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Title: Genome sequencing identifies two nearly unchanged strains of persistent Listeria monocytogenes isolated in two different fish processing plants sampled six years apart

Author
item HOLCH, ANNE - Technical University Of Denmark
item WEBB, KRISTEN - Allegheny College
item LUKJANCENKO, OKSANA - Technical University Of Denmark
item USSERY, DAVID - Technical University Of Denmark
item Rosenthal, Benjamin
item GRAM, LONE - Technical University Of Denmark

Submitted to: International Journal of Food Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/1/2012
Publication Date: 5/1/2013
Citation: Holch, A., Webb, K., Lukjancenko, O., Ussery, D., Rosenthal, B.M., Gram, L. 2013. Genome sequencing identifies two nearly unchanged strains of persistent Listeria monocytogenes isolated in two different fish processing plants sampled six years apart. International Journal of Food Microbiology. 79:2944-2951.

Interpretive Summary: Certain strains of Listeria monocytogenes represent especially important threats to food safety given elevate attack and case fatality rates, and given their ability to persist in food processing environments. It would therefore be useful to understand what endows those strains with the ability to persist, even in environments that are cleaned according to food industry standards. Comparative genomic analysis provides a means to comprehensively evaluate what may uniquely distinguish such strains, and was therefore employed here as a means to compare two strains, isolated at different times and from different fish processing plants in Denmark, which were suspected to be similar or identical. Their genomes were sequenced, compared to each other, and compared to several other available isolates of L. monocytogenes. Doing so confirmed that these two isolates are remarkably similar in gene content and sequence, more so than either is to other reference isolates, and that a particular genomic region has been deleted from these (and a third) ‘persistent’ strain, suggesting that the absence of genes in this region may predispose L. monocytogenes to persistence.

Technical Abstract: Listeria monocytogenes is a food-borne human pathogenic bacterium that can cause infections with a high mortality rate. It has a remarkable ability to persist in food processing facilities and here we report the complete genome sequences for two L. monocytogenes strains (N53-1 and La111) that were isolated six years apart from two different Danish fish processers. Both strains are serotype 1/2a and belong to a highly persistent DNA subtype (RAPD 9) isolated several times in Denmark. We here demonstrate using in silico analyses that both strains belong to the MLST type ST121 that has been isolated as persistent sub-type in several European countries. The purpose of this study was to compare complete genomes and predicted proteomes to identify genes or proteins that could contribute to persistence. In a genome comparison, the two persistent strains were extremely similar and collectively differed from the reference lineage II strain, EGD-e. Also, they differed markedly from a lineage I strain (F2365). On the proteome level, the two strains were almost identical with a predicted protein homology at 99.94% and only 2 proteins separates the two strains. No Single Nucleotide Polymorphisms (SNPs) differences were seen between the two strains, but using EGD-e as a reference 3,942 and 3,471 SNP differences were found for N53-1 and La111 respectively. We included a US persistent L. monocytogenes (F6854) in our comparisons. Two gaps were found in the three persistent strains and not in the two reference strains. This included a gap of 2,472 bp that contains the gene for inlF, and a gap of 3,017 bp covering three genes potentially related to bacteriocin production and transport (lmo2774, lmo2775 and the 3’ terminal part of lmo2776). Further studies of highly persistent strains are required to determine if absence of these genes is indicative of persistence. Whilst the genome comparison did not point to a clear physiological explanation of the persistent phenotype, the remarkable similarity between the two strains indicate that sub-types with specific traits are selected for in the food processing environment and that particular genetic and physiological factors are responsible for the persistent phenotype.