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Resistance of Listeria monocytogenes to the Bacteoricin Nisin.

Davies, Elizabeth Alison. (1995) Resistance of Listeria monocytogenes to the Bacteoricin Nisin. Doctoral thesis, University of Surrey (United Kingdom)..

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Abstract

Resistance of Listeria monocytogenes to the bacteriocin nisin was investigated. Listeria monocytogenes NCTC 5105 and F6861 were evaluated for sensitivity to nisin. The results confirmed those previously published indicating marked differences in the sensitivity of the two strains. Mutants with increased resistance to nisin could be isolated from the less sensitive F6861 strain at a frequency of 10e-6 to 10e-7. The MIC of the mutant strain in broth at pH 5.5, after incubation at 30°C for 47 days, was > 1000 IU/ml as compared with 300 IU/ml for its parent strain. The nisin resistance phenotype of the F6861 mutant strain was completely stable after undergoing ten passages of growth in nisin-ffee media. The growth characteristics of L. monocytogenes F6861 wild type and its nisin-resistant mutant were similar, but the doubling time of the mutant was slightly longer, particularly under sub-optimal conditions such as acid pH (5.5) and low temperature (4°C). There was no obvious difference in the basic morphology of each strain. Adsorption of nisin to the different L. monocytogenes strains was studied by measuring the residual nisin in solution. This was done routinely by means of a nisin-specific ELISA and compared with results obtained from a nisin bioassay. At a range of nisin concentrations, the amount adsorbed was found to reflect the sensitivity of the strain to nisin, with the more sensitive cells showing greater adsorption. These results eliminated the possibility of a nisinase enzyme reported in some resistant bacteria, but indicated that the mechanism of resistance is based on exclusion and inhibition of nisin binding to the cells. Immunogold labelling and transmission electron microscopy did not visibly show the incorporation of nisin into the cell membrane, probably because, if bound, the nisin epitopes were unable to form an antigen-antibody complex. The initial rate of K+ efflux induced by different nisin concentrations was measured in all three L. monocytogenes strains. In each strain, the rate of K+ efflux increased with increasing nisin concentration until it reached a maximum rate. As the nisin resistance of each strain increased, K+ efflux increased at a slower rate and had a lower maximum rate. The saturation kinetics of K+ efflux indicates that the mechanism of resistance involves a reduction in the accessibility or presence of suitable attachment sites, preventing the incorporation of nisin into the membrane. This resistance acquisition could result from adaption of either the cytoplasmic membrane or cell wall or both. Nisin inactivation of protoplast cells showed that the cell wall of L. monocytogenes F6861 plays an active role in the acquired nisin resistance of its mutant. Without its cell wall, the increased nisin resistance of the mutant was lost, with a phenotypic reversion back to the wild type. In contrast, the resistance of the wild type remained unchanged. Further evidence for cell wall involvement was indicated by the fact that cell surface hydrophobicity was shown to correlate with nisin sensitivity, the wild type strain being more hydrophobic than its mutant. The possible role of surface-layer (S-layer) proteins in the acquisition of nisin resistance by L. monocytogenes was eliminated due to the fact that several procedures (freeze-etching, atomic force microscopy, and S-layer extraction/SDS-PAGE) indicated that S-layers did not exist on any of the L. monocytogenes strains studied, when compared with a Lactobacillus positive control. In addition, chloramphenicol did not adversely affect the frequency of isolation of nisin-resistant mutants, indicating that de novo protein synthesis is not involved. Subsequently, the involvement of other cell surface components, namely teichoic and lipoteichoic acids, was investigated. Removal of these components and subsequent inactivation of the cells with nisin, reduced the resistance of both strains when compared with their respective whole cells. The resistance of the mutant strain lacking these components, however, was still significantly greater than whole cells of its wild type indicating the involvement of an extra factor in acquired nisin resistance. Resistance of the wild type lacking these components was significantly lowered when compared with its respective whole cells, and the fact that the resistance of wild type protoplast cells remained the same as whole cells suggests the possible involvement of lipoteichoic acids in intrinsic nisin resistance, conditional on their partial retention in protoplast formation. The role of the cytoplasmic membrane in the acquired nisin resistance of L. monocytogenes F6861 was also investigated. From the results obtained, qualitative and quantitative fatty acid analysis of L. monocytogenes F6861 wild type and its nisin-resistant mutant showed no obvious difference in content between strains. Any slight differences in quantity observed could not be verified from only one analysis. Modification of the lipid composition of L. monocytogenes was conducted by exogenous application of fatty acids (C14:0 or anteiso-C15:0) during growth, in order to determine the resistance response to nisin. Results from nisin inactivation and MIC determinations of these modified strains indicated that fatty acid C14:0 generally increased nisin resistance of both L. monocytogenes F6861 wild type and its nisin-resistant mutant, although MIC determinations of the wild type were similar to the control. Fatty acid anteiso-C15:0 decreased the nisin resistance of the wild type during both experiments, but increased the nisin resistance of the mutant during nisin inactivation only.

Item Type: Thesis (Doctoral)
Divisions : Theses
Authors : Davies, Elizabeth Alison.
Date : 1995
Additional Information : Thesis (Ph.D.)--University of Surrey (United Kingdom), 1995.
Depositing User : EPrints Services
Date Deposited : 30 Apr 2019 08:07
Last Modified : 20 Aug 2019 15:32
URI: http://epubs.surrey.ac.uk/id/eprint/851322

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