Whole-Genome Sequencing Characterization of Virulence Profiles of Listeria monocytogenes Food and Human Isolates and In Vitro Adhesion/Invasion Assessment

Abstract

Listeria monocytogenes (Lm) is the causative agent of human listeriosis. Lm strains have different virulence potential. For this reason, we preliminarily characterised via Whole-Genome Sequencing (WGS) some Lm strains for their key genomic features and virulence-associated determinants, assigning the clonal complex (CC). Moreover, the ability of the same strains to adhere to and invade human colon carcinoma cell line Caco-2, evaluating the possible correspondence with their genetic virulence profile, was also assessed. The clinical strains typed belonged to clonal complex (CC)1, CC31, and CC101 and showed a very low invasiveness. The Lm strains isolated from food were assigned to CC1, CC7, CC9, and CC121. All CC1 carried the hypervirulence pathogenicity island LIPI-3 in addition to LIPI-1. Premature stop codons in the inlA gene were found only in Lm of food origin belonging to CC9 and CC121. The presence of LIPI2_inlII was observed in all the CCs except CC1. The CC7 strain, belonging to an epidemic cluster, also carried the internalin genes inlG and inlL and showed the highest level of invasion. In contrast, the human CC31 strain lacked the lapB and vip genes and presented the lowest level of invasiveness. In Lm, the genetic determinants of hypo- or hypervirulence are not necessarily predictive of a cell adhesion and/or invasion ability in vitro. Moreover, since listeriosis results from the interplay between host and virulence features of the pathogen, even hypovirulent clones are able to cause infection in immunocompromised people.

Keywords: Listeria monocytogenes; Whole-Genome Sequencing (WGS); adhesion and invasion capacity; pork-meat products; premature stop codon (PMSC); virulence genes.

Figure 1

Heatmap of the virulence genes detected in silico using the BIGSdb-Lm scheme. Blue: presence of the gene; red: gene with a mutation that leads to premature stop codons (PMSCs); white: absence of the gene.

Figure 2

Ability of Lm isolates to adhere to and invade Caco-2 human intestinal epithelial cells. Data were plotted as percentages of the starting viable inoculum. Data are the means ± standard deviation (SD) of three separate experiments. p < 0.05. The analyses were conducted using GraphPad Prism 5 Software.* p < 0.05, *** p < 0.001.

Figure 3

Hoechst staining. (A) CTR, uninfected Caco-2 monolayer, and L. innocua ATCC33090, negative control. (B) Caco-2 monolayer infected with clinical strains. (C) Caco-2 monolayer infected with food strains (magnification, 63×). Red arrows highlight Lm infected cells.

Figure 4

Correlation plot of the adhesion and invasion levels of three clinical (490, 566, and 1498) (A) and six food-derived (B) (1484, 1487, 1608, 1643, 1715, and 2018) strains of L. monocytogenes.