Bioluminescence, the emission of light from living organisms, occurs throughout nature. In bacteria, bioluminescence is directly coupled to electron transport making it an excellent reporter of bacterial metabolic activity. The full lux operon from the terrestrial bioluminescent species, Photorhabdus IUl11inescens was transferred via a plasmid to the human pathogenic species, Staphylococcus alfrelfS and Neisseria meningitidis. The resulting transformants produced light constitutively and without the need for addition of exogenous substrate. Bioluminescent S. Ql~lreUS was challenged with the antimicrobial agent linezolid, and the inhibitory effect of the antibiotic on the organism at 6, 13 and 20 mg/L (Cmin, Cint and Cnax) along with its recovery following the withdrawal of linezolid was observable in real-time, by monitoring bacterial light output. Bacterial metabolism recovered more rapidly than bacterial replication (l.5 hours vs 3.5 hours at Cint) and the bacteriostatic nature of linezolid was confirmed since low light levels were detected throughout exposure to the drug. Bioluminescent Streptococcus pneul110niae (transformed prior to this study) was applied to a model blood-brain barrier (BBB) comprising a co-culture of cells grown on opposing sides of a polycarbonate membrane. By monitoring bioluminescence, it was possible to observe the anti-pneumococcal activity of gemifloxacin across the BBB in real-time, and non-invasively. Above the gemifloxacin MIC (0.16 mg/L), inhibition of metabolism was observed and a reduction in bacterial metabolism by sub-MIC gemifloxacin concentrations (0.03 and 0.13 mg/L) across the BBB was also recorded. Bioluminescent S. aureus and S. pneumoniae both demonstrated the potential of lux-expressing bacteria of clinical importance to be used as a novel, rapid and real time method of assessing antimicrobial efficacy when challenged directly, or in the presence of eukaryotic cell lines such as the model BBB. It was also possible to explore, using bioluminescent S. pneumoniae, factors which may damage BBB integrity and which are exploitable by bacteria. Bacterial penetration of the BBB is the key step in the pathogenesis of bacterial meningitis, but remains one of the most poorly understood aspects of the disease. Bioluminescent N. meningitidis was applied to the model BBB and, using low light camera equipment, it was possible to observe meningococcal penetration of the BBB in real time and in situ. By optimising growth conditions, BBB penetration time by N. meningitidis was reduced from 50 hours to 9 hours. This application has shown bioluminescence to be a valuable reporter of bacterial location and activity and has the potential to yield new information about the pathogenesis of meningococcal disease and meningitis in general
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:432309 |
| Date | January 2006 |
| Creators | Robinson, Gareth Michael |
| Publisher | University of the West of England, Bristol |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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