Although nasal colonisation is a well-established risk factor for most types of Staphylococcal infections, several studies have suggested that intestinal colonisation could have important clinical implications. In this study, the effect of S. aureus infection of the human large intestine on resident microbiota was studied using an in vitro human colonic model system. During infection, S. aureus was able to compromise colonisation resistance by the colonic microbiota thus, transiently decreasing resident bacteria. Furthermore, the concentration of short chain fatty acids in the vessel representing the proximal colon was decreased significantly by infection. Bile salts being potent antimicrobial agents in the human intestine represent major obstacles for the survival and colonisation of invading pathogenic bacteria. Hence, the antimicrobial mode of action of various human bile salts on S. aureus was investigated. Comparatively, unconjugated bile salts were found to be more potent than conjugated bile salts in inhibiting S. aureus growth by membrane damaging effect resulting in dissipation of intracellular pH, transmembrane electric potential, leakage of potassium and probably other ions across the cell membrane and also the low molecular- weight metabolites from the cell. This subsequently leads to loss of proton motive force, important ions, and solutes from the cytoplasm, thereby leading to cell death. However, determined MICs of human bile salts were significantly higher than the physiological levels found in the human intestine, raising the possibility of bile salt resistance in the bacterium. S. aureus components involved in bile salt resistance were identified by mutagenesis and functionally characterized in the heterologous host bacterium. MnhF, an efflux pump was found to be involved in the resistance to unconjugated bile salts. Furthermore, survival of S. aureus 6.mnhF was attenuated compared to wild type in the in vitro human colonic model, implicating MnhF in S. aureus colonisation of the human intestine. Thus, infection by S. aureus appears to be able to alter the overall structure of the human colonic micro biota, the microbial metabolic profiles and resists antimicrobial action of bile salts by efflux.

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Staphylococcus aureus is a highly versatile pathogen that causes a wide range of diseases. Appropriate gene expression in various niches within the human body and abiotic environments requires the sensing of environmental conditions. An important environmental parameter affecting S. aureus is NaC1 concentration. This study investigated S. aureus virulence determinant regulation in the presence and absence of NaCl stress. In the absence of NaCl stress, aB was found to repress hla transcription and protease activity, possibly via the repression of agr transcription. The effect of aB on agr probably occurs indirectly, although sarA, sarHi and rot are unlikely to function as intermediates in this pathway. Tn551 mutagenesis identified numerous genes, including lysC, ykuQ, lysA, brnQ and telA, which repress hla transcription and protease activity by upregulating aB activity. These genes are clustered in the SVS (S. aureus virulence and survival) region of the S. aureus genome, in which transposon insertion affected the virulence and survival of mutants isolated in numerous published screens. Other SVS region genes, including asd, dapA, hipO, ac1P and norQ were also found to repress hla transcription and protease activity by upregulating aB activity. In the presence of NaCl stress, virulence determinants (which are normally regulated by agr) come under the control of a novel regulatory system involving an-dependent and aB-independent pathways. Tn917 mutagenesis identified several genes, including citG, opuD, yugT, oppF, ykrP, eprH, yubA, unkl and unk2, which have putative roles in the aB-independent pathway. The SVS region genes analysed may function in the aB- dependent pathway. The sensor saeS was found to upregulate hla transcription in the absence of NaCI stress and may be involved in sensing NaCI stress. The NaCI stress signal may act in concert with other parameters to allow stringent virulence determinant regulation in response to the prevailing environmental conditions.

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'Staphylococcus aureus' ('S. aureus') and especially methicillin-resistant 'S. aureus' (MRSA) have become a major problem in hospital acquired infections worldwide. These organisms may be resistant to multiple numbers and classes of antibiotics making their treatment complicated and challenging. As well as being resistant to antibiotics, 'S. aureus' produces numerous pathogenicity factors including toxins and extra-cellular enzymes. Previous investigations into the relationship between antibiotic resistance and toxin production in both MRSA and methicillin sensitive 'S. aureus' (MSSA) strains have found small or no differences in toxin production. However, no investigation has examined the production of pathogenic enzymes and their relationship to antibiotic resistance. A study panel of 680 isolates of 'S. aureus' was collected from the UK and Malta. This investigation addressed several critical phenotypic characteristics of this clinically important pathogen. The isolates were tested against several significant antibiotics, analysed for selected pathogenic factors, analysed by Pulse Field Gel Electrophoresis (pFGE) to determine molecular epidemiological distribution and subjected to novel differentiation methods. The isolates were tested against a range of fourteen antibiotics. Additionally, the production of three enzymes, DNase, lipase and proteinase, and one toxin, haemolysin was determined for each isolate. The level of antibiotic resistance between the isolate groups for both countries was found to be similar. However, some significant differences were seen in resistance levels to certain antibiotics. No differences in the production of pathogenic factors could be detected between the isolates of MRSA and MSSA from both countries. The isolates were also divided into a strain types via Smaldigestion, with DNA fragments separated by PFGE. Three UK-recognised epidemic MRSA strain profile types were identified and, interestingly, these types were also observed in the Maltese strain panel. Novel chromogenic substrates for detecting lipase activity were tested against the 'S. aureus' strains. Results suggest these substrates would have limited applicability as a diagnostic tool for differentiation between MRSA and MSSA. However, these compounds may have an application as a research tool for the study of 'S. aureus'. A further study examined the effect of antimicrobials on the growth of phenotypically resistant organisms. This pilot study of fourteen isolates, showed significant differences in the fmal cell population and significant decreases in the production of some pathogenic enzymes. The results of this experiment warrant further investigation.