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Elucidation of novel physiological and genetic elements associated with the cold adaptability and survival of Listeria monocytogenes in the food processing continuum Hingston, Patricia

Abstract

Novel physiological and genetic factors associated with the survival of Listeria monocytogenes in the food-processing continuum were investigated, with an emphasis on its cold-growth ability. Food-related L. monocytogenes strains (n=166) were sequenced and subsequently evaluated on their ability to tolerate cold (4°C), salt (6% NaCl, 25°C), acid (pH 5, 25°C), and desiccation (33% RH, 20°C) stress. Stress tolerances were associated with serotype, clonal complex, full-length inlA profiles, and plasmid harbourage. Notably, strains possessing full length inlA (as opposed to a truncated version) exhibited significantly (p<0.001) enhanced cold tolerance and plasmid-positive strains demonstrated enhanced (p=0.013) acid tolerance. Relative gene expression indicated that several plasmid-encoded genes (e.g., NADH peroxidase, clpL, proW) are induced in L. monocytogenes during growth in 6% NaCl and at pH 5. Additionally, a whole-genome sequence phylogeny revealed closely related stress sensitive and tolerant strains, highlighting that minor genetic differences impact strain phenotypes. Strand-specific RNA sequencing showed that L. monocytogenes suppresses 1.3× more genes than it induces at 4°C relative to 20°C. The largest number (n=1,431) and greatest magnitude (>1,000-fold) of differentially expressed (e.g., >2-fold, p<0.05) genes occurred in late stationary-phase cells. A core set of 22 genes were upregulated at all five growth phases investigated and included nine genes required for branched-chain fatty acid (BCFA) synthesis. Correspondingly, BCFA levels increased by 15% during cold stress exposure. Transcription of antisense RNA (asRNA) was 2.5× higher in cells grown at 4°C relative to 20°C, with the most asRNA transcripts upregulated in lag phase cells. Spontaneous L. monocytogenes variants displaying enhanced cold tolerance (ECT) were isolated from a cold-sensitive strain culture following 84 days of storage at 4°C. While the parent strain had an impaired ability to produce BCFAs, the ECT variants were able to overcome this limitation which is hypothesized to be a result of mutations identified in acetyl-coA carboxylase. Collectively this work has improved our understanding of the response of L. monocytogenes to to cold stress and genotypes associated with stress-tolerance phenotypes. This information may be useful for developing biomarkers to quickly predict the risks associated with food isolates, or aid in developing new and/or improved intervention strategies. Supplementary materials available at: http://hdl.handle.net/2429/64422

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Attribution-NonCommercial-NoDerivatives 4.0 International