Lstmed.ac.uk
Effect of environment on the evolutionary trajectories and growth characteristics of antibiotic- resistant Escherichia coli mutants Alasdair T. M. Hubbard1,2, Nazila V. Jafari3, Nicholas Feasey4,5, Jennifer L. Rohn3 and Adam P. Roberts1,2, *1 Department of Tropical Disease Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA 2 Centre for Drugs and Diagnostics Research, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool. L3 5QA3Centre for Urological Biology, Department of Renal Medicine, University College London, London, UK4 Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool L3 5QA, UK.5 The Malawi Liverpool Wellcome Trust Clinical Research Programme, University of Malawi, the College of Medicine, Blantyre, Malawi.*Corresponding author: Adam P. Roberts; Department of Tropical Disease Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA; Adam.Roberts@lstmed.ac.ukAbstractThe fitness cost to bacteria of acquisition of resistance determinants is critically under-investigated, and the identification and exploitation of these fitness costs may lead to novel therapeutic strategies that prevent the emergence of antimicrobial resistance. Here we used Escherichia coli and amoxicillin-clavulanic acid (AMC) resistance as a model to understand how the artificial environments utilised in studies of bacterial fitness could affect the emergence of resistance and associated fitness costs. Further, we explored the predictive value of this data when strains were grown in the more physiologically relevant environments of urine and urothelial organoids. Resistant E. coli isolates were selected for following 24-hour exposure to sub-inhibitory concentrations of AMC in either M9, ISO or LB, followed by growth on LB agar containing AMC. No resistant colonies emerged following growth in M9, whereas resistant isolates were detected from cultures grown in ISO and LB. We observed both within and between media-type variability in the levels of resistance and fitness of the resistant mutants grown in LB. MICs and fitness of these resistant strains in different media (M9, ISO, LB, human urine and urothelial organoids) showed considerable variation. Media can therefore have a direct effect on the isolation of mutants that confer resistance to AMC and these mutants can exhibit unpredictable MIC and fitness profiles under different growth conditions. This preliminary study highlights the risks in relying on a single culture protocol as a model system to predict the behaviour and treatment response of bacteria in vivo and highlights the importance of developing comprehensive experimental designs to ensure effective translation of diagnostic procedures to successful clinical outcomes. Key words: Evolution, antibiotic resistance, fitness, biological cost, urethral organoid, urineIntroductionThe alarming global rise in antimicrobial resistance (AMR) has led to increased interest in resistance development, persistence and fixation within bacterial populations, with a wide variety of studies published in recent years. These include determining whether the prevalence of resistant bacteria is exacerbated by antimicrobials and their metabolites entering the environment following use in humans PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5TaW5nZXI8L0F1dGhvcj48WWVhcj4yMDE2PC9ZZWFyPjxS
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ADDIN EN.CITE.DATA (Singer et al., 2016;Caudell et al., 2018), understanding the evolution of AMR due to selective pressure from antimicrobials, and establishing how the acquisition of AMR genes affects bacterial fitness PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Xb25nPC9BdXRob3I+PFllYXI+MjAxNzwvWWVhcj48UmVj
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ADDIN EN.CITE.DATA (Palmer et al., 2015;Hughes and Andersson, 2017;Suzuki et al., 2017). When bacteria acquire resistance, either through mutation or horizontal gene transfer, they often incur a fitness cost. Many of these evolutionary studies rely on the in vitro development of resistance in the presence of sub-inhibitory concentrations of single, or combinations of, antimicrobials, and investigators employ a range of different media for the generation of resistance PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5TdXp1a2k8L0F1dGhvcj48WWVhcj4yMDE3PC9ZZWFyPjxS
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ADDIN EN.CITE.DATA (Gullberg et al., 2011;Kn?ppel et al., 2017;Suzuki et al., 2017;Westhoff et al., 2017) as well as the subsequent assessment of fitness related to acquired AMR PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5TdGFyaWtvdmE8L0F1dGhvcj48WWVhcj4yMDEzPC9ZZWFy
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ADDIN EN.CITE.DATA (Starikova et al., 2013;Olivares Pacheco et al., 2017). However, the media currently used to grow bacteria in vitro is not necessarily a representative model of the in vivo environment in which the bacteria live. Determining evolutionary trajectories to resistance, and the use of this knowledge as a tool to/ control the emergence and persistence of AMR in bacteria is a growing area of research PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5IdWdoZXM8L0F1dGhvcj48WWVhcj4yMDE3PC9ZZWFyPjxS
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ADDIN EN.CITE.DATA (Hughes and Andersson, 2017;Wong, 2017;Podnecky et al., 2018). One of the prerequisites for translation of this approach into a clinical intervention that optimises patient care whilst leveraging the fitness cost of AMR against a pathogen the establishment of robust and reproducible model systems which convince clinicians and policy makers that what is seen in the laboratory will actually occur in the real world. Therefore, there is a need to analyse the consequences of different experimental conditions thoroughly and develop model systems which have predictive value in order to inform clinical end-point studies.A recent study found that mutations that confer antibiotic resistance can arise following evolution in LB broth in the absence of an antimicrobial, raising questions over whether evolutionary trajectories in vitro will be the same in vivo due to adaption to a specific environment ADDIN EN.CITE <EndNote><Cite><Author>Kn?ppel</Author><Year>2017</Year><RecNum>19</RecNum><DisplayText>(Kn?ppel et al., 2017)</DisplayText><record><rec-number>19</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1516619062">19</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Kn?ppel, A.</author><author>N?svall, J. </author><author>Andersson, D.I.</author></authors></contributors><titles><title>Evolution of Antibiotic Resistance without Antibiotic Exposure</title><secondary-title>Antimicrobial Agents and Chemotherapy</secondary-title></titles><periodical><full-title>Antimicrobial Agents and Chemotherapy</full-title></periodical><pages> e01495-17 </pages><volume>61</volume><number>11</number><dates><year>2017</year></dates><urls></urls><electronic-resource-num>10.1128/AAC.01495-17</electronic-resource-num></record></Cite></EndNote>(Kn?ppel et al., 2017). Therefore, for studies based on in vitro model systems to be used to guide changes in treatment and prescription policy or even inform the design of clinical intervention studies, it is critical that experimental observations made in such systems reflect the real-world; as with mathematical models, all models are wrong, but some are useful. It is known that the fitness cost of a particular mutation conferring resistance to an antibiotic is dependent on the environment it is assessed in, for example growth media PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5MaW48L0F1dGhvcj48WWVhcj4yMDE4PC9ZZWFyPjxSZWNO
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ADDIN EN.CITE.DATA (Maharjan and Ferenci, 2017;Lin et al., 2018), and another recent study investigated whether compensatory mutations that arise following acquisition of AMR genes by Escherichia coli had the same effect in different media; the results showed that such compensation does not always translate in another environment ADDIN EN.CITE <EndNote><Cite><Author>Basra</Author><Year>2018</Year><RecNum>45</RecNum><DisplayText>(Basra et al., 2018)</DisplayText><record><rec-number>45</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1542125262">45</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Basra, P.</author><author>Alsaadi, A.</author><author>Bernal-Astrain, G.</author><author>O'Sullivan, M. L.</author><author>Hazlett, B.</author><author>Clarke, L. M.</author><author>Schoenrock, A.</author><author>Pitre, S.</author><author>Wong, A.</author></authors></contributors><auth-address>Department of Biology, Carleton University.
School of Computer Science, Carleton University.
Research Computing Services, Carleton University.</auth-address><titles><title>Fitness tradeoffs of antibiotic resistance in extra-intestinal pathogenic Escherichia coli</title><secondary-title>Genome Biol Evol</secondary-title></titles><periodical><full-title>Genome Biol Evol</full-title></periodical><keywords><keyword>Antibiotic resistance</keyword><keyword>compensatory evolution</keyword><keyword>epistasis</keyword><keyword>trade-off</keyword><keyword>whole genome sequencing</keyword></keywords><dates><year>2018</year><pub-dates><date>Feb 7</date></pub-dates></dates><isbn>1759-6653 (Electronic)
1759-6653 (Linking)</isbn><accession-num>29432584</accession-num><urls><related-urls><url>;(Basra et al., 2018). This is an import finding, suggesting that a compensatory mutation can be beneficial in one physiological compartment, such as the bladder, but may be deleterious in another, for example the blood ADDIN EN.CITE <EndNote><Cite><Author>Basra</Author><Year>2018</Year><RecNum>45</RecNum><DisplayText>(Basra et al., 2018)</DisplayText><record><rec-number>45</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1542125262">45</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Basra, P.</author><author>Alsaadi, A.</author><author>Bernal-Astrain, G.</author><author>O'Sullivan, M. L.</author><author>Hazlett, B.</author><author>Clarke, L. M.</author><author>Schoenrock, A.</author><author>Pitre, S.</author><author>Wong, A.</author></authors></contributors><auth-address>Department of Biology, Carleton University.
School of Computer Science, Carleton University.
