Colonisation dynamics of Listeria monocytogenes isolates from the food production environment

Listeria monocytogenes is a ubiquitous saprophytic bacterium and human pathogen capable of causing severe disease in at risk population groups. The consumption of contaminated food products, particularly ready to eat products, is the main vector for human listeriosis. L. monocytogenes can enter the food production environment (FPE) through raw ingredients and can be further transmitted throughout the facility through staff movements leading to colonisation and persistence in the production environment. An understanding of the various elements which may contribute to L. monocytogenes' ability to colonise and survive within the FPE is therefore required to minimise the food safety risks of this pathogen. This research assessed biofilm formation as it can be a key contributor to colonising the processing environment; the first aim was to develop a high throughput model biofilm system to assess biofilm formation at conditions reflective of FPEs, including low temperature and low nutrient conditions using common surface material present within processing facilities. This model system successfully enabled rapid screening of biofilm phenotypes, facilitating observation of attachment and biofilm formation. This development led into the second aim which centred on understanding a selection of colonisation dynamics utilising five fast biofilm formers and five slow biofilm formers identified using the model biofilm system. The strain set studied was referred to as the B10 group. No strong associations were identified between the growth rate, exopolymeric substance production and expression of signalling propeptide AgrD with the rapid colonisation phenotype. The global transcriptome suggested that transport, energy production and metabolism genes were widely upregulated during the initial colonisation stages under nutrient limited conditions. However, the upregulation of metabolic systems varied between isolates supporting the idea that L. monocytogenes' ability to colonise the FPE has strain-specific aspects. The L. monocytogenes isolates (n=52) were phenotypically and genotypically assessed for their potential to survive within FPEs, as well as their pathogenicity and response to clinically relevant antibiotics. A vast array of genetic determinants was present across the collection with some strains containing important virulence genes suggestive of hypervirulence. As the strain collection was isolated from foods or the production environment all isolates contained multiple genes that aid tolerance and provide mitigation against a range of stressors. From data here a transposon identified in Enterococcaceae containing a novel L. monocytogenes cadA resistance gene was identified suggesting a horizontal gene transfer (HGT) event may have occurred between Listeria and Enterococcus. A novel insert was also identified in the hypervariable region in which some strains contain Listeria genomic island 1; this novel insertion shared similarity to Tn916 from Bacillus subtilis. All isolates in this study were sensitive to the five clinically relevant antibiotics tested supporting the successful treatment of listeriosis by these antibiotics. The adaptability of L. monocytogenes strains and the presence of antimicrobial resistance determinants opens the door to alternative treatment options within the FPE using biocontrol measures like bacteriophages, endolysins, competitive bacterial species, bacteriocins and plant-derived antimicrobial products. The applicability of bacteriophage has shown significant relevance against bacterial strains with the commercialisation of bacteriophage treatments; analysis of the literature showed bacteriocins and endolysins can offer significant reductions on established biofilms, however, they generally require further research and development. The use of competitive bacterial species can offer customised treatments when antagonist species are identified from the production environment. However, there have been minimal inhouse application of these biocontrol measures and as such further standardisation and infacility assessment is required. In summary, this thesis contributes to further our current understanding on L. monocytogenes' ability to colonise FPEs, and the survival, pathogenicity and treatment potential of strains isolated from representative food environments were investigated. While the ability to colonise stainless-steel surfaces appeared to have some strain specific aspects it is noted that environmental conditions play a large part in this, and as such particular care in design and maintenance of the processing facility is required. In addition, strains isolated from the FPE environment display a vast phenotypic and genetic resistance profile with some isolates capable of hypervirulence. Regular monitoring of the phenotypic and genotypic profile is suggested with the identification of novel genes and inserts indicative of HGT events, which may contribute towards their enhanced fitness in the FPE and influence pathogenicity.