Evaluation of a multiplex PCR assay as an alternative method for Listeria monocytogenes serotyping

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Abstract

Listeria monocytogenes serotyping is commonly used as the first level of characterisation in the epidemiological surveillance of food and clinical isolates and is therefore widely accepted. The aim of this study was to define a scheme for multiplex molecular serotyping of L. monocytogenes based on a previously described PCR assay and then to evaluate and compare this new procedure with conventional serotyping by agglutination. The study included 1204 Listeria strains collected from food products in France, from March 2005 to October 2006. Two multiplex PCR assays were designed to cluster L. monocytogenes strains into five molecular serogroups: IIa, IIb, IIc, IVa, IVb in agreement with the most commonly encountered serotypes. Amplification of the prfA gene was added to the multiplex PCR to check for L. monocytogenes species; forty-eight (4%) of the isolates tested belonged to the genus Listeria but were not L. monocytogenes. Using this first multiplex PCR, the concordance between conventional and molecular methods was 90.6%, 97.8%, 100% and 100%, for 1/2a, 1/2c, 1/2b and 4b serotypes respectively. False results were observed for some atypical 1/2a, 3a and 1/2c strains. Therefore, this lack of specificity was resolved by using an additional PCR assay based on amplification of the flaA gene, a specific target of 1/2a and 3a strains. When applying the second PCR assay to IIa and IIc molecular serogroup strains, total agreement was obtained between molecular and conventional serotyping methods with a lower level of discrimination for the molecular one. This study proposes to define a strategy for molecular serotyping using both PCR assays: a multiplex and the flaA PCR in order to assign the atypical 1/2a, 3a and 1/2c strains. Moreover, prs gene detection was added for Listeria genus recognition as a positive control in association with flaA detection. Indeed, this molecular serotyping scheme could be considered as a useful and rapid method for first-level characterisation of the most frequently encountered L. monocytogenes serotypes.

Introduction

Listeria monocytogenes is an intracellular bacterial pathogen that causes morbidity and mortality in humans and livestock. Ubiquitous in nature, L. monocytogenes can contaminate foods from different sources such as vegetables, milk, cheese, fish, meat products and ready-to-eat (RTE) foods. Listeriosis in adults mostly occurs after consumption of contaminated foods (Farber and Peterkin, 1991, Schlech et al., 1983). Following a large decrease, the number of listeriosis cases has significantly increased in the European Union over the four past years. In 2006, a total of 1583 human cases were reported by the European authority (Denny & McLauchlin, 2008, Goulet et al., 2008). Therefore, the presence of L. monocytogenes is of great concern to the food industry, and tracing isolates within the food chain and the plant environment is of primary importance. For a long time, numerous discriminatory methods have been described for subtyping this organism (Nocera et al., 1993). Pulsed-field gel electrophoresis (PFGE) typing has rapidly become the standard subtyping method for investigating listeriosis outbreaks (Graves and Swaminathan, 2001) and discriminating food strains (Kerouanton et al., 1998). Nevertheless, for epidemiological monitoring of food and human isolates, serotyping is still required as a first level of discrimination. L. monocytogenes serotyping is based on the variation in the somatic (O) and flagellar (H) antigens allowing 13 different serotypes to be differentiated. The four main serotypes identified in food and human sources are 1/2a, 1/2b, 1/2c, and 4b (Jacquet et al., 2002, Wiedmann et al., 1997). Routine analysis of L. monocytogenes by serotyping with traditional agglutination methods is limited by cost, availability and quality of antisera, and typeability of isolates. This method is time-consuming and demands good technical practice and expertise. A multicentric serotyping study conducted as part of the WHO multicentre international typing study pointed out some discrepancies in the results and some problems in the quality of antisera (Schonberg et al., 1996). Several multiplex PCR assays have already been proposed (De Santis et al., 2007, Doumith et al., 2004, Doumith et al., 2005, Zhang and Knabel, 2005) for the rapid separation of L. monocytogenes strains into distinct PCR-types. Nevertheless, using Doumith's method, some atypical 1/2a, 3a, 1/2c strains are not recognised in the expected PCR-types. Here, we presented a proposed molecular serotyping scheme for correctly assigning these atypical strains. This molecular serotyping method has been evaluated in comparison with the conventional serotyping method on a total of 1204 non-human Listeria isolates.

Section snippets

Bacterial isolates

This study included a total of 1204 Listeria isolates, collected from food analysis laboratories, under voluntary monitoring, or mandatory surveillance sampling in France, from March 2005 to October 2006. Among those isolates, 1156 belonged to the L. monocytogenes species. The studied isolates were representative of the diversity of food processing sources: dairy products, fish and seafood products, pork, poultry, and beef products, vegetables and plant environment (Table 1). Reference strains

Results

All 1204 studied Listeria strains had the 370 bp prs and the 274 bp prfA genes, except for 48 strains for which prfA gene amplification remained negative. These strains were confirmed as Listeria non-monocytogenes by the CAMP test and biochemical tests. Thus, the study focussed on the 1156 L. monocytogenes. Serotyping by the conventional agglutination method differentiated isolates into nine serotypes, the most frequently detected being 1/2a (61%), then in decreasing order of frequency 1/2c

Discussion

The two multiplex PCR assays developed in this study provide a rapid and reproducible alternative method to Listeria genus and species recognition and L. monocytogenes serotyping. An interpretable PCR profile was obtained for the 1204 tested strains. Out of these, 48 strains did not give an amplified fragment with prfA primers. They were confirmed as Listeria by the CAMP test and biochemical tests and, among them, 27 were L. innocua, 5 were L. welshimeri, 5 were L. ivanovi and 1 was L. seeligeri

Acknowledgments

This work was conducted as part of the activities of the Community Reference Laboratory for L. monocytogenes and was supported by a grant from the Directorate-General for Heath and Consumers Protection (DG Sanco) of the European Commission.

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    Citation Excerpt :

    We performed PCR using an Eppendorf Mastercycler nexus (Eppendorf Canada) using the following program: lid temperature 105°C, the first denaturation for step 3 min at 94°C followed by 35 PCR cycles consisting of denaturation (94°C for 40 s), annealing (53°C for 45 s), and extension (72°C for 1 min and 15 s). The final step was set at 72°C for 7 min and held at 4°C (Kérouanton et al., 2010). Positive (L. monocytogenes ATCC 63253) and negative (Listeria ivanovii ATCC 19119 and water) controls were used.

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