CLIP 16659 CLIP 16724 Switzerland SLCC 792 Algeria CLIP 2009/00807 CLIP 2009/00810 CLIP 2009/00812 CLIP 2009/00813 CLIP 2009/00815 CLIP 2009/00832 CLIP 2009/00838 CLIP 2009/00839 CLIP 2009/00844 CLIP 2009/00851 CLIP 2009/00852 CLIP 2009/00854 CLIP 2009/00861 CLIP 2009/00868
Originb
Year Multiplex PCR fragment amplificationc Serogroupd Combined PFGE AscI/ApaI Profilee STf isolated prs ORF2819 ORF2110 Lmo0737 Lmo1118 (370 bp) (471 bp) (597 bp) (691 bp) (906 bp)
Human (ICNS) Human (BACT) Human (MN) Human (ICNS) Human (ICNS) Food (Meat product) Human (BACT) Human (BACT) Food (Meat product) Food (Meat product) Food (Milk product) Food (Milk product) Food (Milk product) Food (Milk product) Food (Milk product) Food (Meat product) Food (Milk product) Food (Meat product) Food (Meat product) Food (Meat product) Food (Meat product) Food (Meat product)
2006 2007 2007 1989 1989 1990 1990 1959 2003 2003 2004 2004 2004 2004 2005 2005 2006 2007 2007 2007 2002 2005
+ + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + +
− − − − − − − − − − − − − − − − − − − − − −
4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b
A/A A/A A/A A/A A/A A/A D/D E/E C/A A/A A/A A/B A/A A/A B/C B/C A/A B/C A/A A/A A/A B/C
− − 218 − 218 − 218 240 218 − − 218 − − 218 − − − − − − −
a, CLIP, strain number from Listeria Culture collection of the National Reference Centre and WHO Collaborating Centre for Listeria. SLCC, strain number from Seeliger Listeria Culture Collection. b, MN, maternal/neonatal form; AISNC, Infection of Central Nervous System; BACT, Bacteremia. c, according to Doumith et al. (2004a, 2004b). + or − indicates amplification or not of the DNA fragments. d, according to Seeliger and Höhne (1979). e, according to Pulsenet Protocol of Graves and Swaminathan (2001). f, according to MLST protocol of Ragon et al. (2008). −: Not Done.
assay as well as by simplex-PCR, which confirmed that the 22 strains could not be classified according to the initial scheme of Doumith et al. (2004a). Serotyping of the 22 strains showed clear agglutination with the commercial and WHO-CC for Listeria reference O antisera V and VI and H antisera A, AB, B, and C, which attributed them univocally to serotype 4b, consistent with previous studies (Graves et al., 2007; Huang et al., 2010; Le Monnier and Leclercq, 2008; Moledo de Vasconcelos et al., 2008). Therefore, we named this profile as ‘profile IVb variant 1’ (IVb-v1). Sequencing of a 691-nt fragment of lmo0737 from the 3 strains CLIP 2006/00270, CLIP 2007/00779 and 2007/01070 (Genbank accession No: HQ123583) of ST218 and ST240 showed that they were identical and had only 4 (0.6%) nucleotide polymorphisms compared to the lmo0737 sequence of lineage II strains CIP 104794 of serotype 1/2a (ST 12, clonal complex 7) and CIP 105448 of serotype 1/2c (ST122, clonal complex 9). This low level of divergence contrasts with the average divergence of housekeeping genes (Ragon et al., 2008) between lineage I and II (5%) and suggests horizontal transfer of lmo0737 between these lineages, most probably from lineage II to lineage I strains with the novel profile. The strains with profile PCR serogroup IVb-v1 do not appear to have a recent clonal origin. First, it is not an emergent strain, as it was observed in a strain collected in 1959 (Table 1). Second, the twenty-two strains showed 6 PFGE AscI/ApaI combined profiles (Table 1 and Fig. 1 supplemental). Moreover, MLST revealed two sequence types (ST): ST218 and ST240, which differed by four MLST genes out of seven, excluding a close relatedness between these strains. ST218 was most closely related to the serotype 4b clonal complex 1 (Ragon et al., 2008), differing by three genes from ST1 and ST62, its most closely related MLST profiles. In contrast, ST240 was more related to ST54 (which also includes 4b strains but does not belong to clonal complex 1), differing in genes abcZ and cat by only one single nucleotide polymorphism in each gene. Strains of ST54 had the typical IVb profile, as had strains of clonal complex 1. Phylogenetic analysis of concatenated MLST gene sequences
