Streptococcus uberis is an important bovine mastitis pathogen, which places a significant financial burden upon the dairy industry. Determining the genetic diversity of a collection of field isolates and the mechanisms by which S. uberis colonises the host were the general aims of this project, in particular the determination of the basis for bacterial persistence despite antibacterial therapy. Multi-locus sequence typing identified high levels of recombination within the population, but also a single dominant clonal complex which comprised nearly all sequence types which were isolated from more than one animal. The dominant clonal complex also comprised isolates, derived, however, from both persistent and non-persistent infections, but RAPD typing demonstrated that these isolates can differ in genetic composition elsewhere in the genome. Whole genome sequencing of additional S. uberis isolates confirmed that despite significant homology between much of these genomes, novel genetic material was commonly obtained by phage insertion and horizontal gene transfer. Isolates with identical housekeeping sequences are thus highly likely to differ in their virulence gene repertoires. In this study, the potential for differentiating S. uberis isolates based instead upon protein profiles derived from mass spectrometry of disrupted whole cells was therefore also explored. Differentiation between small numbers of isolates was achieved after optimisation of this protocol, however, discriminatory ability and reproducibility were somewhat compromised when the technique was scaled up to analyse 50 Italian isolates. During the period of study, profile differences between persistent and non-persistent isolates could not be explored. Basic methods were thus also utilised in an attempt to identify factors which promoted bacterial survival in vitro; and a defined medium, representative of the udder environment, was optimised for this purpose. The use of this medium permitted the demonstration that S. uberis was reliant upon magnesium and manganese for proliferation and that, interestingly, the absence of iron did not inhibit bacterial growth. It was also shown that S. uberis had the ability to directly utilise casein, identifying a potential alternative pathway for the acquisition of essential nutrients from nutritionally-limited environments. It was also observed that to a limited extent S. uberis seemed to produce a siderophore. Although this remains to be confirmed, it may correlate with the observation that iron, although not essential for proliferation, improved the growth rate of the bacterium. It was also notable that most novel genes, identified from S. uberis genome sequences, exhibited functions for nutrient metabolism, demonstrating that flexibility in nutrient acquisition is central to the ability of the bacteria to adapt, permitting survival in vastly different environments. The use of the defined medium also demonstrated that S. uberis was able to form biofilms; this ability being variable depending on the growth conditions used and the isolate studied. Most significantly, under conditions representative of the mammary gland, there was an apparent trend for high levels of biofilm formation to correlate with isolates from persistent infections. Biofilm formation by Staphylococcus aureus is considered to be pivotal to the development of chronic mastitis, thus, biofilms may similarly play a role in S. uberis persistence. In an attempt to identify the molecular basis for S. uberis biofilm formation, genes with homology to those of the intercellular adhesion (ica) operon, well described for their involvement in Staphylococcus epidermidis and S. aureus biofilm formation, were identified in the genome sequence of S. uberis 0140J. A targeted mutagenesis protocol was optimised to ‘knock out’ these genes and observe the subsequent effects of these mutations on biofilm formation. During the course of this study, two of these potential biofilm genes (hasA and SUB 0809) were deleted from the S. uberis 0140J chromosome. Surprisingly, deletion of these genes did not retard subsequent biofilm formation, but instead biofilm formation was dramatically improved in the mutant strains. Characterisation of mastitis-causing S. uberis strains and a detailed understanding of the pathogenicity of the organism are required to further the development of a successful vaccine. The research presented in this thesis has increased the knowledge of these important research objectives and optimised techniques which will allow further advancement of knowledge in this field.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:630953 |
| Date | January 2011 |
| Creators | Gilchrist, Tamara Louise |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/2938/ |
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