Studies on the biology of environmentally persistent Listeria monocytogenes strains

Listeria monocytogenes is a saprophytic bacterium capable of causing serious foodborne human disease. Persistent contamination by L. monocytogenes represents a threat to public health and has serious economic implications for the contaminated facility. Actions taken to minimise contamination of the factory environment/food products are expected to cause physiological stress to L. monocytogenes, and it has been shown that the ability to resist stress, and therefore persist within an environment, can augment L. monocytogenes virulence. In this dissertation, the hypothesis that physiological adjustment by L. monocytogenes facilitates persistent food factory contamination, and that some strains are better able to implement this shift than others, is assessed. Isolates recovered from a food factory were characterised using multilocus sequence typing, and attributes of this group thought to enable persistent environmental contamination were compared. These included biofilm production and the mechanisms affording alkaline stress adaptation. Biofilm production by L. monocytogenes strains isolated from multiple environments, including a food processing factory and environmentally persistent/sporadic factory isolates, due to temperature and pH stress was assessed using a colourimetric assay and scanning electron microscopy. A temperature-specific biofilm production response was observed as was environmentally induced homogeneous biofilm production by non-clonal L. monocytogenes strains recovered from the same environment. Observations provided evidence for distinct, inducible biofilm production. Importantly, it was concluded that biofilm production alone does not determine the persistent L. monocytogenes phenotype. To investigate if stress adaptation contributes to persistent factory contamination, protein expression by alkaline adapted persistent and sporadic factory contaminants was compared using high resolution proteomics. Additionally, a well characterised L. monocytogenes strain was studied at pH 9.0, with emphasis on the role of cell membrane proteins. Qualitative and relative protein abundances were compared through functional ontology and determination of spectral abundances. Findings suggested alkaline adaptation involves cytoplasmic acidification by surrogate proton sources, an energy metabolism shift, stabilisation of cellular processes and cell wall modification. This was most pronounced in exponential phase. Although more pronounced in persistent strains, no difference in the mechanisms supporting alkaline tolerance was evident between strains. The results of this dissertation support the notion that exposure to environmental stresses within food facilities can induce or select the persistent L. monocytogenes phenotype. This may be caused by the complexity of operations within these facilities, leading to concentration fluxes of cleansing/sanitising agents and other growth limiting challenges, subjecting the strains to varying forms of sublethal stress.