Characterization of Listeria monocytogenes Plasmids that were Newly Identified in Whole-genome Sequences of Listeriosis Outbreak Isolates.

摘要

Listeria monocytogenes is a Gram-positive bacterium that is found ubiquitously throughout nature and is the etiologic agent of listeriosis. The majority of human listeriosis is foodborne, resulting from the consumption of unpasteurized and ready-to-eat foods that are contaminated during food processing. During the 2008 nationwide outbreak, the Gilmour laboratory performed the first real-time application of high-throughput whole-genome sequencing (WGS) of outbreak strains. Within this genomic data, the 77 kb plasmid, pLM5578, was newly identified in a clinical isolate, and additional Listeria plasmids (the 80 kb pLM5026 and the 60 kb pLM0813) were subsequently identified after WGS was completed on an expanded panel of outbreak isolates. Little was known regarding how plasmids contribute to persistence and virulence of L. monocytogenes, and to investigate these potential relationships, a panel of 147 L. monocytogenes food, environmental, and clinical isolates from Canadian public health events from the last three decades was selected for further study of the plasmids they might contain. Strain carriage of plasmids was determined using conventional PCR targeting known plasmid gene targets. Bioinformatic analyses were then used to predict the functions of individual genes encoded by each sequenced plasmid. These analyses were then used to direct experiments investigating the functions and associated phenotypes conferred by plasmid carriage. Phenotypic analyses included antimicrobial susceptibility testing, heavy metal resistance, and biofilm formation assays. Finally, WGS analyses was performed on isolates with plasmid screening patterns that indicated carriage of potential novel plasmids. Screening revealed that 75 of 147 isolates were positive for the presence of a plasmid, for which WGS analysis identified 24 unique newly identified L. monocytogenes plasmids. Phenotypically, 15 of these plasmids were found to contribute to a decreased susceptibility to the heavy metal cadmium, whereas 4 conferred resistance to the sanitizer benzalkonium chloride. Plasmid carriage was also found to affect biofilm formation. Nine plasmids correlated with stronger biofilm formation phenotypes; whereas 5 plasmids were correlated with weaker biofilm formation phenotypes. No known virulence factors or antibiotic resistance determinants were present in the DNA sequences of these 24 newly identified plasmids. Numerous coding sequences predicted to assist with survival under environmental stress were identified, and it is hypothesized that these plasmids likely contributed to persistence of L. monocytogenes within food processing environments.