Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1375 Mechanisms and Dynamics of Mecillinam Resistance in Escherichia coli ELISABETH THULIN ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-513-0090-0 UPPSALA urn:nbn:se:uu:diva-330856 2017 Dissertation presented at Uppsala University to be publicly examined in A1:111a, BMC, Husargatan 3, Uppsala, Friday, 24 November 2017 at 09:00 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English. Faculty examiner: Professor Laura Piddock ( Institute of Microbiology and Infection, University of Birmingham, UK). Abstract Thulin, E. 2017. Mechanisms and Dynamics of Mecillinam Resistance in Escherichia coli. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1375. 69 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0090-0. The introduction of antibiotics in healthcare is one of the most important medical achievements with regard to reducing human morbidity and mortality. However, bacterial pathogens have acquired antibiotic resistance at an increasing rate, and due to a high prevalence of resistance to some antibiotics they can no longer be used therapeutically. The antibiotic mecillinam, which inhibits the penicillin-binding protein PBP2, however, is an exception since mecillinam resistance (MecR) prevalence has remained low. This is particularly interesting since laboratory experiments have shown that bacteria can rapidly acquire MecR mutations by a multitude of different types of mutations. In this thesis, I examined mechanisms and dynamics of mecillinam resistance in clinical and laboratory isolates of Escherichia coli. Only one type of MecR mutations (cysB) was found in the clinical strains, even though laboratory experiments demonstrate that more than 100 genes can confer resistance Fitness assays showed that cysB mutants have higher fitness than most other MecR mutants, which is likely to contribute to their dominance in clinical settings. To determine if the mecillinam resistant strains could compensate for their fitness cost, six different MecR mutants (cysB, mrdA, spoT, ppa, aspS and ubiE) were evolved for 200-400 generations. All evolved mutants showed increased fitness, but the compensation was associated with loss of resistance in the majority of cases. This will also contribute to the rarity of clinical MecR isolates with chromosomal resistance mutations. How MecR is mediated by cysB mutations was previously unclear, but in this thesis I propose and test a model for the mechanism of resistance. Thus, inactivation of CysB results in cellular depletion of cysteine that triggers an oxidative stress response. The response alters the intracellular levels of 450 proteins, and MecR is achieved by the increase of two of these, the LpoB and PBP1B proteins, which rescue the cells with a mecillinam-inhibited PBP2. Mecillinam is used for UTI treatments and to investigate mecillinam resistance in a more host- like milieu, MecR strains were grown in urine and resistance was examined. Interestingly, this study showed that neither laboratory, nor clinical cysB mutants are resistant in urine, most likely because the cysteine present in the urine phenotypically reverts the bacteria to susceptibility. These findings suggest that mecillinam can be used to treat also those clinical strains that are identified as MecR in standard laboratory tests, and that testing of mecillinam susceptibility in the laboratory ought to be performed in media that mimics urine to obtain clinically relevant results. In summary, the work described in this thesis has increased ourgeneral knowledge of mecillinam resistance and its evolution. Hopefully this knowledge can be put to good use in clinical settings to reduce the negative impact of antibiotic resistance. Keywords: Mecillinam, Antibiotic resistance, Escherichia coli, Urinary tract infections, Fitness, Penicillin binding proteins, cysteine biosynthesis Elisabeth Thulin, Department of Medical Biochemistry and Microbiology, Box 582, Uppsala University, SE-75123 Uppsala, Sweden. © Elisabeth Thulin 2017 ISSN 1651-6206 ISBN 978-91-513-0090-0 urn:nbn:se:uu:diva-330856 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-330856) If there is one thing the history of evolution has taught us it’s that life will not be contained. Life breaks free, it expands to new territories and crashes through barriers, painfully, maybe even dangerously, but, uh… well, there it is. - Dr. Ian Malcolm For Måns, Alvar and Irma Members of the committee Faculty examiner Professor Laura Piddock Institute of Microbiology and Infection University of Birmingham, UK Members of the evaluation committee Professor Roland Möllby Department of Microbiology, Tumor and Cell Biology Karolinska Institute, Sweden Professor Staffan Svärd Department of Cell and Molecular Biology Uppsala University, Sweden Docent Göte Swedberg, Department of Medical Biochemistry and Microbiology Uppsala University, Sweden Chairman of the thesis defence Professor Staffan Johansson Department of Medical Biochemistry and Microbiology Uppsala University, Sweden List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Thulin, E., Sundqvist, M., Andersson, D. (2015) Amdinocillin (me- cillinam) resistance mutations in clinical isolates and laboratory- selected mutants of Escherichia coli. Antimicrobial Agents and Chemotherapy, 59:1718–1727 II Knopp, M., Thulin, E., Ekstrand, E., Andersson, D. (2017) Com- pensatory evolution in mecillinam-resistant Escherichia coli. Manu- script. III Thulin, E., Andersson, D. (2017) An oxidative stress-induced by- pass mechanism confers mecillinam resistance in Escherichia coli. Submitted manuscript. IV Thulin, E., Thulin, M., Andersson, D. (2017) Reversion of high- level mecillinam resistance to susceptibility in Escherichia coli dur- ing growth in urine. EBioMedicine, 23:111–118 Reprints were made with permission from the respective publishers. Work not included in this thesis: Lofton, H., Pränting, M., Thulin, E., Andersson, D. (2013) Resistance mechanisms to antimicrobial peptides LL-37, CNY100HL and Wheat Germ Histones. PLOS ONE, 8:e68875 Contents Introduction ................................................................................................... 13 Antibiotics ................................................................................................ 14 Antibiotic resistance ................................................................................. 16 Mechanisms of resistance .................................................................... 17 Spread of resistance ............................................................................. 20 The cost of being resistant ........................................................................ 21 β-lactam antibiotics ................................................................................... 23 The β-lactam target – the cell wall ....................................................... 23 Penicillin-binding proteins ................................................................... 25 β-lactam resistance ............................................................................... 29 β-lactams in therapeutic use ................................................................. 29 Mecillinam ................................................................................................ 33 Mecillinam resistance ............................................................................... 34 The importance of cysteine biosynthesis in mecillinam resistance ..... 36 Escherichia coli – the accidental pathogen .............................................. 38 Urinary tract infections ........................................................................ 39 Present investigations .................................................................................... 42 Paper I: Amdinocillin (Mecillinam) resistance mutations in clinical isolates and laboratory-selected mutants of Escherichia coli ................... 42 Paper II: Compensatory evolution in mecillinam-resistant Escherichia coli ............................................................................................................ 43 Paper III: An oxidative stress-induced bypass-mechanism confers mecillinam resistance in Escherichia coli ................................................ 45 Paper IV: Reversion of high-level mecillinam resistance to complete susceptibility in Escherichia coli during growth in urine ......................... 48 Concluding remarks ...................................................................................... 51 Svensk sammanfattning ................................................................................. 53 Acknowledgements ....................................................................................... 55 References ..................................................................................................... 59 Abbreviations AUM artificial urine medium bp base pair DNA deoxyribonucleic acid DTT dithiothreitol EAU European Association of Urology ESBL extended spectrum β-lactamase ESCMID European Society of Clinical Microbiology and Infectious Diseases GTase glycosyltransferase HGT horizontal gene transfer HMW high molecular weight ICE integrative conjugative element IDSA Infectious Diseases Society of America kb kilobase LMW low molecular weight Mec mecillinam MecR mecillinam-resistant