In 1967 the International Standards Organisation drew up a method designed to resuscitate a single injured Salmonella cell in 25 g of food. This method takes five days and introduced a pre-enrichment step that allowed injured Salmonella species to recover their resistance to selective agents before they were inoculated in to selective medium. Since 1967 a variety of methods has been developed that shorten the time .taken to detect a positive sample, yet these methods rely on the pre-enrichment step to allow injured Salmonella cells to recover and to allow amplification. This work was carried out to improve recovery ofinjured cells and allow selection during the pre-enrichment phase. Using variants of Buffered Peptone Water as recovery media, it was found that heatinjured Salmonella cells comprised a number of distinct sub-populations, based on their ability to recover in different broths. It was found that anoxic conditions improved recovery of heat-injured cells, while media that allowed rapid growth inhibited recovery. A survey of commercially available BPW found a> 2 10gIO difference in the recovery of heat-injured cells between the best and worst media. To detect Salmonella in food samples it is essential to repress growth of the competitive microflora. An investigation was carried out on autocytotoxic ~galactosides and ~-glucosides based on the biocide, 8-hydroxyquinoline (8HQ). These substrates might be used to selectively inhibit the competitive microflora. Galactosides were chosen to represent substrates transported into the cell by proton symports, and glucosides because they are representative of substrates transported by the phosphotransferase system (PTS). On hydrolysis 8HQ was released into the cytoplasm, but due to its lipophilicity the biocide migrated into the cytoplasmic membrane. The biocide then diffused into the medium until it reached equilibrium on both sides ofthe membrane. This inhibited .the competitive microflora, but would also inhibit the growth of any Salmonellae present. 8HQ glycosides are unsuitable for use in the pre-enrichment phase. In the next stage of this work glycosides of 8-hydroxyquinoline-5-sulphonic acid (8HQ5S) were synthesised. The sulphonate group is ionised at neutral pH and this ensures that on hydrolysis 8HQ5S remains within the cell. It was found that Klebsiella pneumoniae CMCC3077 could take up 8HQ5S-~,D-galactoside and hydrolyse the substrate, but the biocide did not remain within the cell. It is possible that the free 8HQ5S was exported from the cell by efflux pumps. No activity was found for 8HQ5S-~,Dglucoside. However, no free 8HQ5S was found in the medium. It is unknown whether the cell failed to transport and phosphorylate the substrate through the PTS, or failed to hydrolyse the molecule. As these glycosides failed to meet the requirements for autocytotoxic compounds, work was carried out on alaphosphalin. This is a dipeptide analogue that releases Ll- aminoethylphosphonic acid (AEP) on hydrolysis. AEP inhibits alanine racemase and growth. It was shown that alaphosphalin successfully inhibited K. pneumoniae at various inocula, and it was shown that AEP did not inhibit the recovery of heatinjured Salmonella cells. A major limitation to the use of alaphosphalin is that the peptides in BPW competitively inhibit uptake through the di- and oligo-peptide permeases. However, it is possible to synthesise N-linked glycosides that would be taken up by the glycoside permeases. Dr. Bovill of Thermo Fisher Scientific Ltd., has successfully synthesised AEP-~,D-galactoside. This molecule has been shown to be active against coliform bacteria, but inactive against Salmonella species. This molecule successfully meets all the requirements ofautocytotoxic compounds capable of inhibiting the growth of competitive microflora during the pre-enrichment step during detection of Salmonella spp. in foods.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:485755 |
| Date | January 2007 |
| Creators | Druggan, Patrick |
| Publisher | University of Brighton |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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