Research Computing Services, Carleton University.</auth-address><titles><title>Fitness tradeoffs of antibiotic resistance in extra-intestinal pathogenic Escherichia coli</title><secondary-title>Genome Biol Evol</secondary-title></titles><periodical><full-title>Genome Biol Evol</full-title></periodical><keywords><keyword>Antibiotic resistance</keyword><keyword>compensatory evolution</keyword><keyword>epistasis</keyword><keyword>trade-off</keyword><keyword>whole genome sequencing</keyword></keywords><dates><year>2018</year><pub-dates><date>Feb 7</date></pub-dates></dates><isbn>1759-6653 (Electronic)
1759-6653 (Linking)</isbn><accession-num>29432584</accession-num><urls><related-urls><url>;(Basra et al., 2018). It further highlights the importance of the choice of growth media when assessing fitness effects following acquisition of resistance. E. coli is the top urinary pathogen, responsible for upwards of 80% of all community-acquired urinary tract infections in otherwise healthy young women ADDIN EN.CITE <EndNote><Cite><Author>Foxman</Author><Year>2010</Year><RecNum>68</RecNum><DisplayText>(Foxman, 2010)</DisplayText><record><rec-number>68</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1550923496">68</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Foxman, B.</author></authors></contributors><auth-address>Department of Epidemiology, University of Michigan School of Public Health, 1415 Washington Heights, Ann Arbor, MI 48109, USA. bfoxman@umich.edu</auth-address><titles><title>The epidemiology of urinary tract infection</title><secondary-title>Nat Rev Urol</secondary-title></titles><periodical><full-title>Nat Rev Urol</full-title></periodical><pages>653-60</pages><volume>7</volume><number>12</number><edition>2010/12/09</edition><keywords><keyword>Drug Resistance, Bacterial</keyword><keyword>Humans</keyword><keyword>Incidence</keyword><keyword>Prevalence</keyword><keyword>Urinary Tract Infections/complications/drug</keyword><keyword>therapy/*epidemiology/microbiology/transmission</keyword></keywords><dates><year>2010</year><pub-dates><date>Dec</date></pub-dates></dates><isbn>1759-4820 (Electronic)
1759-4812 (Linking)</isbn><accession-num>21139641</accession-num><urls><related-urls><url>;(Foxman, 2010). Enhanced sentinel surveillance of E. coli bacteraemia in England revealed that 51.2% followed urinary tract infection with a large number of study isolates found to be resistant to trimethoprim (48.1%), ciprofloxacin (18.5%) and amoxicillin-clavulanic acid (AMC) (45.4%,) PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5BYmVybmV0aHk8L0F1dGhvcj48WWVhcj4yMDE3PC9ZZWFy
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ADDIN EN.CITE.DATA (Abernethy et al., 2017). Therefore, studying antimicrobial resistance in Enterobacteriaceae with better models is of particular interest as resistance continues to increase within this family to such a degree that carbapenem resistant, ESBL producing Enterobacteriaceae are now considered priority pathogens on the critical list, for which new drugs are urgently required ADDIN EN.CITE <EndNote><Cite><Author>Organisation</Author><Year>2017</Year><RecNum>70</RecNum><DisplayText>(World Health Organisation, 2017)</DisplayText><record><rec-number>70</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1550924737">70</key></foreign-keys><ref-type name="Press Release">63</ref-type><contributors><authors><author>World Health Organisation,</author></authors><secondary-authors><author>WHO</author></secondary-authors></contributors><titles><title>WHO publishes list of bacteria for which new antibiotics are urgently needed</title></titles><dates><year>2017</year></dates><pub-location> Health Organisation</publisher><urls></urls></record></Cite></EndNote>(World Health Organisation, 2017).There have been many in vitro models developed to study bacteria that more closely mimic their natural habitats within the human body; these include the chemostat gut, which has been used to study both planktonic and biofilm growth of Clostridium difficile PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Dcm93dGhlcjwvQXV0aG9yPjxZZWFyPjIwMTQ8L1llYXI+
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ADDIN EN.CITE.DATA (Roberts et al., 2001;Ready et al., 2002). More relevant still are laboratory-grown organoids which allow pathogen-host interactions to be investigated and observed in isolation PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Gb3JiZXN0ZXI8L0F1dGhvcj48WWVhcj4yMDE1PC9ZZWFy
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ADDIN EN.CITE.DATA (Forbester et al., 2015;Leslie et al., 2015;Horsley et al., 2018). A recently described urothelial organoid was shown to be tolerant of urine over extended periods of time, to produce a number of expected biomarkers in the presence of urine, and to respond to bacterial infection in a fashion similar to that of the human bladder ADDIN EN.CITE <EndNote><Cite><Author>Horsley</Author><Year>2018</Year><RecNum>44</RecNum><DisplayText>(Horsley et al., 2018)</DisplayText><record><rec-number>44</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1542104740">44</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Horsley, H.</author><author>Dharmasena, D.</author><author>Malone-Lee, J.</author><author>Rohn, J. L.</author></authors></contributors><auth-address>Chronic UTI Group, Centre for Nephrology, University College London, London, UK. h.horsley@ucl.ac.uk.
Chronic UTI Group, Centre for Nephrology, University College London, London, UK.
Chronic UTI Group, Centre for Nephrology, University College London, London, UK. j.rohn@ucl.ac.uk.</auth-address><titles><title>A urine-dependent human urothelial organoid offers a potential alternative to rodent models of infection</title><secondary-title>Sci Rep</secondary-title></titles><periodical><full-title>Sci Rep</full-title></periodical><pages>1238</pages><volume>8</volume><number>1</number><dates><year>2018</year><pub-dates><date>Jan 19</date></pub-dates></dates><isbn>2045-2322 (Electronic)
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ADDIN EN.CITE.DATA (Musicha et al., 2017b), a country with high rates of bacteraemia caused by E. coli (8.8% of bacteraemia caused by E. coli 1998 - 2016), many of which are multidrug resistant ADDIN EN.CITE <EndNote><Cite><Author>Musicha</Author><Year>2017</Year><RecNum>56</RecNum><DisplayText>(Musicha et al., 2017a)</DisplayText><record><rec-number>56</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1549544443">56</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Musicha, Patrick</author><author>Cornick, Jennifer E.</author><author>Bar-Zeev, Naor</author><author>French, Neil</author><author>Masesa, Clemens</author><author>Denis, Brigitte</author><author>Kennedy, Neil</author><author>Mallewa, Jane</author><author>Gordon, Melita A.</author><author>Msefula, Chisomo L.</author><author>Heyderman, Robert S.</author><author>Everett, Dean B.</author><author>Feasey, Nicholas A.</author></authors></contributors><titles><title>Trends in antimicrobial resistance in bloodstream infection isolates at a large urban hospital in Malawi (1998–2016): a surveillance study</title><secondary-title>The Lancet Infectious Diseases</secondary-title></titles><periodical><full-title>The Lancet Infectious Diseases</full-title></periodical><pages>1042-1052</pages><volume>17</volume><number>10</number><dates><year>2017</year></dates><isbn>14733099</isbn><urls></urls><electronic-resource-num>10.1016/s1473-3099(17)30394-8</electronic-resource-num></record></Cite></EndNote>(Musicha et al., 2017a), including to amoxicillin-clavulanic acid (AMC). For β-lactams to be effective, they need to be maintained at a concentration above the minimum inhibitory concentration for at least 40% of the time between doses in serum ADDIN EN.CITE <EndNote><Cite><Author>WA</Author><Year>1996</Year><RecNum>77</RecNum><DisplayText>(Craig, 1996)</DisplayText><record><rec-number>77</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1563275782">77</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Craig, WA</author></authors></contributors><titles><title>Antimicrobial Resistance Issues of the Future</title><secondary-title>Diagnostic Microbiology and Infectious Disease</secondary-title></titles><periodical><full-title>Diagnostic Microbiology and Infectious Disease</full-title></periodical><pages>213-217</pages><volume>25</volume><dates><year>1996</year></dates><urls></urls></record></Cite></EndNote>(Craig, 1996). However, in 35% patients AMC was found to be at a concentration above the MIC for less than 40% of the time ADDIN EN.CITE <EndNote><Cite><Author>M</Author><Year>2014</Year><RecNum>78</RecNum><DisplayText>(Haeseker et al., 2014)</DisplayText><record><rec-number>78</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1563275785">78</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Haeseker, M</author><author>Havenith, T</author><author>Stolk, L</author><author>Neef, C</author><author>Bruggeman, C</author><author>Verbon, A</author></authors></contributors><titles><title>Is the standard dose of amoxicillin-clavulanic acid sufficient?</title><secondary-title>BMC Pharmacology and Toxicology</secondary-title></titles><periodical><full-title>BMC Pharmacology and Toxicology</full-title></periodical><pages>1-8</pages><volume>15</volume><number>38</number><dates><year>2014</year></dates><urls></urls></record></Cite></EndNote>(Haeseker et al., 2014). Therefore, the selective pressure during treatment with AMC is often sub-inhibitory, to replicate this we used sub-inhibitory concentrations of AMC to study the development of resistance and subsequent associated fitness costs. We included representatives of different media used in previous evolutionary studies, M9 (defined media) PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5TdXp1a2k8L0F1dGhvcj48WWVhcj4yMDE3PC9ZZWFyPjxS
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ADDIN EN.CITE.DATA (Farrell et al., 2011) and LB broth (LB, undefined) ADDIN EN.CITE <EndNote><Cite><Author>Kn?ppel</Author><Year>2017</Year><RecNum>19</RecNum><DisplayText>(Kn?ppel et al., 2017)</DisplayText><record><rec-number>19</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1516619062">19</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Kn?ppel, A.</author><author>N?svall, J. </author><author>Andersson, D.I.</author></authors></contributors><titles><title>Evolution of Antibiotic Resistance without Antibiotic Exposure</title><secondary-title>Antimicrobial Agents and Chemotherapy</secondary-title></titles><periodical><full-title>Antimicrobial Agents and Chemotherapy</full-title></periodical><pages> e01495-17 </pages><volume>61</volume><number>11</number><dates><year>2017</year></dates><urls></urls><electronic-resource-num>10.1128/AAC.01495-17</electronic-resource-num></record></Cite></EndNote>(Kn?ppel et al., 2017) and examined whether MICs and fitness determined in growth media could be used to predict the MIC determined in the more physiological environment of urine, and comparative growth in urothelial organoids. Materials and methodsBacterial strains, media and antibioticsThe ancestor bacterial strain used in this study was the clinical isolate E. coli 10129 which was isolated from a cerebral spinal fluid (CSF) sample in Malawi PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5NdXNpY2hhPC9BdXRob3I+PFllYXI+MjAxNzwvWWVhcj48
