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The Role of the AdeRS Two Component System and the AdeABC RND Efflux Pump in Antibiotic Resistance, Biofilm Formation and Virulence in Acinetobacter baumannii by Grace Emma Richmond A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY Antimicrobials Research Group Institute of Microbiology and Infection College of Medical and Dental Sciences University of Birmingham July 2016 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract Acinetobacter baumannii is a nosocomial pathogen and causes infections in hospitals worldwide. This organism is often multi-drug resistant (MDR), can persist in the environment and forms a biofilm on environmental surfaces and wounds. This thesis describes research that investigates the role of the two component system AdeRS, which regulates production of the AdeABC MDR efflux pump. Its role in MDR, biofilm formation and virulence of A. baumannii was determined in mutants constructed for this study. Deletion of AdeRS or AdeABC resulted in increased susceptibility to antibiotics, decreased biofilm formation on biotic and abiotic surfaces and decreased virulence in a strain dependent manner. RNA-Seq revealed that loss of AdeRS or AdeB significantly altered the transcriptome, resulting in changed expression of many genes, notably those associated with antimicrobial resistance and virulence interactions. This study demonstrated the scope of AdeRS mediated regulation and suggests that inhibition of AdeABC could prevent biofilm formation or colonisation in patients by A. baumannii and so provides a good target for drug discovery. This study also highlighted the differences between A. baumannii strains and shows that conclusions for the species should not be drawn from the study of single strains. Acknowledgements I would like to thank my supervisor Professor Laura Piddock for all her help and advice over the last few years. I would also like to thank my second supervisor Dr Mark Webber for his guidance and for answering all my questions. I am extremely grateful to Professor Marnie Peterson for hosting me at the University of Minnesota and her constant support despite some major setbacks! I would also like to thank Dr Mark Sutton for hosting my visit to Public Health England and for his ongoing input on the project. Thank you also to Al Ivens for sharing his bioinformatics expertise, Michele Anderson for her help with the PVM model and Matthew Wand and Laura Bonney for their assistance with making genetic modifications and the Galleria work. Thank you to all members of ARG, past and present. In particular those who made me so welcome at the beginning and those who got me through the final months. And finally thank you to my friends and family, who always believed in me. Contents 1. Introduction ........................................................................................................... 1 1.1. Acinetobacter species ................................................................................... 1 1.1.1. Natural habitat ......................................................................................... 2 1.1.2. Epidemiology ........................................................................................... 2 1.2. Acinetobacter baumannii complex ................................................................ 6 1.3. Acinetobacter baumannii infection ................................................................ 8 1.3.1. Hospital acquired infections ..................................................................... 8 1.3.2. Community acquired infections .............................................................. 10 1.3.3. Infection in military and disaster zone casualties ................................... 10 1.4. Persistence in the hospital environment ...................................................... 11 1.5. Treatment of Acinetobacter infections ......................................................... 12 1.5.1. Antibiotics .............................................................................................. 12 1.5.2. Novel therapeutics ................................................................................. 18 1.6. Mechanisms of antibiotic resistance............................................................ 19 1.6.1. Target site modification .......................................................................... 24 1.6.2. Drug inactivation .................................................................................... 24 1.6.3. Reduced drug accumulation .................................................................. 26 1.7. Regulation of antibiotic resistance .............................................................. 32 1.8. Pathogenesis and virulence of Acinetobacter ............................................. 36 1.8.1. Biofilm formation .................................................................................... 40 1.9. Biofilm models for Acinetobacter ................................................................. 41 1.10. The role of efflux pumps in biofilm formation and virulence ...................... 44 1.11. Background to this research ..................................................................... 45 1.12. Hypotheses ............................................................................................... 47 1.13. Aims .......................................................................................................... 47 2. Materials and Methods ........................................................................................ 48 2.1. Bacterial strains, growth, storage and identification .................................... 48 2.1.1. Bacterial strains ..................................................................................... 48 2.1.2. Growth and storage ............................................................................... 51 2.1.3. Phenotypic and genotypic identification ................................................. 51 2.2. Primer design and PCR .............................................................................. 51 2.3. Construction of gene deletion mutants ........................................................ 56 2.3.1. Construction of pMo130-TelR-adeBUPDOWN ...................................... 59 2.3.2. Integration of pMo130-TelR-adeBUPDOWN into A. baumannii AYE chromosome ...................................................................................................... 59 2.3.3. Gene deletion ........................................................................................ 61 2.4. Bacterial growth kinetics ............................................................................. 62 2.5. Susceptibility testing .................................................................................... 62 2.6. Measurement of efflux activity ..................................................................... 65 2.6.1. Accumulation of Hoechst 33342 ............................................................ 65 2.6.2. Efflux of ethidium bromide ..................................................................... 66 2.7. Measurement of biofilm formation ............................................................... 67 2.7.1. Biofilm formation on porcine vaginal mucosal tissue ............................. 67 2.7.2. Biofilm formation in a microfluidic flow cell ............................................. 70 2.7.3. Biofilm formation on polypropylene pegs ............................................... 72 2.7.4. Pellicle formation ................................................................................... 72 2.7.5. Biofilm formation on glass cover slips .................................................... 73 2.8. Measurement of twitching and swarming motility ........................................ 73 2.9. Measurement of virulence in Galleria mellonella ......................................... 74 2.10. Statistical analysis ..................................................................................... 74 2.11. RNA-Seq ................................................................................................... 75 2.11.1. RNA extraction ................................................................................... 75 2.11.2. DNase treatment of RNA samples ..................................................... 76 2.11.3. Quantification of RNA ......................................................................... 76 2.11.4. Preparation of RNA-Seq libraries ....................................................... 77 2.11.5. Sequencing of RNA-Seq libraries ....................................................... 78 2.11.6. Analysis of RNA-Seq data .................................................................. 78 3. The Role of the Two Component System AdeRS in Antibiotic Resistance, Biofilm Formation and Virulence ........................................................................... 80 3.1. Summary of background to this
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