Gilchrist, Tamara Louise (2011) Genotypic and phenotypic characterisation of Streptococcus uberis. PhD thesis. http://theses.gla.ac.uk/2938/ Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Glasgow Theses Service http://theses.gla.ac.uk/ [email protected] Genotypic and phenotypic characterisation of Streptococcus uberis Tamara Louise Gilchrist BSc (Hons) A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy at the Faculty of Veterinary Medicine, University of Glasgow August 2011 © Tamara Louise Gilchrist 2011 Abstract 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 ii 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. iii Declaration The work reported in this thesis was carried out under the supervision of Dr Michael C. Fontaine and Professor David G. E. Smith at the Moredun Research Institute and the Faculty of Veterinary Medicine, University of Glasgow. All results presented, unless otherwise stated, are the sole work of this author, as is the composition of this thesis. Signed: Date: iv Acknowledgements When I left university, completing a PhD was the last thing I thought I would ever do, so no one is more surprised than me to find myself submitting this thesis. I feel like I have so many people to thank, my hard work alone would not have got me through this chapter in my life. It was with the direction and support from a great many people that I have achieved something which at times, I did not think I could, but which now, I am proud of. To begin I would like to offer my deepest gratitude to my supervisors, Dr. Mike Fontaine and Prof. David Smith for pushing me to achieve my potential, supporting me academically and providing sound advice and direction. Thanks also to Prof. Ruth Zadoks for considerable MLST and epidemiology advice and Alex Lainson and Frank Wright for bioinformatics/phylogenetics assistance. The staff at the Moredun Research Institute have been incredibly helpful and made this experience, for the most part, an enjoyable one. Heartfelt thanks must also go to Caray, Anita, Eleanor, Flo, Sabrina, Sarah and Henny, who in particular supported me through the tough times and helped me celebrate the good times. I would especially like to thank Caray and Karen for their guidance in the lab. I would also like to mention the other Moredun PhD students who were always a valuable source of encouragement and advice. Further thanks to sporting Moredun for helping vent frustrations. I would also like to thank my past and present Bacteriology colleagues, particularly Colin, Malcolm, Gordon, Sandy, David, Scott and last but not least Chris. I must also specifically mention Kevin Mclean for helping me get my head around the BioTyping software and the IT department for solving numerous PC related crises. I would now like to take a moment to thank my family, particularly my Mum and Dad who have always been incredibly supportive and encouraging, but also Matt and Kate for being the wonderful people they are. Huge thanks also to Mum G and Jason for their advice, company, highland holidays, and for welcoming me into their family so lovingly. Thanks also to my closest friends Louise and Darren for their love, fun and among other things helping me buy a lap top (thanks also to Mum and Dad, Mum G and Aunty Mags for that). To all my other friends and family members not listed (to avoid going on forever, as I am prone to do!) thanks for helping me be the person I am today. My last paragraph is reserved as it should be, for the most important person in my life. I would like to say a massive thank you to my husband and my rock, Gavin. Thanks for your love, understanding, encouragement and kindness, as well as for doing the cooking, cleaning and tidying when it was all too much for me; without your support I don‟t think I would have stuck at it during the tough times. Thanks for putting up with the tears and the tantrums, for taking me out, making me laugh, being there for me and always giving me something to look forward to. v Table of Contents Abstract ...................................................................................... ii Declaration ................................................................................... iv Acknowledgements ........................................................................... v Table of Contents ........................................................................... vi List of Tables.................................................................................. x List of Figures ..............................................................................