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ADDIN EN.CITE.DATA (Musicha et al., 2017b) (Table 1). It was reported as fully susceptible and the genome had previously been resolved to a relatively small number of contigs within this strain collection [19].Cultures were grown in Mueller Hinton broth (MHB), LB (Lennox) (both Sigma, UK), ISO (Oxoid, UK) or M9 (50% (v/v) M9 minimal salts (2x) (Gibco, ThermoFisher Scientific, USA), 0.4% D-glucose, 4mM magnesium sulphate (both Sigma, UK) and 0.05 mM calcium chloride (Millipore, USA)). E. coli 10129 and AMC-resistant isolates were grown on LB agar (Lennox) (Sigma, UK) for colony counting during MIC determination.Amoxicillin trihydrate: potassium clavulanate (4:1) (AMC) was diluted in molecular grade water (both Sigma, UK) to a stock concentration of 1 mg/ml and filter sterilised through a 0.22 ?M polyethersulfone filter unit (Millipore, USA).Determination of minimum inhibitory concentration in laboratory growth mediaMICs were performed in MHB, LB, ISO or M9, using cultures grown in the respective media in the absence of antimicrobial selection, following the Clinical and Laboratory Standards Institute (CLSI) guidelines for broth microdilution MIC and determined visually ADDIN EN.CITE <EndNote><Cite><Author>CLSI</Author><Year>2018</Year><RecNum>53</RecNum><DisplayText>(CLSI, 2018)</DisplayText><record><rec-number>53</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1549405351">53</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Standard">58</ref-type><contributors><authors><author>CLSI </author></authors></contributors><titles><title>Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically</title><secondary-title>11th Ed. CLSI Standard M07</secondary-title></titles><dates><year>2018</year></dates><pub-location>Wayne, Pennsylvania, USA</pub-location><publisher>Clinical and Laboratory Standards Institute</publisher><urls></urls></record></Cite></EndNote>(CLSI, 2018). As per CLSI guidelines, all cultures were normalised to an optical density at 600nm (OD600) of between 0.8 and 1.0 prior to diluting 1/1000 in the appropriate media. CFU/ml of the inoculum for at each isolate in each media was determined at least once by diluting 1/1000 in PBS and 100 ?l plated out to ensure that the cell count was between 2-8.2 x 105 CFU/ml.Determination of minimum inhibitory concentrations in urineOvernight bacterial cultures of E. coli 10129 and AMC-resistant isolates were diluted in urine supernatants derived from the organoids, with the composition of approximately 25% urine and 75% CnT-Prime 3D Barrier medium (CnT-PR-3D) (CELLnTEC, Switzerland), to OD600 of 0.1. Cultures were further diluted 1/1000. Antimicrobial susceptibility was determined by following the CLSI guidelines. Optical density (OD595) was measured at 24 h post-incubation with a microplate reader (Biochrom EZ Read 400). Selection of amoxicillin-clavulanic acid resistant E. coli 10129E. coli 10129 was grown in sub-inhibitory concentrations (4 ?g/ml), as determined by CLSI guidelines in MHB, of AMC in LB, ISO and M9 to select for resistant isolates. Separate cultures of E. coli 10129 were initially grown in 10 ml LB, ISO or M9 for 18 hours at 37°C and shaking at 200 rpm. Following incubation, each culture was serially diluted 1 in 10 in phosphate buffered solution (PBS, pH 7.2, Gibco, ThermoFisher Scientific, USA) and 50 ?l of the neat to 10-7 dilutions were plated out on to LB agar and incubated at 37°C for 18 hours to determine CFU/ml. Using the initial cultures, 10 ?l was diluted in 10 ml of the respective test media containing 4 ?g/ml AMC and incubated at 37°C with shaking at 200 rpm for 24 hours. The cultures were serially diluted in PBS as described above and 50 ?l of the neat to 10-7 dilutions were plated onto LB agar and 50 ?l of neat to 10-2 dilutions were plated onto LB agar + 8 ?g/ml AMC (MIC) and LB agar + 16 ?g/ml AMC (2 x MIC). These plates were incubated at 37°C for 18 hours. Up to 10 single colonies were selected from agar plates from three replicate resistance selection experiments which had growth at the highest concentration of AMC and immediately stored in 1 ml LB + 40% glycerol (Sigma, UK) and stored at -80°C to prevent any further growth and compensatory mutations. The number of colonies was scored at the dilution that grew between 15-100 colonies, except for those that only grew a small number of colonies in the undiluted petitive fitness assaysThe competitive fitness of each of the selected AMC-resistant isolates was determined in comparison to the ancestral isolate, E. coli 10129. Cultures of ancestor and AMC-resistant isolates were initially inoculated in LB, ISO or M9 directly from the -80°C stocks to minimise any further evolutionary events. The ancestor isolate was grown without the presence of AMC, whereas the resistant isolates were grown in the presence of the same concentration of AMC in which they were initially derived to confirm resistance and ensure that only the resistant population grew, i.e. 8 or 16 ?g/ml AMC. All cultures were diluted in the appropriate media to an OD600 of 0.1. The AMC-resistant and ancestor isolates were further diluted 1/1000 and combined 1:1 in the same media, 150 ?l of which was added to a 96 well plate and incubated at 37°C for 24 hours at 200 rpm. The initial combined culture was serially diluted in PBS and 50 ?l of the neat to 10-7 dilutions were plated out onto both LB agar and LB agar plus either 8 or 16 ?g/ml AMC, depending on the concentration of AMC that the isolate was selected on, and incubated at 37°C for 18 hours. Following 24 hours of growth, the combined culture was then plated out on to agar as described above. The number of colonies was scored at the dilution that grew between 15-120 colonies.Relative fitness was calculated using the Malthusian equation ADDIN EN.CITE <EndNote><Cite><Author>Lenski</Author><Year>1991</Year><RecNum>2</RecNum><DisplayText>(Lenski et al., 1991)</DisplayText><record><rec-number>2</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1516287716">2</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Lenski, R.E</author><author>Rose, M.R</author><author>Simpson, S.C</author><author>Tadler, S.C</author></authors></contributors><titles><title>Long-Term Experimental Evolution in Escherichia coli: Adaptation and Divergence During 2,000 Generations</title><secondary-title>The American Naturalist</secondary-title></titles><periodical><full-title>The American Naturalist</full-title></periodical><pages>1315-1341</pages><volume>138</volume><number>6</number><dates><year>1991</year></dates><urls></urls></record></Cite></EndNote>(Lenski et al., 1991):M=Mm(T24T0)/Mwt(T24T0)W=M-1 x 100where W is the relative fitness, M is the Malthusian parameter, Mwt is the Malthusian parameter of the ancestor isolate, Mm is the Malthusian parameter of the resistant isolate, T0 is the log CFU/ml count of the initial culture and T24 is the log CFU/ml count after 24 hours.DNA extractionAMC-resistant isolates and the ancestral isolate were grown in 10 ml LB and incubated at 37°C with shaking at 200 rpm for 18 hours. DNA extraction was performed using the Gentra Puregene Yeast/Bact. Kit (Qiagen, Germany) according to manufacturer’s instructions. The pellet was air-dried at room temperature for 5 minutes. The DNA was dissolved in 50 ?l molecular grade water for 1 hour at 65°C and the DNA concentration determined using a NanoDrop? One/OneC Microvolume UV-vis Spectrophotometer (ThermoFisher Scientific, USA).Comparative growth in urothelial organoidsHuman bladder urothelial organoids were prepared as described previously ADDIN EN.CITE <EndNote><Cite><Author>Horsley</Author><Year>2018</Year><RecNum>44</RecNum><DisplayText>(Horsley et al., 2018)</DisplayText><record><rec-number>44</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1542104740">44</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Horsley, H.</author><author>Dharmasena, D.</author><author>Malone-Lee, J.</author><author>Rohn, J. L.</author></authors></contributors><auth-address>Chronic UTI Group, Centre for Nephrology, University College London, London, UK. h.horsley@ucl.ac.uk.
Chronic UTI Group, Centre for Nephrology, University College London, London, UK.
Chronic UTI Group, Centre for Nephrology, University College London, London, UK. j.rohn@ucl.ac.uk.</auth-address><titles><title>A urine-dependent human urothelial organoid offers a potential alternative to rodent models of infection</title><secondary-title>Sci Rep</secondary-title></titles><periodical><full-title>Sci Rep</full-title></periodical><pages>1238</pages><volume>8</volume><number>1</number><dates><year>2018</year><pub-dates><date>Jan 19</date></pub-dates></dates><isbn>2045-2322 (Electronic)
2045-2322 (Linking)</isbn><accession-num>29352171</accession-num><urls><related-urls><url>;(Horsley et al., 2018). Briefly, primary human bladder epithelial cell (HBLAK) (CELLnTEC, Switzerland) were seeded onto polycarbonate transwell inserts (VWR, UK) at 5 X 105 cells per ml in CnT-Prime medium (CnT-PR, CELLnTEC, Switzerland). An appropriate amount of CnT-PR was added to the basolateral chamber of the inserts so that the medium levels were equal, and the cells were submerged. Once cells reached confluency, CnT-PR was replaced with CnT-PR-3D (CELLnTEC, Switzerland) and incubated for 15-16 h. 3D culture was initiated by replacing medium from the apical chamber with filter-sterilised human urine (BIOIVT, UK), as well as fresh basolateral CnT-PR-3D medium. Urine and CnT-PR-3D medium were changed at regular intervals and organoids were fully established and stratified on day 14-18. All bacterial cultures of E. coli 10129 and AMC-resistant isolates were diluted in urine to OD600 of 0.1, then further diluted 1/1000. Urine from apical chambers were aspirated and replaced with diluted bacterial cultures and fresh CnT-PR-3D was added to the basolateral chambers. Inserts were incubated at 37 ?C, 5% CO2 for 24 h. Cultures from apical chambers were collected and transferred into a 96-well plate. Optical density (OD595) was measured at time 0 and 24 h post-infection using a microplate reader (Biochrom EZ Read 400).Background optical density at 595 nm was accounted for by normalising with a measurement of the urothelial organoids containing the E. coli 10129 ancestor or AMC-resistant isolates at 0 hours, as well as for organoid containing media only. Relative growth was calculated using the following equation:R=OmOwt -1 x 100where R is the relative growth, Owt is the OD595 after 24 hours growth of the AMC-resistant isolate in the urothelial organoid, Om is the OD595 after 24 hours growth of the ancestor isolate in the urothelial organoid.Whole genome sequencing and bioinformatic analysisWhole genome sequencing of E. coli 10129 and AMC-resistant isolates were performed by MicrobesNG (MicrobesNG, UK) using 2x250 bp paired-end reads on the Illumina MiSeq, which also included the trimming of the sequencing reads.De novo assembly of each of the genomes were performed using SPAdes (version 3.12.0) ADDIN EN.CITE <EndNote><Cite><Author>Bankevich</Author><Year>2012</Year><RecNum>34</RecNum><DisplayText>(Bankevich et al., 2012)</DisplayText><record><rec-number>34</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1540992079">34</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Bankevich, A.</author><author>Nurk, S.</author><author>Antipov, D.</author><author>Gurevich, A. A.</author><author>Dvorkin, M.</author><author>Kulikov, A. S.</author><author>Lesin, V. M.</author><author>Nikolenko, S. I.</author><author>Pham, S.</author><author>Prjibelski, A. D.</author><author>Pyshkin, A. V.</author><author>Sirotkin, A. V.</author><author>Vyahhi, N.</author><author>Tesler, G.</author><author>Alekseyev, M. A.</author><author>Pevzner, P. A.</author></authors></contributors><auth-address>Algorithmic Biology Laboratory, St. Petersburg Academic University, Russian Academy of Sciences, St. Petersburg, Russia.</auth-address><titles><title>SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing</title><secondary-title>J Comput Biol</secondary-title></titles><periodical><full-title>J Comput Biol</full-title></periodical><pages>455-77</pages><volume>19</volume><number>5</number><keywords><keyword>*Algorithms</keyword><keyword>Bacteria/*genetics</keyword><keyword>*Genome, Bacterial</keyword><keyword>Metagenomics/*methods</keyword><keyword>Sequence Analysis, DNA/methods</keyword><keyword>Single-Cell Analysis/*methods</keyword></keywords><dates><year>2012</year><pub-dates><date>May</date></pub-dates></dates><isbn>1557-8666 (Electronic)
1066-5277 (Linking)</isbn><accession-num>22506599</accession-num><urls><related-urls><url>;(Bankevich et al., 2012) and assembly statistics were generated using QUAST (version 4.6.3) ADDIN EN.CITE <EndNote><Cite><Author>Mikheenko</Author><Year>2018</Year><RecNum>35</RecNum><DisplayText>(Mikheenko et al., 2018)</DisplayText><record><rec-number>35</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1540992094">35</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Mikheenko, A.</author><author>Prjibelski, A.</author><author>Saveliev, V.</author><author>Antipov, D.</author><author>Gurevich, A.</author></authors></contributors><auth-address>Center for Algorithmic Biotechnology, Institute of Translational Biomedicine, St. Petersburg State University, St. Petersburg, Russia.</auth-address><titles><title>Versatile genome assembly evaluation with QUAST-LG</title><secondary-title>Bioinformatics</secondary-title></titles><periodical><full-title>Bioinformatics</full-title></periodical><pages>i142-i150</pages><volume>34</volume><number>13</number><dates><year>2018</year><pub-dates><date>Jul 1</date></pub-dates></dates><isbn>1367-4811 (Electronic)
