Listeria monocytogenes is a food-borne bacterial pathogen which has the ability to attach to and form biofilm on food-processing surfaces. It is thought that L. monocytogenes biofilm formation in food processing environments is a major source of contamination because, once established, the biofilm appears to have greater resistance to disinfectants and other cleaning agents. In the first stage of this thesis, L. monocytogenes wild type 10403s and Tn917transposon mutant strains were surveyed for their ability to attach to polystyrene surfaces at different temperatures (37°C, 30°C and 18°C). The results indicated that two mutants, B265 and I366, showed a significant reduction in their attachment at 18°C and 30°C but not at 37°C. These data have revealed that there is a temperature-dependent involvement of some genes in surface attachment. Subsequently Arbitrary PCR was used to analyse sequences flanking the transposon insertions in both attachment-deficient transposon mutants. Two open reading frames for a putative NADH oxidase in B265 mutant and for a putative penicillin-binding protein in I366 mutant were identified. In the second stage of investigation, deletion mutants were subsequently made successful in these ORFs. Deletion mutation in each of these ORFs also resulted in a significant decrease in attachment of L. monocytogenes to polystyrene at 18°C and 30°C with no effect at 37°C. In the third stage, L. monocytogenes strains were screened for their ability to attach to polystyrene at different NaCl concentrations and pH values. Both the wild type and deletion mutants showed a decrease in attachment at high NaCl concentrations and a loss of attachment at high pHs. But neither of the mutants differed from wild type which showed a similar pattern of response. The molecular basis of biofilm mechanisms of L. monocytogenes is considered and this has been ascribed to the presence or absence of specific genes. Recently, there has been a growing interest in understanding the molecular basis of these processes. This study has demonstrated that NADH oxidase and penicillin-binding protein may play a role in L. monocytogenes attachment.

This study investigated genes involved in attachment of Listeria monocytogenes to abiotic surfaces. L. monocytogenes has previously been shown to attach to a variety of different abiotic surfaces, including polystyrene, stainless steel and glass. Consequently biofilm formation is a significant problem in food processing industries. An existing attachment assay was modified to allow screening of transposon mutants for a deficiency in attachment of bacteria to wells of a microtitre plate. Using this assay, three mutants were identified that had previously been shown to have reduced attachment to stainless steel and glass. The location of insertion of the transposon into each of the mutants M113, B380 and M237 was mapped to the genes lmo1226, lmo0401 and lmo0501, respectively. Deletion mutants were made in these genes and the gene lmo0402 to better clarify the role of these genes in attachment. The gene lmo1226 was identified as a drug exporter of the RND superfamily, responsible for the streptomycin resistance exhibited by L. monocytogenes strain 10403s. Lmo1226 is also involved in attachment, virulence, and growth of L. monocytogenes in sodium chloride. The genes lmo0401 and lmo0501 were identified as an α-mannosidase and a BglG transcriptional antiterminator respectively, and were identified to be involved in attachment alongside another transcriptional antiterminator (lmo0402). The transcriptional antiterminators were shown to be involved with transcription of the Bgl operon which encodes genes required for phosphoenolpyruvate phosphate transferase systems in L. monocytogenes. The results from these experiments have helped to elucidate the processes involved in L. monocytogenes attachment to surfaces, and have identified targets for future antimicrobials against this bacterium.

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Listeria monocytogenes is a Gram-positive, facultative intracellular pathogen which causes listeriosis, an important food-borne infection, with a high fatality rate. Temperature and growth media constituents have been previously shown to repress virulence factor expression in L monocytogenes but the mechanisms of control surrounding this virulence factor repression are poorly understood. Using both functional and biochemical assays to measure virulence factors, particularly llsteriolysin, and molecular techniques such as real-time PCR and Northern blot analysis to analyse the transcription or Key virulence genes, particularly hlyA, in the presence or absence of carbohydrates it was hoped a better understanding of virulence factor control could elucidated.