Phenotypic and genotypic analysis of persistent and sporadic isolates of listeria monocytogenes

The foodborne pathogen Listeria monocytogenes has the ability to persist for months to years within food-associated environments. These persistent strains maintain a constant risk of contamination and it is vital to identify and eliminate these persistent strains as soon as possible. Previous work has identified and characterized putative persistent and sporadic isolates from 30 retail delis in three regions of the U.S. over six months. Next generation sequencing allowed phylogenetic relationships to identify persistent strains based on the assumption that isolates of a persistent strain were more closely related than isolates of sporadic strains. Those studies provided the isolates and genomic data to investigate additional phenotypic and genotypic properties that may differentiate persistent and sporadic strains. In this present work, two questions were examined. (i) Do isolates of persistent strains contain distinct CRISPR spacers? (ii) Do isolates of persistent strains have higher growth rates or ability to grow in various stressful conditions or carbon sources associated with food environments? CRISPRs, or clustered regularly interspaced short palindromic repeats, are comprised of pieces of foreign DNA (spacers) and are sandwiched between repeated sequences of host DNA (directed repeats). The foreign DNA is integrated into a bacteria’s genome to act as a defense mechanism against foreign invading DNA, like bacteriophages. The integrated DNA, spacers, act like a logbook of which bacteriophages or foreign invaders have collided with a strain. Since persistent strains survive in environments for an extended period of time, and phages are common within food-associated environments, isolates of a persistent strain may contain more or different spacers than sporadic isolates. Viewing the bioinformatic analysis of previously sequenced persistent and sporadic isolates from retail delis, 174 of 175 L. monocytogenes isolates contained CRISPR spacer arrays that contained, at minimum, one CRISPR spacer within the array. Spacers that were found within isolates were conserved within the previously defined phylogenetic clades; these contiguous spacers were assigned to spacer patterns. While spacers were found within L. monocytogenes isolates, and isolates could be subtyped by their spacer patterns, there was no supporting data that persistent isolates could be differentiated from sporadic isolates based on their CRISPR spacer patterns. Can isolates of persistent strains live for longer periods due to an increased ability to adapt to food-associated environmental stresses or carbon sources? The goal was to understand if persistent strains are more likely to grow and how well they grow in response to stress conditions, such as osmotic pressure, acidity, or sanitizer. Specifically, high-throughput growth assays were used to screen for isolates’ ability to tolerate osmotic (5% or 10% NaCl), acidity (pH 5.2 or 9.2), or sub-lethal sanitizer (2 or 5 µg/mL benzalkonium chloride [BAC]) stresses, or to metabolize food-associated carbon sources (25 mM cellobiose, fructose, glucose, lactose, sucrose, or glycogen). At the end, persistent and sporadic strains did not differ from each other in either their ability to grow or their growth rate, if able to grow. Therefore, taken together, these data suggest that there is no strain-specific phenotype that facilitates the persistence of L. monocytogenes in these retail deli environments. The data was able to show that CRISPRs are prevalent in L. monocytogenes and that L. monocytogenes’ reactions to stressful treatments are consistent with previous literature, the persistence phenomenon is not distinguished through mobile genetic elements or responses to stress inducing environments. These data suggest that work should focus on identifying persistent strains of L. monocytogenes instead of understanding phenotypes or genotypes and look into improved environmental monitoring, cleaning and sanitation, or sanitary design