1367-4803 (Linking)</isbn><accession-num>29949969</accession-num><urls><related-urls><url>;(Mikheenko et al., 2018). Annotation of each of the assembled genomes was performed using Prokka (version 1.12) ADDIN EN.CITE <EndNote><Cite><Author>Seemann</Author><Year>2014</Year><RecNum>36</RecNum><DisplayText>(Seemann, 2014)</DisplayText><record><rec-number>36</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1540992103">36</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Seemann, T.</author></authors></contributors><auth-address>Victorian Bioinformatics Consortium, Monash University, Clayton 3800 and Life Sciences Computation Centre, Victorian Life Sciences Computation Initiative, Carlton 3053, AustraliaVictorian Bioinformatics Consortium, Monash University, Clayton 3800 and Life Sciences Computation Centre, Victorian Life Sciences Computation Initiative, Carlton 3053, Australia.</auth-address><titles><title>Prokka: rapid prokaryotic genome annotation</title><secondary-title>Bioinformatics</secondary-title></titles><periodical><full-title>Bioinformatics</full-title></periodical><pages>2068-9</pages><volume>30</volume><number>14</number><keywords><keyword>*Genome, Bacterial</keyword><keyword>Genomics/methods</keyword><keyword>Molecular Sequence Annotation/*methods</keyword><keyword>Sequence Analysis, DNA</keyword><keyword>*Software</keyword></keywords><dates><year>2014</year><pub-dates><date>Jul 15</date></pub-dates></dates><isbn>1367-4811 (Electronic)
1367-4803 (Linking)</isbn><accession-num>24642063</accession-num><urls><related-urls><url>;(Seemann, 2014). Single nucleotide polymorphisms (SNPs), large deletions, mobile genetic element insertions, duplications, amplifications and smaller insertion and deletion (Indels) sequences in each of the E. coli 10129 AMC-resistant isolates were predicated computationally using Breseq ADDIN EN.CITE <EndNote><Cite><Author>Deatherage</Author><Year>2014</Year><RecNum>37</RecNum><DisplayText>(Deatherage and Barrick, 2014)</DisplayText><record><rec-number>37</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1541511574">37</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Deatherage, D. E.</author><author>Barrick, J. E.</author></authors></contributors><auth-address>Department of Molecular Biosciences, Center for Systems and Synthetic Biology, Center for Computational Biology and Bioinformatics, Institute for Cellular and Molecular Biology, The University of Texas at Austin, 1 University Station A5300, Austin, TX, 78712, USA.</auth-address><titles><title>Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq</title><secondary-title>Methods Mol Biol</secondary-title></titles><periodical><full-title>Methods Mol Biol</full-title></periodical><pages>165-88</pages><volume>1151</volume><keywords><keyword>Directed Molecular Evolution/methods</keyword><keyword>Genomics/*methods</keyword><keyword>High-Throughput Nucleotide Sequencing/*methods</keyword><keyword>Microbiological Techniques/methods</keyword><keyword>*Mutation</keyword><keyword>Sequence Analysis, DNA/*methods</keyword><keyword>Software</keyword></keywords><dates><year>2014</year></dates><isbn>1940-6029 (Electronic)
1064-3745 (Linking)</isbn><accession-num>24838886</accession-num><urls><related-urls><url>;(Deatherage and Barrick, 2014) (version 0.32.0) in default mode, with the trimmed sequencing reads produced by MicrobesNG aligned against our assembled and annotated genome of the E. coli 10129 ancestor isolate. Any mutations which were predicted with less than 80% frequency were discounted and stretches of missing coverage which mapped to small, full contigs from the ancestor. Also discounted were a single 192 bp duplication at the end of a contig from LB_5 and a single amplification of 4 x 96 bp also at the end of a contig from ISO_2 which was deemed an assembly artefact as the sequencing read depth were less than 20 reads (Table S1). We therefore focused only on the SNPs within the genome of the AMC-resistant isolates as predicted by Breseq.The ancestral isolate, E. coli 10129, was characterised bioinformatically to determine the sequence type (MLST 2.0, version 2.0.1 ADDIN EN.CITE <EndNote><Cite><Author>Larsen</Author><Year>2012</Year><RecNum>71</RecNum><DisplayText>(Larsen et al., 2012)</DisplayText><record><rec-number>71</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1554450856">71</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Larsen, M. V.</author><author>Cosentino, S.</author><author>Rasmussen, S.</author><author>Friis, C.</author><author>Hasman, H.</author><author>Marvig, R. L.</author><author>Jelsbak, L.</author><author>Sicheritz-Ponten, T.</author><author>Ussery, D. W.</author><author>Aarestrup, F. M.</author><author>Lund, O.</author></authors></contributors><auth-address>Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark, Lyngby, Denmark. metteb@cbs.dtu.dk</auth-address><titles><title>Multilocus sequence typing of total-genome-sequenced bacteria</title><secondary-title>J Clin Microbiol</secondary-title></titles><periodical><full-title>J Clin Microbiol</full-title></periodical><pages>1355-61</pages><volume>50</volume><number>4</number><keywords><keyword>Bacteria/classification/*genetics</keyword><keyword>Bacterial Typing Techniques</keyword><keyword>Contig Mapping</keyword><keyword>Databases, Genetic</keyword><keyword>Genes, Bacterial</keyword><keyword>*Genome, Bacterial</keyword><keyword>High-Throughput Nucleotide Sequencing</keyword><keyword>*Multilocus Sequence Typing</keyword></keywords><dates><year>2012</year><pub-dates><date>Apr</date></pub-dates></dates><isbn>1098-660X (Electronic)
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ADDIN EN.CITE.DATA (Joensen et al., 2015)), resistome (ResFinder, version 3.1.0 ADDIN EN.CITE <EndNote><Cite><Author>Zankari</Author><Year>2012</Year><RecNum>73</RecNum><DisplayText>(Zankari et al., 2012)</DisplayText><record><rec-number>73</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1554450863">73</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Zankari, E.</author><author>Hasman, H.</author><author>Cosentino, S.</author><author>Vestergaard, M.</author><author>Rasmussen, S.</author><author>Lund, O.</author><author>Aarestrup, F. M.</author><author>Larsen, M. V.</author></authors></contributors><auth-address>National Food Institute, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark. east@food.dtu.dk</auth-address><titles><title>Identification of acquired antimicrobial resistance genes</title><secondary-title>J Antimicrob Chemother</secondary-title></titles><periodical><full-title>J Antimicrob Chemother</full-title></periodical><pages>2640-4</pages><volume>67</volume><number>11</number><keywords><keyword>Bacteria/drug effects/*genetics</keyword><keyword>Bacterial Infections/microbiology</keyword><keyword>Computational Biology/*methods</keyword><keyword>*Drug Resistance, Bacterial</keyword><keyword>Humans</keyword><keyword>Internet</keyword></keywords><dates><year>2012</year><pub-dates><date>Nov</date></pub-dates></dates><isbn>1460-2091 (Electronic)
0305-7453 (Linking)</isbn><accession-num>22782487</accession-num><urls><related-urls><url>;(Zankari et al., 2012)) and plasmidome (PlasmidFinder, version 2.0.1 ADDIN EN.CITE <EndNote><Cite><Author>Carattoli</Author><Year>2014</Year><RecNum>72</RecNum><DisplayText>(Carattoli et al., 2014)</DisplayText><record><rec-number>72</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1554450857">72</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Carattoli, A.</author><author>Zankari, E.</author><author>García-Fernández, A.</author><author>Larsen, M. V.</author><author>Lund, O.</author><author>Villa, L.</author><author>Aarestrup, F.M.</author><author>Hasmanb, H.</author></authors></contributors><titles><title>In Silico Detection and Typing of Plasmids using PlasmidFinder and Plasmid Multilocus Sequence Typing</title><secondary-title>Antimicrobial Agents and Chemotherapy</secondary-title></titles><periodical><full-title>Antimicrobial Agents and Chemotherapy</full-title></periodical><pages>3895-3903</pages><volume>58</volume><number>7</number><dates><year>2014</year></dates><urls></urls></record></Cite></EndNote>(Carattoli et al., 2014)) all on default settings.PCR confirmation of predicted single nucleotide polymorphismsPrimers were designed to amplify a 465bp region of the cpxA gene containing the predicted SNP and a 442bp region that contained the predicted SNP within the ampC promoter region (Table 2). The PCR amplification was performed using the Q5? High Fidelity polymerase (New England Biolabs, USA) and the final reaction contained 1x Q5? reaction buffer, 200 ?M dNTPs, 0.5 ?M of the appropriate forward and reverse primers listed in Table 2 and 0.02 U/?l Q5? polymerase in a total volume of 25 ?l. The PCR samples were run using the following protocol: denaturation at 98°C for 30 seconds, followed by 35 cycles of denaturation at 98°C for 10 seconds, annealing at 69°C (cpxA SNP) or 67°C (ampC SNP) for 30 seconds, elongation at 72°C for 20 seconds, followed by a final extension of 2 minutes at 72°C. The PCR products were cleaned up using the Monarch? PCR and DNA clean-up kit (New England Biolabs, USA). The PCR samples were mixed with 125 ?l of the DNA Clean-Up Binding Buffer, transferred to the Clean-Up Columns and centrifuged at 11400 x g for 1 minute. The bound DNA was washed with 200 ?l DNA Wash Buffer and centrifuged at 11400 x g for 1 minute, twice, followed by elution in 20 ?l molecular grade water (Sigma, UK) by centrifuging at 11400 x g for 1 minute after incubating at room temperature for 2 minutes. All PCR products were Sanger sequenced at GeneWiz (Takely, UK). Biofilm assayE. coli 10129 ancestor and AMC-resistant isolates were grown in LB for 18 hours at 37°C and 200 rpm and then diluted 1/1000 in M9. Three 100 ?l technical replicates of the each of the diluted cultures were added to a 96 well microtitre plate alongside three technical replicates of M9 only and three empty wells and incubated statically at 37°C for 24 hours. Following incubation, all culture or media was removed, and the wells washed 4-5 times with 150 ?l PBS and then left to dry upside-down for 10 minutes. The wells were then stained with 125 ?l 0.1% Gram’s crystal violet solution (Sigma, UK) for 15 minutes at room temperature, then washed 4-5 times with 150 ?l PBS and left to dry upside-down for between 60-90 minutes. The remaining stain was dissolved with 125 ?l 30% acetic acid (Sigma, UK) and incubated at room temperature for 15 minutes. Finally, the acetic acid solution was transferred to a fresh 96 well microtitre plate and measured at an optical density of 550nm, with 30% acetic acid used as a blank and the stained empty wells as background.Statistical analysisStatistical analysis of the initial growth of E. coli 10129 in LB, ISO and M9 was performed using Ordinary One-Way ANOVA in GraphPad Prism (version 8.0.0). Statistical analysis of competitive fitness in LB, relative fitness in bladder organoids and biofilm production of the E. coli 10129 ancestor and AMC-resistant isolates was performed using Ordinary One-Way ANOVA plus uncorrected Fisher’s LSD test in GraphPad Prism. Statistical analysis of competitive fitness of LB_2 and LB_5 in LB, ISO and M9 was performed using unpaired t test. Finally, statistical analysis of cell density of E. coli 10129 following challenge with AMC in LB, ISO and M9 was performed using 2-way ANOVA plus uncorrected Fisher’s LSD test, also in GraphPad Prism.ResultsCharacterisation of E. coli 10129E. coli 10129 is a clinical isolate from Malawi and is part of the phylogroup B2 PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5NdXNpY2hhPC9BdXRob3I+PFllYXI+MjAxNzwvWWVhcj48