were in agreement with these relationships, grouping ST218 with clonal complex 1 with 84% bootstrap support, and ST240 with ST54 with 68% bootstrap support. Altogether, current evidence suggests two independent evolutionary origins of profile PCR serogroup IVb-v1, likely by horizontal gene transfer from strains of lineage II (PCR serogroups IIa or IIc) (Doumith et al., 2004b; Nightingale et al., 2005). The identical sequence of fragment lmo0737 in ST218 and ST240 suggests a closely related donor to both STs, or a transfer of lmo0737 between these two STs. Whereas ST240, with a particular combined AscI/ApaI PFGE profile, was found only once (in strain SLCC 792 isolated in 1958 in Switzerland), ST218 isolates, which showed 5 distinct combined AscI/ ApaI PFGE profiles, were isolated in France, Brazil and Algeria (Table 1). Nevertheless, the combined AscI/ApaI PFGE profiles of 19 ST218 strains showed more than 91.4% similarity, consistent with their genetic relatedness indicated by MLST. In conclusion, these 22 L. monocytogenes isolates of PCR serogroup IVb-v1 profile belong to at least two apparently unrelated genomic backgrounds, ST218 and ST240, and do not originate from a recent clonal emergence. This novel PCR serogroup includes food and clinical serotype 4b strains from different parts of the world (Graves et al., 2007; Huang et al., 2010; Le Monnier and Leclercq, 2008; Moledo de Vasconcelos et al., 2008). We propose to name it, profile IVb variant 1 (IVb-v1) and provide in Suppl. Table 1 a revised and updated version of the WHO-CC for Listeria serogroup-related PCR typing scheme. Supplementary materials related to this article can be found online at doi:10.1016/j.ijfoodmicro.2011.03.010.
Acknowledgments We thank all laboratories that have sent strains to the WHO Collaborative Centre for Listeria. This work received financial support from Institut de Veille Sanitaire (France) and Institut Pasteur.
A. Leclercq et al. / International Journal of Food Microbiology 147 (2011) 74–77
References Bille, J., Catimel, B., Bannerman, E., Jacquet, C., Yersin, M.N., Caniaux, I., Monget, D., Rocourt, J., 1992. API Listeria, a new and promising one-day system to identify Listeria isolates. Applied and Environmental Microbiology 58, 1857–1860. Doumith, M., Buchrieser, C., Glaser, P., Jacquet, C., Martin, P., 2004a. Differentiation of the major Listeria monocytogenes serovars by multiplex PCR. Journal of Clinical Microbiology 42, 3819–3822. Doumith, M., Cazalet, C., Simoes, N., Frangeul, L., Jacquet, C., Kunst, F., Martin, P., Cossart, P., Glaser, P., Buchrieser, C., 2004b. New aspects regarding evolution and virulence of Listeria monocytogenes revealed by comparative genomics and DNA arrays. Infection and Immunity 72, 1072–1083. Doumith, M., Jacquet, C., Gerner-Smidt, P., Graves, L.M., Loncarevic, S., Mathisen, T., Morvan, A., Salcedo, C., Torpdahl, M., Vazquez, J.A., Martin, P., 2005. Multicenter validation of a multiplex PCR assay for differentiating the major Listeria monocytogenes serovars 1/2a, 1/2b, 1/2c, and 4b: toward an international standard. Journal of Food Protection 68, 2648–2650. Farber, J.M., Peterkin, P.I., 1991. Listeria monocytogenes, a food-borne pathogen. Microbiological Reviews 55, 476–511. Goulet, V., Hedberg, C., Le Monnier, A., de Valk, H., 2008. Increasing incidence of listeriosis in France and other European countries. Emerging Infectious Diseases 14, 734–740. Graves, L.M., Broeker, R., Garette, N., Swaminathan, B., 2007. Comparison of a multiplex PCR assay and conventional serotyping for sero-classification of Listeria monocytogenes isolates in the USA, 2005–2006, ISOPOL XVI, March 20–23. Proceeding of conference, Poster communication P02. Graves, L.M., Swaminathan, B., 2001. PulseNet standardized protocol for subtyping Listeria monocytogenes by macrorestriction and pulsed-field gel electrophoresis. International Journal of Food Microbiology 65, 55–62. Huang, B., Fang, N., Dimovski, K., Wang, X., Hogg, G., Bates, J., 2010. Observation of a New Pattern in Serogroup-Related PCR Typing in Listeria monocytogenes 4b isolates. Journal of Clinical Microbiology. doi:10.1128/JCM.01207-10. Kérouanton, A., Marault, M., Petit, L., Grout, J., Dao, T.T., Brisabois, A., 2010. Evaluation of a multiplex PCR assay as an alternative method for Listeria monocytogenes serotyping. Journal of Microbiological Methods 80, 134–137.
