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Stenotrophomonas Maltophilia and the Characterisation of Resistant Mutant This electronic thesis or dissertation has been downloaded from Explore Bristol Research, http://research-information.bristol.ac.uk Author: Calvopina Tapia, Karina Title: The efficacy of last-line and experimental antimicrobials against Stenotrophomonas maltophilia and the characterisation of resistant mutant General rights Access to the thesis is subject to the Creative Commons Attribution - NonCommercial-No Derivatives 4.0 International Public License. A copy of this may be found at https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode This license sets out your rights and the restrictions that apply to your access to the thesis so it is important you read this before proceeding. Take down policy Some pages of this thesis may have been removed for copyright restrictions prior to having it been deposited in Explore Bristol Research. However, if you have discovered material within the thesis that you consider to be unlawful e.g. breaches of copyright (either yours or that of a third party) or any other law, including but not limited to those relating to patent, trademark, confidentiality, data protection, obscenity, defamation, libel, then please contact [email protected] and include the following information in your message: •Your contact details •Bibliographic details for the item, including a URL •An outline nature of the complaint Your claim will be investigated and, where appropriate, the item in question will be removed from public view as soon as possible. The efficacy of last-line and experimental antimicrobials against Stenotrophomonas maltophilia and the characterisation of resistant mutants Karina Alexandra Calvopiña Tapia A dissertation submitted to the University of Bristol in accordance with the requirements for award of the degree of PhD in the Faculty of Biomedical Sciences School of Cellular and Molecular Medicine June 2018 Word Count 66675 Abstract In recent years, Stenotrophomonas maltophilia has not only gained importance due to its increasing prevalence in human disease around the world but because of its intrinsic multidrug resistance, which makes this bacterium an interesting model to study mechanisms of drug resistance while informing rational drug design and changes to chemotherapy protocols. Here, we aimed to identify novel mechanisms of resistance to ceftazidime, amikacin, levofloxacin, and minocycline. Overexpression of the chromosomally encoded serine-β-lactamase (SBL), L2, and metallo- β-lactamase (MBL), L1, is known to confer resistance to β-lactams, including ceftazidime. However, the mechanisms behind β-lactamase hyperproduction are not completely understood. Here, a new mechanism responsible for L1/L2 hyperproduction has been characterised: loss of function mutations in mpl which likely lead to accumulation of activator ligand for AmpR, the transcriptional regulator of L2 and L1. Additionally, for the first time a TonB energy-dependent mechanism is proposed for ceftazidime uptake where the alteration of a proline-rich region, deactivating TonB, is responsible for ceftazidime resistance in non-β-lactamase hyper-producers. Although reduced antimicrobial permeability in S. maltophilia due to upregulation of efflux-pumps is well known, little is known about their regulation. Here evidence is provided to associate SmeYZ aminoglycoside efflux pump upregulation with ribosomal damage caused by mutations in the rplA gene, encoding a ribosomal subunit target of aminoglycosides and by ribosomal-acting agents such as gentamicin. Alterations in genes involved in lipid trafficking were found to be associated with SmeDEF efflux pump upregulation. Two novel ABC transporters were shown to be involved in levofloxacin resistance and reduced minocycline susceptibility; each is locally regulated by a two-component system. Minocycline was found to be the most promising therapeutic agent and resistant mutants or clinical isolates could not be found. In addition to characterise last-line antimicrobials, the potential of non-classical β-lactamase inhibitors to restore β-lactam activity was studied. It was found that the diazabicyclooctane avibactam and the new bicyclic boronate 2 have potential to combat β-lactam resistance in S. maltophilia when compared to the traditional β-lactam based inhibitor, clavulanic acid based on microbiological, kinetic and structural evidence. In cases where the non-traditional inhibitors failed to restore β-lactam i antimicrobial activity (e.g. with ceftazidime) new combinations strategies (aztreonam/avibactam or aztreonam/bicyclic boronate 2) or new inhibitors (MBL inhibitors in combination with meropenem) showed significant promise. In addition, the sideromimic modification of γ-lactam antibiotic, lactivicin (LTV-17) was found