Although manuka honey has been shown to inhibit Pseudomonas aeruginosa, an opportunistic pathogen implicated in cutaneous wound infections, the mechanisms of action are not yet defined. The purpose of this study, therefore, was to investigate the inhibitory effects of manuka honey on P. aeruginosa. Initially, a bactericidal mode of action was confirmed using suspension cultures of P. aeruginosa. Laser scanning confocal microscopy, atomic force microscopy and hydrophobicity assays identified physiological changes in manuka honey treated cells, such as reduced surface hydrophobicity, membrane blebbing, cell lysis and the release of extracellular material. The effects of manuka honey on intracellular proteins and the expression of cell envelope components was explored using two dimensional gel electrophoresis and qPCR, respectively. Expression of the gene encoding the structural anchor protein, OprF, was significantly suppressed in manuka honey treated cells. This resulted in a marked reduction in cell envelope stability with a reduced capacity to resist osmotic stress. The effect of manuka honey on P. aeruginosa surface associated functions (motility and biofilm formation/integrity) was also investigated. Transmission electron microscopy and motility assays identified significant de-flagellation and reduced motility of P. aeruginosa cells following exposure to sub-inhibitory concentrations of manuka honey. qPCR of various genes within the flagella regulon, identified suppression of genes encoding master regulators FleQ and FliA. Biofilm cells, inherently more tolerant to antimicrobial agents, required a higher concentration of manuka honey to inhibit biomass and bioactivity than planktonic cells. Reductions in the number of biofilm cells treated with manuka honey and dressings coated/impregnated with manuka honey were monitored using an in vitro cutaneous wound model. qPCR of treated biofilm cells suggested that similar mechanisms to those observed in planktonic cells occurred. These in vitro studies confirm that manuka honey inhibits both planktonic and sessile (biofilm) P. aeruginosa cells through the differential regulation of key genes required for cell envelope stability and motility.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:606638 |
| Date | January 2014 |
| Creators | Roberts, Aled Edward Lloyd |
| Publisher | Cardiff Metropolitan University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10369/5676 |
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