Identification and understanding the roles of biofilm formation-related genes in Listeria monocytogenes isolated from seafood : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Microbiology, Institute of Food Science and Technology, Massey University, Palmerston North, New Zealand

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Listeria monocytogenes is a foodborne pathogen that can lead to severe bacterial infections in immunocompromised people, the elderly and pregnant women and their unborn. Seafood is one of several contamination sources and as the seafood industry is of high economic value to New Zealand, this pathogen needs to be controlled. The main route for contamination is thought to be from biofilms in the seafood-processing environment and their persistence through cleaning and sanitation. Persistent and sporadic strains of L. monocytogenes isolated from mussel-processing facilities were compared using phenotypic assays. Biofilm formation was greater for persistent strains compared to sporadic strains (30°C, 48h) using cell counts and crystal violet staining (CV). The persistent isolate 15G01 exhibited greatest biofilm formation and was therefore chosen to be studied for biofilm formation using transposon mutagenesis. A screen of the transposon library for biofilm-forming ability using the crystal violet assay identified 27 genes to be associated with biofilm formation. Three low biofilm formers (33E11, 39G5, 44D3) and one high biofilm formers (34F11) were analysed with the fluorescent LIVE/DEAD stain and the scanning electron microscope revealing coccoid-shaped cells and long chains for 33E11 and 44D3, respectively. The four mutants and a previously identified fifth (6B4) were investigated for their biofilmforming ability, the surface characteristics of the cells and the influence of cations on biofilm formation. Three different biofilm formation assays were used to assess the composition of the biofilm. The CV assay was used to determine the whole biofilm mass, cell enumeration was applied to calculate viable cells in the biofilm and a formazan based assay (XTT) measured metabolic activity. All three assays showed a significant correlation, however, no correlation with cell surface characteristics was observed. Confocal laser scanning microscopy revealed a unique sandwich structure for the biofilm formed by 44D3, which has not been reported before, and was reversed at higher magnesium concentrations. Magnesium influenced biofilm formation at a concentration of 5 mM resulting in enhanced biofilm formation for the wild-type and the mutant 44D3 and in reduced biofilm formation in 39G5 whereas calcium showed no gene-specific effect on biofilm formation. The research presented in this study provides useful data for the prevention and control of biofilm formation by L. monocytogenes in seafood-processing plants.
Listeria monocytogenes, Genetics, Biofilms, Research Subject Categories::TECHNOLOGY::Chemical engineering::Food technology