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ADDIN EN.CITE.DATA (Musicha et al., 2017b), sequence type 700 and serotype O83:H7. The isolate was found to contain the following acquired resistance genes; sul2 (sulphonamide), dfrA1 (trimethoprim), aadA1, aph(3')-Ia, aph(6)-Id and aph(3'')-Ib (all aminoglycosides), and mdf(A) (macrolide, lincosamide and streptogramin B). Replicon sequences of IncFII (pRSB107), IncFIA and IncFIB, were detected within the genome sequence, as well as 66.5% coverage of the replicon sequence of IncQ1.Selection of E. coli 10129 resistant isolatesThe MIC of AMC for E. coli 10129 was determined to be 4-8 ?g/ml (Table 3). To select for isolates showing decreased susceptibility to AMC, E. coli 10129 was grown in the presence of sub-inhibitory concentrations (4 ?g/ml) of AMC in defined (M9), semi-defined (ISO) and undefined (LB) media for 24 hours. There was no significant difference in the cell densities following growth of E. coli 10129 in the absence of AMC among three different media types (P value = 0.1071, Fig. 1A) and therefore the cell density of the inoculum was also comparable. A significant reduction in cell density of 5.049 log CFU/ml was observed following incubation in M9 containing AMC (P value = <0.0001, Fig. 1B) and we did not recover any isolates from agar containing 8 or 16 ?g/ml AMC. Unlike M9, following exposure of E. coli 10129 to AMC in ISO there was a small but insignificant increase in cell density of 0.592 log CFU/ml (P value = 0.1339, Fig. 1B) and significant growth of E. coli 10129 of 1.03 log CFU/ml in LB in the presence of AMC compared with the initial inoculum (P value = 0.0235). Therefore, using this one-step selection technique, 10 colonies were selected from agar plates containing 8 ?g/ml AMC and 10 from plates containing 16 ?g/ml AMC following growth in ISO and LB broth, respectively. Of the 10 resistant isolates of E. coli 10129 grown in either LB or ISO, three from each media-type were selected at random. The three isolates selected following growth in ISO broth were designated ISO_2, ISO_9 and ISO_10 and those grown in LB were designated LB_1, LB_2 and LB_5. ISO_2 and LB_5 were both derived from independent lineages, whereas both LB_1 and LB_2 were possibly derived from the same lineage and ISO_9 and ISO_10 were also possibly from the same lineage.Minimum inhibitory concentrations of E. coli 10129 ancestor and AMC-resistant isolates in MHBResistance to AMC in the six selected E. coli 10129 derivatives were assessed through MIC determination in comparison to the ancestral isolate in MHB (Table 3). While there was within media-type increased variability in the AMC MIC of the resistant isolates, there was a clear distinction between those resistant isolates selected for in LB and those from ISO (Table 3). There was only a 1 to 3-fold increase in the AMC MIC in resistant isolates selected in ISO, which would be expected as ISO selected isolates where only recovered from agar containing MIC concentrations of AMC. The AMC-resistant isolates selected for in LB broth plated out on LB agar containing 2xMIC concentrations of AMC were all highly resistant compared with the ancestor isolate, resulting in a 3 to 15-fold increase in MIC (Table 3). The most resistant isolates were determined to be LB_2 and LB_5, both with a 7 to 15-fold increase in MIC in comparison to the ancestral isolate (Table 3). Four of the six selected isolates had MICs above the clinical breakpoints according to the EUCAST guidelines for systemic infections caused by E. coli (>8 ?g/ml, Table 3) and therefore deemed AMC-resistant, these were ISO_2, LB_1, LB_2 and LB_5, and were carried forward for whole genome sequencing.Single nucleotide polymorphism predictionSNPs that arose in the genomes of the E. coli 10129 AMC-resistant isolates following selection in sub-inhibitory concentrations of AMC were identified using Breseq, which predicts SNPs in genomes of derivative generations by mapping sequencing reads to the original ancestral isolate ADDIN EN.CITE <EndNote><Cite><Author>Deatherage</Author><Year>2014</Year><RecNum>37</RecNum><DisplayText>(Deatherage and Barrick, 2014)</DisplayText><record><rec-number>37</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1541511574">37</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Deatherage, D. E.</author><author>Barrick, J. E.</author></authors></contributors><auth-address>Department of Molecular Biosciences, Center for Systems and Synthetic Biology, Center for Computational Biology and Bioinformatics, Institute for Cellular and Molecular Biology, The University of Texas at Austin, 1 University Station A5300, Austin, TX, 78712, USA.</auth-address><titles><title>Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq</title><secondary-title>Methods Mol Biol</secondary-title></titles><periodical><full-title>Methods Mol Biol</full-title></periodical><pages>165-88</pages><volume>1151</volume><keywords><keyword>Directed Molecular Evolution/methods</keyword><keyword>Genomics/*methods</keyword><keyword>High-Throughput Nucleotide Sequencing/*methods</keyword><keyword>Microbiological Techniques/methods</keyword><keyword>*Mutation</keyword><keyword>Sequence Analysis, DNA/*methods</keyword><keyword>Software</keyword></keywords><dates><year>2014</year></dates><isbn>1940-6029 (Electronic)
1064-3745 (Linking)</isbn><accession-num>24838886</accession-num><urls><related-urls><url>;(Deatherage and Barrick, 2014).Chromosomal mutations of E. coli that lead to resistance to β-lactams are often found within the promoter region of the ampC gene, resulting in over-production of the chromosomally located β-lactamase AmpC PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5TaXU8L0F1dGhvcj48WWVhcj4yMDAzPC9ZZWFyPjxSZWNO
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ADDIN EN.CITE.DATA (Siu et al., 2003;Tracz et al., 2005;Tracz et al., 2007). Isolates LB_2 and LB_5 contained a predicted SNP at nucleotide position -32 in the -35-box promoter region with 100% frequency within the sequencing reads, which was subsequently confirmed to be present using PCR and sequencing. This mutation converted the natural weak -35 promoter (TTGTCA) to a stronger promoter (TTGACA) ADDIN EN.CITE <EndNote><Cite><Author>Caroff</Author><Year>2000</Year><RecNum>8</RecNum><DisplayText>(Caroff et al., 2000)</DisplayText><record><rec-number>8</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1516612220">8</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Caroff, N.</author><author>Espaze, E.</author><author>Gautreau, D.</author><author>Richet, H.</author><author>Reynaud, A.</author></authors></contributors><titles><title>Analysis of the Effects of -42 and -32 ampC Promoter Mutations in Clinical Isolates of Escherichia coli Hyperproducing AmpC</title><secondary-title>Journal of Antimicrobial Chemotherapy</secondary-title></titles><periodical><full-title>Journal of Antimicrobial Chemotherapy</full-title></periodical><pages>783-788</pages><volume>45</volume><dates><year>2000</year></dates><urls></urls></record></Cite></EndNote>(Caroff et al., 2000) which has been previously linked to 21-fold increase in AmpC production ADDIN EN.CITE <EndNote><Cite><Author>Jaurin</Author><Year>1982</Year><RecNum>7</RecNum><DisplayText>(Jaurin et al., 1982)</DisplayText><record><rec-number>7</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1516612212">7</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Jaurin, B</author><author>Grundstrom, T</author><author>Normark, S</author></authors></contributors><titles><title>Sequence elements determining ampC promoter strength in E. coli</title><secondary-title>The EMBO Journal </secondary-title></titles><periodical><full-title>The EMBO Journal</full-title></periodical><pages>875-881</pages><volume>1</volume><number>7</number><dates><year>1982</year></dates><urls></urls></record></Cite></EndNote>(Jaurin et al., 1982). A second SNP with a 100% frequency was predicted in isolate ISO_2, although there was only a modest increase in AMC MIC when assessed in MHB. This SNP occurred in cpxA, which encodes a change in amino acid from a proline to leucine at position 177. CpxA is the sensor histidine kinase portion of the envelope stress response two-component system, CpxAR. The CpxAR two-component system is involved in the regulation of several proteins including the efflux pumps acrB, acrD, and eefB PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5TcmluaXZhc2FuPC9BdXRob3I+PFllYXI+MjAxMjwvWWVh
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ADDIN EN.CITE.DATA (Srinivasan et al., 2012) and the outer membrane porins ompF and ompC PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5CYXRjaGVsb3I8L0F1dGhvcj48WWVhcj4yMDA1PC9ZZWFy
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ADDIN EN.CITE.DATA (Batchelor et al., 2005) in a wide range of Enterobacteriaceae including E. coli. Therefore, the SNP predicted by Breseq, and subsequently confirmed via PCR and sequencing, present in the cpxA gene may explain the modest increase in AMC resistance in ISO_2. In LB_5, two synonymous SNPs were predicted with 80-90% frequency in vgrG1, which is part of the type VI secretion system of Gram negative bacteria and a significant component for the delivery of effector molecules to other cells ADDIN EN.CITE <EndNote><Cite><Author>Cianfanelli</Author><Year>2016</Year><RecNum>47</RecNum><DisplayText>(Cianfanelli et al., 2016)</DisplayText><record><rec-number>47</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1542627283">47</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Cianfanelli, F. R.</author><author>Alcoforado Diniz, J.</author><author>Guo, M.</author><author>De Cesare, V.</author><author>Trost, M.</author><author>Coulthurst, S. J.</author></authors></contributors><auth-address>Division of Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, United Kingdom.</auth-address><titles><title>VgrG and PAAR Proteins Define Distinct Versions of a Functional Type VI Secretion System</title><secondary-title>PLoS Pathog</secondary-title></titles><periodical><full-title>PLoS Pathog</full-title></periodical><pages>e1005735</pages><volume>12</volume><number>6</number><keywords><keyword>Amino Acid Sequence</keyword><keyword>Bacterial Proteins/*metabolism</keyword><keyword>Chromatography, Affinity</keyword><keyword>Coculture Techniques</keyword><keyword>Escherichia coli</keyword><keyword>Immunoblotting</keyword><keyword>Mass Spectrometry</keyword><keyword>Proteomics</keyword><keyword>Serratia marcescens/metabolism/*pathogenicity</keyword><keyword>Type VI Secretion Systems/*metabolism</keyword></keywords><dates><year>2016</year><pub-dates><date>Jun</date></pub-dates></dates><isbn>1553-7374 (Electronic)
