All human strains sent to the NRC in 1995 were included in this study. Listeriosis surveillance was essentially laboratory based (mandatory notification was established in 1998). Due to the passive nature of this process (strains are sent to the NRC voluntarily by clinical microbiologists), the number of strains collected is probably an underestimate of the true number of cases of listeriosis and its clinical forms. However, this is the only representative set of human listeriosis strains in France. Food strains were randomly selected from strains voluntarily sent by public and private food hygiene laboratories during the same period. Little information concerning these strains was provided. This set of strains was not representative of L. monocytogenes contamination of food at each step of the food chain and consumer exposure. Nevertheless, the variety of foods and the large number of manufacturers in industrialized countries make it impossible to compile a set of L. monocytogenes strains that is fully representative of the population present at each step of the food chain.
It is known that more serogroup 1/2 strains are recovered from foods than from humans (1
). This unequal distribution was observed in the set of strains which was used (OR = 5.05; P
< 1 × 10−7
), which explains why most food strains were in group Ami1. Food may be contaminated by virulent strains, as shown by food-borne outbreaks. However, several studies have indicated that attenuated or nonvirulent populations of L. monocytogenes
may also be found among food isolates. These strains mainly belong to serogroup 1/2 (7
). The statistical comparison of food-borne and human strains in this study suggests that if strains that are attenuated or nonvirulent for humans exist, they are predominantly associated with serovar 1/2c.
All of the virulence determinants in L. monocytogenes
identified to date are chromosome encoded. As in other pathogenic bacteria, virulence genes are organized in genetic islands; hly
, encoding LLO and ActA, respectively, are physically linked in a PrfA-dependent virulence gene cluster, inlB
is in the internalin loci, and ami
is in the autolysin gene region (27
). For a long time LLO was assumed to be a major virulence determinant, and spontaneous nonhemolytic strains, such as transposon-induced mutants, are avirulent in mice (15
). Thus, the lack of polymorphism in LLO apparent molecular weight observed in this study was unexpected. Sequence determination, PCR-restriction fragment polymorphism analysis, mismatch amplification mutation assay-PCR, PCR-single-strand conformation polymorphism analysis, and deducing the amino acid sequence encoded by the hly
gene have revealed some differences between L. monocytogenes
). A comparison of four strains revealed one to five nucleotide differences in the hly
). Furthermore, the hly
genes of serotypes 1/2b and 4b form a cluster, which is not found in serotypes 1/2a and 1/2c. Due to the large number of strains tested in this study (75 clinical isolates and 75 food isolates) strains with such mutations were certainly included. However, slight changes in the LLO gene do not alter the molecular weight of LLO. These results confirm those of Matar et al. (26
), who found no significant difference between L. monocytogenes
serotypes 1/2a and 4b by using immunoaffinity-purified LLO.
Ericsson et al. (12
) showed that the levels of sequence similarity of the inlB
genes of 24 strains belonging to different L. monocytogenes
serovars were between 89.2 to 100%, corresponding to levels of amino acid sequence similarity of 91.9 to 100%. Due to the large number of strains tested in this study (300 clinical isolates and 150 food isolates), strains with these variations were certainly included. Consequently, the lack of polymorphism for InlB in this study was unexpected. Results of the present study showed that there was no difference between strains responsible for CNSI and strains involved in the other clinical forms of disease even though InlB is not an important virulence factor for cerebral listeriosis (39
). The fact that InlB proteins with the same molecular weight are expressed in all strains may be because InlB interacts quite specifically with its receptor (16
). Alternatively, slight differences in inlB
may have no effect on the molecular weight of InlB.
The role of Ami in L. monocytogenes
virulence has been investigated recently (29
). Ami plays a role in adhesion. In this study, strains involved in bacteremia were twice as likely to be associated with group Ami1 as NPRC strains. Thus, it would be interesting to understand the significance of the differences between strains. Ami was not detected in 6% of the human strains examined (serovar 4b). In these strains, ami
is present, as indicated by PCR results, but further experiments are required to determine if the gene is defective or unexpressed.
Niebuhr et al. (33
) found small but clear differences in the molecular weight of ActA in strains belonging to different serovars. In addition, ActA was detected in most strains of L. monocytogenes
tested; the only exceptions were one serovar 3a strain and one serovar 4ab strain. Moriishi et al. (30
) sequenced the actA
gene of 24 strains isolated from healthy humans and patients and found two groups, one of which was characterized by deletion of one proline-rich repeat. No correlation between this classification and serovars was found. In the present study, four groups of strains were identified based on the molecular weight of ActA. These groups were not strictly correlated with serovars. The multivariate analysis used to compare human and food strains indicated that group ActA3 strains were found three times more frequently in food strains than in human strains, after adjustment for other significant markers (OR = 2.90; P
= 1 × 10−4
). This could suggest that attenuated or nonvirulent strains are associated with this marker. However, strains responsible for CNSI are also more likely to be characterized by this marker, which shows that additional markers are required to identify attenuated or nonvirulent strains. The molecular mass of ActA in group ActA3 strains was lower than the molecular mass of LO28, due to a deletion in actA
. As this deletion is detected in human strains, (i.e., pathogenic strains), it is probably not in the amino-terminal region, which is essential for F-actin assembly and movement. This deletion is more likely in the internal proline-rich repeats or the carboxy-terminal domain, which are not essential (22
). This hypothesis is supported by the findings of Moriishi et al. (30
), who located polymorphisms in the proline-rich region, and by the findings of Sokolovic et al. (42
), who found that the lower molecular masses of the serovar 4a and 4e ActA proteins were due to a deletion in the same region. In vitro motility assays showed that this deletion decreases the motility of the bacteria (22
). In the mouse model, a mutant with a mutation in the proline-rich region was less virulent than the wild type (41
). Strains responsible for CNSI were also more likely to be associated with group ActA2. Further studies are required to understand these data and to determine whether the diversity of CNSI strains is correlated with other characteristics, such as phosphatidylcholine-specific phospholipase C, which is known to be a major virulence factor for this clinical form (39
In conclusion, this study was the first step in determining the virulence marker heterogeneity in L. monocytogenes by statistically comparing human strains and food strains. Present results show that there are clear differences in the proteins expressed by different serovars, by strains having different origins (human versus food), and by organisms that cause different clinical forms of listeriosis. However, the markers identified in this study cannot be used to unambiguously identify food-borne strains which are attenuated or nonvirulent in humans. Further studies are required to analyze the group of strains belonging to group ActA3. Further investigations are also required to understand how differences in the expression of Ami and ActA affect the infectious process of the bacterium. It would be interesting to determine whether the observed differences are silent or influence or modulate pathogenicity or pathogenic mechanisms. In addition, more L. monocytogenes virulence proteins should be tested in order to elucidate the pathogenicity of L. monocytogenes strains depending on the human clinical manifestations, as well as the ecology of L. monocytogenes in food.