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This Thesis Has Been Submitted in Fulfilment of the Requirements for a Postgraduate Degree (E.G This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: • This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. • 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. Role of Insertion Sequences in the control of antibiotic resistance in Acinetobacter baumannii Bruno S. Lopes Thesis presented for the degree of Doctor of Philosophy University of Edinburgh 2011. i. Abstract Acinetobacter baumannii is an emerging multiresistant pathogen increasingly known to cause infections in the immuno-compromised patients. Carbapenems and colistin are considered to be the last resorts in treatment of infections involving multidrug resistant strains of A. baumannii . Resistance to carbapenems is well known due to the presence of intrinsic carbapenemase gene bla OXA-51-like, which may be governed by insertion elements, or by acquired carbapenemases like bla OXA-23-like , bla OXA-58-like or bla OXA-40-like genes, most of which are frequently associated with insertion elements. The acquired carbapenemases can be integrated with the host chromosome making the bacterium strongly resistant to a range of antibiotics. Recent reports also suggest that the ubiquitous and intrinsic enzymes encoded by the bla OXA-51-like gene can be mobilized on a plasmid. In this thesis, the prevalence of antibiotic resistance was examined for 96 strains isolated from various parts of the world. The resistances to aminoglycosides, fluoroquinolones and cephalosporins were studied with a major focus on resistance to carbapenems. Section 1 shows the transposition of IS Aba1 and its varied influence in controlling the bla OXA-51-like gene and the bla ADC gene. It explains how IS Aba1 being a strong factor in influencing antibiotic resistance genes contributes to the plasticity of the organism Section 2 is related with a novel insertion element IS Aba125 controlling the bla ADC gene and as an element providing high resistance to ceftazidime in comparison to IS Aba1 . Section 3 analyses the multi-drug resistant profile of strains isolated from Cochabamba, Bolivia. Besides the classification of carbapenem resistance for the clinical strains, the aminoglycoside resistance and ciprofloxacin mechanisms are examined in this project Section 4 relates with the pattern of resistance in strains isolated from the Aberdeen Royal Infirmary. It describes two novel variants of the bla OXA-51-like gene, namely bla OXA-216 and bla OXA-217 and also the acquisition of the bla OXA-23-like gene in two isolates from different years and deemed identical by their PFGE pattern. Section 5 describes the influence of IS Aba825 in controlling the bla OXA-51-like gene and the bla OXA-58-like from clinical isolates Section 6 is related with the insertional inactivation of the bla OXA-132 gene and the carbapenem resistance caused by the activation of the bla OXA-58 gene in isolate Ab244 Section 7 describes the influence of insertion elements in strains having high ciprofloxacin resistance. This project is concerned with the role of efflux pump control system adeRS and how they influence the adeABC operon causing increased and decreased expression of the genes. Section 8 describes the multi drug resistant pattern of 36 strains each isolated from Europe and the United States In conclusion, there are various factors that influence the resistance profile of multi- drug resistant A. baumannii isolates with insertion sequences such as IS Aba1 , IS Aba2 , IS Aba3 , IS Aba825 , IS 1008 , IS Aba125 , IS Aba16 governing the expression or providing alternate mechanisms of resistance for the better fitness of the bacterium. Mutations in the genes identified in this study also have a crucial role in imparting resistance to this bacterium. ii. Declaration. The experiments and composition of this thesis are the work of the author unless otherwise stated. Bruno S. Lopes iii. Dedication This thesis is dedicated to My family who is the source of wisdom, love, support, strength and perseverance And to Catharine McAuley Some great thing which He designs to accomplish would have been too much without a little bitter in the cup………………………..Catharine McAuley iv. Acknowledgements. Firstly I would like to acknowledge my supervisor Professor Sebastian Amyes whose advice and support has been invaluable for the completion of this study. I would like to thank him for not only his advice but also for the freedom he gave me in