Biofilm Forming Capacity, Sanitizer Tolerance and Genetic Characterization of Persistent and Non-Persistent Listeria monocytogenes from Artisanal Cheese Processing Environments

Listeria monocytogenes is known to colonize food production environments and cross-contaminate finished foods. We investigated 30 L. monocytogenes collected from artisan cheese production facilities in Vermont from 2006-2008 for sanitizer tolerance, biofilm formation capacity, biofilm architecture, and tolerance to sanitizers of mature biofilms. Sixteen of these isolates represented a putatively persistent ribotype (DUP-1042B) found in one facility over two years. Isolates of the putatively persistent ribotype all aligned into ST191 and were 0-6 SNPs different, confirming they represented a persistent strain. We found no significant differences in sanitizer tolerance or crystal assay-based attachment capacity between persistent and non-persistent strains. However, using scanning electron microscopy, we found that isolates FML-10 and FML-19 formed substantially denser biofilms after 10 days on stainless steel. Ten-day old biofilms were highly resistant to sanitizers; neither quaternary ammonium nor sodium hypochlorite-based sanitizers achieved an EPA-recommended 6-log reduction. More EPS was found in low-nutrient biofilm conditions; thus, non-food contact surfaces in cheese environments may induce formation of biofilms with high sanitizer tolerance. 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. HighlightsO_LIIsolates from persistent ribotype DUP-1042B/ST191 were within 6 SNPs of each other C_LIO_LITwo isolates from ST191 made dense biofilms in nutrient rich conditions C_LIO_LIMore EPS was produced in nutrient-poor conditions C_LIO_LIMature biofilms of all isolates were highly resistant to QAC and SH sanitizers C_LI ImportanceThis study identifies strategies used by a set of persistently colonizing L. monocytogenes isolated from an artisanal cheese producer in Vermont, finding that some persistently colonizing isolates had high biofilm forming capacity, which may have contributed to their persistence.