Biofilm forming capacity, sanitizer tolerance, and genetic characterization of persistent and non-persistent Listeria monocytogenes from artisan cheese processing environments.

Listeria monocytogenes is known to colonize food production environments and cross-contaminate finished foods. We investigated 30 L. monocytogenes isolates collected from artisan raw milk cheese production facilities in Vermont from 2006 to 2008, Sixteen of which represented a putatively persistent ribotype (DUP-1042B) found in one facility over two years. To determine persistence mechanisms, we evaluated the whole genome of the isolates as well as their sanitizer tolerance and biofilm formation capacity. For a representative subset (six isolates), we evaluated their mature biofilm properties on stainless steel. Isolates of the putatively persistent ribotype all aligned into ST191 and were 0-6 Single Nucleotide Polymorphisms (SNPs) different, confirming they represented a persistent strain. SSI-1 and an enrichment of genes in DNA repair and biosynthetic pathways were also exclusively confirmed in the core and accessory genome respectively, of the persistent strain. However, no significant differences in sanitizer tolerance were found between persistent and non-persistent strains, and none of the 30 isolates carried sanitizer resistance genes (bcrABC or qacH). Some ST 191 isolates formed visually more complex biofilms as shown by SEM and maintained significantly higher biomass over five days on polystyrene but were indistinguishable from non-persistent strains over ten days on stainless steel. No clear persistence mechanism was thus identified, but evidence for biofilm formation was stronger in the ST191 isolates than in the non-persistent strains. Our results highlight the importance of regular environmental testing and strain typing for rapid detection of L. monocytogenes colonization attempts while they can still be removed without major renovations or equipment replacement.