Differentiation of the Major Listeria monocytogenes Serovars by Multiplex PCR

Abstract

A new multiplex PCR assay was developed to separate the four major Listeria monocytogenes serovars isolated from food and patients (1/2a, 1/2b, 1/2c, and 4b) into distinct groups. The PCR test, which constitutes a rapid and practical alternative to laborious classical serotyping, was successfully evaluated with 222 Listeria strains.Listeria monocytogenes is a facultative intracellular pathogen that can cause serious illness in susceptible individuals. Persons with specific immunocompromising conditions, pregnant women, newborns, and the elderly are particularly at risk for listeriosis (9, 23). Although rare, listeriosis remains of great public health concern due to its high mortality rate (20 to 30%) (16). Ingestion of contaminated foods is considered to be the primary source of infection for both sporadic and epidemic human listeriosis cases (19). Because of the importance of L. monocytogenes strain characterizations for epidemiological investigations, a number of discriminatory subtyping methods have been described for this organism (2,4,5,18,20,24,25). Pulsed-field gel electrophoresis (PFGE) typing, which has provided the most sensitive strain discrimination up to now, has rapidly become the standard subtyping method to detect listeriosis outbreaks (4, 11). However, this method is labor-intensive and time-consuming and thus for practical purposes is often preceded by serotyping. Since all major outbreaks of the invasive form of listeriosis are due to serovar 4b strains, an infrequent serovar in foods compared to 1/2a strains (6, 9), the procedure adopted for outbreak investigations relies upon serovar characterization to provide valuable information for rapid screening of groups of strains. Indeed, the serovar information allows discrimination between isolates probably belonging to an outbreak and those that are not part of the outbreak and thus decreases the number of strains which need to be characterized by PFGE in order to improve discrimination beyond the serovar level. Moreover, serotyping is widely used for long-term microbiological surveillance of human listeriosis. For the food industry, where the presence of L. monocytogenes is a big concern, tracing contaminating strains within the food chain and the plant environment is of primary importance. Again serotyping is often used as a first-line typing method. Although 13 serovars are described for the species L. monocytogenes, at least 95% of the strains isolated from foods and patients are of serovars 1/2a, 1/2b, 1/2c, and 4b (12,21,22). Routine analysis of L. monocytogenes by serotyping with traditional agglutination methods is limited by cost, availability, and the need for technical expertise to perform the assay. Furthermore, the reproducibility of serotyping is not always satisfactory. Schonberg et al. (20) concluded in a multicenter study that a critical need exists for high-quality antisera. A new enzyme-linked immunosorbent assay serotyping format used in conjunction with a commercially available kit to make serotyping more efficient and more ...

Gene Scanning of an Internalin B Gene Fragment Using High-Resolution Melting Curve Analysis as a Tool for Rapid Typing of Listeria monocytogenes

“…In contrast to several recently described molecular serotyping approaches, which separate L. monocytogenes isolates of diverse serotypes into four groups, 12,13,28 the inlB HRM curve profiling separated the 11 investigated L. monocytogenes serotypes into at least 15 specific HRM curve profiles comprising 18 different sequence types. Nonetheless, as described for other PCR-based typing methods, 12,13,28 HRM curve profiling does not allow differentiation of serotype 3a isolates from serotype 1/2a inlB ST-7, inlB ST-9, and inlB ST-12 isolates, of serotype 1/2c from serotype 3c isolates, of some serotype 1/2b isolates from serotype 3b and from serotype 7 isolates, of some 2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, 4d, 4e, and 7. The baseline is represented by an inlB ST-2 curve profile.…”

“…8 -11 Recently developed multiplex PCR serotyping methods allow a differentiation of isolates on the PG level. 12,13 Phylogenetic groups have been correlated with serotypes: PG I.1 with serotype 1/2a, 3a; I.2 with 1/2c, 3c; II.1 with 4b, 4d, 4e; II.2 with 1/2b, 3b, 7; and III with 4a, 4c. 10 In cases of outbreak, the discriminatory power of multiplex PCR serotyping methods is insufficient, and it is necessary to type isolates by pulsed-field gel electrophoresis (PFGE), which is the current gold standard for L. monocytogenes typing.…”

Outbreak of encephalitic listeriosis in red-legged partridges (Alectoris rufa)

“…Results of the electrophoresis were visualized under UV light and photographed using a BioRad™ UV Gel-doc© system (Hemel Hempstead, UK). According to the pattern obtained (Doumith et al, 2004;Huang et al, 2011) isolates were classified as being one of the four L. monocytogenes serogroups, as Listeria spp. or as being non-Listeria species.…”

“…Molecular serogrouping of L. monocytogenes isolates was performed using the multiplex gel-based PCR assay (LSER-PCR) described by Doumith et al (2004) using 5 μl of DNA extract. Pre-cast ethidium bromide-stained 1.5% agarose E-gel© (Invitrogen, Life Technologies) were used to separate the multiplex PCR products by electrophoresis.…”

Invasiveness of Listeria monocytogenes strains isolated from animals in Poland

“…All strains were classified into serogroups using multiplex PCR (Doumith et al 2004). Assignment to lineages was based on actA gene partial sequence analysis (Zhou et al 2005).…”