77
Le Monnier, A., Leclercq, A., 2008. Rapport annuel d'activité du Centre national de Référence des Listeria – Année 2007. Institut Pasteur, Paris. Leclercq, A., Clermont, D., Bizet, C., Grimont, P.A., Le Fleche-Mateos, A., Roche, S.M., Buchrieser, C., Cadet-Daniel, V., Le Monnier, A., Lecuit, M., Allerberger, F., 2010. Listeria rocourtiae sp. nov. International Journal of Systematic and Evolutionary Microbiology 60, 2210–2214. Martin, P., Jacquet, C., Goulet, V., Vaillant, V., De Valk, H., 2006. Pulsed-field gel electrophoresis of Listeria monocytogenes strains: the PulseNet Europe Feasibility Study. Foodborne Pathogens and Disease 3, 303–308. Moledo de Vasconcelos, R., Cardoso, Castro, de Almeida, A.E., Hofer, E., Matias da Silva, N.M., Augustus Marin, V., 2008. Multiplex-PCR serotyping of Listeria monocytogenes isolated from human clinical specimens. Memorias do Instituto Oswaldo Cruz 103, 5537–5551. Nightingale, K.K., Windham, K., Wiedmann, M., 2005. Evolution and molecular phylogeny of Listeria monocytogenes isolated from human and animal listeriosis cases and foods. Journal of Bacteriology 187, 5537–5551. Ragon, M., Wirth, T., Hollandt, F., Lavenir, R., Lecuit, M., Le Monnier, A., Brisse, S., 2008. A new perspective on Listeria monocytogenes evolution. PLoS Pathogens 4, e1000146. Rocourt, J., Schrettenbrunner, A., Seeliger, H.P., 1983. Biochemical differentiation of the “Listeria monocytogenes” (sensu lato) genomic groups. Annales de Microbiologie (Paris) 134A, 65–71. Rocourt, J., Seeliger, H.P., 1985. Distribution of species of the genus Listeria. Zentralblatt fur Bakteriologie, Mikrobiologie und Hygiene. Series A., Medical microbiology, infectious diseases, virology, parasitology 259, 317–330. Schlech III, W.F., Lavigne, P.M., Bortolussi, R.A., Allen, A.C., Haldane, E.V., Wort, A.J., Hightower, A.W., Johnson, S.E., King, S.H., Nicholls, E.S., Broome, C.V., 1983. Epidemic listeriosis—evidence for transmission by food. The New England Journal of Medicine 308, 203–206. Schonberg, A., Bannerman, E., Courtieu, A.L., Kiss, R., McLauchlin, J., Shah, S., Wilhelms, D., 1996. Serotyping of 80 strains from the WHO multicentre international typing study of Listeria monocytogenes. International Journal of Food Microbiology 32, 279–287. Seeliger, H.P., Höhne, K., 1979. Serotyping of Listeria monocytogenes and related species. Methods in Microbiology, 13. Academic Press, New York, pp. 31–49.
Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Short Communication
Characterization of the novel Listeria monocytogenes PCR serogrouping profile IVb-v1 Alexandre Leclercq a,⁎, Viviane Chenal-Francisque a, Hélène Dieye a, Thomas Cantinelli a, Rezak Drali b, Sylvain Brisse c, Marc Lecuit a,d a
Institut Pasteur, WHO Collaborating Centre and French National Reference Centre for Listeria, Microbes and Host Barriers Group, Paris, France Service des Entérobactéries et Hygiène de l'Environnement, Laboratoire des Listeria, Institut Pasteur d'Algérie, Route du Petit Staouéli - Dély Brahim, Alger, Algeria Institut Pasteur, Genotyping of Pathogens and Public Health, Paris, France d Université Paris Descartes, Department of Infectious Diseases and Tropical Medicine, Necker-Pasteur Centre for Infectious Diseases, Hôpital Necker-Enfants Malades, Assistance Publique-Hôpitaux de Paris, Paris, France b c
a r t i c l e
i n f o
Article history: Received 10 December 2010 Received in revised form 9 February 2011 Accepted 13 March 2011 Keywords: PCR serogroup Listeria monocytogenes 4b MLST PFGE Atypical pattern
a b s t r a c t The World Health Organization Collaborating Centre for Listeria (WHOCCL) has developed in 2004 a multiplex PCR assay that separates the 4 major Listeria monocytogenes serovars (1/2a, 1/2b, 1/2c, and 4b) into distinct PCR serogroups. A new PCR profile has been recently identified, constituted of amplified DNA fragments of prs, ORF2819, ORF2110 and lmo0737. Here we characterize 22 L. monocytogenes isolates of the WHOCCL collection with this PCR IVb variant 1 (IVb-v1) profile. The 22 isolates belong to the clinically predominant serovar 4b, exhibit 6 distinct pulsed-field gel electrophoresis ApaI/AscI combined profiles, and belong to 2 unrelated multilocus sequence types, indicating that the novel profile does not correspond to a recent clonal emergence. We have updated the WHOCCL serogroup-related PCR typing scheme to include this new profile. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Listeria monocytogenes is a foodborne pathogen that leads to gastroenteritis, maternal/neonatal infections, septicaemia, meningitis and encephalitis (Farber and Peterkin, 1991; Schlech et al., 1983). The populations at risk for listeriosis are pregnant women and neonates, the elderly and immunocompromised individuals. Listeriosis high hospitalisation and mortality rates justify that this rare illness is closely monitored by public health authorities in most Western countries (Goulet et al., 2008). Since 2005, listeriosis incidence has increased in several European countries, for so far unknown reasons (Goulet et al., 2008). In this context, an effective surveillance system of listeriosis and a reliable and rapid microbiological characterization of isolates of food and clinical origins are needed. The microbiological characterization of L. monocytogenes is usually performed in two steps. The first is serogroup determination, either by the classical serotyping method (Seeliger and Höhne, 1979) or by a molecular method known as PCRserogrouping (Doumith et al., 2004a, 2005; Kérouanton et al., 2010). The second step relies on a reference and standardized subtyping method with a higher discriminatory power, pulsed-field gel electrophoresis (PFGE) with enzymes AscI and ApaI (Graves and Swaminathan, 2001; Martin et al., 2006). As PFGE is time-consuming and labor-intensive,
⁎ Corresponding author at: Institut Pasteur, WHO Collaborating Centre for Listeria, 25 Rue du Docteur Roux, 75724 Paris cedex 15, France. Tel.: +33 140613190; fax: +33 140613567. E-mail address: [email protected] (A. Leclercq). 0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2011.03.010
serogrouping is performed as a rapid initial screening in epidemiological investigations. L. monocytogenes serotyping is based on the determination of somatic and flagellar antigens, according to Seeliger and Höhne's scheme (Seeliger and Höhne, 1979). Thirteen serovars have been described, with serotype 4b (phylogenetic lineage I) being more frequent in clinical cases, whereas serovar 1/2a and 1/2c (phylogenetic lineage II) are more frequent in food. Because serotyping is not costeffective, necessitates technical expertise and antisera (Schonberg et al., 1996), it is now often substituted by a quick and reproducible PCR-based method, developed by Doumith et al. (2004a, 2005) and which targets the four DNA fragments prs, ORF2110, ORF2819, lmo1118, lmo0737 (Doumith et al., 2004a; Doumith et al., 2005). All Listeria species but L. rocourtiae possess an amplifiable prs gene fragment (Leclercq et al., 2010). As shown in Fig. 1, PCR serogroup IIa comprises strains of serovars 1/2a and 3a (amplification of prs and lmo0737 DNA fragments); PCR serogroup IIb comprises strains of serovars 1/2b, 3b, and 7 (amplification of prs and ORF2819 DNA fragments); PCR serogroup IIc comprises strains of serovars 1/2c and 3c (amplification of prs, lmo0737 and lmo1118 DNA fragments). PCR serogroup IVb comprises strains of serovars 4b, 4d and 4e (amplification of both prs, ORF2819 and ORF2110 DNA fragments) (Doumith et al., 2004a). Finally, PCR serogroup Listeria species comprises strains of other serovars of L. monocytogenes and other species (amplification of prs DNA fragments), except L. rocourtiae. Interestingly, these groupings are consistent with phylogenetic relationships disclosed among serotypes based on MLST (Doumith et al., 2004b; Ragon et al., 2008).
