Listeria monocytogenes isolated from food samples from a Romanian black market show distinct virulence profiles.

Listeria monocytogenes is a facultative intracellular foodborne pathogen responsible for listeriosis. In a recent study, in which we investigated neglected exogenous routes of transmission of foodborne pathogens into the European Union, we have isolated 15 L. monocytogenes strains in food products, which were imported from the Republic of Moldavia to Romania and illegally sold at a local market. The aim of this study was to characterize the subtype and virulence potential of these 15 L. monocytogenes strains. Multilocus sequence typing revealed that these L. monocytogenes strains belong to six different sequence types (ST2, ST8, ST9, ST20, ST121 and ST155). In addition, in vitro virulence assays using human intestinal epithelial Caco2 and macrophage-like THP1 cells showed a high strain variability regarding the invasion efficiency in Caco2 cells (0.98-2.78%) and the intracellular growth rate in both cell types. Both ST121 strains and the ST9 isolate were unable to invade Caco2 cells, and all ST155 strains showed no proliferation inside macrophages and revealed low cytotoxicity. Furthermore we performed sequence analysis of three main virulence factors: PrfA, internalin A (InlA) and listeriolysin O (LLO). The Romanian food isolates showed a high diversity in the InlA and LLO amino acid sequences, whereas the amino acid sequence of PrfA of all strains was identical. Overall, the amino acid sequences of PrfA, InlA and LLO were identical for strains belonging to the same ST. We detected in total 30 different amino acid substitutions, resulting in seven different InlA variants, two of which have not yet been described. The three strains, which were unable to invade Caco2 cells, harboured a premature stop codon resulting in truncated InlA. Furthermore, we detected four different amino acid substitutions in the LLO sequence, which are present in four variants. The number of LLO mutations correlates negatively with intracellular growth in Caco2 and THP1 cells and subsequently with cytotoxicity. In conclusion, we show that L. monocytogenes isolated from food samples from a Romanian black market show distinct virulence profiles, due to a high diversity in the amino acid sequence of main virulence factors.