Micallef, Christianne (2008) Developing a model system for 'Staphylococcus aureus' respiratory infection in cystic fibrosis patients. (PhD thesis), Kingston University, .
Abstract
For the first time, an in vitro cystic fibrosis (CF) artificial sputum model (ASM) was found to support the growth and survival of a clinical epidemic strain of meticillin-resistant Staphylococcus aureus (EMRSA16-252). Specific components, which included mucin, DNA and others, were removed from ASM and the physiological impact of this was fully explored using viable counts and light microscopy. As CF patients are known to develop cystic fibrosis-related diabetes or CFRD, glucose was added to ASM (GASM), to explore the physiological impact of glucose on the growth and survival MRSA252. Total RNA was extracted from the corresponding log phases of MRSA252 grown in brain heart infusion (BHI) as a laboratory control, as well as ASM and GASM. RNA was extracted in order to conduct micro array analysis. MRSA252 DNA was used as a control. RNA (from the samples) was labelled with Cy5 and control DNA was labelled with Cy3. Once labelled and amplified, the Cy5/Cy3 mixture was then purified and hybridised onto an array containing seven sequenced S. aureus genomes (N315, Mu50, MW2, MRSA252, MSSA476, COL and NCTC8325). The array was then scanned and the raw results were analysed. Differentially expressed genes in ASM equalled 15% of the MRSA252 sequenced genome and those for GASM amounted to 21 %. Normalised results showed that 14 capsular polysaccharide genes were up-regulated significantly in both ASM and GASM, as well as genes coding for enzymes involved in carbohydrate and vitamin metabolism. Genes coding for surface proteins such as spa and IrgA and IrgB, responsiblefor peptidoglycan metabolism, were significantly down-regulated. A similar gene expression pattern was observed with GASM, although the total number of genes which differentially regulated in GASM was greater than for ASM. The micro array data was validated using RT-PCR. Samples of unmodified liquid human CF sputum were then used as a growth medium for MRSA252. These were found to support the growth and survival of this bacterium. RT-PCR was used to investigate gene expression in the liquid humanand the results compared to those obtained with the in vitro models, ASM and Capsular genes were found to be up-regulated in both the human samples as well in vitro media. The work conducted in this thesis presents data which suggest that involved in Staphylococcus aureus-CF respiratory infection may be through genes encoding capsular polysaccharides and offers a possible target for anti-staphylococcal therapy in cystic fibrosis.
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