A. Leclercq et al. / International Journal of Food Microbiology 147 (2011) 74–77
75
Fig. 1. Multiplex PCR assay profiles of the five PCR serogroup of Listeria monocytogenes obtained after agarose gel electrophoresis of amplified DNA fragments. Lane 1, amplified DNA fragments for PCR serogroup IIa obtained from DNA of L. monocytogenes reference strain of serovar 1/2a (CIP 104794); lane 2, amplified DNA fragments for PCR serogroup IIc obtained from DNA of L. monocytogenes reference strain of serovar 1/2c (CIP 105448); lane 3, amplified DNA fragments for PCR serogroup IIb obtained from DNA of L. monocytogenes reference strain of serovar 1/2b (CIP 105449); lane 4, amplified DNA fragments for PCR serogroup IVb obtained from DNA of L. monocytogenes reference strain of serovar 4b (CIP 78.38); lane 5, amplified DNA fragments for PCR serogroup Listeria species (other serovars and Listeria species, except L. rocourtiae) obtained from DNA of L. monocytogenes reference strain of serovar 4a (CIP 74908); lane 6, amplified DNA fragments for PCR serogroup IVb-v1 obtained from DNA of L. monocytogenes strain CLIP 2006/00270 of serovar 4b; lane 7, amplified DNA fragments for PCR serogroup IVb-v1 obtained from DNA of L. monocytogenes strain CLIP 2007/00779 of serovar 4b; lane 8, amplified DNA fragments for PCR serogroup IVb-v1 obtained from DNA of L. monocytogenes strain CLIP 2007/01070 of serovar 4b; lane M, SmartLadder SF molecular weight markers (Eurogentec). Genes corresponding to the amplified fragments are indicated on the right. CIP, Collection of the Institut Pasteur; CLIP, Listeria Culture Collection of the National Reference Centre and the World Health Organization Collaborating Centre for Listeria, Institut Pasteur. Molecular sizes are given in base pairs at the left.
Institut Pasteur (CIP) of serovars 1/2a (CIP 104794), 1/2b (CIP 105449), 1/2c (CIP 105448), 3a (CIP 78.34), 3b (CIP 78.35), 3c (CIP 78.36), 4a (CIP 74908), 4b (CIP 78.38), 4ab (CIP 106065), 4c (CIP 78.39), 4d (CIP 105458), 4e (CIP 105459), and 7 (CIP 78.43).
An atypical and novel PCR profile was identified in 2007 for serogroup PCR IVb L. monocytogenes isolates from France as shown in Table 1 (Le Monnier and Leclercq, 2008), and other countries, including the USA (Graves et al., 2007), Chile (Moledo de Vasconcelos et al., 2008) and Australia (Huang et al., 2010). No outbreak linked to L. monocytogenes strains with this atypical PCR profile has been detected or reported to our knowledge. The aim of this study was to characterize the clonal relatedness of isolates with this atypical PCR serogroup profile, using PFGE with AscI/ApaI endonuclease enzymes and multilocus sequence typing (MLST) (Graves and Swaminathan, 2001; Ragon et al., 2008).
The amplified PCR product of gene lmo0737 was sequenced to confirm its identity, using primers developed previously by Doumith et al. (2004a).
2. Materials and methods
2.5. PFGE analysis
2.1. Bacterial isolates
Molecular typing of strains by PFGE with AscI and ApaI restriction enzymes were performed according to the PulseNet protocol for L. monocytogenes (Graves and Swaminathan, 2001) and analyzed as described by Martin et al. (2006).
A total of 22 Listeria monocytogenes isolates were included, either collected from clinical and food laboratories during French and international listeriosis surveillance activities, or from the culture collections of the National Reference Centre and of the WHO-CC for Listeria (Collections of Listeria from Institut Pasteur, CLIP), and from Seeliger's Listeria Culture Collection (SLCC) (Rocourt and Seeliger, 1985). These isolates have no epidemiological link.
2.4. Sequencing of fragment lmo0737
2.6. Multilocus sequence typing Multilocus sequence typing of strains representing all distinct combined AscI/ApaI profiles (Table 1) was performed as described by Ragon et al. (2008).