designing my own experiments which led me to think independently and because of which I had many fruitful and surprising outcomes. I would like to thank Dr A. Hamouda for making me familiar with the technical skills involved in Molecular Microbiology which helped me to achieve my objectives. Thanks to Prof. Lucía Gallego who worked with me on her summer project with her strains from Cochabamaba, Bolivia leading to a fruitful outcome. I would like to thank Dr Ian Gould for providing me with strains from Aberdeen Royal Infirmary. I would like to thank Dr Kevin Towner for providing me with all the European Acinetobacter baumannii isolates used in this study. I would also like to thank Andres Opazo for having fruitful discussions on various laboratory techniques related with A. baumannii . Special thanks to Juachi, Maher, Leena, Monem and Norya who always wished the best for me and encouraged me to achieve my goals. Thanks to Jacqueline Finday, Ben Evans and Paul Higgins for their expertise. Thanks to Malcolm Baldock for the processing of orders and adding to the stock of chemical and reagents. A very special thanks to Dr Cathy Doherty for allowing me to use all the various reagents and machines required for microbiological and molecular methods without which my work would not have been completed on time. And last but not the least I would like to thank Prof Stephen Hillier and Dr Dorothy Watson for their help in providing me with the University of Edinburgh Overseas Research Scholarship and the College of Medicine and Veterinary Medicine bursary. v. Permission to reproduce papers. Published manuscripts have been reproduced here in accordance with the copyright guidelines of the publishers involved. vi. Publications and presentations. Lopes, B. S., A. Hamouda, J. Findlay, and S. G. B. Amyes. 2011. Effect of frame- shift mutagen acriflavine on control of resistance genes in Acinetobacter baumannii. J. Med Microbiol. 60: 211-215. Lopes, B. S., B. A. Evans and S .G.B Amyes. 2011. The disruption of the bla OXA-51-like gene by IS Aba16 and the activation of the bla OXA-58 gene leading to carbapenem resistance in Acinetobacter baumannii Ab244. J Antimicrob Chemother. 67 : 59-63. (Epub ahead of print on 5 October 2011) Lopes, B. S., I. M. Gould, A.F. Opazo, S. G. B Amyes. 2011. The resistance profile of Acinetobacter baumannii strains isolated from the Aberdeen Royal Infirmary (Accepted in Int J Antimicrob Agents ). Lopes, B. S., and S.G.B Amyes. Role of IS Aba825 in the expression of the bla OXA-51- like gene in clinical strains of Acinetobacter baumannii (Oral, ICAAC, 2011 Chicago) Lopes, B. S., and S.G.B Amyes. The role of Insertion Elements in control of antibiotic resistance in Acinetobacter baumannii isolated from sources throughout the world (Oral, ICAAC, 2011 Chicago). Opazo, A. F., B. S. Lopes, A. Sonnevend, T. Pal, A. Ghazawi, and S.G.B. Amyes. Ceftazidime resistance in Acinetobacter baumannii from the United Arab Emirates (Oral, ICAAC, 2011 Chicago). Lopes, B., A. Hamouda, S. Amyes. The role of IS 30 in the expression of the bla ADC gene in Acinetobacter baumannii (Poster, ECCMID 2011, Milan, Italy) Lopes, B., A. Hamouda, S. Amyes. The utilisation of gluconic acid in certain strains of Acinetobacter baumannii (Abstract, ECCMID 2011, Milan, Italy) Lopes B. S., and S. G. B Amyes. 2011. First report of IS Aba825 governing the expression of bla OXA-51-like gene in clinically relevant strains (Submitted to CMI). Lopes, B. S., and S. G. B Amyes. 2011. The prevalence of IS Aba1 and IS Aba125 , governing the expression of the bla ADC in clinically relevant Acinetobacter baumannii strains resistant to cephalosporins (Submitted to JMM). Lopes, B. S, L. Gallego and S. G. B Amyes. 2011. Multi drug resistance profiles and the genetic features that influences the resilient adaptation in Acinetobacter baumannii isolates from Bolivia (Submitted to JAC). Opazo, A., A. Sonnevend, B. Lopes, A. Hamouda, A. Ghazawi, T. Pál, and S. G.B. Amyes.2011. Plasmid-encoded PER-7 beta-lactamase responsible for ceftazidime resistance in Acinetobacter baumannii isolated in the United Arab Emirates. (Accepted in JAC) vii. Abbreviations ADC Acinetobacter -derived cephalosporinases ARDRA Amplified ribosomal DNA restriction analysis bp Base pairs BLAST Basic Local Alignment Search Tool BSAC British Society for Antimicrobial Chemotherapy CLSI Clinical and Laboratory Standards Institute cm Centimetres CN Gentamicin DNA Deoxyribonucleic acid EC European clone
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