THE ISOLATION AND CHARACTERISATION OF PROTEUS MIRABILIS BACTERIOPHAGES AND THEIR EFFECT ON THE COLONISATION AND BLOCKAGE OF URINARY CATHETERS RICHARD W THOMPSON BSc. A thesis submitted in partial fulfilment of the requirements of the University of the West of England, Bristol for the degree of Doctor of Philosophy This research programme was carried out in collaboration with the Bristol Urological Institute, Bristol Department of Applied Sciences, University of the West of England, Bristol June 2018 Authors Declaration This copy has been supplied on the understanding that it is copyright material and no quotation from the thesis may be published without proper acknowledgement. i Abstract Catheter associated urinary tract infection seriously complicates the care of an already vulnerable patient set and has been estimated to cost the UK National Health Service in excess of one billion pounds per annum. Approximately 50 % of patients catheterised for more than 28 days will experience catheter blockage due to the formation of crystalline biofilm on the eye holes, balloon and lumen of the catheter (Getliffe, 1994) as a result of colonisation by Proteus mirabilis. Blockage can lead to significant complications such as pyelonephritis and septicaemia. To date, strategies to reduce or prevent these infections from occurring have met with limited success. One potential approach to prevent catheter colonisation and blockage is the application of bacteriophages as a catheter coating. Natural parasites of bacteria, bacteriophages offer several advantages over conventional antimicrobial treatment including replication at the site of infection, specificity and, in some cases, biofilm degrading ability. Three novel bacteriophages vB_PmiS_NSM6, vB_PmiP_#3 and vB_PmiM_D3 were isolated from environmental sources and characterised phenotypically and genetically utilising electron microscopy, host range analysis and, for phages vB_PmiS_NSM6 and vB_PmiP_#3, genome sequencing via hybrid assembly. The isolated phages belong to the Caudovirales order and sequence data analysis indicated that they are lysogenic. They possess the characteristic modular architecture of their dsDNA genomes that are densely packed with coding sequence. Both phages displayed terminal redundancy which is indicative of a headful packaging strategy and both appear to be circularly permuted. Putative function was obtained for 63 % of the coding sequences for phage vB_PmiS_NSM6 and 52 % of genes identified in phage vB_PmiP_#3. The effect of these phages, either individually or as a cocktail, on P. mirabilis colonisation of urinary catheters in an in vitro bladder model was investigated. Models were run for 24 h and adhered bacteria used as an indicator of phage activity verses untreated control. A reduction of greater than 3 log10 was observed for phage vB_PmiS_NSM6 treated catheters in comparison to untreated controls across all three sections of catheter analysed. Phage vB_PmiP_#3 reduced bacterial adherence by 1 log10 across all sections and a similar reduction was observed with phage vB_PmiM_D3 of greater than 1 log10. These data were confirmed with scanning electron microscopy (SEM) which showed a significant reduction in crystalline deposits on the phage treated catheters. The time taken for the mineralised biofilm to occlude the catheter lumen in the presence of bacteriophages was also investigated. Time to blockage was extended from 36.2 h to 58.47 h (an increase of 61.49 %) for phage vBPmiS_NSM6, from 41.17 h to 51.73 h (an increase of 25.67 %) for phage vB_PmiP_#3 and from 40.97 h to 62.40 h (an increase of 52.31 %) for phage vB_PmiM_D3. Phages vB_PmiS_NSM6 and vB_PmiM_D3 displayed activity against each other’s isolating strain. This enabled the assessment of a two phage cocktail. The cocktail increased time to blockage by approximately 7 % compared to single phage treatment for both bacterial isolates. These data provide some evidence of efficacy of bacteriophage pre-treatment of urinary catheters in an in vitro model of P. mirabilis infection of the catheterised bladder, despite the lysogenic nature of the phages investigated. As such, this suggests phage treatment of catheters warrants further investigation. ii Acknowledgements Firstly, I would like to thank Adele Long, the former director of the Bristol Urological Institute (BUI), for being instrumental in securing the funding for this project and believing in me and my idea. Thanks also to Karen Evely at the BUI for the additional administrative and organisational support required to allow me to undertake this study part-time. Next I would like to thank my original supervisor Professor Viv Salisbury for agreeing to take me on as a student and undertaking a project in an area of research that she had little prior experience of. Without her the project would never have gotten off the ground. Special thanks also to my current supervisor Dr Shona Nelson, for agreeing to take me on as a student upon Viv’s retirement and for all the help and support she has given me over the duration of the project. The last couple of years have been a particularly challenging time for me personally, Shona has not only supported me as my PhD supervisor but also as a friend and confidant. I am forever grateful for all the guidance she has given me, and also for all the parenting wisdom and clothes she provided for my daughter since her birth! Thanks also to my other supervisors, Dr Roy Pemberton who covered Shona’s maternity leave and Dr Nicola Morris for allowing me to undertake these studies, being flexible with working arrangements and providing much advice and support over the years. Thanks also to Dr Dann Turner for all the help and support he has given me from his time as a fellow PhD student throughout his post-Doctoral study and now as a lecturer. Dann was instrumental in the planning and execution of the sequencing work conducted, his expertise in this field has been invaluable. I feel very lucky to have been surrounded by such interesting and accomplished individuals throughout the project, without their endless help and encouragement it simply wouldn’t have been possible. Thanks also to the laboratory technical team at the University of the West of England for their support and advice, especially Dr Gill Smith, Dr Lee Graham, Dave Corry, and Dr Dave Patton for his assistance with electron microscopy. I would like to extend my thanks to the microbiology department at Southmead hospital for supplying me with Proteus mirabilis isolates, and to Wessex Water for providing me with activated sludge and raw sewage, specifically Alex Jones for his willingness to facilitate my research, and Reuben Hardy for collecting and delivering the samples. Thanks also to Professor Eshwar Mahenthiralingam for his support and assistance with the analysis of PFGE gels at the University of Cardiff. Finally, I would like to thank my wife for her love and encouragement, and for being my best friend. Without her none of this would have been possible. iii Table of Contents Authors Declaration ...................................................................................................... i Abstract ........................................................................................................................ ii Acknowledgements ..................................................................................................... iii List of Figures ...............................................................................................................ix List of Tables .................................................................................................................xi Abbreviations .............................................................................................................. xii Chapter 1 Introduction ................................................................................................. 1 1.1 Overview ........................................................................................................ 1 1.2 Urinary incontinence ..................................................................................... 1 1.3 Urinary catheters ........................................................................................... 3 1.4 Catheter associated urinary tract infection (CAUTI) ..................................... 5 1.4.1 Biofilms ........................................................................................................ 7 1.4.2 Catheter associated mineralised biofilms ................................................. 10 1.5 Proteus mirabilis .......................................................................................... 11 1.6 Attempts at preventing catheter-associated urinary tract infection .......... 17 1.6.1 Antimicrobial coatings ............................................................................... 18 1.6.2 Modification of surface properties ............................................................ 19 1.6.3 Dietary modification .................................................................................. 21 1.6.4 Plant-based antimicrobials ........................................................................ 21 1.6.5 Iontophoresis ............................................................................................. 22 1.6.6 Enzyme inhibitors ...................................................................................... 22 1.6.7 Quorum sensing inhibitors .......................................................................