2.2. Identification 3. Results and discussion Strains were revived by plating onto Columbia Agar (BioRad, Marnes la Coquette, France) and identified as Listeria monocytogenes according to standard methods (Bille et al., 1992; Rocourt et al., 1983). 2.3. Classical serotyping and multiplex PCR serogrouping The strains were serotyped on the basis of somatic and flagellar antigens, according to Seeliger and Höhne (1979) with the use of commercially available antisera (Denka Seiken Co., Tokyo, Japan), except for serum IX, and WHO-CC for Listeria reference antisera (Seeliger and Höhne, 1979). The PCR-multiplex assay was performed as previously described (Doumith et al., 2004a). The specificity and reliability of this PCR-multiplex method was controlled using the 13 Listeria monocytogenes reference strains from the Collection of the
Since 2004, we have applied the PCR serogrouping method to nearly 9000 strains of L. monocytogenes. Of these, 22 strains from Algeria, Brazil, France, and Switzerland (Table 1, Fig. 1) were found to display a novel atypical profile of PCR serogroup IVb composed of the amplified fragments of prs, ORF2819, ORF2110 and lmo0737 (Le Monnier and Leclercq, 2008; Graves et al., 2007; Huang et al., 2010; Moledo de Vasconcelos et al., 2008). The 22 strains were isolated from human listeriosis cases including septicaemia, maternal/neonatal and central nervous system infections, and from milk and meat products including red meat and poultry (Table 1). This atypical profile combined the three PCR products observed in group IVb, with PCR product lmo0737 observed so far only in groups IIa and IIc. The unexpected amplification of DNA fragment lmo0737 (691 bp) was confirmed by multiplex-PCR
76
A. Leclercq et al. / International Journal of Food Microbiology 147 (2011) 74–77
Table 1 Strains of Listeria monocytogenes with PCR serogroup IVb-v1 analyzed in this study. Country
France
Straina
CLIP 2006/00270 CLIP 2007/00779 CLIP 2007/01070 Brazil CLIP 16673 CLIP 16678 CLIP 16659 CLIP 16724 Switzerland SLCC 792 Algeria CLIP 2009/00807 CLIP 2009/00810 CLIP 2009/00812 CLIP 2009/00813 CLIP 2009/00815 CLIP 2009/00832 CLIP 2009/00838 CLIP 2009/00839 CLIP 2009/00844 CLIP 2009/00851 CLIP 2009/00852 CLIP 2009/00854 CLIP 2009/00861 CLIP 2009/00868
Originb
Year Multiplex PCR fragment amplificationc Serogroupd Combined PFGE AscI/ApaI Profilee STf isolated prs ORF2819 ORF2110 Lmo0737 Lmo1118 (370 bp) (471 bp) (597 bp) (691 bp) (906 bp)
Human (ICNS) Human (BACT) Human (MN) Human (ICNS) Human (ICNS) Food (Meat product) Human (BACT) Human (BACT) Food (Meat product) Food (Meat product) Food (Milk product) Food (Milk product) Food (Milk product) Food (Milk product) Food (Milk product) Food (Meat product) Food (Milk product) Food (Meat product) Food (Meat product) Food (Meat product) Food (Meat product) Food (Meat product)
2006 2007 2007 1989 1989 1990 1990 1959 2003 2003 2004 2004 2004 2004 2005 2005 2006 2007 2007 2007 2002 2005
+ + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + +
− − − − − − − − − − − − − − − − − − − − − −
4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b
A/A A/A A/A A/A A/A A/A D/D E/E C/A A/A A/A A/B A/A A/A B/C B/C A/A B/C A/A A/A A/A B/C
− − 218 − 218 − 218 240 218 − − 218 − − 218 − − − − − − −
a, CLIP, strain number from Listeria Culture collection of the National Reference Centre and WHO Collaborating Centre for Listeria. SLCC, strain number from Seeliger Listeria Culture Collection. b, MN, maternal/neonatal form; AISNC, Infection of Central Nervous System; BACT, Bacteremia. c, according to Doumith et al. (2004a, 2004b). + or − indicates amplification or not of the DNA fragments. d, according to Seeliger and Höhne (1979). e, according to Pulsenet Protocol of Graves and Swaminathan (2001). f, according to MLST protocol of Ragon et al. (2008). −: Not Done.
