AbstractA biofilm is a state where bacteria are attached to a surface to form sessile communities. Cells in the sessile communities have a higher tolerance to antibiotics and sanitizers. Correspondingly, the persistence of bacteria can lead to infections and outbreaks of foodborne diseases. Listeria monocytogenes is a Gram-positive Firmicute that causes foodborne infections. Frequently, infection is caused by ingesting food that has been contaminated in processing plants. L. monocytogenes can form biofilms on machinery in food processing facilities, which further leads to outbreaks of infections. This study aimed to understand the molecular mechanism of biofilm formation by Listeria monocytogenes.
It is known that mutations can be unintentionally introduced or accumulate in the chromosome of laboratory-adapted strains and affect the phenotypes displayed by the strains. Therefore, four isolates of a commonly used laboratory-adapted L. monocytogenes strain, EGDe, were collected. It was hypothesised that these variants may have different biofilm-forming abilities. Through the detection of mutations in the genome, the molecular mechanism of biofilm formation by L. monocytogenes could therefore be uncovered. In this project, I first examined five different phenotypes displayed by the EGDe collection and resequenced the genome. Variations in the phenotypes and genotypes between the isolates were discovered.
I next examined the biofilm-forming ability of these four isolates using crystal violet staining and scanning electron microscopy. Among the four EGDe isolates, reduced biofilm formation was detected in one EGDe isolate compared. The analysis of the genomic profiles showed that this isolate contains nonsense SNPs in lmo0184, rsbU, and rmlA. To examine the effect of each SNP on biofilm formation, I constructed single gene deletions in the defined reference strain. The biofilm formed by the mutant strains revealed two genes involved in biofilm development: rsbU and rmlA. RsbU is the upstream regulator of SigB, a sigma factor. Either ΔrsbU or ΔsigB mutant strains had an ‘enhanced’ profile of biofilm formation. On the other hand, removing RmlA, which is the first enzyme for TDP-L-rhamnose biosynthesis, displayed a reduction in the amount of biofilm formed. Further analysis of biofilm formation of the rmlT mutant strain, which still contains TDP-L-rhamnose production but not the rhamnose decorated wall teichoic acid (WTA), showed that the decorated WTA is required for adhesion of cells to surface. Finally, deletion of rmlA can override the effect of rsbU deletion on biofilm formation. In brief, I identified that (1) removing rsbU or sigB induces biofilm formation and (2) sugar decorated WTA enhances cell-to-surface interactions.
|Date of Award||2019|
|Supervisor||Nicola Stanley-Wall (Supervisor)|
Investigation of molecular mechanisms of biofilm formation by Listeria monocytogenes
Hsu, C. (Author). 2019
Student thesis: Doctoral Thesis › Doctor of Philosophy