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Mobile Genetic Elements Associated with Blandm-1 in Acinetobacter Spp

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Mobile Genetic Elements Associated with Blandm-1 in Acinetobacter Spp Mobile genetic elements associated with blaNDM-1 in Acinetobacter spp. and Vibrio cholerae. by Lim Stephen Jones A thesis submitted for the degree of Doctor of Philosophy at Cardiff University 2015 i Summary NDM-producing bacteria are associated with extensive antimicrobial resistance (AMR). This thesis reports on detailed molecular analysis, including whole genome sequencing, of Acinetobacter spp. and Vibrio cholerae isolates. A study of clinical Acinetobacter baumannii isolates from India, demonstrated spread of a single strain containing blaNDM-1 but with evidence of significant genetic plasticity between isolates. A novel plasmid, pNDM-32, was fully characterised in isolate CHI-32. This contained multiple AMR genes including blaNDM-1 and the aminoglycoside methyltransferase gene armA. A repAci10 replicase gene was identified but no conjugation machinery and the plasmid could not be transferred in conjugation experiments. A single isolate of Acinetobacter bereziniae from India contained plasmid, pNDM-40-1, harbouring blaNDM-1, which was closely related to plasmids from NDM-producing Acinetobacter spp. isolated in China, and was readily transferred into Escherichia coli and Acinetobacter pittii by conjugation. Five blaNDM-1 positive Acinetobacter spp. isolated from a faecal screening study in Pakistan also included three, clonal, Acinetobacter haemolyticus isolates harbouring a similar plasmid. Three environmental V. cholerae strains from India and a blood isolate from a traveller returning to the UK from India were found to include three distantly related strains. 2 isolates of a single strain contained an IncA/C plasmid, pNDM-116-17, harbouring AMR genes including blaNDM-1. In one isolate pNDM-116-17 had become integrated into a chromosomal region containing a SXT-like element. In the other isolates blaNDM-1 and other AMR determinants were localised to a large plasmid, pNDM-116-14, with a novel replicase and a full complement of conjugative transfer genes, and a novel genomic island, SGI-NDM-1. Most previous studies have focused on Enterobacteriaceae. The current work contributes to an understanding of the full extent of the genetic diversity of blaNDM-1 contexts, and their dissemination. Such knowledge should help to infer factors which contribute to the spread of AMR in bacterial pathogens. ii Publications and Presentations Publications resulting from data presented in this thesis: Jones L.S., Carvalho M.J., Toleman M.A., White P.L., Connor T.R., Mushtaq A., Weeks J.L., Kumarasamy K.K., Raven K.E., Török M.E., Peacock S.J., Howe R.A., Walsh T.R. (2015). Characterization of plasmids in extensively drug-resistant Acinetobacter strains isolated in India and Pakistan. Antimicrobial Agents and Chemotherapy, 59(2): 923-9. Jones L.S., Toleman M.A., Weeks J.L., Howe R.A., Walsh T.R., Kumarasamy K.K. (2014). Plasmid carriage of blaNDM-1 in clinical Acinetobacter baumannii isolates from India. Antimicrobial Agents and Chemotherapy, 58(7): 4211-3. Darley, E. Weeks, J., Jones, L., Daniels, V., Wootton, M., MacGowan, A. and Walsh, T.R. Overseas traveller presenting with NDM-1 positive polymicrobial infactions including Vibrio cholerae: an increased need for vigilance. The Lancet, 380(9850): 1358. Toleman, M.A., Spencer, J, Jones, L. and Walsh, T.R. (2012). blaNDM-1 Is a Chimera Likely Constructed in Acinetobacter baumannii. Antimicrobial Agents and Chemotherapy, 56(5):2773-6. Manuscripts in preparation: Jones, L., Connor, T., Toleman, M., Mutreja, A., Thomson, N., Howe, R. and Walsh, T. Vibrio cholerae: an environmental reservoir for mobile elements and antimicrobial resistance genes including blaNDM-1? Planned for submission to PLOS Pathogens. Jones L.S., Perry, B., Kumarasamy K.K., Török M.E., Peacock S.J., Howe R.A. and Walsh T.R. Complete sequence of plasmid pNDM-32, harbouring blaNDM-1 and armA, from A. baumannii and sequence variation of plasmids and resistance islands in closely related isolates. Planned for submission to Antimicrobial Agents and Chemotherapy. iii Talks resulting from data presented in this thesis: Origins and spread of New Delhi metallo-β-lactamase 1 (NDM-1): lessons from genomic analysis of Acinetobacter spp. and Vibrio cholera. At the WCAT Annual Training Day, Cardiff University, 18/10/14. Vibrio cholerae: an environmental reservoir for mobile elements and antibiotic resistance genes including blaNDM-1? At the South West & South Wales Microbiology Forum, Bath University, 12/09/13. Vibrio cholerae: an environmental reservoir for mobile elements and antibiotic resistance genes including blaNDM-1? The 2013 Panceltic Summer Meeting, Holland House, Cardiff, 22/06/13. Mobile genetic elements associated