assay as well as by simplex-PCR, which confirmed that the 22 strains could not be classified according to the initial scheme of Doumith et al. (2004a). Serotyping of the 22 strains showed clear agglutination with the commercial and WHO-CC for Listeria reference O antisera V and VI and H antisera A, AB, B, and C, which attributed them univocally to serotype 4b, consistent with previous studies (Graves et al., 2007; Huang et al., 2010; Le Monnier and Leclercq, 2008; Moledo de Vasconcelos et al., 2008). Therefore, we named this profile as ‘profile IVb variant 1’ (IVb-v1). Sequencing of a 691-nt fragment of lmo0737 from the 3 strains CLIP 2006/00270, CLIP 2007/00779 and 2007/01070 (Genbank accession No: HQ123583) of ST218 and ST240 showed that they were identical and had only 4 (0.6%) nucleotide polymorphisms compared to the lmo0737 sequence of lineage II strains CIP 104794 of serotype 1/2a (ST 12, clonal complex 7) and CIP 105448 of serotype 1/2c (ST122, clonal complex 9). This low level of divergence contrasts with the average divergence of housekeeping genes (Ragon et al., 2008) between lineage I and II (5%) and suggests horizontal transfer of lmo0737 between these lineages, most probably from lineage II to lineage I strains with the novel profile. The strains with profile PCR serogroup IVb-v1 do not appear to have a recent clonal origin. First, it is not an emergent strain, as it was observed in a strain collected in 1959 (Table 1). Second, the twenty-two strains showed 6 PFGE AscI/ApaI combined profiles (Table 1 and Fig. 1 supplemental). Moreover, MLST revealed two sequence types (ST): ST218 and ST240, which differed by four MLST genes out of seven, excluding a close relatedness between these strains. ST218 was most closely related to the serotype 4b clonal complex 1 (Ragon et al., 2008), differing by three genes from ST1 and ST62, its most closely related MLST profiles. In contrast, ST240 was more related to ST54 (which also includes 4b strains but does not belong to clonal complex 1), differing in genes abcZ and cat by only one single nucleotide polymorphism in each gene. Strains of ST54 had the typical IVb profile, as had strains of clonal complex 1. Phylogenetic analysis of concatenated MLST gene sequences
were in agreement with these relationships, grouping ST218 with clonal complex 1 with 84% bootstrap support, and ST240 with ST54 with 68% bootstrap support. Altogether, current evidence suggests two independent evolutionary origins of profile PCR serogroup IVb-v1, likely by horizontal gene transfer from strains of lineage II (PCR serogroups IIa or IIc) (Doumith et al., 2004b; Nightingale et al., 2005). The identical sequence of fragment lmo0737 in ST218 and ST240 suggests a closely related donor to both STs, or a transfer of lmo0737 between these two STs. Whereas ST240, with a particular combined AscI/ApaI PFGE profile, was found only once (in strain SLCC 792 isolated in 1958 in Switzerland), ST218 isolates, which showed 5 distinct combined AscI/ ApaI PFGE profiles, were isolated in France, Brazil and Algeria (Table 1). Nevertheless, the combined AscI/ApaI PFGE profiles of 19 ST218 strains showed more than 91.4% similarity, consistent with their genetic relatedness indicated by MLST. In conclusion, these 22 L. monocytogenes isolates of PCR serogroup IVb-v1 profile belong to at least two apparently unrelated genomic backgrounds, ST218 and ST240, and do not originate from a recent clonal emergence. This novel PCR serogroup includes food and clinical serotype 4b strains from different parts of the world (Graves et al., 2007; Huang et al., 2010; Le Monnier and Leclercq, 2008; Moledo de Vasconcelos et al., 2008). We propose to name it, profile IVb variant 1 (IVb-v1) and provide in Suppl. Table 1 a revised and updated version of the WHO-CC for Listeria serogroup-related PCR typing scheme. Supplementary materials related to this article can be found online at doi:10.1016/j.ijfoodmicro.2011.03.010.
Acknowledgments We thank all laboratories that have sent strains to the WHO Collaborative Centre for Listeria. This work received financial support from Institut de Veille Sanitaire (France) and Institut Pasteur.
