Food processing environments as reservoir of Listeria monocytogenes hypo- and hypervirulent clones: use of Whole Genome Sequencing and in vitro assays to characterize persistence

Listeria monocytogenes (Lm) is a significant foodborne pathogen causing human listeriosis, foodborne zoonoses with the highest hospitalization and fatality rate. This mainly due to its abilities to form biofilm, survive and grow under stressful conditions and resist disinfectants, and thus Lm can persist in food processing environments (FPEs) even for years with a continuous risk of food cross-contamination. In this study the role of FPEs as reservoirs of hypo- and hypervirulent clones of Lm was investigated. Whole Genome Sequencing (WGS) and bioinformatics analysis were used to assess the genetic relationships between the strains and to investigate their persistence and virulence profiles. Biofilm formation, sensitivity to Benzalkonium Chloride (BC) as well as adhesion and invasion abilities were assessed in vitro. WGS analysis and in particular cgMLST, identified Lm clones persisting for up to four years in the same food producing plant (FPP) as well as clones contaminating different FPEs of Central Italy. Multidrug efflux-pumps genetic determinants (sugE, mdrl, lde, norM, mepA) were carried by various Lm Clonal Complexes (CCs). All the CC121 strains also harboured the Tn6188_qac gene specific for tolerance to BC. Strains belonging to CC3, CC7, CC9, CC31 and CC191 carried the stress survival islet SSI-1 while CC121 clones harbored the SSI-2. CC9 and CC121 strains presented high-level cadmium resistance genes (cadA1C1) carried by different plasmids and showed a strong biofilm production. Preliminary results on Lm sensitivity to BC in vitro showed that strains belonging to a CC9 long term persistent cluster, despite not carrying specific genetic determinants for tolerance to BC, were less sensitive to low sanitizer concentrations than the other strains. Moreover, if compared with what was reported in recent studies on Lm, our results indicated a lower susceptibility to BC for the CC121 strains harbouring the Tn6188_qac. An investigation of the virulence genetic profiles showed that all the CC9 and CC121 strains presented a premature stop codon in the inlA gene which was complete in the other isolated CCs. The Listeria Pathogenicity Island 1 (LIPI-1) was widespread in all the Lm isolates. CC1, CC3 and CC191 clones also harboured the LIPI-3. The in vitro assessment of Lm virulence showed that the CC1, CC7, CC9 and CC121 tested strains isolated from food presented good adhesive and invasive abilities, with the CC7 clone showing the highest invasiveness and belonging to the epidemic cluster causing a severe listeriosis outbreak. All these findings represented a relevant risk for the consumers’ health. Hypovirulent CCs such as CC9 and CC121, more adapted to FPEs and able to persist after cleaning and sanitation, were the most frequently isolated in the FPP of Central Italy, representing a significant risk of food contamination. On the other hand, in this study hypervirulent clones (CC1 and CC2) were also detected in FPEs with situations in which they warningly persisted for long time in the same plant. A systematic monitoring of Lm in FPEs should be included in Italian food safety surveillance programs performed by the Competent Authorities to improve the management of the pathogen in the food industry minimizing risk of food contamination and recurrence of severe outbreak.