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Investigating Two Component Regulatory Systems for The Investigating Two Component Regulatory Systems for the Determination of Adaptive Responses in A. baumannii. By LAURA PAULINE EVANS A thesis submitted to the University of Birmingham for the degree of MASTER OF PHILOSOPHY. Antimicrobial Agents Research Group School of Immunity and Infection College of Medical and Dental Sciences University Of Birmingham October 2012 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties.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 To investigate the role of the two component systems AdeRS and PmrAB in adaptation to the presence of antimicrobials, adeRS and pmrAB were deleted in multi-drug resistant Acinetobacter baumannii strain AYE. The effect of deleting these genes on antimicrobial susceptibility, growth, accumulation, virulence and ability to form a biofilm was investigated. The deletion of adeRS and pmrAB had no effect on bacterial growth or the accumulation of Hoechst 33342 (bis-benzimide). AYEΔpmrAB, but not AYEΔadeRS, accumulated significantly more norfloxacin than AYE. All strains accumulated more norfloxacin in the presence of the efflux inhibitor carbonyl cyanide m-chlorophenylhydrazone. AYE∆adeRS and AYE∆pmrAB, but not AYE, accumulated more norfloxacin in the presence of verapamil. AYEΔadeRS was more susceptible than AYE to antibiotics and biocides and both strains in biofilm were more tolerant of biocides than their planktonic counterparts. Deletion of pmrAB had no effect on antibiotic susceptibility. AYE was more virulent than both AYE∆adeRS and AYE∆pmrAB. The results of this study suggest that in strain AYE, adeRS are not essential for efflux of norfloxacin or Hoechst 33342 and that pmrAB are involved in the accumulation of some compounds. Both AdeRS and PmrAB are important for virulence and AdeRS has a role in antimicrobial susceptibility. Acknowledgements Nothing could have prepared me for this rollercoaster ride and what an experience! I have worked with many people in ARG and TDG who will continue to inspire me for years to come. Laura, thank you for believing in me, answering my numerous questions and helping me find a higher gear when I already thought I was running at full speed! Thank you also to Vito for your advice and support, I could not have done it without you. Mark and Matt at the HPA, you were full of enthusiasm and encouragement and I thoroughly enjoyed working with you. I must also thank my friends and family who shared my highs and lows and ensured that I started each day smiling. This includes Lucy and Rui who became my second family and gave me an even greater love of cooking! Life is full of opportunities, but it is often the people you meet along the way that make them unforgettable - thanks to you all. For Amelia, my greatest inspiration of all Contents 1. Introduction......................................................................................................................... 1 1.1 Acinetobacter ............................................................................................................... 1 1.1.1 Different Species of Acinetobacter .......................................................................... 2 1.1.1.1 AYE ........................................................................................................................ 6 1.1.2 Physiology ................................................................................................................. 6 1.1.3 Habitat ...................................................................................................................... 7 1.1.4 Disease ..................................................................................................................... 8 1.1.4.1 Pathogenicity ........................................................................................................ 8 1.1.4.2 Virulence ............................................................................................................... 9 1.1.5 Treatment ............................................................................................................... 13 1.2. Antimicrobial resistance ............................................................................................ 18 1.2.1. Antibiotic and biocide resistance in Gram Negative Bacteria................................ 18 1.2.1.1. Chromosomal vs. Transmissible Resistance ....................................................... 18 1.2.1.2. Brief Overview of Mechanisms of Resistance .................................................... 19 1.2.2 Antibiotic Resistance and Mechanisms in Acinetobacter baumannii .................... 22 1.2.2.1 β-lactams ............................................................................................................ 24 1.2.2.2 Fluoroquinolones ................................................................................................ 25 1.2.2.3 Polymyxins .......................................................................................................... 26 1.2.2.4 Aminoglycosides ................................................................................................. 27 1.2.2.5. Tetracycline, Chloramphenicol and Tigecycline ................................................. 27 1.2.2.6. Biocides ............................................................................................................... 28 1.2.2.7. Multidrug Resistance .......................................................................................... 29 1.2.2.7.1. Efflux ............................................................................................................... 30 1.3. Two Component Systems .......................................................................................... 32 1.3.1. Signal Transduction ................................................................................................ 33 1.3.2. Two Component Systems in Acinetobacter ........................................................... 37 1.3.2.1 TCSs in Antibiotic and Biocide Resistance .......................................................... 38 1.3.2.1.1 AdeRS .................................................................................................................. 38 1.3.2.1.2 Other TCSs in Antibiotic and Biocide Resistance ................................................ 43 1.3.2.2 TCSs in Acinetobacter Not Involved in Antibiotic or Biocide Resistance ............ 49 1.4 Methods to Study Acinetobacter ............................................................................... 50 1.4.1 Genetic Manipulation in A. baumannii .................................................................. 50 1.4.2 Virulence Models ................................................................................................... 53 1.5 Background to the Project ......................................................................................... 54 1.5.1 Overview ................................................................................................................ 54 1.5.2. Hypotheses to be Tested ........................................................................................ 54 1.5.3. Aims and Objectives ............................................................................................... 55 2. Materials and Methods ..................................................................................................... 57 2.1. Bioinformatics ............................................................................................................ 57 2.1.1. Comparison of A. baumannii Genomes ................................................................. 57 2.1.2. Identification of TCS genes in AYE .......................................................................... 57 2.2. Bacterial Strains, Growth, Storage and Identification ............................................... 59 2.3. DNA Extraction, Purification, Quantification and Sequencing .................................. 64 All DNA sequencing was carried out by the Functional Genomics, Proteomics and Metabolomics Facility, University of Birmingham. .............................................................. 66 2.4. Inactivation of Histidine Kinase Genes in A. baumannii AYE ..................................... 66 2.4.1. Construction of pMo130-TelR/UPDOWN ............................................................... 66 2.4.1.1. Primer design ...................................................................................................... 66 2.4.1.2. Cloning ................................................................................................................ 70 2.4.1.3. Verification of Construct .................................................................................... 75 2.4.2. Transfer of pMo130-TelR/UPDOWN into S17-1 by Transformation ...................... 76 2.4.3. Transfer of pMo130-TelR/UPDOWN into AYE by Conjugation and Selection of Deletion Mutants .................................................................................................................
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