A. Leclercq et al. / International Journal of Food Microbiology 147 (2011) 74–77
References Bille, J., Catimel, B., Bannerman, E., Jacquet, C., Yersin, M.N., Caniaux, I., Monget, D., Rocourt, J., 1992. API Listeria, a new and promising one-day system to identify Listeria isolates. Applied and Environmental Microbiology 58, 1857–1860. Doumith, M., Buchrieser, C., Glaser, P., Jacquet, C., Martin, P., 2004a. Differentiation of the major Listeria monocytogenes serovars by multiplex PCR. Journal of Clinical Microbiology 42, 3819–3822. Doumith, M., Cazalet, C., Simoes, N., Frangeul, L., Jacquet, C., Kunst, F., Martin, P., Cossart, P., Glaser, P., Buchrieser, C., 2004b. New aspects regarding evolution and virulence of Listeria monocytogenes revealed by comparative genomics and DNA arrays. Infection and Immunity 72, 1072–1083. Doumith, M., Jacquet, C., Gerner-Smidt, P., Graves, L.M., Loncarevic, S., Mathisen, T., Morvan, A., Salcedo, C., Torpdahl, M., Vazquez, J.A., Martin, P., 2005. Multicenter validation of a multiplex PCR assay for differentiating the major Listeria monocytogenes serovars 1/2a, 1/2b, 1/2c, and 4b: toward an international standard. Journal of Food Protection 68, 2648–2650. Farber, J.M., Peterkin, P.I., 1991. Listeria monocytogenes, a food-borne pathogen. Microbiological Reviews 55, 476–511. Goulet, V., Hedberg, C., Le Monnier, A., de Valk, H., 2008. Increasing incidence of listeriosis in France and other European countries. Emerging Infectious Diseases 14, 734–740. Graves, L.M., Broeker, R., Garette, N., Swaminathan, B., 2007. Comparison of a multiplex PCR assay and conventional serotyping for sero-classification of Listeria monocytogenes isolates in the USA, 2005–2006, ISOPOL XVI, March 20–23. Proceeding of conference, Poster communication P02. Graves, L.M., Swaminathan, B., 2001. PulseNet standardized protocol for subtyping Listeria monocytogenes by macrorestriction and pulsed-field gel electrophoresis. International Journal of Food Microbiology 65, 55–62. Huang, B., Fang, N., Dimovski, K., Wang, X., Hogg, G., Bates, J., 2010. Observation of a New Pattern in Serogroup-Related PCR Typing in Listeria monocytogenes 4b isolates. Journal of Clinical Microbiology. doi:10.1128/JCM.01207-10. Kérouanton, A., Marault, M., Petit, L., Grout, J., Dao, T.T., Brisabois, A., 2010. Evaluation of a multiplex PCR assay as an alternative method for Listeria monocytogenes serotyping. Journal of Microbiological Methods 80, 134–137.
77
Le Monnier, A., Leclercq, A., 2008. Rapport annuel d'activité du Centre national de Référence des Listeria – Année 2007. Institut Pasteur, Paris. Leclercq, A., Clermont, D., Bizet, C., Grimont, P.A., Le Fleche-Mateos, A., Roche, S.M., Buchrieser, C., Cadet-Daniel, V., Le Monnier, A., Lecuit, M., Allerberger, F., 2010. Listeria rocourtiae sp. nov. International Journal of Systematic and Evolutionary Microbiology 60, 2210–2214. Martin, P., Jacquet, C., Goulet, V., Vaillant, V., De Valk, H., 2006. Pulsed-field gel electrophoresis of Listeria monocytogenes strains: the PulseNet Europe Feasibility Study. Foodborne Pathogens and Disease 3, 303–308. Moledo de Vasconcelos, R., Cardoso, Castro, de Almeida, A.E., Hofer, E., Matias da Silva, N.M., Augustus Marin, V., 2008. Multiplex-PCR serotyping of Listeria monocytogenes isolated from human clinical specimens. Memorias do Instituto Oswaldo Cruz 103, 5537–5551. Nightingale, K.K., Windham, K., Wiedmann, M., 2005. Evolution and molecular phylogeny of Listeria monocytogenes isolated from human and animal listeriosis cases and foods. Journal of Bacteriology 187, 5537–5551. Ragon, M., Wirth, T., Hollandt, F., Lavenir, R., Lecuit, M., Le Monnier, A., Brisse, S., 2008. A new perspective on Listeria monocytogenes evolution. PLoS Pathogens 4, e1000146. Rocourt, J., Schrettenbrunner, A., Seeliger, H.P., 1983. Biochemical differentiation of the “Listeria monocytogenes” (sensu lato) genomic groups. Annales de Microbiologie (Paris) 134A, 65–71. Rocourt, J., Seeliger, H.P., 1985. Distribution of species of the genus Listeria. Zentralblatt fur Bakteriologie, Mikrobiologie und Hygiene. Series A., Medical microbiology, infectious diseases, virology, parasitology 259, 317–330. Schlech III, W.F., Lavigne, P.M., Bortolussi, R.A., Allen, A.C., Haldane, E.V., Wort, A.J., Hightower, A.W., Johnson, S.E., King, S.H., Nicholls, E.S., Broome, C.V., 1983. Epidemic listeriosis—evidence for transmission by food. The New England Journal of Medicine 308, 203–206. Schonberg, A., Bannerman, E., Courtieu, A.L., Kiss, R., McLauchlin, J., Shah, S., Wilhelms, D., 1996. Serotyping of 80 strains from the WHO multicentre international typing study of Listeria monocytogenes. International Journal of Food Microbiology 32, 279–287. Seeliger, H.P., Höhne, K., 1979. Serotyping of Listeria monocytogenes and related species. Methods in Microbiology, 13. Academic Press, New York, pp. 31–49.