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Polymorphism of actA gene is not related to in vitro virulence of Listeria monocytogenes

https://doi.org/10.1016/j.ijfoodmicro.2009.10.019Get rights and content

Abstract

Listeria monocytogenes is a foodborne pathogen which is able to cause serious disease both in humans and in animals. Several studies have demonstrated variations in the levels of virulence among L. monocytogenes strains.

Invasion and growth ability of L. monocytogenes into cultured cells have been used to evaluate its pathogenicity. In particular, invasiveness and growth ability have been typically investigated using HeLa cell line. This study aimed to provide further insights on the virulence potential as well as on the molecular and phenotypic characteristics of L. monocytogenes isolated both from food sources and food environments. Thirty-eight isolates were tested for cell invasion and intracellular growth. Among the latter, 15 strains exhibited a high invasion index (I.I.); 18 strains showed intermediate II and 5 isolates revealed a low II. Regarding intracellular growth, all tested isolates had a replication time between 2 and 6 h. Furthermore, nine virulence-associated genes (hlyA, actA, inlA, inlB, iap, plcA, plcB, mpl, prfA) were investigated by the multiplex PCR assay. All tested virulence genes were detected in all strains. Interestingly, a polymorphism was observed in the actA gene. However, the polymorphism could not be related to a different level of invasion or intracellular growth. In conclusion, data presented in this study have revealed considerable differences in the ability of L. monocytogenes strains to invade host cells and suggest the presence of additional factors that may contribute to adhesion and invasion. Virulence of L. monocytogenes is still not fully understood in some respects. Further studies focused on the mechanisms of L. monocytogenes pathogenicity together with the development of more reliable and efficient methods for virulence determination in this species are still required.

Introduction

Listeria monocytogenes is a ubiquitous facultative intracellular bacterium which is potentially pathogenic for humans. Ingestion of food contaminated with L. monocytogenes is the primary route of transmission (Dussurget, 2008). Although the incidence of the disease is low, it remains a public health concern because of its high mortality rate (Swaminathan and Gerner-Smidt, 2007).

L. monocytogenes has been found in a wide variety of food and especially in ready to eat (RTE) products (EFSA, 2007). The discrepancy between the widespread distribution of L. monocytogenes in RTE food products and the low incidence of listeriosis supports the hypothesis that the virulence in some strains in food products may be attenuated (Zhou and Jiao, 2005).

L. monocytogenes strains show heterogeneous levels of virulence (Roche et al., 2009). Nevertheless, it is well known that the notion of virulence depends on the tests used and has changed over the last decade (Roche et al., 2009). There are many published methods for determining virulence properties of L. monocytogenes. Among them, the presence of virulence genes, the production and expression of virulence factors and other proteins both in mice assay or in cell-line assay are the most commonly used (Jaradat et al., 2002). The mouse virulence assay is not applied routinely for laboratory determination of L. monocytogenes pathogenic potential, given its constantly escalating cost in addition to its need to sacrifice animals. In vitro cell assays can be an economical alternative to in vivo bioassays for L. monocytogenes virulence. In parallel to in vitro cell assays, molecular approaches (such as the Polymerase Chain Reaction (PCR)) have been developed to assess these characteristics with the additional aim of reducing laboratory costs and times.

From a risk analysis perspective it is important to assess the virulence potential of strains (Jensen et al., 2008). Several publications have reported that 10–20% of strains isolated from the food industry are weakly or non-pathogenic in in vivo (Tabouret et al., 1991) or in vitro tests (Roche et al., 2009).

The aim of this work was to evaluate the possible relationship between in vitro virulence properties of L. monocytogenes strains isolated from food and food environments and the presence of genes encoding key virulence factors, particularly actA. Virulence was tested in vitro by invasiveness and growth of the strains in HeLa cell lines. Moreover, multiplex PCR assay was used to investigate virulence-associated genes.

Section snippets

Bacterial strains and culture media

This study comprised 38 strains of L. monocytogenes previously isolated from different food and working environments. Twenty strains were isolated from meat and meat products (pork, beef, and poultry), 10 were isolated from fish and fish products (salmon, sword fish and black sea bass), and 8 were isolate from the food working environments. Detection was performed according to the EN ISO 11290-1:1996. Identification at species level was performed using a multiplex PCR described below.

To perform

Results and discussion

Numerous methods have previously been applied to determine the L. monocytogenes virulence. The use of either immunocompetent or immunocompromised animals (Roche et al., 2001) or chick embryos (Terplan and Steinmeyer, 1989) is the most reliable of all, but they are expensive and time-consuming. A practical alternative to in vivo studies are in vitro tests, based on continuous cell lines (Larsen et al., 2002). At last, new generation techniques have been devised for the assessment of L.

Acknowledgments

We wish to thank Prof. Rina Mazzette and her colleagues, Dr. Domenico Meloni and Anna Mureddu, from the Department of Animal Biology, Unit of Inspection of Food of Animal Origin, University of Sassari, who have sent strains of L. monocytogenes to the Department of Animal Production in Parma.

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