1553-7366 (Linking)</isbn><accession-num>27352036</accession-num><urls><related-urls><url>;(Cianfanelli et al., 2016). One SNP was found in vgrG1 which mapped to amino acid position 368 (proline) and a second at amino acid 387 (glycine). The SNP in vgrG1, and indeed the only predicted SNP, at amino acid position 368 was also present in AMC-resistant isolate in LB_1 (Table S1) with a frequency of 89.1%. Competitive fitness of AMC-resistant isolates in LB, ISO and M9The relative fitness of three confirmed E. coli 10129 AMC-resistant isolates (ISO_2, LB_2 and LB_5), with identifiable SNPs with 100% frequency and were all from independent lineages were initially assessed competitively in LB with the ancestral isolate. There was a significant increase in the fitness of all three AMC-resistant isolates when assessed in LB. ISO_2 increased by 19.9% (P value = 0.0118; Fig. 3A), LB_2 increased by 14.4% (P value = 0.0463; Fig. 3A) and finally LB_5 increased by 16.6% (P value = 0.0263; Fig. 3A) relative to the E. coli 10129. As we have been able to show that media has an important effect on both the selection of E. coli 10129 AMC-resistant isolates and the MIC of the ancestor and resistant isolates, we determined the relative fitness of the highly resistant and fit LB_2 and LB_5 isolates which contained the same SNP in the AmpC promoter region in three media types in the absence of AMC: LB, ISO and M9. There was no significant difference in relative fitness when LB_5 was assessed in LB, ISO and M9 (P value = 0.4943; Fig. 3C) and no significant difference between the relative fitness of LB_2 and LB_5 when compared in LB (P value = 0.7470; Fig. 3A). However, the relative fitness of LB_2 was significantly different when assessed in M9 media compared with its growth in ISO (P value = 0.0379) and LB (P value = 0.0109) (Fig. 3B). Additionally, there was a significant difference in relative fitness between LB_2 and LB_5 when assessed in either ISO (P value = 0.0216) or M9 (P value = 0.0015) (Fig.3B and 3C). These data suggest that media choice can also affect parative growth rates in urothelial organoidsAs many studies into the evolution or acquisition of AMR and subsequent effects on fitness are ultimately intended to be translated into clinical interventions, we determined whether relative fitness in LB can be used to predict relative growth of the E. coli 10129 AMC-resistant isolates compared with the ancestral isolate in urothelial organoids. Despite relative growth varying among the AMC-resistant isolates, no significant difference in relative growth among all three AMC-resistant isolates was observed (P value = 0.8892) and, in direct contrast to relative fitness assessed in LB, two out of three AMC-resistant isolates grew slower relative to the ancestral isolate. The relative growth of ISO_2 was 5.6% slower compared to the ancestral isolate despite previously being found to have acquired the largest increase in fitness (19.9%) of the AMC-resistant isolates in LB (Fig. 3A and 3D). Although LB_2 and LB_5 increased in fitness in LB (14.4% and 16.6%, respectively), and in the case of LB_5 an increase in fitness in ISO and M9 as well, LB_2 grew 0.3% faster and LB_5 grew 6.1% slower relative to the ancestral isolate in the urothelial organoid (Fig. 3A-D). This indicates that relative fitness assessed in growth media, and in particular LB, has no predictive value to the relative growth in more biologically relevant environments.Minimum inhibitory concentrations of E. coli 10129 ancestor and AMC-resistant isolates in other media and urineFollowing observed differences between media types in the selection of AMC-resistant isolates of E. coli 10129, we assessed the MIC of the ancestor and AMC-resistant isolates in LB, ISO and M9 to determine whether media affects antimicrobial resistance (Fig. 2). MICs of both the ancestor and AMC-resistant isolates were comparable when assessed in MHB and LB; however, there was a slight increase in MIC in the ancestor isolate and ISO_2, LB_2 and LB_5 when assessed in ISO (Table 3, Fig. 2). There was a considerable decrease in MIC of all isolates when assessed in M9 compared with MHB, LB and ISO (Table 3, Fig. 2), which means the MIC of AMC in M9 (1-2 ?g/ml) is actually lower than the sub-inhibitory concentrations of AMC used to select AMR resistant isolates in M9 and therefore causing cell death instead of selecting spontaneous AMC-resistant mutants (Table 3, Fig. 2). Therefore, the concentration of AMC used to select AMC-resistant isolates in M9 was not sub-inhibitory but rather bactericidal, explaining the observed decrease in cell density after exposure to AMC in M9 (Fig. 1B).In order to make in vitro model systems clinically relevant, more sophisticated model growth environments than an agar plate or tube of media are required. We therefore wanted to ascertain if the observed variability in the growth of the AMC resistant E. coli isolates described above is replicated in urine and with organoids. We found that although the growth of E. coli 10129 was the same or better than that of the highly virulent uropathogenic E. coli strain UTI89 ADDIN EN.CITE <EndNote><Cite><Author>Anderson</Author><Year>2003</Year><RecNum>65</RecNum><DisplayText>(Anderson et al., 2003)</DisplayText><record><rec-number>65</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1550923480">65</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Anderson, G.G.</author><author>Palermo, J.J.</author><author>Schilling, J.D.</author><author>Roth, R.</author><author>Heuser, J.</author><author>Hultgre, S.J.</author></authors></contributors><titles><title>Intracellular Bacterial Bio?lm-Like Pods in Urinary Tract Infection</title><secondary-title>Science</secondary-title></titles><periodical><full-title>Science</full-title></periodical><pages>105-107</pages><volume>301</volume><number>5629</number><dates><year>2003</year></dates><urls></urls><electronic-resource-num>10.1126/science.1084550 </electronic-resource-num></record></Cite></EndNote>(Anderson et al., 2003) in 100% urine (data not shown), pure urine is nutrient-poor and overall growth was not robust enough to determine MIC. We therefore used supernatant derived from the apical chamber of urothelial organoids, which consist of urine enriched with cellular exudate and which is more likely to be representative of the host/pathogen interface in the bladder ADDIN EN.CITE <EndNote><Cite><Author>Horsley</Author><Year>2018</Year><RecNum>44</RecNum><DisplayText>(Horsley et al., 2018)</DisplayText><record><rec-number>44</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1542104740">44</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Horsley, H.</author><author>Dharmasena, D.</author><author>Malone-Lee, J.</author><author>Rohn, J. L.</author></authors></contributors><auth-address>Chronic UTI Group, Centre for Nephrology, University College London, London, UK. h.horsley@ucl.ac.uk.
Chronic UTI Group, Centre for Nephrology, University College London, London, UK.
Chronic UTI Group, Centre for Nephrology, University College London, London, UK. j.rohn@ucl.ac.uk.</auth-address><titles><title>A urine-dependent human urothelial organoid offers a potential alternative to rodent models of infection</title><secondary-title>Sci Rep</secondary-title></titles><periodical><full-title>Sci Rep</full-title></periodical><pages>1238</pages><volume>8</volume><number>1</number><dates><year>2018</year><pub-dates><date>Jan 19</date></pub-dates></dates><isbn>2045-2322 (Electronic)
2045-2322 (Linking)</isbn><accession-num>29352171</accession-num><urls><related-urls><url>;(Horsley et al., 2018). MICs of the ancestor isolate, and the AMC-resistant isolates selected in ISO, assessed in this urine were noticeably lower than those assessed in MHB, LB and ISO, and were more comparable to those assessed in M9 (Table 3, Fig. 2). The MIC of the AMC-resistant isolates selected for in LB assessed in urine was markedly higher than those assessed in M9 and are in fact more comparable to the MICs assessed in MHB. Taken together, these data suggest that culture environment can have an unpredictable effect on MIC.Biofilm productionUrinary tract infection (UTI) isolates often form extensive biofilms on catheter surfaces and we wanted to determine whether the fitness of E. coli 10129 AMC-resistant isolates was accompanied by an increase in biofilm production. While biofilm production varied in the AMC-resistant isolates, there was a significant increase in biofilm production for all AMC-resistant isolates in comparison to the ancestor isolate except for LB_5 (P value = 0.4677), which was previously found to acquire a fitness benefit. There was a significant increase in biofilm production compared with the ancestor isolate in both ISO_2 (1.63-fold increase, P value = 0.0092) and LB_2 (1.90-fold increase, P value = 0.0012) (Fig. 4), even though ISO_2 grew more slowly (5.6%) and LB_2 marginally grew faster (0.3%) than the ancestral isolate in urothelial organoids. Therefore, we found no correlation between the fitness of AMC-resistant isolates in different growth conditions and biofilm production.DiscussionIn this preliminary study, we generated resistant isolates in the laboratory using a fully susceptible Malawian clinical isolate E. coli 10129 PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5NdXNpY2hhPC9BdXRob3I+PFllYXI+MjAxNzwvWWVhcj48
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ADDIN EN.CITE.DATA (Musicha et al., 2017b) by exposing it to AMC in vitro as this is a geographically and clinically relevant first line treatment for Enterobacteriaceae infections ADDIN EN.CITE <EndNote><Cite><Author>Musicha</Author><Year>2017</Year><RecNum>56</RecNum><DisplayText>(Musicha et al., 2017a)</DisplayText><record><rec-number>56</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1549544443">56</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Musicha, Patrick</author><author>Cornick, Jennifer E.</author><author>Bar-Zeev, Naor</author><author>French, Neil</author><author>Masesa, Clemens</author><author>Denis, Brigitte</author><author>Kennedy, Neil</author><author>Mallewa, Jane</author><author>Gordon, Melita A.</author><author>Msefula, Chisomo L.</author><author>Heyderman, Robert S.</author><author>Everett, Dean B.</author><author>Feasey, Nicholas A.</author></authors></contributors><titles><title>Trends in antimicrobial resistance in bloodstream infection isolates at a large urban hospital in Malawi (1998–2016): a surveillance study</title><secondary-title>The Lancet Infectious Diseases</secondary-title></titles><periodical><full-title>The Lancet Infectious Diseases</full-title></periodical><pages>1042-1052</pages><volume>17</volume><number>10</number><dates><year>2017</year></dates><isbn>14733099</isbn><urls></urls><electronic-resource-num>10.1016/s1473-3099(17)30394-8</electronic-resource-num></record></Cite></EndNote>(Musicha et al., 2017a). We used a single-step procedure in three different media types that are commonly used in evolutionary studies PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5TdXp1a2k8L0F1dGhvcj48WWVhcj4yMDE3PC9ZZWFyPjxS
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ADDIN EN.CITE.DATA (Farrell et al., 2011;Kn?ppel et al., 2017;Suzuki et al., 2017) to select for AMC-resistant E. coli 10129 isolates with AMC-resistance which were then tested for relative fitness compared with the ancestral strain PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5CYXNyYTwvQXV0aG9yPjxZZWFyPjIwMTg8L1llYXI+PFJl
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ADDIN EN.CITE.DATA (Lenski et al., 1991;Farrell et al., 2011;Kn?ppel et al., 2017;Basra et al., 2018). We were able to determine multiple mutations leading to AMC resistance such as well-characterised SNPs in the ampC promoter region PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5TaXU8L0F1dGhvcj48WWVhcj4yMDAzPC9ZZWFyPjxSZWNO
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Tm90ZT4A
ADDIN EN.CITE.DATA (Jaurin et al., 1982;Caroff et al., 2000;Siu et al., 2003;Tracz et al., 2005;Tracz et al., 2007), and a less well defined evidence of AMC resistance including SNPs in cpxA PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5TdXp1a2k8L0F1dGhvcj48WWVhcj4yMDE0PC9ZZWFyPjxS
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ADDIN EN.CITE.DATA (Suzuki et al., 2014;Dean et al., 2018;Wang et al., 2018). Several separate SNPs in the cpxA gene have previously been linked to aminoglycoside ADDIN EN.CITE <EndNote><Cite><Author>Suzuki</Author><Year>2014</Year><RecNum>39</RecNum><DisplayText>(Suzuki et al., 2014)</DisplayText><record><rec-number>39</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1541758673">39</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Suzuki, S.</author><author>Horinouchi, T.</author><author>Furusawa, C.</author></authors></contributors><auth-address>Quantitative Biology Center (QBiC), RIKEN, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan.</auth-address><titles><title>Prediction of antibiotic resistance by gene expression profiles</title><secondary-title>Nat Commun</secondary-title></titles><periodical><full-title>Nat Commun</full-title></periodical><pages>5792</pages><volume>5</volume><keywords><keyword>Anti-Bacterial Agents/classification/*pharmacology</keyword><keyword>Drug Resistance, Multiple, Bacterial/*genetics</keyword><keyword>Escherichia coli/*drug effects/genetics/growth & development/metabolism</keyword><keyword>Gene Expression Profiling</keyword><keyword>*Gene Expression Regulation, Bacterial</keyword><keyword>*Genes, Bacterial</keyword><keyword>Genotype</keyword><keyword>Microbial Sensitivity Tests</keyword><keyword>*Models, Statistical</keyword><keyword>Mutation</keyword><keyword>Phenotype</keyword><keyword>Transcriptome</keyword></keywords><dates><year>2014</year><pub-dates><date>Dec 17</date></pub-dates></dates><isbn>2041-1723 (Electronic)
