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Significance and Impact of the Study: Listeria monocytogenes 4b serotype strains are responsible for the majority of recorded invasive listeriosis outbreaks. We showed that strains of serotype 4b are not only the most virulent L. monocytogenes strains but also have the best capacity to enhance their invasiveness in response to salt stress. Our results suggest possession of effective stress response mechanisms of 4b serotype strains, which may contribute to the high infection potential of this subpopulation. AbstractOf the 13 serotypes, 4b serotype strains are responsible for the majority of recorded invasive listeriosis outbreaks, although some recent listeriosis outbreaks have been attributed to strains of serotypes 1/2a and 1/2b. Virulence and response to osmotic stress in 41 Listeria monocytogenes strains representing serotypes 1/2a, 1/2b and 4b was investigated. It was found that serotype 4b and 1/2b strains exhibited highest invasion efficiency and formed largest plaques in HT-29 cell monolayer. Invasiveness in response to 10-min exposure to 0Á3 mol l À1 NaCl was the highest in serotype 4b strains. We demonstrated that 4b serotype L. monocytogenes strains not only have the greatest pathogenic potential but also are the most invasive in response to salt stress.

Analysis of actA gene sequence polymorphism has been shown to be an effective and relatively inexpensive method for subtyping Listeria monocytogenes isolates, allowing the division of the population of this species into two deeply separate lineages. This sequence-based method as well as PCR-mediated fingerprinting were applied here for the differentiation of 49 isolates of food and clinical origin. Correlation between these two typing approaches was high. Both methods divided the isolates into two lineages, designated I (33 isolates) and II (16 isolates). All the 33 lineage I isolates were assigned to the same, or closely related, six clusters by both typing methods. For the lineage II isolates, PCR fingerprinting was found to be more discriminatory. The isolates were characterized by cell invasion assay. All highly invasive isolates were assigned to lineage I, which constituted a heterogeneous group also containing low-invasive isolates. High-invasive isolates were not found in the genetically determined lineage II. A particular actA cluster, designated Ha, contained all the isolates showing the lowest invasiveness. A common trait of the isolates belonging to this cluster was the presence of a threonine-441 of the deduced ActA sequence instead of the alanine-441 present in the remaining isolates. Thirteen human isolates were classified to lineage I and five to lineage II. A PCR-based method can therefore differentiate L. monocytogenes isolates in accordance with the current phylogenetic model of the evolution of this species.

During food production and food conservation, as well as the passage through the human gastrointestinal (GI) tract, L. monocytogenes is exposed to many adverse conditions which may elicit a stress response. As a result the pathogen may become more resistant to other unpropitious factors and may change its virulence. It has been shown that low and high temperature, salt, low pH, and high pressure affect the invasion capacity of L. monocytogenes. However, there is a scarcity of data on the duration of the stress effect on bacterial biology, including invasiveness. The aim of this work was to determine the period during which L. monocytogenes invasiveness remains altered under optimal conditions following exposure of bacteria to mild heat shock stress. Ten L. monocytogenes strains were exposed to heat shock at 54°C for 20 minutes. Then both heat-treated and nontreated control bacteria were incubated under optimal growth conditions, 37°C, for up to 72 hours and the invasion capacity was tested. Additionally, the expression of virulence and stress response genes was investigated in 2 strains. We found that heat stress exposure significantly decreases the invasiveness of all tested strains. However, during incubation at 37°C the invasion capacity of heat-treated strains recovered to the level of nontreated controls. The observed effect was strain-dependent and lasted from less than 24 hours to 72 hours. The invasiveness of 6 out of the 10 nontreated strains decreased during incubation at 37°C. The expression of inlAB correlated with the increase of invasiveness but the decrease of invasiveness did not correlate with changes of the level of these transcripts. Conclusions. The effect of heat stress on L. monocytogenes invasiveness is strain-dependent and was transient, lasting up to 72 hours.