with blaNDM-1 in multi-drug resistant Acinetobacter species and Vibrio cholerae. Talk given at WCAT annual training day, Glamorgan Building, Cardiff University, 19/10/12. The Genetic Context of blaNDM-1 in Acinetobacter baumannii and Acinetobacter guillouiae from Chennai, India. Talk given at MITReG: Interdisciplinary Postgraduate Research Day, 21/09/12. iv Poster presentations resulting from data presented in this thesis: Jones, L., Toleman, M., Weeks, J., Kumarasamy, K., Howe, R. and Walsh, T. A broad host range conjugative plasmid carrying blaNDM-1 in an Acinetobacter bereziniae isolate from India: Conjugation experiments and genetic stability. Presented at 23rd ECCMID, Berlin, 28th April 2013, P1296. Jones, L., Toleman, M., Connor, T., Weeks, J., Kumarasamy, K., Torok, E., Howe, R., Peacock, S. and Walsh, T. A broad host range conjugative plasmid carrying blaNDM-1 in an Acinetobacter bereziniae isolate from India: Complete sequence and comparison with related genetic contexts. Presented at 23rd ECCMID, Berlin, 28th April 2013, P1295. Jones, L., Toleman, M., Mutreja, A., Weeks, J., Connor, T., Thomson, N., Howe, R. and Walsh, T. The genetic context of blaNDM-1 in Vibrio cholerae. Presented at 23rd ECCMID, Berlin, 28th April 2013, P1292. Jones, L., Toleman, M.A., Weeks, J, Howe, R., Walsh, T.R. and Kumarasamy, K. The genetic context of blaNDM-1 in Acinetobacter baumannii and Acinetobacter guillouiae from Chennai, India. Presented at the Cardiff Institute of Infection and Immunity Annual Meeting 2012. Jones, L., Toleman, M.A., Weeks, J, Howe, R., Walsh, T.R. and Kumarasamy, K. The genetic context of blaNDM-1 in Acinetobacter baumannii from clinical isolates dating back to 2005 from Chennai, India. Presented at 22nd ECCMID, London 2nd April 2012, P1713. Jones, L., Toleman, M.A., Berry, N., Weeks, J., Daniels, V.E., Dickson, W.A., Davies, D., Wootton, M., Howe, R., Walsh, T.R. and Kumarasamy, K. The genetic context of blaNDM-1 in Acinetobacter baumannii from a burns unit outbreak in Swansea, Wales. Presented at 22nd ECCMID, London 2nd April 2012, P1705. v Acknowledgements I am indebted to many individuals and groups who have made the completion of this thesis possible. Firstly to my supervisors Professor Tim Walsh and Robin Howe, without whom I would never have embarked on this project. I am immensely grateful for their guidance and encouragement throughout my PhD but also for being given the chance to find my own solutions and develop confidence and independence as a researcher. To Mark Toleman for offering me the benefit of his considerable knowledge and enthusiasm. To Janis Weeks for keeping everything running and for tolerating my foibles without any greater censure than the occasional wry smile. To the many colleagues with whom I have worked in the 6th floor microbiology laboratory, but especially Maria Carvalho and Jonathon Tyrell, for all their help and advice but above all for their friendship and humour. To my clinical colleagues in medical microbiology, for their support and understanding whilst this thesis was being written-up. To Tom Connor for performing the assemblies of the Acinetobacter spp., giving invaluable guidance at the start of the assembly projects and performing the phylogenetic analysis of the V. cholerae core genomes. To Ankur Mutreja and Nicholas Thomson at the Welcome Trust Sanger Institute for the sequencing of the Vibrio cholerae strains, performing the genome assemblies and advising on this project. To Estee Török and Sharon Peacock at Cambridge University for their assistance in sequencing the Acinetobacter spp. isolates and for their advice on this project. To Mandy Wootton and the team at the Specialist Antimicrobial Chemotherapy Unit for their help with susceptibility testing and for the use of their MALDI- TOF. Last, but by no means least, to my family. My parents, Steve and Jeanette, for all their support over the years. To my wife Rebecca and our children, Rosie and Dylan, for their patience and love, and for helping to keep all my endeavours in perspective. vi Declaration I declare that this thesis represents my own work, except where otherwise acknowledged. The opinions given are my own and not those of Cardiff University or Public Health Wales. No portion of this thesis has been submitted for any other degree or award at this or any other university or place of learning, nor is it being submitted concurrently in candidature for any degree or other award. Dr Lim S. Jones MBBCh, FRCPath Clinical Lecturer Honorary Registrar, Institute of Infection and Immunity Medical Microbiology Medical Microbiology and Infectious and Virology Diseases Public Health Wales Cardiff University Cardiff April 2015 vii List of Figures: Figure 1.1 Illustration of the “discovery void” of antimicrobial agents. 3 Figure 1.2 Normal structure of the Gram-negative outer membrane 5 and interaction
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