Characterization of genetic determinants for growth and structure of Listeria monocytogenes biofilms / by Nicholas Choy.
Choy, Nicholas Chiau-Yoong
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This thesis investigated wild-type 'Listeria monocytogenes ' EGD and five of its corresponding mutant strains (M1, M4, M7, M11 and M14) for their ability to form biofilms on glass at 22°C and 37°C, with and without shear force. Cell counts of 24 h-old biofilms ranged from 10[superscript 5] to 10[superscript 7]cfu/cm[superscript 2]. For all strains investigated, biofilms grown without shear force at 22°C had higher mean biofilm cell counts than at 37°C (P<0.05). Biofilms of wild-type 'L. monocytogenes' EGD and its mutant M11 ('clpB' gene deleted) were further analyzed with scanning electron microscopy (SEM). SEM imaging of biofilms grown at 37°C without shear force showed that the wild-type strain EGD produced significantly more biofilm cells than its mutant M11. For biofilms grown at 22°C, both strains displayed characteristic three-dimensional biofilms with cells embedded in what is believed to be extracellular polymeric substances (EPS) when dehydrated with hexamethyldisilizane (HMDS). With the aid of confocal scanning laser microscopy (CSLM), biofilm structures of wild type Listeria monocytogenes EGD and its corresponding mutant strains were quantitatively described with PHLIP using the image analysis program. Strains were then characterized with a statistical method known as principal components analysis (PCA). Results from this analysis revealed three strains (M14, M11 and M7) having high thickness and surface-to-biovolume ratio when grown under three conditions (22°C with shear, 37°C with and without shear). A complementation in trans was then performed on mutant M11 to ascertain that the phenomenon observed regarding biofilm production and stucture on glass, could be attributed to the absence of the clpB gene.