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ADDIN EN.CITE.DATA (Dean et al., 2018), Ala-183-Gly, Ile-95-Phe, Met-22-Arg and Trp-184-Gly ADDIN EN.CITE <EndNote><Cite><Author>Suzuki</Author><Year>2014</Year><RecNum>39</RecNum><DisplayText>(Suzuki et al., 2014)</DisplayText><record><rec-number>39</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1541758673">39</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Suzuki, S.</author><author>Horinouchi, T.</author><author>Furusawa, C.</author></authors></contributors><auth-address>Quantitative Biology Center (QBiC), RIKEN, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan.</auth-address><titles><title>Prediction of antibiotic resistance by gene expression profiles</title><secondary-title>Nat Commun</secondary-title></titles><periodical><full-title>Nat Commun</full-title></periodical><pages>5792</pages><volume>5</volume><keywords><keyword>Anti-Bacterial Agents/classification/*pharmacology</keyword><keyword>Drug Resistance, Multiple, Bacterial/*genetics</keyword><keyword>Escherichia coli/*drug effects/genetics/growth & development/metabolism</keyword><keyword>Gene Expression Profiling</keyword><keyword>*Gene Expression Regulation, Bacterial</keyword><keyword>*Genes, Bacterial</keyword><keyword>Genotype</keyword><keyword>Microbial Sensitivity Tests</keyword><keyword>*Models, Statistical</keyword><keyword>Mutation</keyword><keyword>Phenotype</keyword><keyword>Transcriptome</keyword></keywords><dates><year>2014</year><pub-dates><date>Dec 17</date></pub-dates></dates><isbn>2041-1723 (Electronic)
2041-1723 (Linking)</isbn><accession-num>25517437</accession-num><urls><related-urls><url>;(Suzuki et al., 2014) whereas the non-synonymous SNP we identified within cpxA in E. coli 10129 was an amino acid change of Pro-Leu at position 177. Complementation of SNPs within cpxA and vrgG in order to determine functionality in AMC resistance will form the basis of further study.In a clinical setting, MICs are standardised and are determined in MHB/cation-adjusted MHB according to either CLSI ADDIN EN.CITE <EndNote><Cite><Author>CLSI</Author><Year>2018</Year><RecNum>53</RecNum><DisplayText>(CLSI, 2018)</DisplayText><record><rec-number>53</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1549405351">53</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Standard">58</ref-type><contributors><authors><author>CLSI </author></authors></contributors><titles><title>Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically</title><secondary-title>11th Ed. CLSI Standard M07</secondary-title></titles><dates><year>2018</year></dates><pub-location>Wayne, Pennsylvania, USA</pub-location><publisher>Clinical and Laboratory Standards Institute</publisher><urls></urls></record></Cite></EndNote>(CLSI, 2018) or EUCAST ADDIN EN.CITE <EndNote><Cite><Author>ISO</Author><Year>2006</Year><RecNum>54</RecNum><DisplayText>(ISO, 2006)</DisplayText><record><rec-number>54</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1549444773">54</key></foreign-keys><ref-type name="Standard">58</ref-type><contributors><authors><author>ISO</author></authors></contributors><titles><title>ISO 20776-1:2006 Clinical laboratory testing and in vitro diagnostic test systems — Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices</title><secondary-title>Part 1: Reference method for testing the in vitro activity of antimicrobial agents against rapidly growing aerobic bacteria involved in infectious diseases</secondary-title></titles><dates><year>2006</year></dates><pub-location> International Organization for Standardization</publisher><urls></urls></record></Cite></EndNote>(ISO, 2006) guidelines. Various evolutionary studies assess MICs in the same media that is subsequently used for selection of spontaneous AMR mutants, such as M9, ISO and LB PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5GYXJyZWxsPC9BdXRob3I+PFllYXI+MjAxMTwvWWVhcj48
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ADDIN EN.CITE.DATA (Hu et al., 2010) or MHB ADDIN EN.CITE <EndNote><Cite><Author>Birosova</Author><Year>2009</Year><RecNum>49</RecNum><DisplayText>(Birosova and Mikulasova, 2009)</DisplayText><record><rec-number>49</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1542987455">49</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Birosova, L.</author><author>Mikulasova, M.</author></authors></contributors><auth-address>Department of Biochemistry and Microbiology, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinskeho 9, 81102 Bratislava, Slovak Republic. piryyy@</auth-address><titles><title>Development of triclosan and antibiotic resistance in Salmonella enterica serovar Typhimurium</title><secondary-title>J Med Microbiol</secondary-title></titles><periodical><full-title>J Med Microbiol</full-title></periodical><pages>436-41</pages><volume>58</volume><number>Pt 4</number><keywords><keyword>Anti-Bacterial Agents/*pharmacology</keyword><keyword>Anti-Infective Agents, Local/*pharmacology</keyword><keyword>Ciprofloxacin/pharmacology</keyword><keyword>*Drug Resistance, Bacterial</keyword><keyword>Gene Expression Regulation, Bacterial</keyword><keyword>Microbial Sensitivity Tests</keyword><keyword>Mutation</keyword><keyword>Salmonella typhimurium/*drug effects/genetics</keyword><keyword>Triclosan/*pharmacology</keyword></keywords><dates><year>2009</year><pub-dates><date>Apr</date></pub-dates></dates><isbn>0022-2615 (Print)
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ADDIN EN.CITE.DATA (Hu et al., 2010) and LB ADDIN EN.CITE <EndNote><Cite><Author>Birosova</Author><Year>2009</Year><RecNum>49</RecNum><DisplayText>(Birosova and Mikulasova, 2009)</DisplayText><record><rec-number>49</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1542987455">49</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Birosova, L.</author><author>Mikulasova, M.</author></authors></contributors><auth-address>Department of Biochemistry and Microbiology, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinskeho 9, 81102 Bratislava, Slovak Republic. piryyy@</auth-address><titles><title>Development of triclosan and antibiotic resistance in Salmonella enterica serovar Typhimurium</title><secondary-title>J Med Microbiol</secondary-title></titles><periodical><full-title>J Med Microbiol</full-title></periodical><pages>436-41</pages><volume>58</volume><number>Pt 4</number><keywords><keyword>Anti-Bacterial Agents/*pharmacology</keyword><keyword>Anti-Infective Agents, Local/*pharmacology</keyword><keyword>Ciprofloxacin/pharmacology</keyword><keyword>*Drug Resistance, Bacterial</keyword><keyword>Gene Expression Regulation, Bacterial</keyword><keyword>Microbial Sensitivity Tests</keyword><keyword>Mutation</keyword><keyword>Salmonella typhimurium/*drug effects/genetics</keyword><keyword>Triclosan/*pharmacology</keyword></keywords><dates><year>2009</year><pub-dates><date>Apr</date></pub-dates></dates><isbn>0022-2615 (Print)
0022-2615 (Linking)</isbn><accession-num>19273638</accession-num><urls><related-urls><url>;(Birosova and Mikulasova, 2009). We found that media can have a direct effect on the MIC, most notably an obvious drop in MIC when assessed in M9 compared to MHB, LB and ISO, and there were also more subtle differences between the latter three media. A recent study has found that supplements added to MHB, such as human serum or lung surfactant, directly affected the activity of antibiotics and therefore the determination of the MIC ADDIN EN.CITE <EndNote><Cite><Author>Kavanagh</Author><Year>2018</Year><RecNum>59</RecNum><DisplayText>(Kavanagh et al., 2018)</DisplayText><record><rec-number>59</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1549570948">59</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Kavanagh, A.</author><author>Ramu, S.</author><author>Gong, Y.</author><author>Cooper, M.A.</author><author>Blaskovich, M.A.T</author></authors></contributors><titles><title>Effects of Microplate Type and Broth Additives on Microdilution MIC Susceptibility Assays</title><secondary-title>Antimicrobial Agents and Chemotherapy</secondary-title></titles><periodical><full-title>Antimicrobial Agents and Chemotherapy</full-title></periodical><pages>e01760-18</pages><volume>63</volume><number>1</number><dates><year>2018</year></dates><urls></urls><electronic-resource-num>10.1128/AAC.01760-18 </electronic-resource-num></record></Cite></EndNote>(Kavanagh et al., 2018). While we understand that the use of MHB in a clinical setting is entirely to ensure standardisation across different clinical laboratories to be able to monitor AMR, there is currently no comparable standardisation to use for evolutionary studies. We have found that media not only has a direct effect on the selection of spontaneous mutants conferring resistance to AMC in our experiments, but it also affects the competitive fitness of AMC-resistant strains selected for in LB, as has previously been acknowledged PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5MaW48L0F1dGhvcj48WWVhcj4yMDE4PC9ZZWFyPjxSZWNO
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ADDIN EN.CITE.DATA (Maharjan and Ferenci, 2017;Lin et al., 2018). Although both LB_2 and LB_5 contain the same mutation in the ampC promoter region which conferred resistance to AMC ADDIN EN.CITE <EndNote><Cite><Author>Caroff</Author><Year>2000</Year><RecNum>8</RecNum><DisplayText>(Jaurin et al., 1982;Caroff et al., 2000)</DisplayText><record><rec-number>8</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1516612220">8</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Caroff, N.</author><author>Espaze, E.</author><author>Gautreau, D.</author><author>Richet, H.</author><author>Reynaud, A.</author></authors></contributors><titles><title>Analysis of the Effects of -42 and -32 ampC Promoter Mutations in Clinical Isolates of Escherichia coli Hyperproducing AmpC</title><secondary-title>Journal of Antimicrobial Chemotherapy</secondary-title></titles><periodical><full-title>Journal of Antimicrobial Chemotherapy</full-title></periodical><pages>783-788</pages><volume>45</volume><dates><year>2000</year></dates><urls></urls></record></Cite><Cite><Author>Jaurin</Author><Year>1982</Year><RecNum>7</RecNum><record><rec-number>7</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1516612212">7</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Jaurin, B</author><author>Grundstrom, T</author><author>Normark, S</author></authors></contributors><titles><title>Sequence elements determining ampC promoter strength in E. coli</title><secondary-title>The EMBO Journal </secondary-title></titles><periodical><full-title>The EMBO Journal</full-title></periodical><pages>875-881</pages><volume>1</volume><number>7</number><dates><year>1982</year></dates><urls></urls></record></Cite></EndNote>(Jaurin et al., 1982;Caroff et al., 2000), it is likely that either LB_5 has a compensatory mutation that allows it to grow better in ISO and M9 or there is a mutation elsewhere within the genome of the strains which results in a negative (for LB_2) or positive epistatic (for LB_5) interaction(s) only detectable during growth in ISO and M9 (Table S1). It is noteworthy that any compensatory mutations or mutations resulting in epistatic interactions identified in the genome would not be able to be linked to the growth phenotype unless comparative fitness assays were carried out in the different media. A similar phenomenon has previously been observed when competitive fitness was assessed in LB, M9 and tryptone soya broth following adaption of E. coli K12 MG1655 to LB to select for mutations that compensate for a loss of fitness following a mutation in either gyrA or marR ADDIN EN.CITE <EndNote><Cite><Author>Basra</Author><Year>2018</Year><RecNum>45</RecNum><DisplayText>(Basra et al., 2018)</DisplayText><record><rec-number>45</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1542125262">45</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Basra, P.</author><author>Alsaadi, A.</author><author>Bernal-Astrain, G.</author><author>O'Sullivan, M. L.</author><author>Hazlett, B.</author><author>Clarke, L. M.</author><author>Schoenrock, A.</author><author>Pitre, S.</author><author>Wong, A.</author></authors></contributors><auth-address>Department of Biology, Carleton University.
