Abstract. Recent studies have indicated that macrolide antibiotics, in doses well below their minimum inhibitory concentration (MIC), may have a specific effect on quorum regulated systems in Gram negative bacteria. Vibrio fischeri was used in this study as a paradigm to observe the effects of sub-MIC erythromycin on quorum sensing. Concentrations of 1/100th of the MIC inhibited bioluminescence but did not affect growth in V. fischeri. Reduction of bioluminescence was reversible with the addition of exogenous acyl-HSL. To elucidate the effects of macrolides on this quorum regulated system, reverse-transcriptase PCR was used to compare transcription of luxD, luxR and a 'housekeeping gene', 16s rRNA, when grown with sub-MIC macrolides with/without acyl-HSL.The luxR and 16s rRNA genes were unaffected but transcription of luxD was reduced by sub-MIC erythromycin, suggesting that sub-MIC erythromycin affected genes under quorum sensing control. As both acyl-HSL and erythromycin contain a lactone ring structure, competitive inhibition could explain these results. An Escherichia coli strain that overproduced LuxR protein was used to investigate whether acyl-HSL and macrolide antibiotic competed for a site on or near to LuxR. Radiolabelled acyl-HSL was produced by adding tritiated methionine to growing cultures of V. fischeri. Radiolabelled acyl-HSL bound to the LuxR protein when compared to the control, supporting the hypothesis that macrolides may block binding of acyl-HSL to LuxR transcriptional activator. Clinical strains of P. aeruginosa were transformed with luxCDABE genes under a constitutive promoter and used to investigate the effect of sub-MIC macrolides on growth and light output. In contrast to results obtained from V. fischeri, where the lux genes are quorum regulated, sub-MIC macrolides did not have a differential effect, indicating that the low dose macrolide results observed in V. fischeri are due to an effect on regulation of the lux genes. Sub-MIC macrolides have been reported to decrease virulence production in P. aeruginosa; to investigate their in-vivo effects a Caenorhabditis elegans P. aeruginosa model was developed. Worms fed on a clinical isolate of P. aeruginosa were killed in 24 hours and this lethal effect was reversed in the presence of 0.4 mg/L erythromycin. Further experiments with a selfbioluminescent construct of the clinical P. aeruginosa isolate demonstrated that 0.4 mgll erythromycin did not alter bacterial viability withilJ worms, whilst clearly inhibiting virulence factor production. The results of this study demonstrate that sub-MIC erythromycin affects quorum regulated systems in V. fischeri and P. aeruginosa and indicate a possible competitive inhibition effect between sub-MIC erythromycin and acyl-HSL.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:495519 |
| Date | January 2008 |
| Creators | Smith, Gillian Patricia |
| Publisher | University of the West of England, Bristol |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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