| Summary: | 碩士 === 國立臺灣大學 === 醫學檢驗暨生物技術學研究所 === 96 === Staphylococcus aureus is an important human pathogen, which can cause a variety of diseases, for example, wound infection, food poison, life-threatening osteomyelitis, bacteremia and endocartidis. Biofilm infections associated with S. aureus account for serious clinical problems, especially in medical device-related infections. Biofilms are multi-cellular communities consist of surface-attached microorganisms encased by extracellular matrix. Bacteria living in biofilm mode can escape from host immune systems and resist to adverse stress in the environment. Biofilm cells are more tolerant to antimicrobial agents thus are notoriously difficult to treat.
In this study, a transposon mutant library was generated by transposon mutagenesis and the biofilm activity was screened by microtiter plate assay. One mutant strain with significantly reduced biofilm capacity has been selected. Further analysis was performed to identity the transposon disrupted gene and it’s role in S. aureus biofilm formation. Single transposon insertion was confirmed by Southern blot analysis. Comparison with S. aureus NCTC8325 whole genome sequence, the region disrupted by transposon encodes a putative esterase, which belongs to the α/β hydrolase family.
There was no significant difference in growth rate between wild type and the mutant, suggest that the reduced biofilm phenotype was not due to insufficient cell accumulation. In the initial attachment experiment, the mutant displayed weaker adherence to polystyrene surface than wild type. The mutant strain also exhibited lower hydrophobicity compared to wild type. These results implied that the surface attachment capacity of the mutant was impaired.
Gene complementation and deletion experiments were conducted to verify the altered biofilm phenotype resulted from est gene disruption. We used 2D electrophoresis to compare the protein expression profile between wild type and mutant, and attempted to identify proteins possibly involved in biofilm formation.
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