School of Computer Science, Carleton University.
Research Computing Services, Carleton University.</auth-address><titles><title>Fitness tradeoffs of antibiotic resistance in extra-intestinal pathogenic Escherichia coli</title><secondary-title>Genome Biol Evol</secondary-title></titles><periodical><full-title>Genome Biol Evol</full-title></periodical><keywords><keyword>Antibiotic resistance</keyword><keyword>compensatory evolution</keyword><keyword>epistasis</keyword><keyword>trade-off</keyword><keyword>whole genome sequencing</keyword></keywords><dates><year>2018</year><pub-dates><date>Feb 7</date></pub-dates></dates><isbn>1759-6653 (Electronic)
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ADDIN EN.CITE.DATA (Maharjan and Ferenci, 2017;Lin et al., 2018;Yokoyama et al., 2018), highlights that mutations, and the associated fitness effects, can have different consequences in different environments.To determine if any of the data we derived from growth in laboratory media could have predictive value in a more clinically relevant environment, we assessed growth in urine and urothelial organoids. We found that MICs of the E. coli 10129 AMC-resistant isolates, when determined in urine, were not directly comparable to any media type we tested in this study and, more importantly, that the relative fitness of the AMC-resistant isolates was not comparable to relative growth in urothelial organoids. The human bladder urothelial organoid model system used in this study was deliberately chosen as it closely represents the stratified and differentiated bladder urothelium, which is a common, and clinically important, in vivo environment for E. coli ADDIN EN.CITE <EndNote><Cite><Author>Horsley</Author><Year>2018</Year><RecNum>44</RecNum><DisplayText>(Horsley et al., 2018)</DisplayText><record><rec-number>44</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1542104740">44</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Horsley, H.</author><author>Dharmasena, D.</author><author>Malone-Lee, J.</author><author>Rohn, J. L.</author></authors></contributors><auth-address>Chronic UTI Group, Centre for Nephrology, University College London, London, UK. h.horsley@ucl.ac.uk.
Chronic UTI Group, Centre for Nephrology, University College London, London, UK.
Chronic UTI Group, Centre for Nephrology, University College London, London, UK. j.rohn@ucl.ac.uk.</auth-address><titles><title>A urine-dependent human urothelial organoid offers a potential alternative to rodent models of infection</title><secondary-title>Sci Rep</secondary-title></titles><periodical><full-title>Sci Rep</full-title></periodical><pages>1238</pages><volume>8</volume><number>1</number><dates><year>2018</year><pub-dates><date>Jan 19</date></pub-dates></dates><isbn>2045-2322 (Electronic)
2045-2322 (Linking)</isbn><accession-num>29352171</accession-num><urls><related-urls><url>;(Horsley et al., 2018). The availability of cellular constituents secreted into the urine from the epithelial cells of the urothelial organoid, as well as nutrients and attachment factors present on the urothelial surface itself, along with its elaboration of a glucosaminoglycan layer, are all likely to have a direct effect on bacterial growth, therefore affecting the growth and MIC of bacteria, neither of which would be accurately predicted by growth in laboratory media. As biofilm-producing bacteria are a major cause of catheter-associated UTIs PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5TYWJpcjwvQXV0aG9yPjxZZWFyPjIwMTc8L1llYXI+PFJl
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ADDIN EN.CITE.DATA (Sabir et al., 2017), of which E. coli is the amongst the most common cause PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5TYWJpcjwvQXV0aG9yPjxZZWFyPjIwMTc8L1llYXI+PFJl
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ADDIN EN.CITE.DATA (Sabir et al., 2017), we sought to determine whether there is any association between biofilm formation, MICs and fitness assessed in the different growth media, including urine and urothelial organoids. We found no correlation between fitness, MICs and biofilm production in our E. coli 10129 derivative strains suggesting that this lack of predictive value between MIC and fitness in different media is also evident between MIC, fitness and biofilm forming ability for this strain. We acknowledge there are limitations to this study, not least the limited amount of different E. coli isolates, bacterial strains and antimicrobials tested, and the limited number of AMC-resistant derivative isolates tested following selection. Whilst we had a very limited number of fully susceptible clinical isolates from Malawi to choose from PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5NdXNpY2hhPC9BdXRob3I+PFllYXI+MjAxNzwvWWVhcj48
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ADDIN EN.CITE.DATA (Manges et al., 2001;Yamaji et al., 2018).ConclusionsThese results highlight the importance of not only assessing aspects of evolutionary studies in several media types, but also the importance of using in vitro model systems, such as urothelial organoids, to ensure the phenomenon that is observed occurs in a range of different settings rather than in a single environment. For example, a diagnostic laboratory using MHB to determine sensitivity of the ISO-derived strains in this study would have deemed them resistant to AMC, whereas the organoid result would report that the strains were actually sensitive. If the MHB-derived information were deployed in a clinical setting, this could lead to incorrect treatment of the patient, resorting to a less-optimal alternative or delaying the administration of a more suitable drug. Indeed, in clinical experience, mismatches between predicted and real-world response outcomes of antibiotic treatment have been observed; our results could in part explain these discrepancies.AcknowledgementsThis work was supported by the Antimicrobial Resistance Cross-Council Initiative through a grant from the Medical Research Council, a Council of UK Research and Innovation, and the National Institute for Health Research. This award is part of the EDCTP2 programme supported by the European Union [Grant numbers MR/R015074/1, MR/S004793/1]. This manuscript has been released as a Pre-Print on BioRxiv ADDIN EN.CITE <EndNote><Cite><Author>Hubbard</Author><Year>2019</Year><RecNum>75</RecNum><DisplayText>(Hubbard et al., 2019)</DisplayText><record><rec-number>75</rec-number><foreign-keys><key app="EN" db-id="a0aw9dezoe9xpsefeep5a22wvezp2vwsps00" timestamp="1556794890">75</key><key app="ENWeb" db-id="">0</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Hubbard, Alasdair T. M.</author><author>Jafari, Nazila V.</author><author>Feasey, Nicholas A.</author><author>Rohn, Jennifer L.</author><author>Roberts, Adam P.</author></authors></contributors><titles><title>Effect of environment on the evolutionary trajectories and growth characteristics of antibiotic resistant Escherichia coli mutants</title><secondary-title>BioRxiv</secondary-title></titles><periodical><full-title>BioRxiv</full-title></periodical><pages>1-37</pages><dates><year>2019</year></dates><urls></urls><electronic-resource-num>10.1101/262691</electronic-resource-num></record></Cite></EndNote>(Hubbard et al., 2019).Accession numbersThis assembled genome of the ancestral isolate, E. coli 10129, has been deposited at GenBank under the accession SIJF00000000. The version described in this paper is version SIJF01000000. 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ADDIN EN.CITE.DATA (Musicha et al., 2017b)ISO_2AMC-selected derivative of E. coli 10129 selected for in Iso-sensitest brothThis studyISO_9AMC- selected derivative of E. coli 10129 selected for in Iso-sensitest brothThis studyISO_10AMC- selected derivative of E. coli 10129 selected for in Iso-sensitest brothThis studyLB_1AMC- selected derivative of E. coli 10129 selected for in LB brothThis studyLB_2AMC- selected derivative of E. coli 10129 selected for in in LB brothThis studyLB_5AMC- selected derivative of E. coli 10129 selected for in in LB brothThis studyTable 2: Primer names and primer sequences for confirmation of the SNPs in the cpxA gene and ampC promoter region of E. coli 10129 ancestor and AMC-resistant isolates as predicted by breseq.TargetPrimer NamePrimer SequencecpxA SNPEc10129_F1TGAACGCAGCGAAATGCAGAEc10129_R1GTGCGCAGTTCGTGAGAGATampC promoter SNPEc10129_F2GGTATTCTGCTGCCGCTAGGEc10129_R2CCGGGGATCTTTTGTTGCTCTable 3: Minimum inhibitory concentrations of the E. coli 10129 ancestor and AMC-resistant isolates assessed in MHB, LB, ISO, M9 and urine following the CLSI guidelines.MediumE. coli 10129ISO_2ISO_9ISO_10LB_1LB_2LB_5MHB4-8 ?g/ml16 ?g/ml8-16 ?g/ml8-16 ?g/ml32 ?g/ml64 ?g/ml64 ?g/mlLB4-8 ?g/ml16 ?g/ml---64-128 ?g/ml64 ?g/mlISO8 ?g/ml16-32 ?g/ml---128 ?g/ml64-128 ?g/mlM91-2 ?g/ml2-4 ?g/ml---32 ?g/ml16 ?g/mlUrine2 ?g/ml4 ?g/ml---64 ?g/ml64 ?g/mlTable 4: Summary table of relative fitness assessed of the E. coli 10129 AMC-resistant derivatives compared to the ancestral isolate in LB, ISO, M9 and urothelial organoids, biofilm production measured as optical density at 550nm and the nucleotide, amino acid position and gene in which there were identified SNPs. Error represents the standard error of the mean.ISO_2LB_2LB_5LB19.9 ± 5.814.4 ± 6.416.6 ± 0.5ISO-8.7 ± 2.920.1 ± 1M9--6.6 ± 0.718.6 ± 3.2Urothelial organoids-5.6 ± 9.90.3 ± 12.8-6.1 ± 7.4Biofilm production0.101 ± 0.0080.118 ± 0.10.054 ± 0.002SNPCCG→CTGGTC→GACGTC→GACAmino acid positionP177LV111DV111DGenecpxAampC protomoterampC protomoterFigure LegendsFigure 1: Difference in log CFU/ml of E. coli 10129 (A) after growth in LB, ISO and M9 and (B) following 24-hour exposure with sub-inhibitory concentrations of AMC in LB, ISO and M9 compared to the initial inoculum. Error bars represent standard error of the mean.Figure 2: Circular barplot chart displaying the MIC values of the E. coli 10129 ancestor with three evolved derivative strains. The inner circle shows which bars belong to which strains and which media the MICs were tested. Clinical breakpoint for sensitivity and resistance are 8 ?g/ml and 16 ?g/ml respectively and are indicated by a circle. The scale represents the MIC value. Figure 3: Relative fitness and growth of E. coli isolates; error bars represent standard error of the mean A; Relative fitness of E. coli 10129 AMC-resistant isolates ISO_2, LB_2 and LB_5 compared to the ancestral isolate in LB B; Relative fitness of E. coli 10129 AMC-resistant isolate LB_2 in LB, ISO and M9 compared to the ancestral isolate in the absence of AMC. C; Relative fitness of E. coli 10129 AMC-resistant isolate LB_5 in LB, ISO and M9 compared to the ancestral isolate in the absence of AMC D; Relative fitness of E. coli 10129 AMC-resistant isolates ISO_2, LB_2 and LB_5 compared to the ancestral isolate in urothelial organoids.Figure 4: Biofilm production of E. coli 10129 ancestor and AMC-resistant isolates in M9. Error bars represent standard error of the mean. ................
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