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
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.