tp increase its antimicrobial activity 1000-fold against S. maltophilia. Although LTV-17 induces L1//L2 production, lactivicin is only slowly hydrolysed by β-lactamases. Therefore, LTV-17 antimicrobial activity is still strong against β-lactamase and efflux pump hyper-producing mutants, and extensively-drug resistant clinical isolates. Mutants with reduced LTV-17 susceptibility were identified, and were found to have the same loss of the TonB energy-transducer seen in ceftazidime resistant mutants. This work has given insight into the mechanisms of resistance in S. maltophilia to assist the optimisation and identification of possible candidates to combat this species in the clinic. ii Acknowledgments First I would like to thank Dr. Matthew Avison. Thanks for believing in me and giving me this opportunity. An amazing opportunity that started before coming to Bristol and that has changed my life in so many ways. Thank you for being always supportive, for all your time and dedication in making me a better student and person. I will keep trying my best for not letting you down. I would also like to thank Dr. James Spencer. Thanks for inviting me to face new challenges. Thanks for all the beautiful experiences you made me live by sharing our work. Most of what I am about to live will be because of you. Phil Hinchliffe, thank you for sharing your knowledge and expertise during the execution of this work. Thanks for always being ready to answer my questions, thank you for your patience and care during this time. Thank you D60, you were not only my friends and teachers in the lab, you became my family during this time. Professor Christopher Schofield (University of Oxford) for letting me carry out part of my work in the Chemistry Research Laboratory. Dr. Jurgen Brem for your supervision and patience during my short stay in Oxford. Dr. Kate Heesom (University of Bristol) for her help in the proteomics facility. Secretaria de Educacion Superior, Ciencia, Tecnologia e Innovacion (SENESCYT) for providing the funding for my PhD studies. Friends I met in Bristol: Ramya, Juan Carlos, Leonor, Fiizh, Ji who made this city even more wonderful than it is. Friends in Ecuador and/or around the world who are pursuing similar dreams: Mabe, Lottyta, Mariel, Cris, Rose, Ale, Che, Sagu, Lili y Andrey. Thank you all, for being an inspiration, for always teaching me something valuable, for giving me a reason to smile and for being with me through this adventure. Last but not least, God and my loving and unconditional family that has always been with me regardless time and distance. Dedication To the memory of my grandparents and especially to the memory of my uncle Juan who instilled love and passion for biology in our family. iii Author’s Declaration I declare that the work in this dissertation was carried out in accordance with the requirements of the University’s Regulations and Code of Practice for Research Degree Programmes and that it has not been submitted for any other academic award. Except where indicated by specific reference in the text, the work is the candidate’s own work. Work done in collaboration with, or with the assistance of, other, is indicated as such. Any views expressed in the dissertation are those of the author. iv Publications and Presentations Published literature Calvopina K, Avison MB. 2018. Disruption of mpl Activates beta-Lactamase Production in Stenotrophomonas maltophilia and Pseudomonas aeruginosa Clinical Isolates. Antimicrob Agents Chemother doi:10.1128/AAC.00638-18. Hinchliffe P, Tanner CA, Krismanich AP, Labbe G, Goodfellow VJ, Marrone L, Desoky AY, Calvopina K, Whittle EE, Zeng F, Avison MB, Bols NC, Siemann S, Spencer J, Dmitrienko GI. 2018. Structural and Kinetic Studies of the Potent Inhibition of Metallo- beta-lactamases by 6-Phosphonomethylpyridine-2-carboxylates. Biochemistry 57:1880-1892. Calvopina K, Hinchliffe P, Brem J, Heesom KJ, Johnson S, Cain R, Lohans CT, Fishwick CWG, Schofield CJ, Spencer J, Avison MB. 2017. Structural/mechanistic insights into the efficacy of nonclassical beta-lactamase inhibitors against extensively drug resistant Stenotrophomonas maltophilia clinical isolates. Molecular Microbiology 106:492-504. Calvopina K, Umland KD, Rydzik AM, Hinchliffe P, Brem J, Spencer J, Schofield CJ, Avison MB. 2016. Sideromimic Modification of Lactivicin Dramatically Increases Potency against Extensively Drug-Resistant Stenotrophomonas maltophilia Clinical Isolates. Antimicrobial Agents and Chemotherapy 60:4170-4175 Oral presentation Role of mpl mutations in the beta-lactamase hyperesistant phenotype in Stenotrophomonas maltophilia. Calvopina K, Avison MB. (2017). 27th ECCMID, the European Congress of Clinical Microbiology and Infectious Diseases, Vienna-Austria. Presenting author. Poster presentation Proteomic-mediated
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